Handbook of Enology
Volume 1
The Microbiology of Wine and Vinifications
2 nd Edition
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Handbook of Enology
Volume 1
The Microbiology of Wine and Vinifications
2 nd Edition
Pascal Ribe>eau-Gayon
Denis Dubourdieu
Bernard Doneche
Aline Lonvaud
Faculty of Etiology
Victor Segalen University of Bordeaux If, Talence, Fit
Original iran.sl.mon by
Jeffivy M. Branca. Jr.
Wine maker
M.S., Faculty i>f Enology. University of Bonleaux II
i translated by
CJ/ristiiie Rythlewski
Atfiitaine Traduction, Bonleaux, Fri
John Wiley & Sons, Ltd
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Contents
Remarks Concerning Ihc Expression of Certain Parameters of Musi and Wine Composition vii
Preface K> Ihc First Edition ix
Preface K> Ihc Second Edition xiii
1 Cytology. Taxonomy aid Ecology of Grape and Wine YcasR 1
2 Biochemistry of Alcoholic Ecrmcntioou and Mclibolic Pathways of Wine Ycass 53
3 Conditions of Yeast Dcvclopmcnl 79
4 Lactic Acid Bacteria 1 15
5 Metabolism of Lactic Acid Bacteria 139
6 Lactic Acid Baclcria Dcvclopmcnl in Wine 161
7 Acetic Acid Bacteria 183
8 The Use of Sulfnr Dioxide in Must and Wine Treatment 193
9 Pioducls and Methods Complementing Ihc Effect of Sulfnr Dioxide 223
10 The Grape and ifc Maturation 241
11 Harvcsl and Prc-EcmicnEiuOn Treatments 299
12 Red Wincntaking 327
13 While Wincmaking 397
14 Other Wincmaking Mclhods 44S
Index 481
Remarks Concerning the Expression
of Certain Parameters of Must
and Wine Composition
UNITS
Metric system units of length (m). volume (1) and
weight (g) arc exclusively used The conversion of
metric nnit> Ion Imperial units (Inches, feci, gal-
lons, ponds, clc.) can be Ibind In the folbwing
etiological work: Principles and practices cfwine-
itKik/ng.R B . Bonltoi.V.L. Singleton. L.F. BLsson
and K.I!. Kunkcc. 1995. The Chapman & Hall
Etiology Libiaiy. New Yort.
EXPRESSION OF TOTAL ACIDITY
AND VOLATILE ACIDITY
Although EC regulations recommend the expres-
sion of total acklity in the equivalent weight of tar-
taric acid, the French ensuni is u give this expres-
sion in the equivalent weight of sulfuric acid. The
morc corrcct expression in in 111 (equivalents per
IlKr has not been embraced in France. The expres-
sion of total and volatile acidity in the equivalent
weight of sulfuric acid has been used predomi-
nantly throughout these works. In certain cases, the
corresponding weight in tartaric acrd.ofkrn ised in
other countries, has been given.
Using the weight of the milliequivalcnt of the
various acids, the below table permit! the conver-
sion from one expression to another
More particularly, to convert from total acidity
expressed in H.S() 4 to its expression in tartaric
acid, add half of the valnc to the original value
14 g/l H;SOj — 6g/l tartaric acid) In the other
direction a third of the value mist be subtracted.
The Ficnch also continue to express votulite
acidity in equivalent weight of sulfuric acid. Mote
generally, in other countries, volatile acidity Is
Desired Expression
Known Expression
tmqfi
HiSOj
tartaric acid
tft
acetic acid
mqfl
1.00
0.049
0075
0.060
g/l H ? S0 4
20.40
1.00
153
122
g/l tartaric arid
UJJ
0.65
1.00
g/l acetic mill
16.67
0.82
1.00
Multiplier u> pass from one expression of total or rolatile acidity lo another
Remarks Concerning the Expression of Certain Parameters of Mum and Wine Composite
expressed in acetic acid. Il Is rarely expressed
Id millicquivalcnft per liter. Tic below tabic also
allows simple conversion from one expression fc>
another.
The express [i m in acetic acKI Is approximately
2&i higher than in sulfuric acid.
EVALUATING THE SUGAR
CONCENTRATION OF MUSTS
This measurement is important for tracking grape
maturation, fermentation kinetic and if necessary
determining the eventual need forchaptali/arion
This measurement is always determined by
physical. densinctric or icfrac lame trie analysis.
The expression of the results cat be given acconl-
ing to several scales: some arc rarely nsed. i.e.
degree Baantf and degree Occhslc. Presently, two
systems exist (Section 10.4.3):
1. The potential alcohol content Wire tilctxmet-
nupie potential or TAP. in French) of musts
can be read directly on equipment, which is
graduated using a scale corresponding to 175
or IT g/l of sugar for i'f volume of akohol.
Today, the BC recommends using 16.83 g/l as
the conversion factor. The mustimctcr' is a
hydrometer containing two graduated scales:
one expresses density and the other gives a
direct reading of the TAP. Different methods
varying in precision exist to calculate the TAP
fiont a density reading. These methods lake var-
ious clement* of must compositKin into account
<Boilioi <■<()/.. 1995).
2. Degree Brix expresses the pcrccniagc of sugar
in weight By multiplying degree Brix by 10.
the weight of sugar in I kg. or slightly less
than I liter, of must is obtained A conversion
table between degree Brix and TAP exists in
Section 10.4.3 or this book. 17 degrees Brix
correspond to an approximate TAP of Hfi and
20 degrees Brix correspond to a TAP of about
1 2'i Within the alcohol range most relevant m
enology. degree Brix can be multiplied by 10
and then divided by 17 to obtain a fairly good
approximation of the TAP.
In any case, the determination of the Brix or TAP
of a must is approximate First of all. il Is not
always possible to obtain a representative grape
or must sample for analysis Secondly, alihoagb
physical, dcusimctric or refractomciric mcasarc-
mcn&arc extremely prccBC and rigorously express
the sagar concentration of a sugar and water mix-
lire, these mcasurcmcnbi arc affected by other sub-
stances released into the sample from the grape
and other sources. Furthermore, the concentrations
of these substances are different for every grape
or grape must sample. Finally, the conversion rale
of sagar inlo alcohol (approximately 17 to 18 g/l)
varies and depends on fermentation conditions and
yeast properties. The widespread use of selected
yeast strains has lowered the sugar ct
Measurements Using Visible
and Ultraviolet Spectrometry
The measurement of optic density, absorbance. is
widely used to determine wine color (Volume 2.
Section 64.5) and total phenolic compounds con-
centration (Volume 2. Section 6.4 I) In these
works, the optic density is noted as OD. OD 420
(yellow). OD 520 ( red). OD 620 ( Not) orOD 280
(absorption in ultraviolet spectrum) to indicate the
optic density al the indicated wavelengths.
Wine color intensity is expressed as:
CI = OD 420-F OD 520-F OD 620.
Or is sometimes expressed in a more simplified
fom: CI = OD 420 + OD 520.
Tint is expressed as:
_ OP 420
~OD520
The total phenolic compound concentration is
expressed by OD 280.
The analysis methods are described in Chapter 6
of HniHlbtkit tf Etiology MAtmie 2. The Chemistry
r;T HVuf .
Preface to the First Edition
Wine has probably Inspired nunc research and
publications than any oilier beverage or food In
fad. through ihcir passion for winc.grcal scientists
have not only eonlributcd to ibe dcvck>pn>cnt of
practical cnology bui have also made discoveries
in ihc general tickl or science.
A forerunner of modern cnology. Louis Pasteur
developed simplified contagious infection mod-
eLs for humans and animals based on lus obser-
vations of wine spoilage. The following quote
clearly expresses his Ihcoiy in his own words:
when profound alterations of beer and wine arc
observed because these liquids have given refuge
to microscopic organisms, introduced invisibly and
accidentally into the medium when: they then
proliferate, how can one not be obsessed by the
thought that a simitar phenomenon can and must
sometimes occur in humans and animabt.'
Since Ihc 19th century, our understanding of
wine, wine composition and wine transformations
has greatly evolved in function of advances in rel-
evant scientific fields ic. chemistry. biochcmisUy.
microbiology. Each applied development has lead
to better control of wincmaking and aging con-
ditions and of course wine quality. In onler to
continue this approach, researchers and wincmak-
ers must strive to remain up to date with the latest
scientific and technical developments in cnology.
For a long lime, the Bordeaux school of cnology
was largely responsible for the communication of
progress in cnology through the publication of
numerous works (8 (ranger Publications and later
Do nod Publications):
Wine Analysis U. Gayonand J. Laborac(l912):
Treatise on Enology J. Ribctran-Gayon (1949):
Wine Analysis J. Ribc'icau-Cayon and E. Pcynaud
t!947and I958X Treatise m Enology (2 Wilumcs)
J. RitWrcau-Gayon and E. Peynand (I960 and
19611. Woe ami Winemaking E. Pcynaud (1971
and 198 1 ); Wine Science ami technology (4 volu-
mes) J. Ribcrcan-Gayot. E. Pcynaud. P. Ritrfrcau-
Gayon and P. Sudraud 1 1975- 1982).
Hot an understanding of current advances in
cnokigy. the authors propose this book Hamlbooi
if Enology Witiene I: The Microbiology <jf Wine
ami Mm'/k'aluna and the second volume of the
Hamlhoak of Enology Wihinie 2 The Chemistry of
Wine: Stabiliziiion ami Treatments.
Although written by researchers. Ihc two vol-
umes arc not spccilically addressed to this group
Young researchers may. however, find these books
useful to help situate their research within a par-
ticular field of cnokigy Today, the complexity of
modem cnology does not permit a sole researcher
to explore the entire field.
These volumes arc also of use to student, and
professionals Theoretical interpretations as well
as solutions arc presented In resolve Ihc problems
encountered most often at wineries. The authors
have adapted these solutions to many different sit-
uations and wincmaking methods In older to make
Ihc best use of the informauou contained in these
works, cnologists should have a broad understand-
ing of general scientific knowledge, l-or example.
Ihc undcrstinding and application of molecular
biology and genetic engineering have become
indispensable in Ihc iickl of wine microbiology.
Similarly, structural and quantitative physiochem-
ical analysis methods such as chromatography.
Preface to the First Edilion
NMR and mass spectrometry must now Ik
mastered )■ oidcr to explore wine chemistry.
The goal of these iwo works was not fc> create
an exhaustive bibliography of each subject The
authors strove to choose only the most relevant and
significant publications lo their particular Meld of
research A large nunber of refcicnccs Kt French
cnological researeh has been included in these
works in order to make this information available
to a larger non-French-speaking audience.
In addition, the uufhois have (tied lo convey
a French and more particularly a Bordeaux per-
spective of enology and the an of wincmaking.
The objective of this perspective is lo maximize
the potential quality of grape ciops based on the
specific natural conditions that constitute their icr-
roir'. The role of enology is u> express the char-
acicristics of the giape specific not only to variety
and vineyard practices but also maturation condi-
tions, which are dictated by soil and climate.
It woald. however, be an crmr to think that the
worlds greatest wines arc exclusively a rcsnlt of
tradition, established by exceptional natural con-
ditions, and thai only the most ordinary wiles,
produced in giant processing facilities, can ben-
clil from scientific and technological progress.
Certainly, these facilities do benefit the most from
high performance installations and antomalion of
operations. Yet. history has unequivocally shown
thai the most important caokrgical developments
in wine quality (for example. italolaclK fermenta-
tion) have teen discovered in ultra premium wines.
The corresponding techniques were then applied to
less prestigious products
High performance technology is indispensable
for the production of great wines, since a lack
of control of wincmaking parameters can easily
compromise their quality, which wonkl be lessor
a problem with lower quality wines.
The word vindication' has been used in Ibis
work and is part of the technical language of
the French tradition of wincmaking. Vindication
describes the tiisl phase of wincmaking. It com-
prises all technical aspects from grape maturity
and harvest to the end of alcoholic and some-
times malofciciic fermentation The second phase
of wincmaking wincmalu ration, stabilization and
treatments' is completed when the wine Is bolUed.
Aging specifically refers to the transformation of
bottled wine.
This distinction of two phases Is certainly the
rcsnlt of commercial practices Traditionally in
France, a vine glower farmed the vineyard and
transformed grapes into an unfinished wine. The
wine merchant transferred the bulk wine to his cel-
lars, finished the wine and marketed the product,
preferentially before bottling. Even though most
wines are now bottled at the winery, these long-
standing practices have maintained a distinction
between wine grower enology and wine mer-
chant enology . In countries with a more recent
viticuliaral history. generally English speaking, the
vine giowcr is responsible for wincmaking and
wine sales. For this reason, the Anglo-Saxon tradi-
tion speaks of wincmaking. which covers all oper-
ations from harvest reception lo bottling.
In Ihesc works, the distinction between vinifi-
eatron and stibilizaikm and treatments' has been
maintained, since the first phase primarily concerns
mKrobtotogy and the second chemistry. Ii Ihls
manner, the Individual operations coukl be linked
to iheir particular sciences. There are of coarse lim-
its lo this approach Chemical phenomena occur
during vindication, the stabilization of wines dur-
ing storage includes the prevention of microbial
contamination.
Consequently, the description of Ihc different
steps of enology docs not always obey logic as
precise as the lilies of these works may lead
to believe. For example, microbial contamination
during aging and storage are covered in Vol-
ume 1 The antiseptic properties of SO. incited the
description of its use in the same volume This line
of reasoning lead to Ihc descripttou of the antioxi-
dant related chemical properties of this compound
in Ihc same chapter as well as an explanation of
adjuvants to snlfur dioxide: sorbK acid (antisep-
tic) and ascorbic acid (antioxidant). In addition,
lie on Ices aging of white wines and the result-
ing chemical transformations cannot be separated
from vindication and arc therefore also covered
in Volume I . Finally, our understanding of pheno-
lic compounds in red wine Is based on complex
chemistry. All aspects related to Ihc nature of the
Preface K> Ibc First Fdition
corresponding substances, their propcrticsaid ihclr
evolution during grape maluiurion. vinifkaltjn and
aging arc therefore covered in Volume 2.
These works only discuss the principles of
equipment used for varions cnotogical operations
and their effect on product quality For example.
Kinpcralirc control systems. dcstcmmciN. crushers
and presses as well as tillers, inverse osmosis
machines and ion exchangers arc nol described in
detail. Bollling is noi addressed al all An ln-dcplh
description of cnological equipment would merit a
detailed work dedicated to the sabject
Wine lasting, another essential rote of the
wincmakcr. is not addressed in Ihese works.
Many related publications are. however, readily
available Filially, wine analysis Is an essential tool
lhat a wincmakcr should master It is. however, not
covered in these works except in a few particular
cases i.e. phenolic compounds, whose different
families ate oficn defined by analytical criteria
Thc authors thank the following people who
nave contributed to Ibc creation of this work:
J.F. Casts Lncas. Chapter 14. Sherry. A. Brtgl-
rani. Chapter 14. Sweet wines: J.N. dc Almeida.
Chapter 14. Port wines. A. Manjcai. Chapter 14.
Champagne. C Poupot for the preparation of
material in C hapten* 1. 2 and 13. Miss F. l.nyc-
Tanct for her help with typing.
They also thank Madame B Masctefin paitica-
lar for her Important part in the typing, preparation
n of the final manuscript.
Pascal Ribtficau-Cayon
Bordeaux
Preface to the Second Edition
The rwo-volomc Etiology Handbook was pub-
lished simultaneously In Spanish. French, and 1 ui-
ian in 1999 and has been reprinted several limes.
The Handbook has apparently been popular with
students as an educational reference book, as well
as with wincmakcrs. as a source of practical solu-
tions b their specific technical pioblcms and sci-
entific explanations of the phenomena involved
ll was felt appropriate at tins stage Id prepare
an updated, reviewed, corrected version, including
the latest cnological knowledge, to reflect the many
new research findings in this very active tickl The
outline and design of both volumes remain the
same Some chapters have changed relatively little
as the auihois decided there had not been any sig-
nificant new developments, while othcis have been
modified much more extensively, either to clarify
and improve the txl. or. more usually, to include
new icscaich findings and their practical applica-
tions Entirely new sections have been inserted in
some chapters.
We have made every effort to maintain the same
approach as we did in the first edition, reflecting
the ethos of enotogy research in Bordeaux. We use
indisputable scientific evidence in microbiology,
biochemistry, and chemistry to explain the details
of mechanisms involved in grape ripening, fermen-
tations and other vvincmaking operations, aging,
and stabilization The aim is to help winentakcrs
achieve greater control over the various stages in
wincmaking and choose the solution best suited
to each situation Quite remarkably, this scientific
approach, most intensively applied in making the
finest wines, has rcsulkd in an enhanced capac-
ity to bring out the full quality and character of
individual terrain. Scientific wincmaking has not
resulted in standaidi ration or leveling of quality*.
On the contrary, by making it possible to correct
defects and eliminate technical imperfections, it
has revealed the specific qualities of the grapes
harvested in different vineyards, directly related to
the variety and lerroir. more than ever before.
Interest in wine in recent decades has gone
beyond considerations of mere quality and taken
on a truly cultural dimension This has led some
people to promote the use of a variety of tech-
niques that do not necessarily represent significant
progress in vvincmaking Some of these are sim-
ply modified forms of processes thai have been
known for many years. Others do not have a suf-
ficiently reliable scientific interpretation, nor ate
their applications clearly defined. In this Hand-
book, we have only included rigorously tested
techniques, clearly specifying the optimum con-
ditions for their utilization
As in the previous cdiuon. we deliberately
omitted three significant aspects of cnotogy: wine
analysis, lasting, and winery engineering. In view
of their importance, these topics will each be
coveted in separate publications
The authors would like to take the opportunity
of the publication of this new edition of \blumc I
lo thank all those who have contributed to updating
this work:
— Marina Hcly for her work on fermentation
kinetics (Section 3.4) and the production of
volatile acidity (Sections 23.4 and 1425)
— Isabclle Masncuf for her investigation of the
yeasts' mi."-"' 11 supply (Section 3.4.2)
Preface to the Second Edition
(lilies dc Revel for clncKlaling Ibc chemistry
of SOj. particularly, details of combination
reactions (Section 8.4)
Cillcs Masson for ihe section on irac wines
(Section 14.1)
- Cornells Van Lccnwcn lor data on the impact
of vineyard water supply on crape ripening
(Section 10.4.6)
Andre Brugiraid for the section on French
fortified wines— vim ihmx mntrels (Section
14.42)
— Paulo Bams and Joa Ntcolan de AlnKida for
tneir work on Port, Section 14.43)
— Jnsto. F. Casas Lucas for the paragraph on
Sherry (Section 1432)
— Alain Man jean for his In-depth revision of the
section on Champagne (Section 143).
March 17. 2005
Professor Pascal RIBEREAU-GAYON
Corresponding Mcnihcrof the InslltnK
Member of the French Academy of Agriculture
Cytology, Taxonomy and Ecology
of Grape and Wine Yeasts
11 Introduction
1 2 The cell wall
I J The plasm ic membrane
1 .4 The i y lopLisni and its organelles
1 5 The nucleus
1.6 Reproduction anil the yeast biological cycle
1.7 The killer phenomenon
18 Classification at yeast species
1 .9 Identification of wine ycasl strains
1 10 Ecology ofgiapc and wine ycasK
1.1 INTRODUCTION
Man has been making bread and fcniKnlcd lev-
erages since Ihe beginning of iccordcd hLsiory.
Ycl ihe mlc of yeast, in alcoholic fermentation,
particularly In ihe transformation of grapes into
wine. «;is only clearly established in the middle
of Ihe nineteenth century The ancients explained
Ihe boiling during fcmicitalion (from the Lalin
feivere. lo boil) as a reaction between substances
that come inlo contact wilh each other during
crushing. In 1680. a Dutch clolh merchant. Anionic
van Lccuwcnhock, liisl observed ycas& in beer
worf using a microscope that he designed anil
produced. He did not. however, establish a rela-
tionship between these corpuscles and alcoholic
fcmicitalion. It was not until the end of the eigh-
teenth century that Lavoisier began the chemical
study of alcoholic fermentation. Gay-Lussac con-
tinued LavoRicr's research into the next century
Handbook of Hnology: The Microbiology of Wine and Vindications
As early us 1785. Fabron. an ! Lilian scientist, was
the first *> provide an interpretation of Ibc chem-
ical com position of the ferment responsible for
alcoholic fermentation, which he described as a
pian I -animal substance According li> Pabroni. In is
material, comparable lo ibc gluten in flour, was
kKakrd In special utricles, particularly on grapes
and wheal, and alcoholic fermentation occurred
when il came inio contact with sugar in the mist. In
1837. a trench physicist named Charles C;ninanl
dc La Tour proved for the tiist time lhat Ihc ycusi
was a living organism. According lo his findings,
it was capable of multiplying and bckingcd to the
plan! kingdom, its vital activities wen: at Ibc base
of Ihc fermentation of sugar-con tailing liquids.
The Gentian naturalist Schwann con filmed his the-
ory and demonstrated lhat beat and certain chem-
ical piodncls were capable of stopping alcoholic
fermentation He named ihc beer yeast sicker-
p»V;. which means sugar fungus — Staclhimmres
in Latin. In 1838. Mcycn used this nomenclature
for the first lime.
This vltallst or biological viewpoint of the role
of yeasts in alcoholic fermentation, obvious lo
os today, was not readily supported. Lie big and
certain other organic chcmpK were convinced thai
chemkal reactions, not living cellular activity,
were responsible for the fermentation of sugar.
In his famous studies on wine (1866) and beer
(1876). Louis Pasteur gave definitive credibility
lo the vitalrst viewpoint of alcoholic fermentation.
He demonstrated that the ycasls responsible for
spontaneous fermentation of grape must or crushed
grapes came from Ihc surface of the grape:
he isolated several races and species. He even
conceived the notion that the nature of the yeast
carrying out the alcoholic fermentation could
influence the gustatory characteristics of wine. He
also demonstrated the effect of oxygen on the
assimilation of sugar by ycasfr. Louis Pasienr
proved that the yeast produced secondary products
such as glycerol in addition to alcohol and carbon
dioxide.
Since Pasteur, ycasb and akoholic fermen-
-in. 'n have incited a considerable amount of
research, making use of progress in microbiology.
biochemistry and now genetics and molecular
biology.
In taxonomy, scientists define yeast, as unicel-
lular fungi that reproduce by budding and binary
fission. Certain pericellular fungi have a unicellu-
lar stage and arc also grouped with ycasfc Yeasts
form a complex and heterogeneous group found
in three classes of fungi, characterized by their
reproduction mode: the sac fnngi (AsconiyccKs).
the club fungi (Basidiomyccics). and the imper-
fect fnngi (Dcuteromycctcs). The yeasts found on
the surface of Ihc grape and in wine belong lo
Ascomycclcs and Dculcromyeclcs The haplold
spores or ascosporcs of Ihc Ascomycclcs class arc
contained In Ihc ascus. a type of sac made from
vegetative cells. Aspoiifcrous yeasts. Incapable of
sexual reproduction, arc classified with the imper-
fect fungi.
In this first chapter, the morphology, repro-
duction, taxonomy and ecology of grape and
wine ycasrs will be discussed. Cytology Is the
morphological and functional study of the struc-
tural components of the cell (Rose and Harrison.
1991).
I\il j 11* (jipfcrnfiliHc
I'iH 1.1. A ycari cell (Oriilanlia jnd HeikH. 198?)
Cytology, Taxonomy anil Ecology of Grape and Wine Yeasts
Yosts arc the most simple of Ihc cuearyoics.
The yeast cell contains cellular envelopes, a
cytoplasm wild various organelles, and a nucleus
surrounded by a membrane and enclosing Ihc
chromosomes (Figure 1.1). Like all plain cclb>.
Ihc yeasl cell has two cellular envelopes: ihc
cell wall and Ihc membrane. The periplasm*:
space Is Ihc space between ihc cell wall and
the membrane The cytoplasm and Ihc membrane
make ip Ihc protoplasm. The lerm proloplasl
or sphacroplast designates a cell whose cell
wall has been artificially removed. Yeasl cellular
envelopes play an essential role: ihey contribaic
to a SKCCssfal alcoholic fermentation and release
certain constituent which add to ihc resulting
wine s composition. In order to take advantage of
these properties, the wiucmakcr or cnologist must
have a profound knowledge of these organelles
1.2 THE CELL WALL
1.2-1 The General Role
of (he Cell Wall
During the lasl 20 years, researchers (Fleet. 1991:
Kits. 1994: Stratford. 1999. Klis el til.. 2(102) have
grcatiy expanded our knowledge of Ihc yeast cell
wall, which represent. 15-25'.* of the dry weight
of the cell ll essentially consist* of polysaccha-
rides It is a rigid envelope, yet endowed with a
certain elasticity
lis lirsl function Is to protect the cell. Without
its wall, the cell would burst under the internal
osmotic pressure, determined by the composition
of ihc cells environment. Protoplast, placed in
pure water arc immediately lyscd in this manner
Cell wall elasticity can be demonstrated by placing
yeasts, taken during their log phase, in a hypertonic
(NaCI) solution. Their cellular volume decreases
by approximately 5<fA . The cell wall appears
thicker and is almost in contact with the membrane
The cells rcgaiu their initial form after being placed
back into an isotonic medium.
Yet the cell wall cannol be considered an inert,
semi-rigid armor'. On the contrary, il Is a dynamic
and multifunctional organelle Its composition and
functions evolve during the life of the cell, in
response to environmental factors. In addition lo
its protective role, the cell wall gives Ihc cell
its panic nlar shape through its maciomolccular
Olgani<ioa. It is also Ihc site of molecules
which determine certain ccllutir interactions such
as sexual union, fhxculation. and the killer
factor, which will be examined in detail later in
this chapter (Section 1.7). Fiially. a number of
enzymes, generally hydrolases, arc connected lo
the cell wall or situated in Ihc pciiplasmic space
Their substrates arc nutritive substances of the
environment and the macromolccules of the cell
wall itself, which is constantly reshaped during
cellular morphogenesis.
1 .2.2 The Chemical Structure
and Function of the Parietal
Const iluenfs
The yeast cell wall is nude up of two prin-
cipal constituents: fl-glucans and mannopiotcins
Chilin represents a minute pari of its composi-
tion The most detailed work ot Ihc yeasl cell
wall has been carried out on Sacehumntwes cere-
risiite — the principal yeasl responsible for Ihc
alcoholic fermentation of grape must.
Glucan represents aboul6oy of the dry weight
of the cell wall of S. cerevisuie. It can be
chemically fractionated into three categories:
1. Fibrous 0-13 glucan is insoluble in water,
acetic acri and alkali It has very few branches
The branch points involved arc 0-1.6 linkages
Its degree of polymerization is 1500. Under
the electron microscope, this glucan appear,
fibrous. It ensures the shape and the rigidity of
the cell wall. It is always canceled to chitin.
2. Amorphous 0-13 glucan. wilh about 1500
glucose units, is insoluble in water bul soluble
in alkalis. II has very few branches, like the
prcccding glucan. In addition to ihc.se few
branches, it is made np of a small number of
fMoglycosidic linkages. It has an amorphous
aspect under the electron microscope. It gives
Ihc cell wall its elasticity and acts as an anchor
for the mannoproleins. It can also constitute an
cxtraprotoplasmic reserve substance.
Handbook of Fnology: The Microbiology °f Wi»e anil Vinifications
3. The (1-1.6 glucau is obtained front alkali-
insolublc glucans by extracting in acclK acid.
The resulting product is amorphous, water sol-
uble, and extensively ramified by ;i* IJ glyco-
side linkages. In decree or polymcrizalkti is
14o It links the diffcrcnl constituents of the
cell wall together. It is also a receptor site for
the killer rack*.
The fibrous /I- J J glucan (alkali- insoluble* proba-
bly results from the incorporation of chitin on the
amorphous fi-l 3 glucan
MannoprDtcinsconslililc 25-50* of the cell
wall of S. ceivviiiae. They can be cxtracicd from
the whole cell or fmni the isolated cell wall
by chemical and enzymatic methods Chemical
methods make use of antocktving in the pres-
ence of alkali or a citrate buffer solution at
pH 7. The enzymatic method frees Ue ntanno-
prolcins by digesting the gUcan. This method
docs not denature the structure of the manuopro-
Kins as much as chemical methods Zyniolyasc.
obtained from Ihc bacterium Arifmilxtcter luleiis.
is the enzymatic preparation ntost oflen used to
extract the parietal ntannoprotcins of S ceirvisiue.
This cn/ymatic complex is effective primarily
because of ifc. (I- 1 J glucanasc activity. The actio*
of prolcasc contaminant! in the zyniolyasc com-
bine, with the aforementioned activity to liberate
the mannoprolcins. Glucancx. another industrial
preparation of the (l-glucanasc. produced by a fun-
gus {Trkhttdfmm haryimim). has been recently
demonstrated to possess endo- and cxo-f-IJ and
cndo-fl-l .6- glucanasc activities (Dubounlicu and
Mont. 1995). These activities also facilitate the
extraction of the cell wall ntannoprotcins of the
S ctreiiiiar cell.
The mannopiotcins of S cerevisilie have a
molecular wcighl between 20 and 450 kDa Their
degree of glycosykitwn varies. Ccrctln ones con-
tuning about <XKf ntannosc and l(« peptides axe
hy pc rman n osy la led
Four forms of glycosyfctlion arc described
(Figure 1.2) but do not necessarily exist at the
same unit- in all of the ntannoproicins.
The ntannosc of the ntannoprotcins can consil-
ium short, linear chains with one to live residues
They are linked to Ihc peptide chain by 0-glycosyl
linkages on serine and threonine residues These
glycosidic s*lc-chain linkages arc a- 12 and a- 1 J.
The glucidic pari of the mannoproiein can also
be a polysaccharklc. It is linked to an asparaginc
residue of the peptide chain by an jV-glycosyl
linkage. This linkage consist, of a double unit of
A 1 -accty (glucosamine (chitin) linked in (M.4. The
mannan linked in this manner to the asparaginc
includes an attachment region made up of a do/en
mannosc residues and a highly ramified outer
chain consisting of 150 to 250 ntannosc unit*
The attachment region beyond the chitin residue
consist of a ntannosc skeleton linked in »-IJi
with side branches possessing one. two or three
ntannosc residues with a-12 and/or o-U bonds.
The outer chain is also nude up of a skeleton of
mannosc .mi- linked in . r- 1 6 This chain bears
short side-chains constituted of mannosc residues
linked in a-12 and a terminal ntannosc in a-
I J Some of these side-chains possess a branch
attached by a phosphcKlicsicr bond.
A Ihinl type of glycosykition was described
more recently. It can occur in ntannoproicins.
which make up the cell wall of the yeast ll consists
of a glncontannan chain containing essentially
mannosc residues linked in u-1.6 and glucose
residues linked in a- 1 .6. The nature of the glycan-
peptide point of attachment Is not yet clear, but il
may bean asparaginy I -glucose bond This type of
glycosyfctlion characKri/cs the proleins freed from
Ihc cell wall by Ihc action of a (l-IJ glucanasc.
Therefore, in vivo, the glucomannan chain may
aLso comprise glucose residues linked in 0- 1 J.
The fourth type ofglyeosylalion of yeast ntanno-
protcins is Ihc ply cosy I phosphatidyl -inositol
anchor (GPI). This attachment between the ter-
minal carboxylic gmup of Ihc peptide chain aid
a membrane phospholipid permits certain ntanno-
protcins. which cross the cell wall, to anchor
themselves in Ihc pfcismic membrane. The region
of attachment is characterized by Ihc following
sequence (Figure 1 .21: clhanolaminc-phosphalc-
6- mannosc-..- 1 2-mannosc-a- 1 .6-mannosc-o- 1 .4-
glucosaminc-a-16-inositol-phospbolipid. A C-
phospholipasc specific to phosphatidyl inosilol
and therefore capable of realizing this cleavage
Cytology. Taxonomy ami Ecology of Grape ami Wine Yeasts
M|t 4CNA4* — 4aH\ip — > v " — -^
l'l£ 1.2 The lour Iv pet ill ' |f lutoit} btl »■ ill faricl*! vosl I
GN Bgbrouainc:GXAt = iV-icay^lucDumlnc: Ins = li
X» =lk nature of the bond b no known
*; Asa = aip4 opine;
was demonstrated in lac 5. <
Thoner. 1993). Several GPI-typc a
proteins have been Ulcnlilicd in the cell wall of
Chit In is a linear polymer of ,\'-accly (glucos-
amine linked in 0-1.4 and is not generally found in
large quantities in yeast cell walK InS. ceiwiaae.
chitin constitutes I -2'i of the cell wall and is
found for the most part <bnl not exclusively) in
hnd scar /ones These awes arc a type of raised
crater easily seen on the mother cell under the
election microscope (Figure 1.3). This chilinic scar
is formed essentially to assire cell wall integrity
and cell survival. YcasC^ treated with D polyoxinc.
an antibiotic inhibiting the synthesis of chitin. arc
not viable: they bnrsl after budding
The presence of lipids in the cell wall has not
been ckraiiv demonstrated It Is trie that cell walls
.i-;' |il.iivi;iji)i at
relB. The budding ku di
Handbook of Etiology: The Microbiology of Wive anil Vindications
prepared in the laboratory contain some lipids
12-15'* for S. cervruBv) bnl il ts most likely
contamination by Ihc lipids of Ihc cytoplasmic
membrane, adsorbed by the cell wall daring their
volition The cell wall can also adsorb lipids from
us external cnvlronnieit. especially the different
fatty acids Inat activate and inhibit Ihc fcmienuiioa
(Chapter 3).
Chitin arc connected to the cell wall or sit-
■atcd li the peri plasm ic space. One of the
■mm characteristic enzymes is the iivcrtasc 10-
fniclofurauosldasc). This enzyme catalyzes the
hydrolysis of saccharose into glucose and fruc-
tose. It is a thcrmosGiblc niannoproicin anchored
to a 0-1.6 glncan of the cell wall, its molecular
weight is 2700CO Da_ It contains approximately
50* iiannosc and 5<« protein The pcriplasaiic
acid phosphalase is cquall)' a mannoprotcln
Other pcriplasmic en/ynies thai have been noted
arc 0-glucosidasc. a-gatactosidasc. mclibtasc. tre-
halasc. aminopcplidasc and esterase. Yeast cell
walls also contain endo- andcxo-0-glucaiascs(£-
1.3 and 0-1.6). These enzymes arc invoked in the
reshaping of the cell wall diring the growth aid
bidding of cells Their activity is al a maximim
diring the exponential log phase of the population
and diminishes notably afterwards. Ycl cells in the
stationary phase and even dead yeasts contained
in Ihc Ices still retain 0-glucanascs activity in
their cell walls several months alter the completion
of fcrmcnlation. These endogenous enzymes ate
involved in the autolysis of the cell wall during the
ageing of win
be covered in
(Chapter 13)
s on Ices. This ageing method will
the chapter on while wiacmaking
.i ion of the Cdl
t Affecting ils
1.2.3 (Jcncral Organi
Wall and Factoi
Composition
The cell wall of 5 cerevisiae Is made up of an
outer layer of ntannoproleins. These maiiopro-
tcins arc connected toamainxofamorphoasjI-U
glican which covers an inner Liver of fibrons (>-
I J glucan The inner layer is connected to a small
quantify of chilli (Figure 1.4). The rM.o glican
probably acts as a cement between the two lay-
ers The rigidity and Ihc shape of the cell wall
arc die to Ihc internal framework of the fi- 1 J
fibrous glucan. Ik elasticity is due to the outer
amorphous layer. The iukrmolccular stricture of
Ihc man no proteins of Ihc outer layer t hydrophobic
linkages and disulfur bonds) cqially determines
cell wall porosity aid impermeability to macro-
molccilcs (molecular weights less than 4500). Thus
impermeability can be affected by Healing the
cell wall with certain chemical agents, such as
fJ-iKicaptoclhaiol This substance provokes the
rupture of the disulfur bonds, thus destroying the
inicrmokxuLu network between the mannoptovin
chains
The composilioi of the cell wall is strongly
influenced by nitrilivc conditions and cell age.
The proportion ot glucan ii the cell wall increases
* ilatiirterlR an\i-
^ .!Edf|tl1]^ftA3l
|- Uflno
JU
< a>pBRin. 11.1H..1.
I-ifi L4. CelkihtDBi
m.flhc cell wall ol S i-
Cytology. Taxonomy anil Ecology of Grape and Wine Yeasts
with respect to Ihc amount of sugar in the euF
(arc medium Certain deficiencies (for example,
ii mcsoliasiul) also result in an increase in Ihc
proporlion of glucan compared with mainopro-
tcins Tic cell walls of older cells aic richer in
glucansand in chitin aid less tarnished ii manno-
prole ins For ihis reason, they arc more resistant
to physical aid en a malic agents used lo degrade
them. Finally, ihc composition of (he cell wall is
profoundly modified by morphogcnclK alterations
(conjugation and sporibilion).
1.3 THE PLASMIC MEMBRANE
1.3.1 Chemical Composition
and Organization
The plasmK membrane is a highly selective harrier
conirolling exchanges between Ihc living cell and
its external environment. This organelle ts essential
to the life of Ibc yeast.
Like all btotogKal membranes, the yeast plasmK
membrane is priicipally made up of lipids and
proiciis The ptasmK mcmbraic of S cetrvisiae
con tuns aboil 41 Ki lipids aid 5l/» proteins
Glucans aid mannans arc oily present ii small
quantities (several per cent!
The lipids of ihc membrane arc essentially
phospholipids and sterols They arc amphiphilic
molecules, ie. possessing a bydropbilK and a
hydrophobic pari
Thc in" principal phospholipids (Figure 13)
of Ihc plasmic membrane of yeast arc pbos-
pbalidylclianolaniiic (PI:), phosphatidylcholine
IPC) and phospbatidylinosilol (PI) which repre-
sent 70-85'i of the total. Phospbatidylscrinc (PS)
and dipbospbalidylglyecrol orcardiolipii(PG)are
less prevalent Free fatty acids and phosphalklK
ackl arc frequently reported in plasmic membrane
analysis They aie probably extraction artifacts
caused by tic activity ofccrciln lipid degradation
The fatly acids of the membrane phospholipids
contain ai even umber ( 14 to 24) of tatbn atoms.
The hum abundant arc C lt aid C| h acids. They
can be saturalcd. such as palmitic acid (Cis) and
stearic acid (Cm), or unsaturated, as with oleic
acid (Cih. double bond in position 9). linolcic acid
(C| h . two double bonds in positions 9 and 12) and
liiolcnic acid (C lh . three double bonds in positions
9. 12 aid IS). All membrane phospholipids skate
a common characteristic: they posses a pofcir or
bydropbilK part made up of a phosphorylatcd
alcohol and a noi-polar or hydrophobic part
comprisiig two morc or less puiullcl fatly acid
chains (Figure 16) In ai aqueous medium, the
phospholipids spontaneously form bimolccular
Dims ora lipid bilaycr becansc of their ampbipbilK
characteristic (Figure 1.6). The lipid bilaycrs arc
cooperative bit lon-covalcnl structures They
arc maintained in place by mutually reinforced
intcraciiois: hydrophobic interactions, van dcr
Wauls attractive forces between the hydrocarbon
tails, hydrostatic interactions and hydrogen bonds
between the polar beads and water molecules
The examination of cross- sec tiois of yeast
plasmic membrane under the electron microscope
reveals a classic lipid bilaycr stricture with a
thickness of about 73 nm. The membrane sirfacc
appeals sculped with creases, especially during
the stationary phase. Hiivcvcr. the physiological
meaning of this anatomic character remains
unknown. The plasmic mcmbraic also has an
underlying depression oa the bud scar.
Frgosterol is the primary sterol of the ycasl plas-
mic membrane In lesser quantities. 24(28) deby-
drocrgostciul aid zymosterol also exist (Figure
1.7). Sterols are exclusively pitKluccd in Ihc mito-
chondria during the yeast tog phase As with phos-
pholipids, mcmbraic sterols arc amphipalhic. The
bydropbilK pan is made up of hydroxy! groups
in C-3. The rest of the molecule is hydrophobic,
especially Ihc flexible hydrocarboi tail.
The plasmK membrane also contains numerous
proteiis or glycoproteins prcscniiig a wide range
of nokcilar weights (from 10000 to 120000).
The available information indicates that the orga-
nization of the plasmic iKmhranc of a yeast cell
resembles the fluid mosaK model This model,
proposed for biological membranes by Singer and
Nicolsoa ( 1972). consists of two-d) incisional soli-
tkus of proteins aid oriented lipids Certain pro-
teins arc embedded in the membrane: they arc
called iitcgral proteins (Ftgitc 1.6). They interact
Handbook of Etiology: The Microbiology of Wine anil VhUialima
ryr — o — r — o-cn,-c — mi,*
nfi—o—t
iuc — o — c — t
K C O CHj
nfi— o— r— o— c
o-
n»i(nau\id»ii)
, — err, — n'ich,),
D P O CM,
?1>-|(I(.MI.IMm1
M^T — O— C— K
l^(i«|4» tl> I y l\ ■ <ti il i .
Fift 1.5. Ycuu mc-hi
strongl) with Ihc non-polar part of Ihc lipid bilaycr.
The peripheral proteins arc linked k> the precedent
by hydrogen bonds Their location is asymmetrical,
al cither the inner or Ihc outer side of Ihc plasmic
membranc The molecules of proteins and mem-
braac lipids, constantly in lateral moventcm. aic
capable of rapidly diffusing in the membrane.
.Some of the yeast membrane proteins have been
studied in greater depth These include adenosine
triphosphatase t ATPasc). solute (sugars and amino
acids) transport proteins, and enzymes involved in
the production of glncans and cbilin of Ihc cell
wall.
The yeast possesses three ATPascs: in the mito-
chondria, the vacuole, and the ptasaiK membrane.
The plasmic membrane ATPasc Pi an integral pro-
tein with a molccnlar weight of aronnd 100 Da. It
catalyzes the hydrolysis of ATP which furncthes
the necessary energy for the active transport of
solutes across the membrane (Note: an active
Cytology, Taxonomy ami Ecology of Crape ami Wine Ycasls
Fift I. ft. A membrane lipid bih»cr. The inlcpal
fnHcim Ijijk uronglv luixtiKd In the non-polai
naibn of ibe bibyer. The pcriphcal piuciiu (b)aic
linked In Ibe iir.--ji.il fmlciiu
transport mows a compoiid againsl ihc eoncci-
Iralion gradient ) Sliialtmcossly. Ihc hydrolysis oi
ATP creaks an cfllix of protons towards ihc cxlc-
rior of Ike cell.
The pcnclralion of amino acids ;wd sugars
ink) ihc ycasl activates membrane transport sys-
tems called permeases The gcnciul amino acid
permease (GAP) contains ihtcc membrane proteins
and ensues Ihc transport of a umber of neutral
amino acids The cultivation of yeasts )■ Ihc pres-
ence of an easily assinilalcd liirogci- based nun-
ent such as ammonium icprcsscs Ihts permease.
The membrane composition in laity acids and
its proporlioi ii stcioK control iLs HuHJiiy The
hydmcaibou chains of fatly acidsof the membrane
phospholipid bilaycr cai be in a rigid and orderly
State or in a relatively disorderly aid ftuRI stale. In
Ihc rigid slat, some or all of Ihc carbon bonds
of Ihc fatly acids ate from. In Ihc fluid stile,
some of Ihc bonds become rir. The transition
from Ihc rigid state to the fluid stile tikes place
when Ihc temperature rises beyond the fusion
Icmpcraliic This transition tcmpcralutc depends
on Ihc krnglh of the fairy acid chains and their
degree of uusalnraltoa. The rectilinear hydiucarbon
chains of ihc saturated fatly acids interact sliungly
These interactions intensify wilh their length. The
transition Icmpcraliic therefore increases as Ihc
tally acid chains become longer. The doibk
bonds of the insaturated fatly acids arc generally
cis. giving a curvature to Ihc hydrocarbon chain
(Figure IS) This cirvatirc breaks ihc orderly
I M nil m ji.nl el i*
Handbook of Hnology: The Mic ro biology of Wine anil Vlnifieations
ng i*
Molecular model* npmcalif
ihc Ihrcc-di-
il summit ol ucaric and oleic ;
i. ill. TW rfi
"'"£,!
lion of (be double bo*l of oleic i
Kcoftbccaihoncbiln
icld jrodicej,
sticking of ibe fall)' acid chains and lowers ihc
transition temperature lake cholesterol in lie nils
of mammals, cigostcrol R aLso a fundamental
regulator of the membrane fluidity in yeasts.
Ergostcrol is InscrKd in the hilaycr perpendicularly
lo Ihc membrane, lis hydroxy! group joins, by
hydrogen bonds, wilh the polar head of ihe
phospholipid and Its hydrocarbon oil Is inserted
in ihc hydrophobic region of ihc bilaycr. The
me in hi. i iic sterols intercalate themselves between
the phospholipids. In this manner, they inhibit
the crystallization of Ihc fairy acid chains at low
temperatures, inversely, in reducing the movement
of these same chains by stcric cncumbcniKnl. they
regilalc an excess of membrane fluidity when the
kmpcraturc rises.
1.3.2 Functions of the Pkismic
Membrane
The plasm* membrane constitutes a stable.
hydrophobic barrier between the cytoplasm and
the cnvlronmcni ouLsHle the cell, owing to its
phospholipids and sterols This barrier present* a
certain impermeability 10 solutes in function of
osmotic properties.
Furthermore, through Its system of permeases.
Ihc plasm*.* membrane aLso controls Ihc exchanges
berween Ihc cell and the medium The function-
ing of these transport proteins is greatly influenced
by Ik lipid composition, which affects membrane
fluidity. In a defined environmental model, the
supplementing of membrane phospholipids with
unsaturated fatty acids (oleic and linoIcK) pro-
moted the penetration and accumulation of ccrttin
amino acids as well as Ihe expression of the gen-
eral amino acid permease (CAP). (Hcnschkc and
Rose. 1991 ) On Ihc other hand, membrane sterols
seem in have less Influence on the transport of
amino acids than Ihc dcgicc of atsaturallon of
Ihc phospholipids The production of unsaturated
fatty acids B an oxidative process and requires the
aeration of the culture medium at Ihc beginning
of alcoholic fermentation In semi-anaerobic winc-
making conditions, the amount of unsaturated rally
acids in Ihc grape, or In the grape must, probably
favor the membrane transport mechanisms of fatty
acids
The transport systems of sugars across Ibe mem-
brane arc far from being completely elucidated.
There exists, however, at least two kinds of trans-
put, systems: a high affinity and a low affinity
system (Kn times less important) tBisson. 1991).
The k>w altinily system is essential during Ihc log
phase and it> activity dccicascs during the station-
ary phase. The high aflinlty system is. on Ihc con-
trary, repressed by high concentrations of glucose,
as in Ihc case of grape must (Salmon el irf . 1993)
il-ii-iire 1.9). The amount of sterols in Ihc mem-
brane, especially crgoslcrol. as well as Ihc degree
of unsataration of the membrane phospholipids
favor the penetration of glucose in the cell. This
is especially true during the stationary and decline
phases This phenomenon explains the determining
influence of aeration on Ihe successful completion
of alcoholic fermentation during Ihc yeast multi-
plication phase.
The presence of ethanol. in a cnllnrc medium,
slows the penetration speed ofarginitc and glucose
into the cell and limits Ibe efflax of protons
Cytology. Taxonomy anil Ecology of Grape and Wine Yeasts
11
(Section 1.4.2). They an: ihcn transported by vesi-
cles which fuse "ill the plasm ic membrauc
and deposil their contents at (he cxlcrior of the
membrane
Finally, certain membrane prolcins acl as cel-
lular spec ilk rcccptois. They permit Ihc ycasl lo
react to various external stimuli such as sexual hor-
mones or changes in the conccnimlion of external
niilricBls The activation of these membrane pn>
Icins inggcn> ihc liberation of compounds such as
cyclic adenosine monophospbaK (cAMP) in the
cytoplasm. These compounds serve as secondary
messengers which set off other infcrccllular reac-
tions. The consequences of these cellular mecha-
nisms In the alcoholK fcraicniation process merit
further slndy.
Fig 1.9. Evolution of pkM-w
of X omiaW ICnucMlnu ■ medium Wodcl iSjiUo'ii
erf.. IWJ). II =Umsh of ihc ferae Hat kin as a
cinul of total liatc GP = Gh>co*c pcnciraiina ipceil
i mo I'll V of Jr. weight) = Low alhni;. liantpon
Mem nlhiiv rsllqth alhiut lianpon avMcm
liviy
resulting from membrane ATPasc activity (Alexan-
dre eltil.. 1994. Charpenticr. 1995). Simulta-
neously, ihc presence of clhanol increases the
synthesis of membrane phospholipids and their
percentage in unsaturated fatty acRls (especially
oleic). Temperature and clhanol act in syncigy to
affeel membrane ATPasc activity The amount of
clhanol required to slow the proton efflux decreases
as the temperature rises. However, this modifica-
tion of membrane ATPasc activity by clhanol may
nol be Ihc origin of the decrease in plasmic mem-
brane permeability in an alcoholic medium The
idIc of membrane ATPasc in ycasl resistance lo
clhanol has noi been clearly demonstrated
The plasmic membrane also pioduces cell
wall glncan and chilin. Two membrane enzymes
are involved: 0-IJ glucanasc aid chilli syn-
thetase These two enzymes catalyze Ihc poly-
merization of glucose and .V -acetyl-glucosamine,
derived from Iheir activated forms (uridine
diphosphates— UDP). The mannopnrtcins arc
essential!) produced In Ihc endoplasmic reticulum
1.4 THE CYTOPLASM AND ITS
ORGANELLES
Bcrvvcci ihc plasmic membrane and Ihc nuclear
membrane, the cytoplasm contains a basic
cytoplasmic substance, or eytosol The organelles
(endoplasmic reticulum. Colgi apparatus, vacuole
and mitochondria) are isotilcd from ihc eytosol by
membranes.
1.4.1 Cytosol
The eytosol is a buffered solution, with a pH
between 5 and 6. conciining soluble enzymes,
glycogen and ribosomes
Glycolysis and alcoholic fermentation enzymes
(Chapter 2) as well as trehalose (an enzyme cat-
alyzing Ihc hydrolysis of trehalose) are present
Trehalose, a reserve drsaccharidc. also cytoplas-
mic, ensures ycasl '.lability during ihc dehydration
and rehydration phases by maintaining membrane
integrity.
The lag phase precedes Ihc log phase in a
sugar-con lain I ng medium ll is marked by a rapid
degradation of trehalose linked to an increase in
irchalasc activity. This activity is Itself closely
related to an increase in Ihc amount of cAMP in
the cytoplasm This compound is produced by a
membrane cn/ymc. adenylate cyclase, in response
12
Handbook or Etiology: The Microbiology of Wine and Vindications
10 the stimulation of a membrane icccptor by ai
environmental factor.
Glycogen is the principal yeast glucklic reserve
substance. Animal glycogen is similar in structure.
11 accumulates dnring the stationary phase in the
form of spherical grainles of about 40 |tm in
diameter.
When observed under the electron microscope,
the yeast cytoplasm appeals rich in ribosomes.
These tiny granulations, made up of ribonucleic
acids and proteins, an: the center of piotcin
synthesis. Joined to polysomes, several ribosomes
migrate the length of the messenger RNA. They
translate it simultaneously so that each one
produces a complete polypeptide chain.
1.4.2 The Endoplasmic Reticulum,
(he Golgi Apparatus
and the Vacuoles
The endoplasmic reticulum (ER) is a double
membrane system partitioning the cytoplasm II is
linked to the cytoplasmic membrane and nuclear
membrane It is. in a way. an extension of the
killer Although less developed in yeasts than in
exocrine cells of higher cucaryoics. the ER has
the same function It ensures the addressing of
the proteins synthesized by the attached ribosomes.
As a matter of fact, ribosomes can be either free
in Ihc cylosol or bound lo the ER. The pro-
Kins synthesized by free ribosomes remain in the
cylosol. as do Ihc enzymes involved in glycolysis.
Those pioduced in Ihc ribosomes bound lo Ihc ER
have three possible destinations the vacuole, the
plasmic membrane, and Ihc external cnviionmcnl
(secretion). The presence of a signal sequence (a
particular chain of amino acids) at the .V- terminal
extremity of Ihc newly formed piolcin determines
the association of the initially free ribosomes in
the cylosol with the ER The synthesized proKin
crosses the ER membrane by an active transport
process called translocation. This process requires
the hydrolysis of an ATP molecule. Having reached
the inner space of the ER. Ihc proteins undergo ccr-
tiin modifications including the necessary excising
of the signal peptide by the signal peptidase. In
many cases, they also undctgo a glycosylation.
The yeast glycopiotclns. in particular Ihc struc-
tural, parietal or enzymatic mannoprotcins. con-
lain glncidic side chains (Section 1.22). Some of
these are linked to asparaginc by A'-glycosidic
bonds. This oligosaccharidic link is constructed in
Ihc Interior of Ibe ER by the sequential addition
of activated sugais (in the form of UDP deriva-
tives) 10 a hydrophobic, llptdic transporter called
dolic hoi phosphate The entire unit is transferred in
one piece lo an asparaginc residue of the polypep-
tide chain The dolKholphosphatc Is regenerated.
The Golgi apparatus consists of a slack of
membrane sacs and associated vesicles, it is an
extension of the ER. Transfer vesicles transport
the proteins issued from the ER to the sacs of the
Golgi apparatus The Golgi apparatus has a dual
function It is responsible for Ihc glycosyfciuon
of piotcin. then sorts so as to direct them vEt
specialized vesicles cither into the vacuole or into
Ihc plasmic membrane An N- terminal pcpodK
sequence determines the directing of proteins
towards the vacuole. This sequence is present in
the prccuisors of two vacnolar-oricntatcd enzymes
in the yeast: Y carboxypcplidasc and A proteinase.
The vesicles that transport the proteins of the
plasmic membrane or the secretion grannies, such
as those that transport the pcriplasmic invertasc.
are still the default destinations
The vacuole is a spherical organelle. OJ lo
3 pin in diameter, surrounded by a single nicm-
bmne. Depending on the stage of the cellular
cycle, yeasts have one or several vacuoles. Before
budding, a large vacuole splits into small vesi-
cles Sonic penetrate into the bud. Others gather
at Ihc opposite extremity of the cell and fuse
to form one or Iwo huge vacuoles The vacuo-
lar membrane or lonoplasi has the same general
structure (fluid mosaic) as the plasmic membrane
bnl it Is more elastic and its chemical com-
position is somewhat different. It Is less rich
in sterols and contains less protein and glyco-
protein but more phospholipids wilh a higher
degree of nnsaturalion. The vacuole sucks some
of the cell hydrolases, in particular Y carboxypep-
tidasc. A and B protases. I aminopeptidasc.
X-piopyl-dlpcplldylanitnopeplidase and alkaline
phosphatase. In this respect, the yeasl vacuole can
Cytology. Taxonomy anil Ecology of Grape and Wine Yeasts
i:
be compared to an animal cell lysosomc. Vacuolar
proteases play an essential role )■ the turn-over
of cellular proteins lu addition, the A protease
Is indispensable in Ihc matuialion of other vacuo-
lar hydrolases. It excises a small peptide sequence
and thus converts precuisor forms (proenzymes)
into active engines. The vacuolar proteases also
autolyzc the cell after its death. AuiolysR. white
ageing white wine on its lees, can affect wine qual-
ity and should concern the wlacmaker.
Vacuoles also haw a second principal function:
they stuck metabolites before their use. In fact,
they contain a quarter of the pool of the amino
acids of the cell, including a lot of argininc as well
its 5-adcnosyl methionine In this organelle, there
is also pousslnm. adenine, isoguaniic. nric acid
and polyphosphate ctystds These arc involved
In the fixation of basic amino acids. Specific
permeases ensure the transport of these metabolites
across the vacuolar membrane An ATPasc linked
to the uaoplast furnishes Ihc necessary energy
for the movement of stocked compounds against
the concentration gradient. It is different from the
plasmic membrane ATPasc. but also produces a
proton efflux
The BR. Gouji apparatus and vacnolcs can
be considered as different component of an
internal system of membranes, called the vacnomc.
participating in Ihc flux of glycoproteins to be
excreted or sucked.
1.4.3 The Mitochondria
Distributed in Ihc periphery of Ihc cytoplasm, the
miuchondria (ml) ate spherically or rod-shaped
organelles sarronnded by two membranes. The
inner membrane is highly folded lo form eristic
The general organization of mitochondria is the
same us in higher plants and animal cclK. The
membranes delimit two compartments: Ihc inner
membrane space and the matrix. The mitochon-
dria arc true respiratory organelles for ycasti In
acrobiosis. the S. eererisiue cell contains about
50 mitochondria In anacrobiosis. these organelles
degenerate, their inner surface decreases, and the
eristic disappear. Ergcslcrol and unsaturated fatly
.Mils supplemented in culture media limit the
degeneration of mitochondria in anacrobiosis. In
any case, when cells formed in anacrobiosis ate
placed in acrobktsis. Ihc mitochondria regain their
normal appearance Even in aerated grape mast,
the high sagar concentration represses the synthe-
sis of respirator)* enzymes As a result, the mito-
chondria no lotgcr function This phenomenon,
calabolic glucose repression, will be described in
Chapter 2.
The mitochondrial membranes arc rich in phos-
pholipids-- principally phosphatidylcholine, phos-
phatidyl I msl to! and phaspbatidylclhanolaminc
(Figure 1.5) Cardiolipin (diphcsphalldylglyccrol).
In minority in the plasmic membrane (Figuie 1.4).
is predominant In the Inner mitochondrial mem-
brane The fatty acids of the mitochondrial phos-
pholipids ate in CI60. CI6:I. CI8:0. Cl8:i\
In acrobiosis. the unsaturated residues predomi-
nate When the cells arc grown In anacrobiosis.
without lipid supplements, the short-chain satu-
Mi.'.l residues beconte predominant cardiolipin
and phospbalidylclhanolaminc diminish whereas
Ibc proportion of phosphabdylinosiiol increases. In
acrobiosis. the temperature during Ihc tog phase of
Ihc cell influences the degree of unsaluialion of the
phospholipids- more saturated as the temperature
dec teases.
The mluchondtial membranes also contain
sterols, as well as numerous proteins and enzymes
(Gacrin. 1991). The two membranes, inner and
outcr.coatain en /ymes involved in ihc synthesis of
phospholipids and sterols The ability to synthesize
significant amounts of lipids, characteristic of yeast
mitochondria. Is not limited by respiratory deficient
mutations or catibolic glucose repression.
The outer membrane Is permeable lo most
small mettbolitcs coming from the cyusol since it
contains porinc. a 29 kDa transmembrane protein
possessing a large pore Porinc is present in
the mitochondria of all the cucaryotcs as well
its in Ihc outer membrane of hactria The
in ic membrane space con tuns adenylate kinase,
which ensures inlcrconvcision of ATP. ADP and
AMP. Oxidative phosphorylation takes place in the
inner miKKhoudrial membrane. The matrix, on the
other hand. Is the center of the reactions of the
tricarboxylic acids cycle and of the oxidation of
fatty acids.
14
Handbook or Etiology: The Microbiology of Wine and Vlnifications
The majority of mitochondria proteins aic coded
by the genes of the nucleus aid arc synthesized by
the free polysomes of the cytoplasm The mito-
chondria, however, also have their own machinery
for protein synthesis li fact, each mitochon-
drion possesses a circular 75 kb (kikibasc paits)
motccitc of doublc-slmndcd AND and ribcftomcs.
The mtDNA is extremely rich in A (adenine) aid
T (thymine) bases. It contains a few do/cn genes,
which code in particular for the synthesis of cer-
tain pigment! and rcspiralory enzymes, such as
cytochrome 6. and several sub- units of cytochrome
oxidase and of the ATP synthetase complex. Some
mutations affecting these genes can rcsilt in the
yeast becoming resistant to certain mitochondrial
specific inhibitor such as olivomycin This prop-
er!)' has been applied in the genetic marking of
wine yeast strains Some mitochondrial mutants
ate respiratory deficient and form small colonics
oi solid agar media. These petit' mutants arc not
used in wincmaklag because it is impossible to
produce them industrially by inspiration.
1.5 THE NUCLEUS
The yeast nucleus is spherical. It has a diameter
of I -2 mm and Is baicly visible using a phase
contrast optical microscope It is located near the
principal vacuole in non- proliferating cells. The
■•clear envelope is made iipola double membrane
attached to the ER It contains many ephemeral
poics. their locations continually changing. These
potcs permit the exchange of small proteins
between the nucleus and the cytoplasm. Contrary
*> what happens in higher encaryotcs. the yeast
nuclear envelope is not dispersed during mitosis
In the basophilic part of the nucleus, the crcsecit-
shaped nucleolus can be seen by using a unclear-
specific stoning method As in other cncaiyotcs. it
Is responsible for the synthesis of ribosomal RNA.
During cellular division, the yeast nucleus also
contains rudimentary spindle threads composed of
microtubules of tubulin, some discontinuous and
others continuous (Figure I 1(1). The continuous
microtubules arc stretched between the two
spindle pole bodies (5PB) These corpuscles arc
permanently included in the nuclear membrane and
l'i(i I. III. Thc;.«u*iHkXK<\VilUim.s..B. I»>1|| .Si'E! =
Spindle pole body:NUC = NuckohniP = Pbic;CHB =
Cbwrnjiin: CT = Ci>uiimxii» tubules: DCT = DtKou-
lioUDintulHllcs:CTM BC'yioplumK ■ fcivluhulo.
coricspond with Ihc ccntriolcs of higher organisms.
The cytoplasmic microtubules depart from the
spindle pole bodies towanls the cytoplasm.
There is little nuclear DNA in ycasis compared
with higher cncaiyotcs— about 14000 kb in a
hapkiid strain. It has a genome almost three limes
larger than in Escherichia ratf. but its genetic
material is oigani/cd into true chromosomes. Each
one contiins a single molecule of linear double-
stranded DNA associated with basic proteins
known as his tones. The Imtoncs form chromatin
which contains repetitive nulls called nnclcosomcs.
Yeast chromosomes ait too small to be observed
under the microscope.
Pulse- field electrophorcsLs (Carle and Olson.
1 984: ! Schw.ui/ and Cantor. 1984) pcraiils the sep-
aration of Ihc 16 chromosomes in S. cerevisiae.
whose be range from 200 to 2000 kb. This
species has a very laigc cbromosomK polymor-
phism This characteristic has made karyotype
analysis one of Ihc principal criteria for the iden-
tification of S ceievisiae slrains (Section 19.3).
The scientific community has nearly established
Ihc complete sequence of the cbromosomK DNA
of S cereviatte. In Ihc futnrc. this detailed knowl-
edge of Ihc ycasl genome will constitute a powerful
tool, as much for understanding is molecular phys-
iology as for selecting and improving winemaking
strains.
The ycasl chromosomes contain relatively few
repealed sequences. Most genes arc only present
Cytology. Taxonomy anil Ecology of Grape ami Wine YcasLs
ii a single example in lie haploid genome, bat the
ribosomal RNA genes air highly repealed laboui
ICO copies).
The genome of S. cenrrisiar contains traitspos-
able clement*, or Iransposons — specifically. TV
tlransposon yeast) elements. These comprise acct-
iral i- icgion (5.6 kbl fiunied by a direct repealed
sequence called ihc A sequence (0.25 kb). The &
sequences have a tendency to recombinc. resulting
in Ihc loss of ihc ccninil legion and a A sequence
As a fcsali. ihcie air aboui 100 copies of Ihc &
sequence )■ ihc ycasl genome The Ty elements
code for uon-infecuoas retrovirus panicles This
retrovirus contains Ty messenger RNA as well as
a tevcrse transcripcisc capable of copying the RNA
into complementary DNA. The Litier can reinsert
itself into any sile of ihc chromosome. TV ran-
dom excision and insertion of Ty clcmenis in the
yeast genome can modify the genes and ptay an
important role in .strain evolution
Only one plasmid. called Ihe 2 pit plasmid. has
been identified in the yeast nucleus ll Is a circular
molecule of DNA. containing 6 kb and there ate
50-100 copies per cell Ik biological function is
nol known, bui it is a very useful tool, ascd by
molecular biologists to construct artificial plasm ids
and genetically transform yeast strains.
1.6 RKPRODUCTION AND THE
YEAST BIOLOGICAL CYCLE
Like other sporiferons ycasls belonging to the
class Ascomyccts. S ceiwisiae can multiply
eilher ascxualfy by vegetative multiplication or
sexually by foming ascosporcs. By definition,
yeasts belonging to the imperfect fnngi can only
icproducc by vcgctilivc multiplication.
1.6.1 Vegetative M nil ip lie at ion
Most ycasLs undcigo vcgctilivc multiplication by
a process called badding. Some ycasis. sach as
species belonging u lie genus Schizpsaetlui-
mm««. multiply by binary tension.
Eigurc I. II represent! Ihc life cycle of S ceitvi-
siiie divided into four phases: M. CI. S. and G2.
M corresponds with mitosis. CI Is the period
<§>
1'ii; I. II. S.tereiiuir cell cycle (icgcuiivc mul-
tiplinlkiii) (Tunc and Oliver. 1991). M=aaOM;
CI = period preceding DNA v. m Ik-.i. . S = DNA i,y»-
lho.it; G2 = pcrkid f receding mit<i>B.
preceding S. which Is the synthesis of DNA and G2
is the period before cell division. As soon as the
bud emerges, in the beginning of S. Ihc splitting
of Ihc spindle pole bodies ISPB) can be observed
in the nuclear membrane by electron microscopy
At the same time. Ihc cytoplasmic microtubules
orient themselves toward the emerging bud. These
microtubules seem to guide numerous vesicles
which appear in Ihe budding /one and air involved
in Ihe reshaping of the cell wall. As the bud
grows kugcr. discontinued nuclear mKrotabalcs
begin to appear The longest microtubules form
the mitotic spindle between the Iwu SPB At the
end of G2. the nucleus begins to push and pull
apart in order to penetrate Ihe bud. Some of the
mitochondria also puss with some small vacuoles
into the bad. whereas a large vacuole Is tomed
at the other pole of the cell. The expansion of
Inc Litter seems to push the nucleus into the
bud. During mitosis. Ihc nucleus stretches to its
maximum and Ihe mother cell separates from the
daaghtcrccll This separation tikes place only after
Ihc construction of Ihe separation cell wall and
if'
i taadbonk of Fnology: The Microbiology of Wiie anil Vinifieaiions
ibe deposit of a riig of chliin on the bnd scar of
ibe moihcr tell Tic movement of chromosomes
daring miktsis is difficult to observe in yeasts.
but a mKiotubulc-ccnlfomcR: link mnsl guide
ihc chromosomes In grape mnsl. the duration of
■'.ililiiiu is approximately 1-2 houiv As a result,
the papulation of the cells double during Ihc ycasl
log phase during fcmicitalion.
1.6.2 Sexual Reproduction
When sporifcious ycasl diploid cells arc ii a
hostile nutritive medium (for example, depleted
of fermentable sugar, poor in nitrogen aid very
aerated) Ihcy stop multiplying Some liansform
inu a kind of sac with a thick cell wall. These
sacs arc called asci. Each one cot tains four haploid
ascospoits Ktucd from iKiotic division of the
nucleus Grape must and wine arc not propilioas
to yeast sporulation and. in principal, it never
occurs in this medium. Ycl Mortimer*/ itf (19941
observed ihc sporulation of certain wine ycasl
strains, even in sugar-rich media. Our researchers
have often observed asci in old agarcullurc media
surcd for several weeks in Ihc rcfrigcralor or at
ambient tempcralurcs (Figure 1.12). The natural
conditions it which wild wine yeast, sportkilc aid
the frequency of this phenomenon arc nol known.
In the laboratory, the agar or liquid medium
I'. Si.innlnu clcdmn mk'RncofC pfeotafgnpl ol
.till pUccll OB i MrJ.i-jJ.i =ciln=
tir icvcr.l wcclA. Ami iiinliiniiij' jni>i((in can be
conventionally used k» provoke sporulallou has
a sodium acclalc base tl'it It S. eeremitir.
sporulation aptitude varies grcally from strain to
strain. Wine yeasts, both wild and selected, do
nol spurn lak' easily, and when they do they often
produce non-viable sporcs.
Mciosis in ycasls and in higher cucaiyotcs
(Figure 113) has some similarities Several houis
after the transfer of dipmid vcgclativc cells to
a sporulatou medium, the SPB splits during the
DNA replication of the S phase. A dense body
(DB) appears simullaicously in Ihc nucleus near
lie nucleolus The DB evolves into synaploncmal
complexes— strictures pcrmilling thccouplingand
recombination of homologous chromosomes Alter
8 -9 hours it the sporulation medium, the two SPB
separate aid Ihc spindle begins to form. This stage
is called mctiphasc I of mciosis At this stage, the
chromosomes arc not yet visible. Then, while the
nuclear membrane remains intact, the SPB divides.
At me biphase II. a second mitotic spindle stretches
itsclt while Ihc ascosporc cell wall begins ki form.
Nuclear buds, cynptasm aid organelles migrate
into (he ascospoics At this point. edificatoi of (he
cell wall is com pic led The spindle then disappears
when the division Is achieved.
Placed in favorable conditions, i.e. nutritive
sugar-enriched media, (he ascospoits germinate,
breaking the cell wall of the ascus. aid begii to
multiply In S. cerevishie . lie haploid cells have
two mating types: a aid a The ascus con talis two
a ascospoics and two a ascospoics (Figure 1.14).
Sign a i MA 'a i cells produce a sexual phcromonc
a. This peptide made up of 12 amino acids is
called sexual factor a In Ihc same mailer, sign o
cells produce Ihc sexual factora. a peptide made
np of 13 amino acids. The a factor, emitted by
the XlAIa celLs. slops Ihc multiplication of MATo
cells in CI. Reciprocally, the a factor produced
by o cells stops the biological cycle of a cclK
Sexual coupling occurs bctwcci two eclbt of the
opposite sexual sign. Their agglutination permits
cellular and nuclear fusion and makes use of
parietal glycoproteins and a and a agglutinins.
The vegetative diploid cell that Is formed la/a)
can no longer produce sexual pheromoacs and is
insensitive to their ac tion. il multiplies by budding.
Cytology. Taxonomy anil Ecology of Grape and Wine Ycasls
<4>itibpi>icKKiy
+0 Of i
Ffc MX Mem b
SC=» f - f WDC-.l™- P lc„
Id) * |i.i i,u i ■ r. of (be SP8; let
II of ncH«n: (hi end uf men
lie and Olh
MICcllbcfuic nciov
imkutbonf ipindfc la
i; liiiimitlin o( umpa
:i. 1991). srit=s ? i*llc pule body: Ull =<lcnst body;
; <t>) dividing of SPB: (c) lyoaploocBuiI complete* appear.
:i*pha*e I of mcb%B);(0 dhkli«p of ihe SPB; <p> mcuikmc.
Some strains, turn a ntonospork culture, can he
maintained in a stable haploid stile. Their scxul
sign remains coastinl during many generations.
They arc hctcrothallic Others change sexual sign
during a cellular division. Diploid cells appeal
in the descendants of ai ascosporc. TVy arc
fturaoirtalllcand have an HO gene which inverses
sexual sign at an elevated frequency diring
vegetative division Thiscnangcovcrd-igurc I 15)
occurs in the mother cell at the G I stage of the
/ \ / \ / \ / \
00 00
Fig, 1.15. Sc.u.1 *tgn ..■■Ji>i»n mxk\ of hupkikl
>c*u well. In .i bnmothallk: Main tllcnkouiw eiti..
1992) (« dn^un celK capable of cbanpiotr >tvuil
%ig* Ji the Kit ecll divaioa. or eclb already having
undeigooe balding). . =lniUI eell canyingibe HO
|Nk: Fl. F2 = daughter eclb of I; Fl.i. = daughter
cell of F I
18
Handbook or Etiology: The Micmbiology of Wine anil Vindications
btotogkal cycle, after the tirst building hut before
the DNA rcplKalKii phase. It this manner, a sign
a ascospore S divides to prodncc two a cells
(S and lie lirsl daughter cell. PI). Doling the
following cellular division. S produces rvvo cells
(S and F2) thai nave become a cclK In the same
manner, the PI cell produces wo a cells after the
first division and two a eclbt during lis second
budding. Laboratory strains that arc deficient or
missing the //■ ' gene have a stable sexual sign.
Hckrrothallrsm can therefore he considered the
result of a mutation of tne HO gene or of genes that
control its functioning (Hcrskowitzer of . 1992).
Most wild and selected wincntaking strains that
belong to the S eerevisiae species arc dipk>id
and homothallic. It is also true of almost all of
the strains that have been isolated in vineyards
of tne Bordeaux region. Moreover, recent studies
carried out by Mortimcrer til 1 1994) in Califoraian
and Italian vineyards have shown that the majority
of strains iHi'fi I are homozygous for the HO
character IHO/HO): hctcrozygose (HOlho) is
in minority Hclerolballic strains fhttlho) arc
rare (less than Mi) Wc have made the same
observations for yeast strains isolated in the
Bordeaux region For example. Ike PIO strain fairly
prevalent in spontaneous fermentations in certain
Bordeaux growths Ls HO/HO. In other words, the
four spores issued from an ascusgivc monoparcnl
diploids, capable of forming asci when placed in
a pure culture. This generalized bomozygosis for
the HO character of wild winemaking strains is
probably an important (actor in their evolution,
according to the genome renewal phenomenon
proposed by Mortimer ei <il. (l994)IFigurc 1.16).
in which the continuous multiplication of a yeast
strain in i& natural environment accumulates
nctcro/ygolic damage to the DNA. Ccrttin
slow-growth or functional loss mutations olccrtiin
genes decrease strain vigor in the heterozygous
state. Sporu ration, however, produces haploid
celrs containing different combinations of these
betcrozygolic characters. All of these spores
become homozygous diploid cells with a scries
of genotypes because of the homozygosity of the
HO character. Certain diploids which prove to be
more vigorous than others will in time supplant
the parents and less vigorous ones This very
Cytology. Taxonomy anil Ecology of Grape and Wine Ycasls
templing model is reaffirmed by die characteristics
of the wild wincmaking strains analyzed. In these,
the spore viability rat is the inverse function
of Ihc heterozygosis rale for a ccruin number
of mutations. The completely homozygous strains
present the highest spore viability and vigor.
In conclusion, sporuUlion of strains in natural
conditions seems indispensable It assarcs their
growth and fermentation performance. With this in
mind, the conservation of selected strains of: him
dry yeasts as yeast stirrers should be questioned. It
may be necessary to regenerate them periodically
to eliminate possible mutinous from their genome
which conld diminish their vigor.
1.7 THE KILLER PHENOMENON
1.7.1 Introduction
Ccruin yeast strains, known as killer strains <K).
secrete proteinic toxins into their environment that
are capable of killing other, sensitive strains (S)
The killer strains arc not sensitive 10 their toxin hut
can be killed by a toxin thai they do not produce
Neutral strains IN) do not produce a loxin but ate
icsistint The action of a killer strain on a sensitive
strain is easy to demonstrate in the laboratory on an
agarculturc medium at pH 4.2-4.7 at 20 : C. The
sensitive strain is inoculated into the mass of agar
before it solidifies, then Ihc strain ki be tested is
inoculated in streaks oa the solidified medium. If it
is a killer strain, a clear zone in which the sensitive
strain cannot grow encircles the inoculum streaks
(Figure 1.17)
This phenomenon, ihc killer facur. was dis-
covered in S. eeterisiae but killer strains also
exist in other yeast genera such as Htmitnula.
CumUiki. Kliieckeru. Htimtniuspora. Pkhia. limi-
Ivpsis. Kluytewnrxes and Delxayimyret. Killer
yeasts have been classified into 1 1 groups accord-
ing to the sensitivity reaction between strains as
well as Ihc nature and properties of Ihc toxins
involved The killer factor is a cellular interaction
model mediated by the proteinic toxin excreted
It has given rise to much fundamental research
(Tipper and Bostian. 1684: Young. 1587). Banc
(1984. 19921. Radlcr( 1988) and Van Vuurcn and
I'V, 1.17. Idcnitcaibn ol ihc K2 killer fhenoivpc ia
S. «rnmwir. Tbc pkkocc of a halo inxind the loo
uicak* of ihc killer Kali U due to ihc dcuth of the
icamftivG Main cull rated on ihc medium
Jacobs (1992) have described the technological
imp I ha (ions of this phenomenon for wine yeasK
and Ihc fermentation process
1.7.2 Physiology and Genetics
of the Killer Phenomenon
The determinants of Ihc killer factor aie both
cyuptasmK and nuclear. In S cereYisiae. Ihc killer
phenomenon is assocEilcd with ihc presence of
double- stranded RNA particles, virus-like panicles
(VLP). in the cytoplasm. They arc in Ihc same
category as non-infectious mycovirus. Thctc arc
two kinds of VLP: M and L. The M genome
(IJ-l.9kb) codes for the K toxin and for the
immunity factor (R). The L genome (45 kb) codes
for an RNA polymerase and the proteinic capsid
that encapsulates Ihc two genomes Killer strains
iK'R' I secrete Ihc toxin and arc immune to it.
The sensitive cells (K~R~) do noi possess M VLP
bul most of them have L VLP. The two types of
viral particles arc necessary for ihc yeast cell to
express ihc killer phcnolypc <K 'R '). since ihc L
mvcovirns is ncccssarv for the maintenance of the
Mtype.
:■:;
i taadbonk or Hnology: The Microbiology of Wine and Vnifkatkws
There are Ihicc kinds of killer activities ii
S ctreiiiine sir.iins. They correspond win ihc K I .
K2and K3 toxins coded, respectively, by MI.M2
and M3 VLPs(19. 15 and 1J kb. respectively).
According u Winglicld afar. (1990). the K2 aid
K3 types arc very simitar. M3 VLP results from a
mutilion of M2 VLP. The K2 slrains arc by far
the most widespread In the 5. cerevisiue strains
encountered in wine. NcDtr.il strains IK R") air
insensitive k> a given toxin without being capable
of producing it They posses* M VLPs of normal
dimensions that axle oily for Ihc immunity
factor. They cither do not produce Knits or arc
inactive riccausc of munitions allccting the M-typc
RNA.
Many chroniosomic genes aic involved in the
maintenance and rcplicalKM of I. and M RNA
particles as well as )■ lie maturation and transport
of the toxin prodKcd.
The Kl toxin is a small piolcin made np of
mo sub-units (9 and 95 kDa>. It Ls active aid
sttblc in a very narrow pH range 142-4.6) aid
Is therefore inactive in grape must The K2 toxin,
a 16 kDa glycoprotein, produced by bomolhallic
strains of X ceievisiiie encountered in wine, is
active at between pH 2 8 and 4X with a maximum
activity between 4.2 and 4.4. It is therefore active
at the pH of grape must and wine.
Kl and K2 toxins attick sensitive cells by
attaching themselves to a receptor located in the
cell wall — a ("1.6 glucan. Two chroniosomic
genes. KREl and KRE2 (Killer resistant), deter-
mine Ihc possibility of this linkage. The kre\ gene
produces a parietal glycoprotein which has a ff-
1.6 glucan synlhccisc activity The krel mutants
arc resistant to Kl and K2 toxins because they
arc deficient In tin enzyme and dcvxiRI of a ("1.6
glncan icccplor. The KRI'2 gene is also involved
in the fixation of toxins lo the parietal recep-
tor; the kirl ii in i.i ■ is are aLso icsistant The K>xin
linked to a glucan receptor Is then transferred to a
membrane receptor site by a mechanism needing
cncigy. Cells in the log phase arc. therefore, more
sensitive lo the kllkrrcffccl than cells in the station-
ary phase. When the sensitive cell plasmK mem-
brane Is exposed lo the toxin, it manifest* serious
functional alterations after a lag phase of about
+ ) minutes. These alterations include the interrup-
tion of the coupled transport of amino acids and
protons, the acidification of Ihc cellular content,
and potissium and ATP leakage. The cell dies in
2-3 hours after contact with the toxin because of
the above damage, due to the formation of pores
in the plasmic membrane.
The killer effect exerts itself exclusively on
\ casts and has no effect on hnmans and animals
1.7.3 The Role or the Kill*
Phenomenon in Wi
king
Depending on Ihc authors and viticultural regions
studied, the frequency of Ihc killer character varies
a lot among wild wincmaking strains Isolated on
grapes or in fermenting grape must In a work by
II aire 1 1978) stndying 908 wikl strains. 504 man-
ifested the K2 killer character. 299 were sensitive
and 95 ncnlral Cninicrand Grost 1983) reported a
high Ircqucncv (65 -9OT lof K2 strains in Mediter-
ranean and Bcaujotals region vineyards, whereas
none of the strains analyzed in Tourralnc mani-
fested the killer effect. In the Bordeaux region, the
K2 killer character is extremely widespread. In a
study carried out in 1989 and 1990 on the ecol-
ogy of indigenous strains of S. eerevisiiie in several
tanks of red must inaPcssac-Lc'ognan vineyard, all
of Ihc tsoLilcd strains manifested K2 killer activity,
about 30 differentiated by their karyotype (Frcuer.
1992) Rossini el ul (1982) reported an extremely
varied frequency (I2-80** I of K2 killer strains
in spontaneous fermentations in Italtin wineries.
Some K2 killer strains were aLso isolated in the
southern hemisphere (Australia. South Africa and
Brazil). On the other hand, most of the killer
strains isolated in Japan presented the Kl char-
acteristic Most research on the killer character of
wine ycasR concerns Ihc species 5 cererisitte. Lit-
tle information exeats on the killer effect of the
alcohol-sensitive species which essentially make
np grape microflora Heard and Fleet 1 1987) con-
tinual Bancs (1980) observations and did nol
establish the existence of Ihc killer effect it C«n-
ih'tlu. tfauentuparn, HamemHu and Timdtisp/xti.
However, some killer strains of Hansemupora
itrimim and Pichui kluyvtri have been identified
by Zocg <•( <rf (1988).
Cytology, Taxonomy anil Ecology of Grape and Wine Yeasts
21
Banc (1992) studied Ibc activity and stability
of ibc K2 killer toxin In cnologlcal conditions
(f-ignic 1.18). The killer uxin only manifested a
prouoanccd activity on eclh In the k>g phase Cells
In Ihc stationary phase were relatively Insensitive.
The ..mi "mi of nil. mi 1 or SO; In Ihc wine has
practically no effect on the killer toxin activity.
On the other hand, it is qnickly destroyed by beat,
since lis half-life is around 30 niimiKs al 32"C.
It is also qnickly inactivated by the presence of
phenolic compoands and is easily adsorbed by
ben ton lie.
I'iH I.IK Ycaw ,"■■■ lb and ■
la Ci[oiK«lul pki>c intimhxcil
/i'kc (ttiucicd bv heated imiccn
ml ,nncs i= * -j'"F julrr moUum coniilning killer Mh (Banc. 1992): «. \0T.
M;0. Ill', Mipcrnii.nl jmclivalcd by heal nutmcnt I... White «ikc. r 11 34; eclb
nine =0.(b)Sunic]iifcc .,-tlb hiUbmi} phase Irriniducctt at liac =0.«iRid
Mm. pit Jjt; cclk in ctpnncHnl phaac introduced at time =
22
Handbook of linology: The Microbiology of Wive anil Vindications
Scientific literature has reported a divcislty of
findings on the role or the killer factor in the com-
pclilioa between strains during grape most fermen-
uiHin In an example given by Banc (1992). killer
cclK inoculated at 2'i can coniplclcly supplant the
sensitive strain during the alcoholic fcraicnunon
of must In other works, the kilter yeast/sensitive
yeast ratio able to effect the implaitalion of sensi-
tive yeasts in wincataking varies between l/KKX)
and 100/1. depending on the author. This con-
siderable disciepancy can probably be attributed
to implantation aid fententation speed of the
strains present. The killer phenomenon seems more
important to intcrslraii competition when the killer
strain implants itself qaKkly and the sensitive
strain slowly. In the opposite situation, an elevated
percentage of killer yeast* would be necessary k>
eliminate the sensitive population Some authors
have observed spontaneous fermentations domi-
nated by sensitive strains despite a non- negligible
proportion of killer strains (2—25%). In Bordeaux,
we have always observed that certain sensitive
strains implant themselves in led wine fermenta-
tion, despite a strong presence of killer yeasts in
the wikl microflora (for example. 522M.an active
dry yeast starter! In walk: vvincmaking. the neu-
tral yeast VL1 and sensitive strains snch as EG8.
a slow-growth strain, also successfully implant
themselves The wikl killer popalalion does not
appear to compete with a sensitive yeast starter and
therefore is not an important cause of fermentation
difficulties in real- life applications.
The high frequency of killer strains antoag
the indigenous yeasts in many viticultuial regions
con fee. little advantage to the strain in terms
of implanution capacity. In other wonts, this
character is not sufficient to guarantee the implan-
tation of a certain strain during fcnticntalion over
a wikl strain equally equipped. On the other hand,
under certain conditions. Inoculating with a sensi-
tive strain will fail because of the killer effect of
a wikl population. Therefore, the resistance to the
K2 toxin (killer or neutral phenotype) should be
included among the selection criteria of etiologi-
cal strains. The high frequency of the K2 killer
character in indigenous wine yeasts facilitates this
strategy
A medium that contains the toxin exerts a
selection pressure on a sensitive etiological strain.
Stable variants survive this selection pressure and
ran be obtained in this manner (Banc. 19H4).
This is the most simple strategy for obtaining a
killer cnological strain. However, lac development
of molccalar genetics and biotechnology permits
scientists to construct cnological strains modified
k> contain one or several killer characters
Cytodaction can achieve these modifications.
This method introduces cytoplasmic determinants
t mitochondria, plasm ids) Issacd from a killer strain
into a sensitive cnological strain without altering
lac karyotype of the iaitial cnological strain.
Seki etui. (1935) nscd this method to make
the 522M strain K2 killer. By another strategy,
new yeasts can be constructed by integrating the
toxin gene into their chromosomes Boone el id.
(1990) were able to introduce the Kl character
Into K2 vvincmaking strains in this manner.
The Kl killer character among wine yeasts is
rare, and so the cnological Interest of this Last
application is limited. The acquiring of mnltl-
klllcr character strains presence little cnological
advantage. Sensitive selected strains and current
K2 killer strains can already be inipkinlcd without
a problem On the other hand, the dissemination
of these newly obtained mulll-klllcr strains In
nature could present a non- negligible risk. These
strains could adversely affect the natural microflora
population, although we have barely begun to
inventory its diversity and exploit its technological
potentials It would be detrimental to be no longer
able to select wild yeasts because they have
been supplanted in their natural environment by
genetically modified strains— a transformation that
has no cnological interest.
1.8 CLASSIFICATION OF YEAST
SPECIES
1.8.1 Gc
■a I Rci
rks
As mentioned in the Introduction to this chapter,
yeasts constitute a vast group of unicellular
fungi— economically heterogeneous and very
complex. Hansen's first classification al the
Cytology. Taxonomy and Ecology of Grape and Wine YcasLs
beginning or Ihis ccnluiy only distinguished
between sporifcrous and asporifcrous ycasLs. Since
lien, yeast taxonomy has incited considerable
research. This icseaich has been regrouped
in successive works progressively creating (he
classification known today The last cnotogical
ucaly of the University of Bordeaux (Ribercan-
Gayonela/.. 1975) was based on Loaders (1970)
classification. Between Ihis monograph and the
previous classification (Loddcr and Kreggcr-Van
Rij. 1952). the designation and classification of
yeasts had already changed profoundly In this
book, tic last two classifications by Kreggcr-Van
Rij ( 1984) and Banetl el til. (2000) are of interest
These con tun even mure significant changes in the
delimitation of species and genus with respect to
earlier systcmalics.
According to the current classification . yeasts
belonging to Asconiycctcs. Basidioniyccles and
imperfect fungi tDcutcromycctcs)arc divided into
81 genera, to which 590 species belong Taking
into account synonymy and physiotogKal races
(varieties of the same species), at least 4000 names
lor yeasts have been used since the nineteenth cen-
tury. Fortunately, only 15 yeast species exist on
grapes, are involved as an alcoholic fermentation
agent in wine, and arc responsible for wine dis-
eases. Table 1 .1 lists the two families to which cno-
togical yeasts belong: Sacchimmytelacetie in the
Ascomycctcs (sporifcrous) and Cryptacacenceae
in the Dcutcromycctcs (asponfcrousl Fourteen
genera to which one or several species of grape
or wine yeasts belong ate not listed in Table II
1,8.2 Evolution of the General
I 'iliu i|il.s of Yeast Taxonomy
and Species Delimitation
Yeast taxonomy (from the Greek taxis putting in
order), and the taxonomy of other microorganisms
for that ni.iik.-f. includes classification and
identification. Ckr.sitK.u«>n groups organisms into
laai according to their similarities and/or their
tics to a common ancestor. The basic (axon is
species. A species can be defined as a collection of
strains having a certain number of morphological,
physiological and genetic characters in common
This group of characters constitutes the standard
description of the species. Identification compares
an unknown organism to individuals already
classed and named thai have similar characteristics
Taxonamists first delimited yeast species using
morphological and physiological criteria. The first
classifications were based on the phcuotypK dif-
ferences between yeasts: cell shape and size, spote
formation, cultural characters, fermentation and
assimilation of different sugars, assimilation of
ni Irak's, growth-factor needs, resistance to cyckv
bcximide. The treaty on etiology by Ribcreau-
Gayon etal. (1975) described the use of these
methods on wine yeasts in derail. Since then,
many rapid, ready- louse diagnostic kits have been
Tabic 1.1. I'buikuiu
v VCttl t-vritrc i. Kic-.ycr-Yja Rij. 1984)
s.
.■In,
(
ipongcaa
,.- ini
in)
lily
(4!
rwptooRMK
family
iporngcueaui
F O
......
fa
ipoa
mil>'
ipcneomi
Sub-f»-ily
Sub-fa muv
Sub-fan
lily
—
Sehk-x
rKiAivnni icrti>it!ei#
\.«<M»(/«if«v*"
.V«*"<»7iriii , .v , .i"<lW«Vn-
1 li-JllS
Geoub
Genu
Gcmb
Gen*
aedumvmjea
HuiiniiiBpotn
Drbtr-umw.
licktrr
Haaamla
FidlSa
TbmioMpora
uoiera
Villllltlli
Klonke/a
Rlmlinomlti
:-
Handbook or Etiology: The Microbiology of Wive anil Vinifkaiions
proposed l«> determine yeast response U> different
physkdogkal tests Lafon-Lafourcadc and Joycux
(1979) and Cninicr and Lcvcan (1979) designed
Ibc API 20 C system for the klcnlifkation of cno-
k>gKal yeasts II contains eight fermentatkm tests.
10 assimilation tests and a cyckiacximidc resis-
tance test For a nniic complete idcniifkation. ihc
API 50 CH system contains 50 substrates for fer-
mentation (under paraffin) and assimilation tests
Heard and Reel ( 1990) developed a system thai
uses Ihc different tests listed in the work of Bamcll
el <il. ( 1990).
Due to Ihc relatively limited number of yeast
species significantly picscn ton grapes and in wine.
these phenotypic tests identify etiological yeast
specks in certain genera without difficulty Certain
species can be idcnllficd by observing growing
cells under the microscope Small apkulatcd
cells, having small lemon-like shapes, designate
the species Haitsewitsptrn wiiiitm and its
impelled form Kloeckeru iipiculttiti (Figure 1 .19).
Stwchammwodes luthrigji is characterized by-
much larger (10-20 |im) apkulatcd cells Since
most ycasR mulliply by budding, the genus
Sduzpsacthimmxcfs can be recognized because
of is vegetative reproduction by binary fission
(Figure 1.20). In modern taxonomy, this genus
only contains the species Schizpsacch pmihe.
Finally, the budding of Ctmluki slellnla (fomicrly
known as Tbndapsii siellma) occurs in the shape
of a star.
According to Harnett rial. (1990). the physio-
kigkal characlcristks lisKd in Table 1.2 can be
used to distinguish between ihc principal grape
and wine yeasts. Yet some of these characters
( fur example, fermentation profiles of sugars) vary
within the species and arc even unstable for a
given strain during vegetative multiplication lax-
onomists rcali/rd that they could not differential:
species based solely on phenorypic discontinuity
criteria They progressively established a delimita-
tion founded on Ihc biological and genetic defini-
tion of a species.
In theory. aspccicscan be defined asacollcclion
of intcrfcrlik: strains whose hybrids arc themselves
fertile —capable of producing viable spores. This
btologkal definition runs into several problems
Cytology. Taxonomy and Ecology of Grape and Wine Ycasls
Tabic 1.1 i'h>t Kilo? Kilt ha CKlCrnlK* of Ihc prin. ifal Jjnft J"l 'vine ;,Ci*Li (Ilincil tt <i.. 1990)
Ii!lii
Sill ill i II llliiffli 1 1«! i
IjlH
Sill^ :».lllK1i»M, l f»WiL-
Mil
fl M f ii liiiJIIl J Mi
siiilill iiiiiiiii ms
iij
lit
>J [unlive -: ii'U iii.ine;
ittaa X aro.mu .1 pmmiucf
26
Handbook or Etiology: The Microbiology of Wive anil Vlnif Stations
when applied to yeast*. First of all. a latgc num-
ber of yeasts (Dculeiomycclcs) aic not capable of
sexual tcprcxluction. Secondly, a lol of /\scomy-
cclcsycastSarchomolhallic: nybrNlicition lists arc
espec ially fastidious and dlflkull for routine iden-
tification. Finally, ccrttin wine yeast strains have
little or to sporulalion aptitude, which makes ihe
■sc of strain infertility criteria even more difficult.
To overcome these diflicillies. researchers have
developed a molecular taxonomy over the List
IS years based on (he following tests DNA recom-
bination: Ihe similarity of DNA base composition:
the similarity of enzymes: ullrastructurc chatuc-
■eristics: and cell wall composition. The DNA
recombination tests have pioven to be effective
for delimiting yeast species. They mcasuie the
recombination percentages of denatured nuclear
DNA (mono- stranded) of different strains. An
elevated recombination rale between two strains
(SO- ifXfi 1 indicates that they belong K> the same
species. A low recombination percentile ilcss than
2fXf of the sequences in common) signilics that
the strains belong to different and very dKtanl
species. Combination rales between these extremes
are more d)flkull to interpret
1.8.3 Successive Classifica lions
of the Genus Saccharomyces
and the Position of Wine Yeasts
in the Current Classification
Due to many changes in yeast classilicalion and
nomenclature since the beginning of taxonomic
studies, cnological yeast names and their positions
in the classification have often changed. This
has inevitably resulted in some confusion for
cnologisls and winemakers. liven the most recent
cnological worts I Fleet. 1993: Dclfini. 1995: Boul-
mi'.'J.. 1995) use a number of different epithets
Weievisiiie. baynnus. iimnmi. etc ) attached to the
genus name Saccnannrycet lo designate yeasts
responsible for alcoholic fermentation Although
still in use. this cnological terminology is no
longer accurate to designate the species currently
delimited by laxonomists.
The evolution of species classification for
the genus Sacebaromxces since the early 1950s
(Table 1.3) has created this difference between the
designation of wine yeasts and current taxonomy.
By ciking a closer look at this evolution, the origin
of the differences may be understood.
In Loddcr and Kreggcr-Van Rij (1952). the
names cererisiae. <mfi#mis. buyatms. itvtmeit. etc.
referred to a number of the 30 species of the
genus Sacchwtmnees. Ribfrcau-Cayon and Pcy-
naud (I960) in the Treatise of (Etiology consid-
ered fhat two principal fermentation species weir
found in wine: S. ceretisiae (fonnerly called ellip-
undtia) and S. ovifurntis. The latter was encoun-
tered especially towards the end of fermentation
and was considered more clhanol resistant The
difference in their ability to ferment galactose dis-
tinguished the two species S. ceretisiae (Cal r )
fermented galactose, whereas 5. arffomtu (Call
did not According u the same authors, the species
S. bayaaiis was rarely found in wines. Although
it possessed the same physiological fermentation
and sugar assimilation chatuctcis as S. firifontiis.
Its cells were more elongated . Ik fermentation was
slower, and it bad a particular behavior towards
growth factors The species S. unman was iden-
tified in wine by many authors It differed from
eererisioe. nvifoimis and buyanus because it conld
ferment melibiosc
In Loddcr s following edition (Loddcr 1970). the
number of species of the genus Soirlionirnyres
increased from 30 to 41. Some species formerly
grouped with other genera were Integrated into the
genus Slice hnnmxces. Moreover, several species
names were considered to be synonyms and
disappeared altogether. Such was the case of
5. otiformis. which was moved to the species
bayanus. The treatise of RiWrcau-Cayon el ill.
(1975) considered, however, that Ihe distinction
between nvifonnis and baxamu was of cnological
interest because of the different technological
characteristics of these two yeast. Nevertheless,
by the beginning of the 1980s most cnological
work had abandoned Ihe name ovifoniiis and
replaced it with bayanus. This name change began
the confusion that currently exists.
The new classilicalion by Krcggcr-Van Rij
(1984). based on Yarrows work on base per-
centages of guanine and cylosinc In yeast DNA.
Cytology, Taxonomy ami Ecology of Grape ami Wine Yeasts
I I
I i
I 1
i is
I ..*!-§-!: s :-' i 'lilll-J'l- i -«1 !
mmmnmmmmmmnmmm)
LSI I J 1
imJ
iiiiilillllllilii Hi fi,,
s.iiiii;};;;;;;;;) >>) ii.i.i.i.f.1.
;;
Handbook of Hnology: The Microbiology of Wine and Vindications
brought forth another important chaise in ihc des-
ignation of ihc Sticc IttHtniyrei species Only seven
species continued to exist, while 17 names became
synonyms of S ceievisiae. Ccnain authors con-
sidered them (o be races or physiological vari-
eties of lie species S cerevisiae. As wiln Ihc pre-
ceding classification, these races of S. cerevisiae
were differentiated by Ihcirsug ar utilization pndile
(Tabic 1.4). However, this method of cfctssilka-
tion was nothing nunc than an artificial taxonomy
wilbonl biological significance. Sonologists took to
the habit of adding the varietal name Id S cere-
visiae to designate wine yeasts: S. cerevisiae var.
cerevisiae. var. bayainis. var. uvanoti. var eheva-
lieri. ck. In addition, two species. bailii and ivsei.
were removed fiom the gcins Saccharan/yces aid
integrated into another genus to become Zygoirtr-
clkHi'imes India and Toniiaspara tlelbnieckii.
respec lively.
The latest yeast classification (Harnett el til..
2000) r> based on iccent advances in genet-
ics and molecular laxotom> ' — In panicnlar. DNA
recombination tests reported by Vaughan Martini
and Martini 1 1987) and hybridization experiments
between Strains (Nanmov. 1987) It has again
throwi the species delimitation of the genus
Sticcl/anmmes into confusion. The species now
number 10 and ate divided into three grotps
(Table I J). TV species S. cerevisiae. S. btiytimu*
S. ptradoxus and S. ptisltiriamis cannot he differ-
entiated from one another by physiological tests
hul can be delimited by measuring Ihc degree of
homology of ihcir DNA (Tabic 15). They form the
group Sacclumniyces setau siricio. S. ptisltiriamis
replaced ihc name S. carlsber&ensis. which was
given to brewers ycasl strains used for bot-
tom fermentation (lager) and until now included
in the species cerevisiae. The recently delimited
S. paradti.aa species includes sliains initially n>
lalcd from treccxudalcs. insects, and soil (Naumov
eta).. 1998) ll might conslilnlc the natural com-
mon ancestor of three other yeast species involved
in the fcmicitibon pmccss. Recent genomic anal-
ysis (Rcd/cpovic el nl . 2002) identified a high
percentage of S. partitta.au in Croatian grape
microflora. The occurrence of this species in other
vincyanls around the world and its wlncmaking
properties certainly deserve further investigation.
TuMc 1.4. Phjinlogkal R
single »pccfc% Sirtiuromw
of Sttrtiuromyri
winV* bj Yam
IcnnezU
iba
',.,
Su
Mil
Ra
Me
si
Satiluromyces
baymiM
-
t
-t-
t
-
-
capaifas
-
t
-
t
-
-
-t-
t
-t-
t
rtiewtieri
-t-
t
-
-t-
—
-
coreiim*
i-
-t-
-
t
t
—
dua/ttieus
-t-
t
-t-
t
-
-t-
globo**
i-
-
-
-
-
-
liticrogrnicat
-
t
-t-
-
—
~
lilenipimat
-
-
-t-
-
t
~
t
-t-
t
t
nartoniit
-
-
_
—
t
—
nlaxciti
-t-
-
-
t
t
—
ole.iffno.'uii
i-
-
-t-
t
t
-
protiosmltnu
-
-
-t-
—
—
-
utineri
-t-
t
-t-
-
—
~
«'>»«•"
+
+
+
+
+
Cytology, Taxonomy ami Ecology of Crape ami Wine Yeasts
Tnhlc 1.5. DNA/DNA
F« WvW«.mvce* *
°T
da (Vauphan Mali
hel
ind Martini. 1987)
a bdnngfeaj to
S. i
v S. hnmlL
S ^i-.-i,.,.^
S. piriitoiHt
S. caetmoe
.V pmafanu
100
20
58
S3
KB
70
24
100
24
Id
A second group. Saccltannryces seitsu largo, is
made op of the species eaguxa, ctulelli. servazz
ami Mtui/Kviu The ihinl group consists oily of
Ihe species iliryveri Only toe first group coat-
prises species of cnologicul interest: S. cerevisiae.
S. bayanus, and, pcwibly. S. paraikiais. if its suit-
ability for wine making Is demonstrated This new
classification has created a lot of confusion in
Ihe language pertaining to Ibe cpithcl bayaaut.
Hot raxonomists. 5. bayamu Is a species dlsllucl
fromS. cerevisiae. l-orcnologis&audwiucmakcrs.
bayamis lex ovijiwmis ) dcsignaKs a physiological
race of S. cerevisiae thai does not ferment galac-
tose and possesses a stronger resistance lo etbanol
than Saccharomyces ceievisiae var. cerevisiae.
By evaluating the infertility of strains (a basic
species delimitation criterion). Naumov el al.
(1993) demonstrated lhat most strains fermenting
mclibiosc (Mel 1 1 isolated in wine, and until now
classed as S. cerevisiae var. iivanan, belong to
Ihe species S. bayamis. Some strains, however,
can be crossed with a reference S. cerevisiae lo
produce fertile descendants They arc therefore
attached lo5. cerevisiae These results confirm, bul
nevertheless put inlo perspective. Ihe past works
of Rossini ei ai. I I9S2) and BKknell and Douglas
I l*B2>. which were based on DNA recombination
tests The DNA recoaibinalion pcrcentiges arc
k>w bclwccn the tmnm and cerevisiae strains
tested, but they arc elevated between these same
uvanini slrains and the S bayanus slruin (CBS
380) In other words, most cnokigKal strains
formerly called itvanen belong lo the species
S. bayanus This relationship, however, is nol
complete. Certain Saccharomyces Mel' found in
Ihe spontaneous fermentations of grapes belong lo
cerevisiae. The ycasls thai cnologlsts commonly
called S. cerevisiae var. bayanus. formerly S. ovi-
fomtis, were studied to determine If they belong to
the species bayanus or lo the specie
the majority of inanan slrains. In Ibis case, their
designation only leads to confusion.
All of the result of molecular Gixonomy pre-
sented above show thai the former phenotypic
classifications, based on physiological Identifica-
tion interta. are nol even suitable for delimiting the
small number of fermentative species of Ihe genus
Saccharm/yces found In wincmaking. Moreover,
specialists have long known about Ihe instability of
physiological properties of Sacchammyces strains
Rossini el al I 1982) reclassified a thousand strains
from Ihe ycasl collccuon of Inc Microbiology Insti-
tute of Agriculture al the University of Pcrousc
During this research, they observed thai 23 out of
591 5. cerevisiae strains conserved on mall agar
losl the ability to ferment galactose Twenty three
slrains became' bayamis, according lo 1. odder s
(1970) classification. They found even more fre-
quently thai, over Unte. slrains acquired the ability
to ferment certain sngars Fox example. 29 out of
1 13 strains of Sacchanntyces ovifannis became
capable of fermenting galactose, thus becoming
cerevisiae. According to these authors, this physi-
ological instability is a specific property of strains
from the Saccharomyces group se iisu siricto. In the
same collection, no noticeable change In fermen-
tation profiles was observed In 150 strains of Sac-
cbiotmyces rosei t today Tonilaspora delbrueck/'ii
or in 300 strains of Kloectera apkiilala. Gcncllc
methods arc therefore Indispensable for identifying
wine yeasts. Yet DNA recombination percentage
measures or Infertility tests between homoihallK
slrains. a long and fastidious technique, arc nol
practical for routine microbiological controls. The
amplification of genome segments by polymcriia-
lion chain reaction (PCR) Is a quicker and easier
method which has recently proved lo be an excel-
lent Uol for discrimination of wine ycasl species
30
IKunllxx.k of Hnology: The Microbiology of Wive anil VnificatxMS
1.8.4 Delimitation of Wincmakmg
Specks of S. cererisiae and
S. bayanus by PCR
Since its discovery by Salkl end. (1685). PCR
has oflcn been used k> identify different plani ant
baclcna species. This icchniqnc consists of cn/y-
mabcally amplifying one or several gene fragments
in vino The reaction is bused on the bybndi&i-
lion of two oligonucleotides which I'raatc a taigcl
regui on a double-stranded DNA or leniplalc.
These oligonucleotides have different sequences
and aic coin pie ■icniury k» Ihe DNA sequences
which fraite ihe strand to amplify. Ftgnic 1.21
illustrales the different stages of lie amplification
process The DNA R first denatured ai a high tem-
perature (95 C) The rcaclional mixture Is then
cooled u a Kmpcraturc between 37 and 55 "C. per-
mitting the hybridization of these oligonucleotides
on the denatured strands The strands serve as
primers from which a DNA polymerase pennies the
stage- by-stagc addition of dcsoxyribonuclcoodK
uniS in Inc 5'-3' direction. Tic DNA polymerase
(Figure 1.22) requires lbirdeoiyribonuclcosidc-5'
«■"-*.— -- »»a.,tc |
^™— 1
— 1
~
'
.\mfKB,i «-«■
Kifi 1.21. Principle of ihe poly-
Cytology, Taxonomy ami Ecology of Crape ami Wine Ycasls
31
Vi& 1.22 Mode of Mibn of* DNA jnlvmceoc
triphosphates (dATP. dGTP. dTTP. dCT?|, A
phosphodicstcr bond Is formed between the 3'-OH
end of the primer ami the Innermost pfcosphoras of
tic activjtcd dcoiyribouuclcosidc: py rophosphak'
Is thus liberated. The newly synthesized sir.mil is
fomed on the template model. A Ihcrmorcsislanl
enzyme, the TAQ DNA polymerase, is derived
from Inc Ihcrmofcsntant bacteria Theivms mpuiti-
eus. It permit! a large umber of amplification
cycles (23—10) In vitro wilnonl having to add the
DNA polymerase after each dcnalirauou In this
manner, the DNA fragment amplified during the
first cycle serves as the template for tic follow-
iig cycles. Ii conscqicncc. each successive cycle
doubles the target DNA frag men I —amplified by
a factor of 10' H> 10* duriig 25-30 amplification
cycles.
Hansen ami Kiclland-Brandl (1994) proposed
Mi'.il gene PCR amplification to differentiate
between S cerevisiiie and S. baytuna. while work-
iig on strain rypes of the species ceievisiiie
ami btiyamn and on a strain of the variety
S. uninm. This gene, which codes for the synthe-
sis of the homoscrine acetyl transferase, has differ-
ent sequences in the two species. Part of lie gene
Is initially amplified by using two complementary
oligonucleotides of the scq Knees which border the
fragment to be amplified Tie amplllicat ob timed,
aboil 600 b.p. is the same size for the strains of
the species cerevisiiie and btiytimis tested, as well
as for the Isolate designated S immnt. Different
restriction cmloinclcascs. which recognize certain
specific DNA sequences, then digest the ampli-
fied fragment. Eignic 1.23 gives an example of the
mode of action of the EcoRI restriction cndoiiclc-
asc. ThR ci/ymc recognizes the base sequences
GAATTC and cuts at the localioi indicated by
the arrows. Electrophoresis is used to separate
the obtained fragments As a result, the rcslric-
Itoi fragment length polymorphism (RELP) can
be appreciated. The restriction profiles obtained
ditTcr between cerevisiiie and bayama. They arc
Identical for Inc strain types /wtow/j and uvimoit
tested
This PCR-RELP technique associated to the
MET2 gcic has beci developed and adapted for
rapid analysis The whole cells arc simply heated
in water (95 ! C). 10 minutes before amplification
Only two restriction enzymes arc iscd: EcoRI and
Psti (Masaeuf el ill. 1996a.b). The MET2 ampli-
fkat (580 bp) is cut into two fragments 1369 and
211 bp) by EcoRI in S cerevisiiie. Psll icstriction
creates two fragments for slrali type S. ■'■-.■■ .-.'■•■
EcoRI does not cnl the MET2 amplifkat of
5. hnyianis. nor docs Psti cit the S ceieviiiiie
ainplilkat (Figure I 24) Masncif (1996) dcmoi-
stratcd thai 5 paratkmis can be idcnlilicd by this
method lis MI'.TZ gene amplifkat prodiccs one
fragment of the same sin: as with the two other
species. This one. however. B not cleaved by
EcoRI or Psti. but rather bv Mac III.
Handbook of Fnology: The M km biology of Wine anil Vindications
tnyjBi S.caohn
* S.l*«*«
'■■■-■■.'■ i
xapt
>ft5]P
ZI9|A
i ?I5|*«
Fig 1.24. IdcMifUtfiun principle* fm (he specie*
.V. cm-n»'>- »*1 £ »...fi UI by ihc ,W£7"2 gene
IVR-RI'l E* Ic.Einiipic. ..ilci ullim Ike .imp Il6cal by
i'.'..>KI j ml /'nl irsifkikm tinmo
Fig 1.25. .V.MIlKlck.ll<>pb>ltNSllS'.tl>l(J|/:.-.<|{l
**d I'vl dpcuk.ni of ihc MET2 gene anpli6cats of ihc
Melt MiaiMMudtedby Nmimo. n ..V. <l99.t|. Baud I:
$.Otiuma SCU ll:biad 2: S. Oaunm SCU IJ:b»nd
1: S. ftmwui* SCt! 71: bund 4: i iniytiMt 1.19: band
5: X tu»Mi UW: band 0: S omnbv L 579: band
7: 5. (VTCiin.* L 1425; band 8: S. amisitm VKM-Y
502: band 9: S. Iwvwiat VKM-Y 1 146. M = mokxubt
■>cifil .-.h!.-
By applying ihis relatively simple and quick
technique to difTcirnl cnological strains of 5. imi-
n«i studied by Naumov el al 11993). strains
allacbcd lo Ihc specks bayanus by hybridi/atioa
teste have been clearly demonstrated lo present
the same profile characteristic as bayanus (two
bands after restriclion with Psll. no restticlion with
EcoRI). On Ihe other hand. Ihc unman strains
included in the species cerevisiae, according k>
hybridization teste, effectively have a rcstrktioa
profile characteristic of S. cerevisiae (Figure 1.25
and Table 1.6). The delimitation of the species
cerevisiae and bayaims by these two methods
produced Identical results for the 12 strains
analyzed.
This type of PCR-RFLP analysis of Inc. W£T2
gene has been extended lo different selected > c ;tsi
strains available in Ihc trade and currently used
in wiftcmakiig. Dcpcidiig on ihcir ability lo
ferment galactose, wine professionals in Ihc entire
work! still call these strains cerevisiae or bayanus
(Table 1.7). Hoc all of these strains, restriclion
profile characteristics of the species S cerevisiae
have been obtained.
In the same manner, the species of 82 indige-
nous Sacclianmyces strains teolatcd in wines in
fermentation and on grapes has been determined
(Table 18). For the eight Gal' Mel strains and
lie 47 Cal~Md" strains analyzed, called cere-
visiae and bayamis respectively by cnologiste. the
restriction profiles of the Ml'.TZ gene amplifical
arc characteristic of the species S. cerevisiae Sim-
ilar result, were obtained for 2 elievalieri strains
fermenting galactose bul nol maltose (Ma), as
well as for the capeiuis strain (GalMa >. Mosl
of Ihc Mcr strains, called uvanm until now.
(II oat of 12 for Ihc Isolates from Santerncs
and 1 1 onl of 1 1 for the isolates from Satccne).
belong to Ihc species 5. bayaiau Ycl certain Mcr
arc 5. cerevisiae (one strain from Santerncs and
two strains from Ihc Laltcmaud collection). To
summarize, the classification of the main wine-
making yeasts (Section 18.3) has gone fhrongh
three stages. Initially, several separate specks
were envisaged: S cerevisiae. S. baytuna and/or
5. avifonnis. and S. uvanmi. Subsequently, all of
these were thought to belong u a single species:
S. cerevisiae The current classilkatKan idcnlilks
three dtetinct species on the baste of motecu-
i.i biological data: ' cerevisiae. S bayanus. and
5. pariub ion As the strains of 5 bayama used in
wincmaking belong exclusively to the S. itvanim
varkly (or sub-specks), the remainder of this
Handbook will consider just two specks of wine-
making yeasts. 5. cerevisiae and S. ummn. The
involvement of S. paradoxus in grape fermentation
microflora has yet lo he confirmed.
Finally. PCR-RFLP associated with the MET!
gene can be used to demonstrate Ihc extetence
of bybrkls between the specks 5. cerevisiae and
S. iHiyanus. This method has been used to prove
the existence (Masncuf end.. 1998) of snch a
natural hybrid (strain S6U var Hvarumi among
dry yeasts commercialized by Lallemand Inc.
(Montreal. Canada). Ciolli 0992. 1994) isolated
Cytology. Taxonomy and Ecology of Grape and Wine Yeasts
1'iibk- LA ChanclcriaibnbvPCR-RFLP
llflbc METlfiCK Of 12 S.i:
nntm (He
1 n.hrifrc.1. ifto hybridiraiio*
i«ly by \:iiini..w.' tJ <mi>.,isit* spo-s
S trniimae and 5. btiuti
II IMUMKU
1990)
Sinin '1 III number Origi
Auhor
Hybrid.™
ton km PCR- RPLP of itae
MBT2 gene
SCI II
SCU 13
SCU 74
L 19
L99
LJ90
DBVPG 1042
DBVPG 104-3
DBVPG 1089
Ku»a |
HIB m
rrv>c v
rrvs v
m r s v
rrvTw
rrvTw
rr\'T«
UPGpi
UPG^i
UPC pi
Sape.-Dom
Pouranl
ruubid
Poubnl
Vauphia Mitfia
Vauplaa MjBi.
Vjn^hia Mini*
1 lii(Mi.(V.ik^n,iiii,iiii.
Si aim.
Comacicul brand
Ilnohijiii'il dci^ikUlon
Spcoc,
VLI
Am.lloic VLI
FttB
S. antfto
S. wniw
VL3c
Zvmalloic VL3
REB
X ritri )«.«•■
5. twtiHHJt-
WCT 130
SUra k»Mlif 3
Dorm Midi
X <*trno<»
£ cwe\iv,ie
TIB
AailoR primcur
1XRA Naritonnc
X cerniiaae
5. .vrmif.*"
Pin
AniiDoic PID
REB
S DajDHM
X ( «ni™
R2
Vkkvuir KD
■-a
£ fciMwiM
X twniii.jr'
B02I3
AdiloR htivamn
ImllU ItiMcur
S. rMiimu.i
S <W<*MU«-
CH158
Siba lcndiT4
D-J
£ tW.u»™.v
X tweiiau*-
QA23
Ulvln QA23
LTM
£ bajumM
X <w<tiau«-
IOC 182007
HI'-
X (wniniw
DV10
Vilnuic DVIO
CIVC
S bayami
S (wninur-
i na
Ulvin OI0
UB
S. tqjnli
X nww
EpcnuyZ
Uvufetm CEG
»*
5. buuiias
X mill'
mi . .!>
im <h- H-vurcs d htatt riMnlii-k* >|tc (• olkii til i>! iM ix
tX4iirmtmi-.il DcloU) 1N.VPC.
• iimHi limic
lie <lr Ituwa Mitlrv Pulutil.
OlDkMftflC 1* CTHM(Of)K< 1 rill
iii^iV"
c ntti|ai>kMUirl dra vlf. dc t
■fit Ilmim.il c DIM I l!JU
UllfaalE (tl)CI)l(lfcM«DI <
unpipK CtilUDiMccI Ipnyy. It
i landbook of Etiology: The Microbiology *>f Wive and Vinifii.iiions
Tabic 1.8. Ctuaciei
IhGgBpGkKlBWn
, (Mi
a by PCR-RF1.P
incuf. 19%)
itl'lbc mT2$cm
rofvirk-B^pcck!
1 "t - 'III .Vi.l (',-(.! -<ii. V ■: i"
i iuibiol on
Number ofdiftcicm
Malnamhwd
Oripi
Collect bo
££"'.
Specie*
Sauctnn wine*
ra ii
[ir. «hic Boidcaui ■■.me*
Saute rac* wine*
ra i!
Dr. uhHc Boidcaux 'Im>
KEB
Saueracs » iae%
KEB
I al.i>...iii
Ulb
Sane iocs wine*
FtEB
Saucrao. wine*
KEB
Sjnccnc anil
ra ii
Pouuly/I.oue Valley wines
Sanvene grapes
I" ■!:«)«■
KEB
Ullei
fjurtvv. cank.
Kift 1.2ft. i:k.ln.pfc)iHB ib agan.se gel I IS'. ( of
(■)£»R1 and <b)Pul digest kins of ihc anpliScats of
(he MI.T2 gene of ihc Main hybrid. Bant. I. 2. 3:
sub-ebon, of ihc hybrid Unia; ba*d X: hybrid Unia;
bund 5: 5. omfaar VKM-V 502; band Ik: 5. Imhmiij
VKM-Y I1U.M = molecular weight ■a.ker
this yeast in an Italian winery Ii was selected
for certain cnological properties, in particular us
aptitude to ferment al km temperatures. Its low
produclioa of acetic acid, and IS ability K> preserve
must acidity. The MET! gene restriction profiles
of this strain by EcoRI and Psll. constituted
by three bands, air identical (Figure 1.26). Ii
addition to Ihc amplified fragment, two bands
characteristic of S. cfrerisiiie with EcoRl and two
bands characteristic of Ihc species S biniunis with
Pstl arc obtained The hands are not artefacts due to
an impurity in the strain, because the amplification
of the StETl gene carried out on subclones
(obtained from the multiplication of unique cells
isolated by a micromanipulator) produces identic al
icsalis Furthermore, alter spontathn of the strain
in the laboratory. 10 tetrads were equally Isolated by
a micromanipulator after the digestion of the ascus
cell wall. Note of the 4D ascosporcs analysed could
germinate The non-viability of the ascosporcs
concurs with the hypothesis that this strain is
an interspecific hybrid. Hansen of the CarLsbcig
laboratory (Denmark) sequenced two of the MBT2
gene alleles from Ihts strain. The sequence of one
of the alkies Is identical to that of (he S ceieritiae
MET2 gene, with the exception of one nucleotide.
The sequence of the other allele is 98 5'i similar
ioih.il of 5'. Ixiytuais. The presence of this allele Is
thus probably due loan interspecific cross.
Recent research tNaumov etal.. 2000b) has
shown (hat (he Soil strain is. in fact, a Wlraploid
interspecific nybrRI. Indeed, the percentile germi-
nation of spores from 24 tetrads, isolated using a
micromanipulator, was wry high (94't ). whereas
i( ■■. ■ hi ...i haw been very low fora normal*' diploid
interspecific hybrid The monosporc clones in this
first generation (Fl ) were all capable of sporulat-
ing. while none of the ascosporcs of the second-
generation tetrads wcic viable The hybrid nature
of ihc nKMosporc clones produced by Fl was con-
lirmcil by the presence of the S. eerwisiae and
S. munmi MET2 gene, identified by PCR/RFLP
Filially, measuring the DNA content per cell
using flux cytometry estimation continued that
Ihc descendants of S6U were interspecific diploids
Cytology, Taxonomy anil Ecology of Grape and Wine Yeasts
35
anil that S6U itself was ai allolctraploid. Natu-
ral S. eenr*isitie/S. mantm hybrids have been iso-
lated on grapes and )■ sponuncoasly I'cniKnling
ninsis in Alsace (Lcjcunc and Nfasncuf. unpub-
lished results)
Several other methods using PCR/RR.P have
been applied to typing SacctuBtmrvces itself.
The fragments amplified were ribosomal DNA
sequences* DNArMGuillamonW al.. 1998; Nguyen
Wo/.. 2000).
1.9 IDENTIFICATION OF WINE
YEAST STRAINS
1.9.1 General Principles
The principal yeast species involved in grape
must fermentation, particularly 5. ceretist'iir. com-
prise a very large number of strains with varkd
technological properties. The yeast strains which
arc involved during wiucmaking influence fermen-
tation speed, the nature and quantity of secondary
products formed during alcoholic fermentation,
and the aromatic characters of the wine. The abil-
ity Undifferentiate between the different strains of
5. eerevisiae is required for tic following lields
the ecological study of wild yeasts responsible for
the spontaneous fermentation of grape must; the
selection of strains presenting the best cnological
qualities, production and marketing controls, the
verification of the implantation of selected yeasts
used as yeast starter, and the constitution and main-
tenance of wild or selected yeast collections
Bouix elal. (198 1 1 (cited in Van Vuuren and
Van DcrMccr. 1987) conducted the initial research
on infraspecific differentiation within S. cerew'siiir
They attempted to distinguish strains by clcc-
Irophoretic analysis of their cxoccllular proteins
and later (1987) used the separation of Intra-
cellular proteins Other teams proposed identi-
fying Ihc strains by the analysis of long-chain
fatty acids using gas chromatography (Tredonx
end.. 1987:Aogustyn«7(ir. 1991; Bcndova<7(tf.
1991: Ro/cs el ul.. 1992). Although these dif-
ferent techniques differentiate between certain
strains, they are irrefutably less discriminating
than genetic differentiation methods. They also
present the major inconvenience of depending
on the physiological slat of the strains and
the cultural conditions, which mnsl always be
identical.
In Ihc late 1980s, owing to the development of
generics, certain techniques of molecular biology
were successfully applied to characterize wine
yeast strains. They arc based on the clonal
polymorph ism of the mitochondrial and genomic
DNA of 5. eererisiae These genetic methods
arc independent of the physiological state of the
yeast, unlike Ihc previous techniques bused on Ihc
analysis of metabolism byproducts
1.9.2 Mitochondrial DNA Analysis
The mtDNA of S. ctrevisitie has two remarkable
properties: it Is extremely polymorphous, depend-
ing on the strain: and its is stable (it mutates very
little) during vegetative multiplication Restriction
endon iic leases (such as /.'roRS.i cut this DNA at
specific sites This process generates fragment of
variable size which arc few in number and can be
separated by clcclrophofcs&t on agarose gel.
Aiglc eltd (1984) first applied Ibis technique
to brewers yeasts. Since 1987. it has been
nsed for the charactcri ration of cnological strains
of S. cavriaae (Dubonidicu elal., 1987: Hallct
ruit.. 1988)
The extraction of mtDNA comprises several
stages The protoplasts obtiincd by enzymatic
digestion of the cell walls ate lyscd in a hypo-
tonic buffer The mtDNA is then separated from
the chromosomic DNA by ullraccnlrifugaiion in a
cesium chloride gradient, in Ihc presence of bis-
ben/imide which acts as a fluorescent iMcrcakll-
ing agent This agent amplifies the difference in
density between chromosoniic and mtDNA The
mtDNA has an elevated amount of adenine and
thymine base pairs lor which Ihc bisben/imide has
a stiong affinity. Finally, the mtDNA is purified
by a phcnolcukHolorm- based extraction and an
ethanol- based precipitation
Dcfontainc el ill. 1 1991 ) and Qucrol el ill. ( 1992)
simplified this protocol by separating Ihc mito-
chondria from the other cellular constituents before
extracting the DNA. In this manner, they avoided
36
Handbook or Etiology: The Microbiology of Wine and Vindications
ibe nliraccnlrifugalion step. Tbc coarse cellular
debris is eliminated from the yeast lysatc by ccn-
irifuging at ICOOg. The supernatant is nxen-
trifuged a( 1500 g m obtain Ihc mitochondria. The
mitochondria aic then lyscd in a sai table bolter b>
liberate Ihc DNA
Unlike iadastrial brewer strains analyzed by
Aiglc etal. (1984). which have ihc same mlDNA
reMricllon prolilc. implying lhai (hey arc of
common origin. Ihc cnological ycasi strains have a
huge MlDNA diversity Thct method differentiates
between most of the sctectcd ycasls used in
wine making as well as wild strains of 5. cereviskie
found in spontaneous fermentations (l-ig arc 1.27).
This techniqac is very discriminaling and nol
too expensive, but il is long and requires several
complex manipulations. It is ascfal for Ihc subtle
characterization of a small nambcr of strains.
Inoculation effectiveness can also be verified by
this method To verify an inocihtlion. a sample
Is taken during or uwards the end of alcoholic
fermentation. In the kiboralory. Ihc Ices arc placed
in a liquid medium culture. The mlDNA restriction
profile of this total biomassand of the yeast starter
strain arc compared. If Ihc restriction prolilc of the
sample has no sapcrnumcrary bands with respect
to Ihc ycasi starter strain profile. Ihc yeast sculer
has been properly implanted, with an accuracy of
yi f* . In facl. in the case of a binary mixture,
the minority strain must represent artinnd U'< of
the total population to be detected (Mallei el of.,
1989).
h'iH 1.27. Rcsirkibn pmtte by r7<»R5 of an DNA of
diflciciH urn ins of S. ambit*. Bind I: F10; hand 2:
B02l3:band J: VU;haiKU ; 522; band S: Sia h bind
6: VLJc.M =mari*r
1.9.3 Karyotype Analysts
5. cetetisiae has 16 chromosomes with a size
range between 250 and 2500 Kb. Its genomic
DNA Is very polymorphic: thus it B possible U>
differentiate strains of Ihc species accotding to the
size distribution of their chromosomes Pulse- tie Id
electrophoresis is used to separate 5. ceievisiiie
chromosomes and permit ihc comparison of tbc
karyotypes of Ibe strains This technique uses
two electric liclds oriented differently (90 to 120
degrees). The electrodes placed on Ihc sides of the
apparatus apply the fields alternately (Fignrc 1.281
Fit; 1.28. ''II:.! puhed bckl ekcimphon:
clamped cfcxtrophumu fickl)
Cytology, Taxonomy ami Ecology of Crape ami Wine Yeasts
V
*>
1'iH Lift Principle <if DNA molctulc »c pint bit by puked field cktiroptort
The user can define Die duration of ihc electric
curreni thai will be applied in each direction
ipulse). With each change in direction of the
cIcctrK field. Ihc DNA molecules tcorlcntaK
themselves. The smaller chromosomes reorientate
themselves more quickly than Ihc larger oacs
(Figure 1.29).
Btaadia and Vcyhiact (1988). Edcrsca et id.
1 1988) and Dnbourdicu and Frczicr 1 1990) applied
this technique to identify cuologKal yeast strains.
Sample preparation Is relatively easy. The yeasts
arc cultivated in a liquid medium, collected during
Ihc log phase, and then placed in suspension in
a warm agarose solution that is ponied mm a
partitioned mold to form small pings.
Figure 1.30 gives an example of the iden-
tification of 5. eerevisiiie strains isolated from
a grape must in spontaneous fcmicitilion by
Kifc LJft IX.mptc of clc.lmphinoi. i.pulwd fcckl)
piulilr of S. cvi'iii...' unrio ciryotypci
this method Vc/hiact et id (1990) have shown
thai karyotype analysis can distinguish between
strains of X ceivvisiae as well or better than
Ihc use of mtDNA restriction profiles Funhcr-
more. karyotype analysis is much quicker and
easier n use than mtDNA analysis. In Ihc case
of ecological studies of spontaneous fermentation
niicmtVira. pulse- 1 kid electrophoresis of ehromo-
somes is extensively used today to characterize
slralns of S eerevtsiae (Frcacr and Dubourdicu.
1992; Vcrsavand el til.. 1993. 1995)
Very little research on the chiuatasoatk
polymorphism in other species of giapc and
wine yeasts is currently available. Naumov et id
I 1993 ) suggested that S. btmatus and S eerevisiiie
karyotypes can be easily distinguished. Other
authors (Vanghaa- Martini and Mania). 1993:
Masacuf. 1996) have confirmed his results. In
fact, a specific chtoatosomic band sysKmalically
appeals in S. btnunus. Furthermore, there are only
Iwochromo&omes whose si?es are less thaa 400 kb
ia 5. btmima bul generally atoic in S. eererisiae.
in all of Ihc strains lhat we have analyzed.
Species olhcr than Saciluvtmycts. ia partic-
ular apiculatcd yeasts illaiuciiiutixra uvimm,
Kltieckeni aphidiitn). are presenl on Ihc grape
and are sometimes found at Ihc beginning of fcr-
mcnuiKws. These species have fewer polymor-
phous karyotypes aad fewer bands than in Sac-
ebtwmycet. \'cisavaud el ill. 1 1993) differentiated
between strains of apiculatcd yeast species and
Ciiiulidiifiamna byusiag reslriclioacndonuclcascs
at rare sites (Not I aad Sri I). The cadonuclcascs
cut the chromosomes into a limited numbcrof frag-
ments, which were then separated by pulse-field
electrophoresis.
Handbook of Hnology: The Microbiology of Wive anil Vindications
1.9.4 Genomic DNA Restriction Profile
vnalysis Associated with DNA
Hybridization by Specific Probes
(Fingerprinting)
The yeast genome con Gilts DNA sequences which
repeal front 10 np to 100 lines, snch as ihc A
or Yl sequences of Ihc chromosome trlomcrcs.
The dLsiribulion. or more specifically, ihc number
and location of Incse elements, has a ccmin
Iniraspccific variability. This gcnciK fingcrprinl Ls
bsciI id identify strains iPedciscn. 1986: Dcgre
end. I989>.
The yeasts arc cultivated in a liquid medium.
Samples are laken during the log phase, as in
(be preceding techniques The entire DNA Is iso-
lated and digested by rcstnclKw endonue leases.
The generated fragments are scparalcd by elec-
trophoresis on agarose gel and then transferred
to a nylon membrane (Soaihcra. 1975). Complc-
mcniary radioactive probes (nucleotide sequences
taken from A and Yl elements) are used K>
hybridize with fragments having homologous
sequences. The result gives a hybridization profile
containing several bands.
Cenclic fingerprinting is a more complicated
and involved mclbod than mlDNA or karyotype
analysis. It is. however, without donbl the most
discriminating strain idcnlilication method and
may even dlscrinrinalc too well. II has correctly
indkaKd minor differences between very closely
relaled strains. In fact, in the Bordeaux region.
S ctretiiiae clones isolated from spontaneous
fcrmcnuiKws in diffcrenl wineries have been
cnconnlered which have ihc same karyotype and
the same mdJNA restriction profile Yet their
hybridization profiles differ accenting li> sample
origin (Frczicr. 1992). These strains, probably
descendants of Ihc same mother strain, have
therefore undergone minor random modifications,
maintained during vegetative multiplication.
1.9.5 Polymerization Chain Reaction
<PCR) Associated to & Sequences
This method consist of asing PCR hi amplify cer-
tiin sequences of the yeast genome (Section I 8.4).
ocenrring between the repealed A elcnKnis. whose
separation distince docs ml exceed a certain
valued kb). Ness eial. (1 992 1 and Masncul and
Dubourdieu ( 1994) developed this method to char-
acterize S ceirvisiiie strains. The amplification is
carried out directly on whole cells. They arc simply
healed to make Ihc cellular envelopes permeable.
The resulting amplification fragments arc sepa-
rated according h> their size by electrophoresis in
agarose gel and viewed using ultraviolet fiuorcs-
ccncc (Figure 131).
PCR profile anal>sis associated w|ih<S sequences
can distinguish between most S ceirvisiiie active
dry yeast strains lADY) used in witcmaking
(Figure 1.32): 25 out of the 26 selected commer-
cial yeast strains analyzed. Lavallcc ei <tt. ( 1994)
also observed excellent discriminating power with
this method while analyzing industrially produced
commercial strains from Lalkrmand Inc (Mon-
treal. Canada! In addition, this method permits
Ihc identification of 25 lo 50 strains per day. if
Is Ihc quickest of Ihc diffcrenl strain identifica-
tion lechniqnes currently available. When used for
— MHpbl mi !-.!<■■
itl\ clMIt{tll«tb II 1431 irl
l-'i(i L3I. Principle of idcniiti.iiini
univ. by PCR auueatcd «hn ..W.'.i
Cytology. Ta\onomy and Ecology of Grape and Wine Yeasts
lift 1.32 Heel n-nbuinB. m -pr gel (H I K", i of
iiuplllicd Iriun-cnis iit»:uncil (mm virum commcmil
ci.i Mnlnv Band I: F10; bund 2: 80213: band 3:
VL3c: band 4: UP3DYS;band S: 522 D: band 0: EG8:
band 7: L-1597; hind 8: WET 130. M = moktubi
uc^ki marker: T = ■cpam c cuMnil
Elft 1.33. Ele«H>pllOie*B. in aglt gel (IS*) of
amplified fnigincnu ilkiiinting cvampk-i of verifying
;.«■:.' i implantation |ua-ce->*ful: yca-.li. B and C;
un-u.vcM.ful: yca-A A. D and la Bind I: negative
control: band 2: Lee* A: band 3: ADY A: hand 4: Lees
B: bind 5: ADY B; hind 6: Ira C; bind 7: ADY C;
bind*: Lcc* D; bind 9: ADY D:bind ID: Lees, E: bind
II: ADY E. M = molecular weight miiie<
indigenous sir.iin identification in a given viticul-
innil region, however. It seems in he less dis-
criminating than karyotype analysis. PCR profiles
of wild yeasts isolated in a given location oflcn
appear simitar. They have several constant bands
and only a small number of variable discriminating
bands Certain strains have the same PCR amplifi-
cation profile while having different karyotypes. In
a given location, the polymorphism witnessed by
PCR associalcd with A sequences is less important
than that of Ihe karyotypes. This method is there*
fore complemcnlary to other methods for charac-
terizing wincmaking strains. PCR permits a rapid
primary sort of an indigenous population. Kary-
otype analysis refines this discrimination.
S. '', ■'..'".>'■ strains cannot be distinguished by
this technique because their genome contains only
a few Ty elements
Finally, because of its convenience and rapidity
PCR associated with A sequences facilitates verifi-
cation of Ihe mi plan Li lion of yeast starters used in
wincmaking. The analyses arc effectuated on the
entire btomass derived from lees, placed before-
hand in a liquid medium in a laboratory calturc.
The amplification profiles obtained are compared
with inoculated yeast strain profiles They arc iden-
tical with a successful implantation, and different
if Ike inoculation fails. Figure 1J3 givescxampks
of successful (yeasts B and C) and unsuccessful
Eift 1.31. Dctcrmiaaibn ot Ihe detection l km ho Id of
i contamlnaiing main. Band I: main A 70S. umin
B 30'r;bind 2: -4 ram A HOTi, urain B 20-:; : bind 3:
uiainAW^.uninB I0*i;hamll: Main A 99'.. bind
B I'.; band 5: Mum A 99.9 1 ;. unin B D.1*S; bind
ft: urala A: bind 7: *inln B. M = ■okiutai ..cuhi
i. yeasts A. D. and F> implantations Contaminating
strains haw a different amplilication piotile than
Ihe yeast starter The detection thrcshokl of a coa-
laminating sltain was studied in the laboratory by
analyzing a mixture of two strains in variable pro-
portions. In the example given in Figure I. .14. the
contaminating strain Is easily detected at \'< In
winery fermentations, however, several minority
indigenoas strains can coexist wilh the inoculated
40
H-imllxx-l »>r Etiology: The Microbiology *>f Wive anil Vitrifications
strain When miisi ■■ fermentation or lees is ana-
lyzed by PCR. the ycasl implauution rate sat Icasl
y< H when ihe amplilkation profiles of lie Ices and
ihc ycasl starter arc Mien deal.
In light or various icscaicb. different DNA
analysts methods should be combined to identify
wine yeasl strains
1.9.6 PCR with Mfcrosatdlites
Mkiusatcllilcs arc undent repeal miiis of shon
DNA sequences (I -10 nucleotides), i.e. in the
same direction and deepened throtgbout the
cukaiyotc genome (Field and Wills. 1998). The
number or motif rcpetitions is exlrcmely tri-
able fn>m one individual to another, making these
sequences highly polymorphous in size. These
legions air easily identified, thanks ki the full
sequence of Ihc 5. cererisiue genome, available
on ihc Interact in Ihc Saeclummyces Genome
Dauhase. Approximately 275 sequences have been
listed, mainly AT dinuclcolidcs and AATand A AC
trinucleotides (Perez el til.. 2001). Furthermore.
these sequences arc allelic markers, transmitted
Ri the offspring in a Mcndelian fashion Conse-
quently, these arc ideal genetic markers for iden-
tifying specilic yeast strains, making it possible
not only u distinguish between strains bat also to
arrange Ihcm in related groups This KchniqK has
many applications in man: paternity tests, forensic
medicine, etc. In viticulture, this molecular identi-
fication method has already been applied to Vlits
vinifera grape varieties (Bowcis el al., 1999).
The technique consists of amplifying Ihc region
of Ihe genome containing these mkrosalcllilcs.
then analyzing Ihc size of the amplified portion ma
level of detail of one nucleotide by electrophoresis
on acrylamRIc gel. This varies by a certain number
of base pairs I approximately 8 —JO) from one strain
Ri another, depending on the number of times the
motif Ls repeated. A yeast strain may be hclcrozy-
gous for a given locus, giving two diffcrcm-si/cd
amplified DNA fragments Using 6 mkrosalcllilcs.
Perez el al (2001 ) were able to nlentify +4 differ-
ent genotypes within a population of 5 I strains of
5 crrrvisuie nscd in wincmaking. Other authors
(Gonzalez elal . 2001: Hcuncquin rial.. 2001)
have shown that Ihe strains of S. rermnae used
in wincmaking are weakly heterozygous for the
loci studied However, iniersirain variability of
Ihc uticrosalcllilcs is very high The results are
expressed in numerical values for the size of the
mKrosatellilc in base pairs or Ihc number of rep-
etitions of the motifs on each allele. These digital
data are easy to inkiprct. unlike the karyotype
images on agarose gel. which arc not really compa-
rable from one laboratory k> another. Micmsatcl-
litc analysis has also been used to identify the
strains of S. wtinmi used in wincmaking (Masncuf
and Lcjcunc. unpublished) As the S. manmi and
S. cereYi'siiir mkrosalcllilcs haw different amplifi-
cation prime is. this method provides an additional
means of distinguishing between these species and
their hybrids.
In future, this molecular typing method will cer-
tainly he a useful tool in identifying wincmaking
yeast strains. ccoU^kal surveys, and quality con-
trol of industrial production bathes
Finally, another KchniqK has recently been
proposed for identifying Stxchnnmiyces strains
with PCR by amplifying nitrons of the COX I
mitochondrial DNA gene, which varies in number
and position in different strains It is possible
m amplify cither purified DNA or fermenting
must This technique has been used to monitor
yeasl development during fermentation (Lopez
rial.. 2003).
1.10 ECOLOGY OF GRAPE
AND WINE YEASTS
1.10.1 Succession of Grape and Wine
V-isi Species
Until recently, a kiige amount of research focused
on the description and ecology of wine yeas*.
Il concerned the distribution and succession of
species found on the grape and then in wine
during fermentation and conservation (Ribcrean-
GayoDelal 1975: Lafoi-Lafourcadc 1983).
The ecological study of grape and wine ycasl
species represents a considerable amount of
research. De Rossi began his research in Ihc 1930s
iDc Rossi. 1935) Castclli 11955. 1967) pursued
Cytology, Taxonomy and Ecology of Grape and Wine Yeasts
41
yeast ecology In ItalEin vineyards Pcynaud and
Domcrcq (1953) and Domcrcq (1956) published
lie liisi results on the ecology or cnological
ycasls in France. They described nol only Ihc
species found on the giape and during alcoholic
fermentation, bnl also contaminating ycasls and
diseases. Among the many publications on this
theme since Ihc 196th in vilicullnru) regions
around the world, the following works arc worth
noting: Brechot eiol. (1962). Minarik (1971).
Baractl end. (1972). Park (1975). Cninicr and
Gucrincau(1976). Souflcros (1978). Helm (1979.
1981). Poniard eiol. (1980). Poulanl and Lccocq
(1981). Bureau rt of. (1982), Rossini el of. (1982).
YcasLs arc widespread in nature and air found in
soils, on the surface of vegetables and in the diges-
tive tract of animals. Wind and insects disscminaKr
them. They arc distributed irregularly on the sur-
face of Ihc grape vine: found in small quantities
on leaves, the stem and unripe grapes, they col-
onize Ihc grape skin during main ration Observa-
tions under the scanning electron microscope have
identified Ihc kKation of yeasts on the grape They
arc lately found on Ihc bloom, but multiply pref-
erentially on exudates released from mkrolcsions
in roues situated around the stoualal apparatus.
Bmrytis dnereii and lactic acid and acetic acid bac-
teria spotes also develop in the proximity of these
perisuniatic fractures (Figure 1.35).
The number of yeasts on Ihc grape berry, jusl
before Ihc harvest, is between 10' and 10*. depend-
ing on the geographical situation of the vineyard.
climatic conditions during maturation. Ihc sani-
tary stale of the harvest, and pesticide treatments
applied lo the vine The most abundant yeast pop-
ulations are obtained in warm climatic conditions
(lower latitudes, elevated temperatures) Insecti-
cide treatments and certain fungicidal treatments
can contribute to the rarefaction of indigenous
grape microflora Quantitative results available on
this subject, however, arc few. After the harvest,
transport and crashing of the crop, the number
of cells capable of forming colonies on an agar
medium generally attains I" - ' cclls/uil of must.
The number of yeast species significantly
present on the grape is limited Strictly oxida-
tive metabolism ycasK. which belong to the genus
kihHlniiviilii and a few alcohol-sensitive species,
arc essentially found there. Among the taller,
the apicnlated species IKIiieekem apkutaia and
it. sporifciDus form Haatem'tupnra imnwn) arc
Ihc most common. They comprise np to 99 t .f
of Ihc yeasts isolated in certain grape samples
The following arc generally found bnt in lesser
proportions: Meischnikimiii ptdeherrmui. Cantlhkt
fiomila. C<mti<ki stetlata. Pichiiinietiibninefiicieia.
Pic hia jermentiim. HinaeinHu tintxnitla.
All research confirms the extreme rarity of
S. cerensitte on grapes. Ycl these ycase* arc
nol totally absent Their existence cannot be
proven by spreading out diluted must on a solid
medium prepared in aseptic conditions, bul their
presence on grapes can be proven by analyzing
the spontaneous fermentative microflora of grape
samples placed in sterile bags, then aseptically
crushed and 'milled in the laboratory in the
absence of all contamination Red and white grapes
from Ihc Bordeaux region were treated in this
manner At mid- fermentation in Ihc majority of
cases. 5 cerew'siae represented almost all of ihc
yeasts isolated. In some rare cases, no yeast of this
species developed and apiculaled ycasls began Ihc
fermentation.
Ecological surveys carried oul at Ihc Bordeaux
Faculty of Enology from 1992 to 1999 (Nan-
mov elal., 2(XXXn demonstrated the presence of
S. immwi yeasts on grapes and in spontaneously
fermenting white musLs from the Loire Valley.
Jurancon. and Saulcrncs The frequency of the
42
Handbook or Etiology: The Microbiology of Wine and Vindications
presence of this species alongside S. i
varies from 4-I(XW. On one estate in Abacc.
strains of S. iiranmi were Klcnlilicd on grapes,
in lie ptess. and in vats, where they rcpicsentcd
■p to 9tH of the ycase* involved throughout
fcrmcntiiioa in two consecutive years (Lcjcaac.
■npoblished work). More icccnlly. Nanniov eiiil.
(2002) showed that S. iminmi. identified on grapes
and in fermenting hum. was involved in making
Tokay wine.
The adaptation of S itvitrtm to relatively tow
temperatures (6- 10 C i cerciinly explains its pres-
ence in certain ecological niches: northerly vine-
yards, late harvests, and spontaneous cool" fer-
mentation of while wines. In contrast, this strain
Is sensitive to high tcnipcralnres and has not been
found in spontaneous fermentations of red Bor-
deaux wines.
Recent observations also report the presence of
natural S. eeteriskielS. wiirum hybrids on grapes
and in wineries where both species arc present
(Lcjcunc. unpublished work).
Between two harvests, the walls, the Hoots,
the equipment and sometimes even the winery
baikling are colonized by the alcohol- sensitive
species previously cited Wincmakcrs believe,
however, that spontaneous fermentations are more
difficult to initial: in new tanks than in tanks which
have already been used .This empirical observation
leads to the supposition that S. certrisiae can
also survive in the winery between two harvests.
Moreover. In is species was found In non- negligible
proportions In the wooden fcniicnlers of some of
the best vineyards In Bordeaux during the harvest.
Just before they were tilled.
In the (list hoars of spontaneous fermentations,
the first Ginks filled have a very similar microflora
to that of the grapes. There is a large proportion
of apKulalcd ycase* and M puh'herritia. After
about 20 hours. S. eerevisae develops and coexists
with the grape ycasK. The latter quickly disappear
at the start of spontaneous fcrnten ration. In red
wineniaking in the Bordeaux region, as soon as
must density drops below 11170- 1 060. the colony
samples obtained by spreading oat diluted must
on a solid medium generally isolate exclusively
- (I0 1 to 10* cells/ml). This species
plays an essential role in the alcoholic fermentation
process. Environmental conditions influence its
selection. This selection pressure is exhibited by
four principal parameters: anacrobtosis; must or
grape sulfillng. the sugar concentration: and the
increasing presence of clhanol. In wineniaking
where no sulfur dioxide is used, sach as white
wines for tic production of spirits, the dominant
grape microflora can still be found. It is largely
present at the beginning of alcoholic fermentation
(Figure IJ6). Even in this type of wineniaking.
the presence of apicutalcd yeasts is almost non-
existent at mid alcoholic fermentation.
During dry while wineniaking. the separation of
the marc after pressing combined with clarification
by racking strongly redaccs yeast populations,
at least in the first days of the harvest The
yeast population of a severely racked must rarely
exceeds 10* to lo' cells/ml.
A few days into tic harvest, the alcogencons
5. ceiwisiiir ycass contaminate the harvest
material, grape transport machinery and especially
the harvest receiving equipment, the crusher-
steinmer. and the wine press, lor Ihis reason, it
is already largely present at the time of tilling the
tanks i around 50*i of yeasts Isolated during the
first hoinogcni/ation pumping-ovcr of a red-grape
lank). Fermentations are initiated more rapidly In
the course of the wineniaking campaign because
A
B
Kifi l-Jfi. ro«pirB.i>n ol vcau ipccir* pnunl al iIh
uanol alcoholic kimcnutUm id = 1.00). |A) iaalaal
ofMiMicd ledgnpcj, )• Bonkaux (Frcoc*. I992);<B) )i
a lank of unwilled whie =mt. Strike clahoiatnn o:
Cognac (Ve&avaud. 1994)
Cytology. Taxonomy anil Ecology of Grape and Wine Yeasts
43
of this increased percentage of S. i
fact. Ibc lasi tanks filled offcn complete their
fermentations befocc the liistoncs Similarly, statu
racking in dry whiK wincmaking Is becoming
in' !■: and more difficult to achieve, even al
low lempcralnics. from the second week of the
harvest onward. especially in hoi years The entire
installation inocafcilcs the must with a si/cabk
alcogencou.s yeasi population General weekly
disinfection of the pumps, the piping, the wine
presses, the racking tanks, clc is Ibciefoir strongly
nrcom mended
During the final part of alcoholic fermenta-
tion (tic yeasi decline phase), lie population of
S. eetefiskie progressively decreases while Mill
remaining greater than \(f cclLs/ml. In favorable
wincmaking conditions, characterised by a rapid
and complete exhaustion of sugais. no other yeast
species significantly appeals at the end of fermen-
tation. In poor condi lions, spoikigc ycasK can con-
taminak: Inc wine. One of the most freqient and
most dangerous con tarn inalions is dnc to the devel-
opment of Breihtiumxcts itvermedna, which is
responsible for serious olfactivc flaws (\blumc 2.
Section 8.45).
In the weeks Inal follow the completion of
alcoholic fermentation, the vEihlc populations of
5. cerwisiae drop rapidly, falling below a few hun-
dred cells/ml. In many cases, other yeast species
(spoilage yeasft) can develop in wines during age-
ing or Untie storage. Some yeasts have an oxida-
tive ntclabolism of clhanol and form a veil on the
surface of the wine, sach as Pichia or Candida, or
even certain strains of S. cererisiiw — soaghl after
in inc production of specialty wines By topping
off regularly, the development of these respiratory
metabolism yeasts can be prevented Some other
yeasts, such as Brtttananwes or Dekkera. can
develop in anacrobiosis. consuming trace amounts
of sugars thai have been incompletely or nol
fermented by S. cerevisiae Their population can
attain Ml 1 lo 10' cells/ml In a contaminated red
wine In which alcoholic fermentation is other-
wise completed normally These con Cim inalions
can also occur in Ibc Umk Rcfcrmcntation yeasts
can develop stgnifkrautly in sweet or botryll«:d
sweet wines during ageing or Untie storage: the
principal species found arc Saccbmmycndes hut-
M'ij«. Zxpniicclxaimytet liiiiii. and also some
strains of X leiev/'siiie that arc particularly resis-
tant to clhanol and sulfur dioxide.
1.10,2 Recent Advances in the Study
of the Ecology of S. cererisiae
Strains
The ecological slady of the clonal diversity of
ycasLs. and in particular of 5. cerevisiiie dur-
ing wincmaking. was inconceivable for a long
time because of a lack of means to distin-
guish yeast strains front one another Such
research has become possible with the devctop-
ment of molecular yeast strain identification meth-
ods (Section 1.91 This Section focuses on recent
advances in this domain.
The alcoholic fcrmentiliot of grape must or
gtupes is essentially carried out by a single yeast
species. S. i ■(vvm/i.'c. Therefore, an understanding
of the clonal diversity within this species is
much more important for Ibc wincmakcr than
investigations on the partially or non- fermentative
gtupc microflora
The analysis in this Section of 5. cerevisiae
strains in practical wincmaking conditions in par-
ticular intends to answer the following questions:
• Is spontaneous fermentation carried out by a
dominant strain, a small nnmbcr or a very large
number of strains?
• Can the existence of a sacccssiou of strains
during alcoholic fermentation be proven'' If so.
what Is their origin: grape, harvest material, or
winery equipment?
• During wincmaking and from one year to
another in the same winery or even the same
vineyard. Is spontaneous alcoholic fermentation
carried out by Ibc same strains?
• Can the practice of inoculating with selected
strains modify the wild microflora of a vineyard* 1
During reccnl research (Dnbourdlcu and Fre/icr
1990; Fierier 1992: Masncnf 1996). many samples
of yeast microflora were laken at the vincyaid and
Handbook or Etiology: The Microbiology of Wine and VnUkatkas
ibc winery from batches of while and red wines
spontaneously fermenting or iaoeulalcd with active
dry ycasrs. Several coKlusions can be drawn from
Ibis research, carried out on several thousand wild
strains of S ctrevisitie.
In the najority of cases, a small umber
of major strains (one to Ihrccl representing up
to 7O-80f of the colonics isolated, rarry onl
Ibc s]- hi.iik-1 m> fermentations of red and dry
while wines Tbcsc dominant strains arc found in
comparable proportion-! in all of the fenncnurs
from the same winery f iobi start to end of alcoholic
fcmiciGilion. This phenomenon is illustrated by
the example given in Figure 137. describing ibc
indigenous microflora of several tanks of red mnsl
)■ a Pcssac-Leognan vineyard in 198V. The strains
of S cetvvitiae. possessing different karyotypes,
arc identified by an alphanumeric code comprising
the initial of the vineyard, the tank number, the
time of the sampling, the Isolated colony number
and the year of the sample. Two strains. l-/lbl
1 1939) and FzIK 1 1939). arc encountered in all of
the tanks during the cnlirc alcoholic fermentation
process.
Fig 1.37. Breakdon* of S roriimr caryoiyfo, during ak-ohuli
trcMiic-Lcognan. Fa*cc) in 1989 (Frc.ier. 1992) (b.c. and d ddqjmiic the >
fcrmcriiiian . re*fc«ivel) i. 1.:1a I and II ( Mcriot) and III and IV ICubcwei-Sauv
**d J.lnl diy of Ibc hurvcw. respectively
red .-ii[iL- i,. ■!■- In F/ vine van!
. middle, und end of ultuholic
mi ire filled on Ike l.i. Int. 7ih
Cytology. Taxonomy and Ecology of Grape and Wine Ycasls
The spontaneous fcrmcntalioi of dry while
wines in (he sonic vineyard is also carried oul
by (he vliij! dominant ycasl strains in all of the
tunc Is
The tmk filling order and the grape variety
have little effect on the clonal composition of the
populations of S. cerevisiae spontaneously found
in the winery. The daily practice of pumping-
over (he red grape must with pumping equipment
used for all of the Links probably ensures the
dissemination of the same strains in the winery.
In white wincmaking. the wine press installation
plays the same rote as an inoc ulaior
In addition. in Figure IJ7. all of the strains ana-
lysed arc K2 killer. The two dominant strains do
not ferment galactose (phenotypc Gal J Their for-
mer denomination was thenrforc S ireifonnis or
5. cerevisiae (race bayiunisi in previous classifi-
cations Domcrcq 1 1956) observed a lesser propor-
tion of S. orjfarma in the spontaneous microflora
of Bordeaux region fcrnicn unions in the 195th
tone-filth at the beginning of fermentation to
onc-thinl at the end). In the indigenous fermenth
live microflora of Bordeaux mists, certain strains
of S. cerevisiae Gal - which dominate from the
start of alcoholic fermentation were selected over
time. The causes of this change in Ihc microflora,
remain unknown On the other band, a systematic
increase in (he proportion of Gal - strains during
icd or dry while wine fermentation has km been
observed (Table 1.9) In botryti/cd sweet wines
from Sanlernes. the succession of strains is more
distinct
The same major strain is frequently encountered
for several consecutive vintages in Ihc saute
vineyard in spontaneous- fermentation red-grape
must tanks In 1990. one of the major strains was
ihc same as the previous year in the red grape must
tank of (he !■/ vineyard. Other strains appeared,
however, which had not been isolated in 1989.
When sterile grape samples are taken, pressed
slcrilcly. snllitcd at wincmaking levels and
fermented in (he laboratory in sterile containers,
one or several dominant strains responsible for
spontaneous fcrmcntaiKws in the winery exist in
some samples. These strains arc therefore present
at the vincyaid. In practice, they probably begin
Table 1.9. Rumple uf ph>skilo£kal race bieuU
t'il of St*t1><ri>micei inetiair tfcin«g tpontia
akoholk fciincmaiion (Freucr. 1992)
77 — —
90 — —
02 —
00 —
02 —
ofeku
» M |y.
l-"l,
JO
1991
mi
1992
85
1993
7J
1 ■■u
79
,i -..in 1,1 :<■-. '[].■■. .ill
Tnble 1.10. Rule oloccurn:iH.c<if ihc dnmliuv F/IB2-
K9 caiyiMypc In mkinvinilRaiioiu carried oul on uiiik
(jopeMimplcMl.il. till In the r7 vincyaid
7VZ
it';
to multiply as soon as (he grapes arrive at lie
winery. A few days into ihc harvest, (hey infest
Ihc winery equipment which in turn ensures a
systematic inoculation of Ihc fresh grape crop
The presence each year of ihc same dom-
inant strain in Ihc vineyard is not systematic
tTable 1.10) In the Ft vineyard. Ihc Fzlb2-89
strain could noi be isolated in 1991 although il
was present in certain vineyard samples in 1990.
1992 aid 1994. (n 1993. another strain proved to
be dominant In spontaneous fermentations of sler-
ilc grape samples
46
Handbook or Enology: The Microbiology of Wine anil Vlnificabons
The spontaneous microflora of S. i
seems (u fluctuate Al present, the factors involved
in this fluctuation have nol been identified. Ii a
given vineyard, spontaneous fermentation is nol
syslcnialically catricd out by the same strains each
yean strain specificity docs nol exist and therefore
docs not participate in vineyard characteristics.
Ecological observations do not confirm the notion
of a vineyard- spec ilk yeast. Furthermore, sonic
indigenous strains, dominant in a given vincyaid.
have been found in other nearby or distant
vineyards. For example, the F/Jb2-89 strain,
isolated for the first lime in a vineyard in Pcssic-
Lcognan. was later identified not only in the
spontaneous fermentation of dry white and red
wines of other vineyards in the sane appellation,
but also in relatively distant wineries as far away as
the Medoc. This strain has since been selected and
commercial i/cd under the name Zymaflorc 110
In sonic cascslFigurc 1.38). 5. ceterisiae popu-
lations with a large clonal diversity carry out spon-
tincons must fermentation. Many strains coexist.
Their proportions differ from the start u the end
of fermentation and from one winery to another.
In the Bordeaux region, this diversity causes slow
fermentations and sometimes even stuck fermen-
tations. No strain is capable of asserting itself. On
the other band, the presence of a small number
of dominant strains generally characterizes com-
plete and rapid spontaneous fermentations. These
ii' ■linn. mi strains are found from the scut to the
end of the fcrnicnoiion.
In normal red wincmaking conditions, the inoc-
ulation of the lirst tanks in a winery influences
the wild microflora of non-inoculaKd links. The
strairKs) used for inoculating lie first tanks arc fre-
quently found in majority in the killer Figure 1.39
provides an example comparing the microflora of
a lank of Mcriot from Pomcrol. inoculated wilb
an active dry yeast strain (522M) on the first day
of the harvest, with a not- inoculated tank tilled
later. From the start of alcoholic fermentation.
Ike selected strain is successfully implanted in the
inoculated tank Even in Ihc non- inoculated tank.
the same strain is equally implanted throughout the
fermentation. II is therefore difficult lo select the
dominant wikl strains in red wincmaking tanks,
when siimc of Ihc tanks have been inoculated.
An early and massive inoculation of the must,
however, permit, the successful implantation of
tiH 1.38. BitiiLifciu-
ct. 19921 |b. c. <™d d do^oaic the u
ml. Fiance) in 1989
lively)
Cytology. Taxonomy and Ecology of Grape and Wine Ycasis
Samples
Tank I. inoculated with F5
Samples
Tank II. inoculated with FID
□ <
r carywyfo, m
1990 (b.t. awl
:nd of akoholk
IniKuUicd *Hh
KiH LM Breakdown at S. tm\i
link I a*d lank II P vincjaid U
d .L-M!-ii.il. (he v. in. rrn..lk i it.
kfintnljiliii. Ilifcaivcly) Tank I
S22M dry yenti 4*1 lank II uodcrxem *po«a-
fcimcntarion lb. c and d detlginaic the unit, miiklk
end of akoholk fc mental ion. mpcciivcly)
difTcicnl sclccfcd yeasts in several tanks al the
sunic winciy (Figure 1.40).
In while wiucniaking. iiocuktliig rarely influ-
ences the microflora of spontaneous fermentations
ii wineries Rn the ntost pail, dominait indige-
nous strains in non-i>oculatcd barrels of foment-
ing diy white wine are observed, whereas in the
same wine cellar, other hitches were inoculated
'■:■:■
r
'■'■/.
■■+■
1,
Samples
Ta
Ik I]
ulatf
dw
Hi 52
:\:
a
iHHeinl \«ul viair-
I'< I 40. BmLdown of .S caeiiuiK earyoiypci ■■
lank* I. II and III in F viacynid i* 1990. with mauivc
cad> inocublbn with FS. FID and 522M. (cipccllvcly
(Frc/kr. I992)(b.ca*d d iksqJMic Ike uait. middk.
and end of aleukia: tcimcotaiiin. itifctflvdy)
Handbook of linology: The Microbiology of Wine and Vindications
witn different sc Ice it'll ycasis Toe alwicc of
pumping-ovcrs probably hindcis Ihc dissemination
of Ihc same ycasls In all of Ihc fermenting barrels.
This situation permits Ihc fermentative behavior
and cnological interest of difTcrcnl selected strains
u be compared with each olhcr aid wilh indige-
nous strains in a given vineyard The barrels ait
filled wilh thesame must some arc inoculated with
the yeast u be compared A simple of Ihc biomass
is taken at mKI Icrmcn union The desired implan-
tation is then verified by PCR associated with <t
sequences. Due ■> ihc case of use of this method,
information on characteristics of selected strains
and their influence on wine quality can be gathered
al the winery
Vczhinct ei ill. (1992) and Vcisavaud el ill.
(1995) have abo studied the clonal diversity of
yeast microflora in other vineyards Their results
confirm Ihc polyclonal character of fermentative
populations of S. cererisii/e. The notion of
dominant strains (one to two per fermentation) is
obvious in the work carried out in the Charcots
region As in Champagne and the Loire Valley,
sonic Charcutcs region strains arc found for
several ycaa in a row in the same winery The
presence of these dominant strains on the grape
has been continued before any contact with winery
equipment during several harvests.
Why do some S. cfrerisiiw strains issued Mod
a very heterogeneous population become domi-
nant during spontaneous fermentation? Why can
they be found several years in a row at the same
vineyanl and wine cellar? Despite their practi-
cal interest, these questions have not often been
studied and there arc no definitive responses. II
seems that these strains rapidly starl and com-
plete alcoholic fermentation and have a good
rcsRcincc to sulfur dioxide (up to 10 g/nl). Fur-
thermore, during mixed inoculations in the lab-
oratory of cither %'&■■ cthanol or non-fermented
must*, these strains rapidly become dominant
when placed in the presence of other wild
•on- dominant strains of S ceiwitiae totaled al
the starl and end of fermentation. This sub-
ject merits further research. Withonl a doubt,
it woukl be interesting u compare the genetic
characteristics of dominant and non-dominant
yeasts and their degree of hclcro/ygosity Con-
sidering ihc genome renewal theory of Mortimer
rut). (1994) (Section 1.62). dominant strains arc
possibly more homozygous than non-dominant
strains.
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Biochemistry of Alcoholic Fermentation
and Metabolic Pathways of Wine Yeasts
2 1 Introduction
12 Sugar degradation pathways
2 .1 Regulation of sagar-ulilizing metabolic palbw
2 4 Metabolism of nitrogen compounds
2.1 INTRODUCTION
The synthesis of living material R cndcigonic.
requiring the consumption of energy Chloro-
phy lions piano, called pbotoUophs. collect solar
energy. Some bacKria obtain energy from the oxi-
dation of minerals: they arc chcmolitkolrophs. Like
most an i mats and bacteria, fnngi. including yeast,
arc chcmootganolropks: they draw their necessary
eneigy from the degradation of organic nutrients
In a growing oiganism. energy produced by
degradation rcacttoastcabibolisni) is transferred to
the chain of synthesis icac lions ianabolt.ni i Con-
forming to the laws of thermodynamics, energy
furnished by the degradation of a substrate is only
partially convened into work: this r. called free
energy (the rest ts dissipated in the form of hcatl
Part of this free energy can be used for transport,
movement, or s\n these* In most cases, the ftcc
energy transporter particnlar to bkitogical sysiems
is adenosine triphosphate (ATP) This molecule is
rich in energy because its triphosphate nit con-
tains two pbosphoan hydride bonds (Figure 2 I)
The hydrolysis of ATP into adenosine diphosphate
(ADP) results in the liberation of a large quan-
tity of free eneigy (7.3 kcal/mol). Biosynthesis and
the active transport of metabolites make use of
free energy.
Handbook or Etiology: The Microbiology of Wiac anil VnifkatkMS
Fit; £'• Sinmuic of aik*a»)nciriphii»phMc (ATP)
ATP + HiO = ADP+Pi+H'iiG"
= -7.3 ncal/mol (2.11
In Ibis icaciKii. AG" is Ike change in five cncigy.
ATP is considered to be ihc univcis.il money of
free energy in biological systems* (Sliyct. 1992).
In realily. microorganism growth or. in this ease,
yeast growth is directly related lo the quantity of
ATP famished by metabolic pathways used for
degrading asubstralc. It is indiircily* fcUttcd to the
...hi ni> of substrate degraded
In Ihc living cell, there arc two processes which
produce ATP: substrate- level phosphorylation aad
oxidative phosphorylation. Poih of these pathways
exist in wine yeasts.
Substrate- level phosphorylation can be cither
aerobic ot anaerobic. Diring oxidation by electron
loss, an csicr- phosphoric bond is limned. II Is
an energy-rich bond between Ihc oxidiccd carbon
of Ihc substrate and a molccalc of inorganic
phosphate. This boad is then transferred lo the
ADP by iraaspnosphoryljiion. thus foraiing ATP.
This process lakes pfctcc daring glycolysis.
Oxidative phosphorylation is an aerobic pro-
cess The production of ATP is linked b> the
transport of electrons to an oxygen molecule by
the cytochroaiic respiratory chain This oxygen
mokreak is Ihc final acceptor of the elections.
These reactions occur in the mitochondria.
Tbischapler describes the principal biochemical
reactions occarring during grape must fermenta-
tion by wiac ycasLs. It covers sagar avclabojisms.
i.c the biochemistry of alcoholic fermentation, aad
nitrogen mctibolisms Volatile sulfur-con tuning
com pan nds and volatile phenol formation mecha-
nisms will be discussed in Volume 2. Chapter 8 in
the section coaccming olfactory flaws The influ-
ence of yeasts on varietal wine aromas will be
covered in Volume 2. Chapter 7
2,2 SUGAR DEGRADATION
PATHWAYS
Depending on aerobic conditions, yeast can de-
grade sugars using two metabolic pathways: alco-
holic fermentation and respiration. These two
processes begin in the same way. sharing the com-
mon Hank of glycolysis
2.2-1 Glycolysis
This scries of rcactioiis. transforming glucose into
pyruvate with the formation of ATP. constitutes
a quasi-universal pathway in biological systems.
The elucidation of lac different steps of glycolysis
Is intimately associated with the birth of modern
biochemistry. The starting point was the fonu-
itaas discovery by Hans and liduaid Bachncr. in
1897. of the fermentalioB of saccharose by an accl-
lalar yean extract Studying possible ihcrapcatK
applications for their yeast extracts, the 8 uebners
discovered that the sugar used K> preserve their
yeast extract wits rapidly fermented into alcohol.
Several years later. Harden and Young demon-
strated that inorganic phosphate must be added to
acellular yeast extract to assure a constant glu-
cose fermentation speed The depletion of inor-
ganic phosphate daring hi vliro fcrmentillon krd
them to believe that it was incorporated into a
sagar phosphate. They also observed that the yeast
extract activity was due to a aoa-dialysiblc com-
ponent, dcaatutable by heal, and a thermostable
dialy/ablccoaiponenl They named these two com-
ponents zymase and lo/ym.isc .Today. nmiKT
is known to be a scries of cn/ymes and co/ymasc
is composed ol their cofacloisas well as me Eil Ions
and ATP. The complete description of glycolysis
dates back « the 1944b. due in particular b the
coutribalioBs of Embdcn. Mcycrhoff and Ncubcrg.
Biochemistry of Alcoholic Fermentation anil Metabolic Pathw;rys of Wine Ycasfc
SS
For thai reason, glycolysis is often called Ihc Fmb-
dcn-Mcycrhoff pathway.
The transport of must hexose (glucose and fruc-
tose) across Ihc plasm ic membrane activates a
complex system of proteinic transporters not fully
explained (Section 1.32). This mechanism facili-
tates the diffusion of must hexoscs in the cyto-
plasm, where they are rapidly metabolized. Since
solilc moves in the direction of the concentration
gradient, from the concentrated outer medium to
lie diluted inner median, it is not an active Irats-
port system requiring energy
Next, glycolysis (Figure 2.2) is carried out
entirely In Ihc cylosol of the cell It includes a
first stage which converts glucose ilk) frucUsc
1 .6- bi phosphate, requiring two ATP molecules.
This transformation itself comprises three steps:
an initial phosphorylation of glucose into glu-
cose 6- phosphate. Ihc isomer) /ation of the latter
into fructose 6-phosphatc and a second phcopho-
rylation forming fructose 1.6-biphosphatc. These
three reactions arc catalyzed by hexokinase. phos-
phoglucosc isomcrasc and phosphoglKokinase.
icspcc lively.
In fact. SacchiB»myces eeretisioe has two bcx-
okinases (PI and Pll) capable of phosphorylating
glucose and fruclosc. Hexokinase Pll Is csscn-
ml and is active predominantly during the yeast
log phase in a medium with a high sugar con-
centration Hexokinase PI. partially repressed by
glucose. Is not active until Ihc stationary phase
(Bisson. 1991).
Mutant strains devoid of phosphoglncoR)-
mcrasc have been isolated Their inability to
develop on glucose suggests that glycolysis is
Ihc only cauboltc pathway of glucose In Sac-
eharomyces ceiwitiae (Canbct el til.. 1988). The
oxidative pentose phosphate pathway, by which
some organisms utilize sugars, serves only as a
means of synthesizing rlbose 5 -phosphate, incorpo-
rated in nucleic acids and in reduced nicotinamide-
adenine dinuclcotidc phosphate (NADPH) in
Smrhanmyces.
The second stage of glycolysis forms glyccr-
aldcbydc 3- phosphate . Under the catalytic action
of aldolase, fructose 1.6-biphosphatc is cleaved
thus forming two Iriose phosphate
dihydroxyaceunc phosphate and glyccraktchydc
3-phosphate. The tiiosc phosphate isomcrasc cat-
alyzes the isomeri nation of these two compounds
Although at equilibrium the kctonic form Is more
abundant. Ihc transformation of dihydmxyacc-
lone phospbaK inioglyccraldchydc 3-pbosphatc is
rapid, since this compound is continually climl-
nakrd by Ihc ensuing glycolysis reactions In other
words, a molecule of glucose leads to Ihc formation
of two molecules of glyceraldcbydc 3- phosphate
The third phase of glycolysis comprises two
steps which recover part of the energy from glyc-
craldchydc 3-phosphale (G3P). Initially. GA3P
is converted into 1 J-biphosphoglyccraic (13-
BPG). This reaction is catalyzed by glyccraldc-
hydc 3- phosphate dehydrogenase. It isan oxidation
coupled with a substrate- level phosphorylation.
Nicotinamide -adenine dinnclcotldc (NAD ' i is (he
cofactDrofthc dchydrogc nation. At thrssugc.it is
in its oxidised form: nicotinamide is the reactive
pari of the molecule (Figure 2.3). Simultaneously,
an energy- rich bond is established between the oxi-
dized carbon of the substrate and the inorganic
phosphate The NAD* accepts two electrons ;ind
a hydrogen atom lost by the oxidized substrate
Next, phusphoglycc rate kinase catalyzes the trans-
fer of the phosphoryl group of the acylphosphaic
from 1.3-BPG to ADP: and 3-phosphoglyccralc
and ATP arc formed
The last phase of glycolysis transforms 3-
phosphoglyccraie into pyruvate. Phosphoglyccro-
muiasc catalyzes the conversion of 3-phospho-
glyccralc Into 2-pbosphoglyccratc. Enolasc
catalyzes the dehydration of Ihc latter, forming
phosphie no! pyruvate This compound has a high
phosphoryl group transfer potential By phospho-
rylation of ADP. pyruvic acid and ATP arc formed:
Ihc pyruvate kinase caulyzes this reaction. In this
manner, glycolysis creates four ATP molecules
Twoarc immediately used toactivakrancwhcxosc
molecule, and the net gain of glycolysis is there-
fore two ATP molecules per molecule of hexose
metabolized. This stage marks Ihc end of the com-
mon trunk of glycolysis: alcoholic fermentation,
g lyce ropy ru vie fermentation or icsplration follow.
depending on various conditions.
I LiiullwiL i>r Isnology: The Microbiology *>f Witc anil VnifkatkMS
<£-"B=c ,a cb.«-4>
Jr
1 . .; . J ■!: h ;. A- 1 f h» (*U ,
1 *""
run -.£»
Kift 22 c;hcoJy.(. and akoholk fcnncMMiun palhoi)
Biochemistry of Akoholic letmcniation anil Metabolic Pathways of Wine Ycas&
I'iii 2.X (a) Siuictuic
>vMi*d<\AD umlitduccd (NADH) lorn
n.lifcd d>nn (NAD' ). rt>) EqulliirR
2.2.2 Alcoholic Fermentation
The reducing power of NADH. produced by gly-
colysis, must be transferred t> an electron acceptor
K> regenerate NAD' In alcoholic fermentation, il
is noi pyruvate bit ralhcr aceciktch)\lc. its decar-
boxylation product, that scrvesas the terminal elec-
tion acceptor With respect to glycolysis, alcoholic
fermentation contains two additional ciftnuK
reactions, the fiist of which (cattly/cd by pyru-
vate decarboxylase), dccarboxykilcs pyruvic acid.
The cefaclor is thiamine pyiophosphalc ITPP)
(Figure 2.4) TPP a ud pyruvate form an interme-
diary compound. More picciscly. the carbon atom
located between the nitrogen and the sulfur of the
TPP thiamlc cycle is knifed. It forms a carbon ion.
which readily coat bines with the pyruvate carbonyl
group. The second step reduces acctaldcbydc inlo
etnanol by NADH. This rcactkii is catalysed by
the alcohol dehydrogenase whose active sin- coa-
lainsa Zn ! " ion.
olihi-niK pymphosftaieaFPl
Handbook or Etiology: The Microbiology of Wive anil VnificatxMS
pyruvate decarboxylase (PDC)
comprises two isoenzymes a major form. PDCI.
representing 80* of the decarboxylase activity,
and a minor form. PDC5. whose friction remains
nn certain
From un energy vicwpoinl. glycolysis followed
by alcoholic feme illation supplies the ye;isi with
two molecules of ATP per molecule of glucose
degraded, or 14.6 biologically usable kcal/mol
of glucose fermented Fiom a thermodynamic
viewpoint, the change in live cncigy during ihc
dcgradalioi of a mole of glucose inlo clhaiol and
CO2 is -40 kcal The difference (254 kcal) is
dlssipaled in ihc form of heal.
fermentation
2.2.3 (ilyccropyr
In (he presence of sulfite (Ncubcrg. 1946). ihc
fermentation of glucose by yeast* produces equiv-
alent quantities of gl)ceml. carbon dioxide, aid
acclakUrhyde ii let bisulfilK form. This glyccropy-
nivic fermentation lakes pint in Ihc following
manner. Since Ibc acclaldcbydc combined with
salfilc cannoi be reduced inlo clkanol. dibydroxy-
acclone- I - phosphate bccouKs Ihc terminal electron
acccplor. Ii is derived from lie oxidation of give-
craldchydc 3-phosphalc and reduced lo glycerol
.l-phosphalc. which is itself dcphosphorylalcd iilo
glycerol. This mechanism was used for the indus-
trial production of glycerol. In Ibis fcrmcniation.
oily two molecules of ATP arc pioduced for every
molecule of nexose degraded. ATP is required to
activate the glucose in the first step of glycolysis
l Figure 25). Glycc ropy ru vie fermentation, whose
net gain ii ATP is ill. docs not furnish biologically
assimilable energy Tor yeasts.
Glycc ropy ru vie fermentation docs not occur
uiiqucly ii a highly sulfite: environment. In the
beginning of Ihc alcoholic fcrmcntalioa of grape
must, the inoculum consists of yeasts initially
grown iu the presence of oxygen. Their pyru-
vate decarboxylase and alcohol dehydrogenase
arc weakly expressed. As a result, cthanal accu-
mulation is limited. The rcoxidaliou of NADH
docs lot involve cthanal. bnl rather dihydroxy-
acctonc. Glycerol, pyruvate and some secondary
fcrmcuttlMM products arc formed These secondary
products arc pyruvate derivatives— including, bnl
not limited to. succinate and diacctyl.
2.2.4 Respiration
When sugar is used by the tcspiralory pathway.
pyruvic acid (originating in glycolysis) undergoes
an oxidative decarboxylation in the presence of
coenzyme A (CoA) (Figure 2.6) and NAD' Thus
process generates carbon dioxide. NADH and
accryl-CoA:
pyruvate 4- CoA -I- NAD r
acetyl CoA 4- COj + NADH + rT (12)
The cu/ymatK system of the pyruvate dehydroge-
nase catalyzes this reaction. It tikes place in the
interior of the mitochondria Thiamine pyrophos-
phate (TPP). lipoamRle and flavin -adenine diu-
uclcolidc (PAD) participate in this reaction and
serve as catalytic cofaciors.
The acetyl unit issued from pyruvate is activated
in Ihc form of acetyl CoA. The reactions of the
citric acid cycle, also called the tricarboxylic acids
cycle aid Krcbs cycle (Figure 2.7). completely
oxidi/c the acetyl CoA into CO,. These reactions
also occur iu the mitochondria.
This cycle begins wilh the condensation of
a 2-curbon acetyl unit with a 4-iarbon com-
pound, oxaktacctale. to produce a tricarboxylic
Bloc hen bury of Alcoholic Fcmicniation and Metabolic Pathways of Wine Ycasfc
I— CM,— CM,- N— C— Ctl,-C
V V»
cofeoco/yme A. The
11.. I ili ■■! ..- ■■ -. - 1'
acid wiih 6 carbon atoms: citric acid. Poor oxt-
dation- reduction reactions regenerate the oxaloac-
ctaic. The oxidative pathway dccarboxylatcs
Isocitratc.an isomer of citrate, intoa-kctoglutaralc
The isocltralc dehydrogenase cataly/cs this reac-
tion The a-ketogluuratc. a 5-carbon ami com-
pound, undergoes an oxidative decarboxylation to
become succinate by the a-kcloglutaratc dehy-
drogenase. In these luti reactions. NAD' is
Ihc hydrogen acceptor. The fumaraK dehydroge-
nase is responsible for the reduction of succinate
into fumaralc: PAD Is the hydrogen acceptor
(Figure 2.8). Finally, fumaralc is hydrakrd into
i.-malatc. The latter n reduced iiKioxaloaceuie by
Ihc nuila ic dehydrogenase In this case, the NAD*
Is an electron accepur once again.
From ate la I: . each complete cycle produces two
COj molecules, three hydrogen ions transferred to
three NAD * molecules (six electrons) and a pair of
hydrogen annus (two electrons) transferred to an
PAD molecule The cytochrome chain transports
these electrons towards the oxygen. AT? is formed
during this process. This oxidative- phosphoryla-
tion (Figure 2.9) tikes place in the mitochondria
This process makes use of three enzymatic systems
(the NADH-Q reductase, the cytochrome reduc-
tase and the cykxhroatc oxidase). Two electron
transport sysfcms( ubiquinone, or coenzyme Q. and
cytochrome • i link these enzymatic systems.
Oxidative phosphorylation yields three ATP
molecules per pair ofclcctrois transported between
toe NADH and the oxygen— two ATP nwlccnlcs
with FADH<. In the Krcr* cycle, substrate- level
phosphorylation forms an ATP molecule during the
transform. 1 1 ion of succinyl CoA into succinate.
The respiration of a glucose molecule (Table
2.1) produces 36 to 38 ATP molecules. Two
originate from glycolysis. 28 from the oxidative
Tunic!!. Knciuy haUnt
.-.. .Liu-
Si*ge
Rcditlnncocan«e
Sumherol molecules
of ATP formed
Grycoly*B
2NADH
4ot0
Nei juin ia ATP from pJ-.iol.Mi
2
Pyaivnc • acetyl CoA
NADH
6
boenrate - — -• a- Kei op hi a rate
NADH
6
u-Kctofi tin rate succinyl CoA
NADH
6
Succinyl CoA — — Mnviiratc
2
Succinic famine
KADH,
4
Mabtc ■ oxibaccutfc
NADH
6
No yield fmm phx-ciM?
36-38
H.iiklhx'k i>r Etiology: The Microbiology of Wme and VnifkalkMB
Pit; 27. Tricaiboxvlic Mil or Kicta* cycle. I =cintc *Yirtku.c: 2-3 =
■n*c: 5 =compki a-Lcm^liiJcuc <lcby<liogcn*bc: ft = wjccieyl-CoA ..nta;
8 = tiimjiiM: « = arable dehydrogenase: CnTspwacuinr triphmplalc: GDP =gl
= I--- ll.il. .k--;. ,!.-.,'.-
tciaaic dchvdfnjicmsc:
diphosphate
Biochemistry of Alcoholic Fcrnicn union anil Metabolic Pathways of Wine Ycmfc
niC-c* c
I II
ri(C-c c c N-ti
i ii I I
Klemkk(FAD): (atoxtdiwd fura iFAI)K(b) reduced torn 1FADH,)
(m
Fifl -"■ Oxidant phn*fhoi%bi»« Juii«f ckciiun
laBpiiit In ihc mp inituiy chaia
phosphorylation of NADH and FADHj generated
by the Krchs cycle, anil two from substrate-
level phosphorylation during Ihc fomiation of
succinate Four to six ATP molecules icsult fmm
Ihc oxidative phosphorylation of two NADH
molecules produced in glycolysis The ptcclsc
number depends on Ihc transporl system used lo
move the electrons of the cytosolic NADH lo
Ihc respiratory chain in the mitochondria The
respiration of the same amonnl of sugar produces
IS lo 19 times more btotogkally usable cncigy
available fe> yeasts than fermentation Respiration
Is used for industrial yeast production.
2.3 REGULATION OF
SUGAR-UTILIZING
METABOLIC PATHWAYS
2.3.1 Regulation Between
Fcrmcnlation and Respiration:
Pasteur Effect and Crabtrcc
Effect
Pasteur was the lirsi lo compare ycasl growth in
aciobktsn and anacrohiosis For h>w concentrations
of glucose on mimic iKdia. yeasts utill/c sugars
through cilher respiration or fermentation Aera-
tion induces an increase in biomass formed (lolal
and per unil of sugar degraded) aid a decrease in
Handbook of Etiology: The Microbiology of Wine anil VinUicalions
alcohol production and sugar consumption Pas-
Knr therefore deduced thai respiralKau inhibits
fermentation
The Pasteur effeef h;is been interpreted in
several ways. Two enzymes contpclc lo catily/e
cilbet Ihc respiration or fermenurion of pyruvate.
This competition explains the respiratory inhibition
of fc mien tit ion Tic pyruvate decarboxylase is
involved in the fermentative pathway h has a
lower affinity towards pyruvaic than pyruvate
dehydrogenase Furthermore, oxidative phosphory-
lation consumes a lot of ADP and inorg mic phos-
phate, which morale k> the mitochondria. A lack
of ADP aid inorganic phosphalc in the cytoplasm
ensues This deficit can limit the phosphorylation
and thus stow the glycolilic flux. The inhibition
of glycolysis enzymes by ATP explains the Pas-
Kur effect for the most part The ATP Issued
from oxidative phosphorylation inhibits pbospho-
fruclokinasc in particular PhosphorylaKd hexoscs
accumulate as a icsull. The transmembrane trans-
port of sugats and thus glycolysis is slowed.
For high glucose concentrations — for example,
in grape must — S. eeterisiae only mctibolijrs
sugars by the fermentative pathway. Even in the
presence of oxygen, icspiiutioa Is impossible Dis-
covered by Crabtrcc < 1929) on tumoral cells, this
phenomenon is known by several names catabolic
repression by gUcose. the Paslcnr contrary effect
and the Crabtrcc effect. YcasR manifest the fol-
kiwing signs during this effect: a degeneration of
the mitochondria, a decrease in the pioportion of
cellular sterols and fatty acids, and a repression
of both the synthesis of Krebs cycle mitochondrial
enzymes and constituents of Ihc respiratory chain.
With S cererisiae. there musl be at least 9g of
sagar per lilcr for Ihc Crabtrcc effect to occur The
catabolic repression exctvd by glucose on wine
yeasts rs very strong In grape must, at any level
of aeration, yeasts arc only capable of fermenting
because of the high glucose and frnctosc concen-
trations. Prom a technological viewpoint, yeasts
consume sugars by Ihc respiratory pathway for the
industrial pnxlnction of dry yeast, but not In wine-
making. If must aeration helps the alcoholic fer-
mentation process (Section 3.7.2). Ihc tally acids
and sterols synthesized by yeast, proliferating in
the presence of oxygen, are responsible bul not
respiration.
S. cerevia'iie can rnctiboli/c ctbanol by the
respiratory pathway in the presence of small
quantities of glucose Aflcr alcoholic fermentation,
oxidative yeasts develop in a similar manner on
Ihc surface of wine as part of the process of
making ccruin specialty wines (Sherry. Yellow
Wine of Jura).
23.2 Regulation Between Alcoholic
Fermentation and
Clyccro pyruvic Fermentation;
(•Ivccrol Accumulation
Wines conetin about 8 g of glycerol per 100 g
of clhanol During grape must fermentation, about
8W of the sugar molecules undergo glyceropyru-
vie fermentation and 92'i undergo alcoholic fer-
mentation The fermentation of the hist 100 g of
sugar forms the majority of glycerol, after which
glycerol production slows but is never nil. Glyc-
cropyruvK fermentation is therefore more than an
inductive fcrmcntibon which regenerates NAD'
when acetaklchydc. normally reduced inu clhanol.
is not vet present Alcoholic fermentation and give-
cropyruvic fermentation overlap slightly through-
out fermentation.
Pyruvic acid is derived from glycolysis. When
this molecule is not used by alcoholic fermentation,
it participates in the formation of secondary
products In this case, a molecule of glycerol is
formed by Ihc reduction of dibydroxyaccianc.
Glycerol production therefore equilibrates the
yeast cndocellukir oxul.ition- reduction potential,
or NAD*/NADH balance. This relief valve
eliminates surplus NADH which appears at Ihc end
of amino acid and protein synthesis
Some wincmakcrs place too mnch importance
on the oiganoleptical role of glycerol This com-
pound has a sugary flavor similar to glncosc. In the
presence of other constituents of wine, however,
the sweetness of glycerol is piactically impercepti-
ble. For the majority of lastcis. even well trained,
the addition of 3-6 g of glycerol per liter to a
red wine is not discernible and so the pursuit of
wincmaking conditions that are more conducive
Biochemistry of Akoholic Fermentation anil Metabolic Pathways of Wine Ycasfc
63
to g lyce ropy nt vie fermentation has no cnological
interest On lie contrary, lac wiKmakct should
favor a pure alcoholic fcnticnttuon and skonld
limit glyecropyrnvic fcmicitalion. The produc-
tion of glycerol is accompanied by Ihc formation
of oiher secondary products, derived fiom pyru-
vic acid. Ike incicascd presence of which (such
;is carbon y I fanction compounds and acetic acid)
decreases wine qaalily.
2.3.3 Secondary Products Formed
from Pyruvate by Clyccropyruvie
Fermentation
When a atolcculc of glycerol R formed. a atolcculc
of pyruvate cannot be transformed into cfhaaol fol-
lowing its decarboxylation iniocthanal. In anaero-
bic conditions, oxatoacctav is the means of entry
of pyruvate into the cyanotic citric acid cycle
Although the mitochondria arc no longer func-
tional, thccnzynicsof the tricarboxylic acids cycle
arc present in Ihc cytoplasm Pyruvate carboxylase
(PCI catalyzes the carboxylation of pyruvate into
oxakiaccbitc The prosthetic group of this cu/ymc
isbiotin: itscrvcsasaCO; transporter. Tkc reaction
makes use of an ATP axilcculc:
biotia-PC + ATP + COi
COi-biotia-PC + ADP + liPI (2.3)
C02-biotiB-PC + pyruvate
bioiin-PC + oxaloacctatc (2.4)
In these anaerobic conditions. Ihc citric acid cycle
cannot be completed since Ihc succinodebydro-
genase activity requires the presence of PAD. a
strictly respiratory coenzyme Tkc chain of reac-
tions is therefore interrupted at succinate, which
accumulates (03-13 g/1). The NADH generated
by this portion of the Krcbs cycle (from oxakvac-
cute to succinate) is rcoxidi/cd by Ihc formation
of glycerol from dihydroxyaccn>ne.
The a-kcttgluttratc dehydrogenase has a very
low activity in anacrobiosis~ some authors there-
fore believe that the oxidative reactions of the
Kill's cycle arc interrupted at a-kcb>glutaralc
In their opinion, a reductive pathway of ihc
citric acid cycle forms succinic acid ia anacr-
obiosis: oxaloacetic — mutate — fumaraK —
succinate. Bacteria have a similar median ism In
yeast, this is probably a minor pathway since only
the oxidative pathway of the Krcbs cycle can main-
tain the NAD "/NAD! I icdox balance during fer-
mentation (dura. 1977) Furthcratore. additional
succinate is formed during alcoholic fermentation
on a glutimalc-cnrichcd medium Tkc glntamaK
is dcaminatcd to form a-kcloglutaratc. wkich is
oxidised into succinate
Among secondary products, kctonic function
compounds (pyruvic acid. o-kcloglntarK acid) and
acctaklchydc predominantly combine with sulfur
dioxide in wines made from healthy grapes. Their
excretion is significant during the ycasl prolifcra-
(ioa phase and decreases towards Ihc end of fer-
mentation Additional acctildcbydc is liberated in
the presence of excessive quantities of snlfur diox-
ide in must An ctcvaicd pH and fermentation tem-
perature, anaerobic conditions, and a deficiency in
thiamine and pantothenic acid increase production
of kctonic acids. Thiamine supplementing of must
limits the accumulation of kctonic compounds in
wine (Figure 2.10).
[■iK - '«' Kllcci of J thiamine addition no pvni-
vic uHl fnidudiin during » to hoik' fcimcMillun
lUfea-Laluurcadc. IWIJ). I s carnal must: □ =tfch-
idIbc Mippkmcncil bum
64
1 1.imlKx'k of HnoUigy: The Microbiology of Wive anil Vinitications
Olncr secondary products of fermentation air
aLso derived fron pyruvic acid: ace lie acid,
tactic acid, buciiediol. diacclyl aid acckHn Their
forttalion processes arc described in ihc Ibllowiig
paragraphs.
2.3.4 Formation and Accumulation
of Acetic Acid by Yeasts
Acclic acid is the principal volatile acid of witc. II
Is produced in particular during bacterial spoilage
(acclic spoilage and lactic disease) bul is always
foratcd by yeasts during fermentation Beyond
a ccrtuii limit, which varies depending on ihc
wine, acclic acid has a detrimental organolcptical
c lice i on wine qaalily. In bcallhy grape must
with a moderate sagar concentration (less than
220 g/l). S. cerevisiite produces relatively small
qiantities (100-300 mg/l). varying according to
ihc strain. In certain wincmakiug conditions, even
without bacterial contamination, yeasl acclic acid
production can be abnormally high and become a
problem for the wincmakcr
The biochemical pathway for Ihc formation
of acetic acid in wine yeasts has not yet been
clearly Identified. The hydrolysis of acclylCoA can
produce acetic acid. The pyruvate dehydrogenase
produces acetyl CoA beforehand by the oxidative
decarboxylation of pyravic acid. This reaction tikes
place ii the matrices of the mitochondria but is
limited in anacrobtosrv Aldehyde dehydrogenase
can aLso form acetic acid by Ihc oxidation of
acclaklchydc (Figure 2.11). This enzyme, whose
cofacior is NADP'. is active daring alcoholic
fermentation. The NADPH Idas formed can be used
to synihcsl/r lipids When pyruvate dehydrogenase
b. repressed. In is pathway fonnsacctyl CoA through
ihc use ofacclyl CoA synthetase. Inanacrobioslson
a model mediim. yeast strains producing the least
amount of acetic acid have Ihc highest acetyl CoA
synlhctisc activity ( Vcrdhuyn el id.. 1990).
The accciklchydc dehydrogenase in 5. cerevisine
has live Reforms, three located in Ihc cylosol
(Section 1.4.1} ( Aldop. Ald2p. and Ald3pl and the
remaining two (Atd4p and Akl5p) in Ihc mito-
chondria t Section 1.43). These enzymes differ by
ihcirspccifkascof Ihc NAD' or NADP* cefaclor
(Table 2.2).
Mi- 1 **"
Kifi 111. Acetic jcBI fernuiiou pa)h«i>«. in ycauv
l=fyamtG dccaibtxyhsc: 2 =*knb»l dcfcydiopc-
nuc: 3 = tviuvaie dchj dmpcin.%e: 4 s aldehyde dchy-
dnij-em-.e: 5 =*cayhCoA hy<tiob>,c; =icovI-CdA
ivathciuc
Tabic 2.2.
Uofoirn), of acclaklchydc dchydnijKU-NC In
■<■ (Navino- Avian n a/.. 19991
C'hiuniciui.
nc Gene
Location
Co factor
XIII
AU>2
CvlOWll
NAD*
XIII
AWS
C\n*ol
NAD*
XV
ALDJ
Yliochunilrii
NAD' and
NA DP-
V
AWS
Mioihoadrii
NAD P*
XVI
\ !.'■■'>
Cytowl
NADP*
Remize mil. (2030) aid Bk-ndin ■-/■;/. (2002)
stadicd Ihc impact of ihc deletion of each gene
and dcmonslraKd thai the NADP-dcpcndcut cyto-
plasmic isoform AUX> ptuyed a major role in the
formation of acetic ackl during the fermentation
of dry wines, while the AIJ)5 isoform was aLso
involved, bat to a lesser extent (Figure 2.I2).
Practical wincmaking conditions likely to lead
to abnormally high acetic acid production by
S. ctrevisitte arc well known As is Ihc case
with glycerol formation, acetic acid production
is closely dependent on Ihc initial sugar level
of the mast, independent of the qaantity of sug-
ars fcmicnlcd (Table 2 Jl The higher Ihc sugar
consul of Ihc must the more acclic acid (and
glycerol) ihc yeast produces during fermentation.
This Is due lo Ihc yeasts mechanism for adapt-
ing to a medium with a high sugar concentration:
Btochciiistiy of Alcoholic Fcmicniation and Metabolic Pathways of Wine Ycasfc
I* Hi Accuic pmduetiin by unlet
(VS) following dclciiin »f different utnc coiling for
Rotii(miofMttaUlc»yck.knyditigen*M;(Biondin*<,rf..
2002)
Ulilc acidly prodic-
II popubiiin in ht|rh-ui|&ii.
Tabic 2.3. Kflcd of Inial m.j.i concent nit kin uf ihc
muil tin the foimiliun uf stuoixLiiy fioducl* of IDC
fcimcauikiniLafua-Lafuurodc. !"N.ij
1ml .il
l"cmc»
igl)
Sec
Dftdaiy pmducu
(g/ll
Acetic acid
tf/1)
Gly««>l
Succinic acid
221
211
02b
4.77
0.20
206
226
045
S.33
0.2S
318
211
002
S.70
0.20
321
178
084
5.95
0.20
248
152
1.12
74H
0.28
KtH 214. Kfteci of the
350 p/ll
^pk-.c.
rcascs its intracellular accumula-
tion olgkecrol U counterbalance Ihc (Mitotic prcs-
suit of Ihc medium (BkMnbcrg and Alder. 1992).
This regulation mechanism is conliollcd by a cas-
cade of signal transmissions leading loan increase
ii the transcription level of genes involved in the
production of glycerol (GPP/ 1, but also of acctilc
\AW2 and AID3) (Attfickl el id . 2«X)). Acctilc
formation pfctys an important physiological rote
in the intracellular redox bulaicc by regCKiating
reduced equivalents of NADH. Thus, it is clear
thai an incicav in acctitc production is Inherent
Hi Ihc fermentation of high-sugar must* However.
Bcly el al. (2003) dcmonslnitcd lhat it was possi-
ble in reduce acctilc pmduclion by supplying more
NADH *i Ihc redox balance process. This may be
done indirectly b> sun iu latin u bioniass formation,
which generates an excess of NADH during amino
acid synthesis. Available nitrogen in the must plays
a key role in this process Thus, in high-sugar
mnsis. acclatc production Is inversely correlated
with the maximum cell population (Figure 2.13).
which is. in rum. retried to the available nitrogen
content of the must. It is strongly recommended
Id monitor the available nitrogen content of botry-
il/cd musts and supplement Ihcnt with ammonium
sulfate, if necessary The optimum available nitro-
gen concentration in Ibis type of must u minimize
acetic acid production is approximately 190 mg/l
(Figure 2.14). The best time for adding nitrogen
supplements Is at the very beginning of fermenta-
tion, as Liter additions arc less effective and may
Handbook or Enology: The Microbiology of Wine and Vinifieations
: parductioH liHlccd. i:
of the unpredictable increase in acetic acid produc-
tion that M>iiie u ik's occurred in botryli/cd musts
sapplcmcnlcd wlih ammoniam saltan?, many cnoV
ogis*> had given up Ihc practice entirely. It is now
known that, provided the supplement is added ai
the very beginning of fermentation, adjusting the
available nitrogen contnl lo Ihc optimum level
(191) itg/1) always minimizes acetic acid produc-
tion in botryli/cd wines.
In wines made from noble rented grapes, cciuln
sahstanccs in the must inhibit yeast growth and
increase the production of acetic acid aid glyc-
erol during fermentation. Boiiytis cinerea secretes
these botrylicinc sahstanccs (Ribcrcau-Cayon
Hal., 1932. 1979). Fractional precipitation with
clhanol partially purities these compounds from
mast and cnlturc media of Boiniis cinerea. These
beat stable glycoproteins have molecular weights
between 10 and 50CO0. They comprise a peptidic
( It'i i and glucidK pan containing mostly man-
nose and galactose and some rhamnosc and glu-
cose (Dubounlicu. 1982). When added to healthy
grape must, these compounds provoke an increase
in glycciopymvic fermentation and a significant
excretion of acetic acid at lie end of fermentation
(Figure 2.15). TV mode of action of these glyco-
proteins on yeasts has not yet been identified. The
physiological stile of yeast populations at the time
of inoculation seems lo pray an important rote in
the fermentative development of boUytiycd grape
must. Industrial dry yeast preparations arc much
nunc sensitive to alcoholic fermentation inhibitors
than yeast stirrers obtained by prcculturcin healthy
grape mast..
Other wincmaking factors favor the production
of acetic acid by S. cerevisiite anacrobiosis. very
low pH (<3.l) or very high pH (>4). certiia
amino acid or vilamin deficiencies in tic must,
and kx> high of a temperature (25-30'C) dur-
ing the yeast mulliplication phase In red wine-
making, temperature is the most impottani faclor.
especially when the mast has a high sugar concen-
tration. In hot climates, the grapes should be cooled
when tilling the vats. The temperature should not
exceed 20'C at the beginning of fermentation The
l'i(i 115. Kited of 40 a kvi ho I- Induced pmlpialc of
a boliytircd grape mi-i tin glycc*>l and acetic --■!
foratatbn during ihc alcoholic fermentation of bcalhy
pope muu (Dubounlicu. 1982). (II Evohftbn of ackt
«c kl acetic coaccnitatiua In the muM lupplcmcntcd «. feh
the fKcre-dricil ficcipiaic: <r)cvoknba ol glycerol
glycerol coKCMraibn in the muu MipptemcMcd with
Ihc liee/e-dried precipitate
same piocedure shoakl be followed in thcrmovini-
I nation immediately folkiwing the healing of the
grapes.
In dry white and rose wincmaking. excessive
masl clarification can also lead lo the exces-
sive production of volatile acidity by yeast This
phenomenon can be particularly pronounced wilb
certain yeast strains. Therefore, must turbidity
should be adjusted b> the lowest possible level
which permit! a complete and rapid fermen-
tation (Chapter 1.1). Solids sedimentation (must
Ices) furnishes long-chain unsaturated falt>* acids
(CI8:I. CI8:2). Yeast lipidic alimentation greatly
Biochemistry of Akoholk f>cimcniation anil Mctabolk Pathways of Wine Ycasft
67
m m
lift 1.10. Ilflctl Of the lip.lk focib* of mu.l kn
■id ..i.i.i ■. .l-.'i pnxfcicl ■'■ by .-.I-'-. -In. .Hi- .1 li i ■ ■■ ■ !i.
Ic>mcnmic>n<U'4!«c. 1990)
influences acetic acid production during white and
nisc wincmakiug
The experiment in Figure 2.16 illustrates the
important rote of lipids in acetic acid metabolism
lDclliniandCctvcui.l992;Alcxaiidrcff<i/..l9**).
The volatile acidily of thirc wines obGiincd from
lie same Sauvignon Blanc mast was compared
A her iilir.iti.tn. must turbidity was adjusted » 250
Nephelometric turbidity units |NTV) before inoc-
ulation by three diffcicnl methods: reincorporating
fresh Ices (control): adding cellulose powder: and
supplementing the same quantity of Ices adsorbed
on Ine cellulose powder with a lipidK extract
(methanol- chloroform ) The volatile acidities of the
control wine and the wine that was suppkrmcnfcd
with a lipid ic extraction of Ices before fermentation
are identical and perfectly normal. Al thong h the fer-
mentation was normal, the volatile acidity of the
wine made from the must supplemented with cel-
lulose (therefore devoid of lipids) was practically
twice as high tLavignc. 1996). Supplementing the
medium with lipids appears u favor the penetra-
tion of amino acids into the cell, which limits the
formation of acetic acid.
During the alcoholic fermentation of red or
slightly i Unified u hikr wines, yeasts do not contin-
uously produce acetic acid. The yeast metabolizes
a large portion of tie acetic acid secreted in must
during the fermentation of the lirsl 50-100 g of
sugar. It can also assimilate acetic acid added to
must at the beginning of alcoholic fermentation
The assimilation mechanisms are not yet clear
Acetic acid appears lo be reduced to acctaldchydc.
which favors alcoholic fermentation *> the detri-
ment of glyce ropy ru vie fermentation. In fact, the
addition of acetic acid to a must lowers glycciol
production but increases the formation of acctoin
and butancdiol-2.3. Ycasbsccm to use Ihc acetic
acid formed at Ine beginning of alcoholic fermen-
tation tor added to musl) via acetyl CoA in the
lipid- producing pathways.
Ccrtitn wincmaking conditions produce abnor-
mally high amount> of acetic acid Since this
acid is nol used during the second half of the
fermentation, it accumulates until the end of
fermentation. By rcfermenting a tainted wine,
yeasts can lower vofcttite acidity by metaboliz-
ing acetic acid. The wine Pi incorporated into
freshly crushed grapes at a proportion of no more
than 20- l&i. The wine should be salfitcd or
filtered before incorporation to eliminate bacte-
ria. The volatile acidity of this mixture should
not exceed 0.6 g/l in H?SO* The volatile acid-
ity of this newly made wine rarely exceeds 0.) g/l
in H.SO, The concentration of ethyl acctalc
dec teases sim u I tancously.
2.3.5 Other Secondary Products
of the Fermentation of Sugars
Lactic acid is another secondary product of
fermentation. It is also derived from pyruvic
acid, dirccdy reduced by yeast tl+) and tK— )
laclKodcbydrogenases. In anactobiosis (lie case
in akoholk fcrmenttlioa). Ine yeast synthesizes
predominantly ri — i lacucodcbydrogcnasc Ycasft
form 200-300 mg of rX-) lactic acid per liter
and only about a dozen milligrams of U+) lac-
Ik acid. Tic latter is formed essentially at the stirt
of fermentation By determining the rX— ) lactic
acid concentration in a wine, it can be ascertained
whether the origin of acetic acid is yeast or lactic
bacteria (Section 14.2 J). Wines thai nave under-
gone malotactic fermentation can contain several
grams per liter of exclusively tl+) tactic acid. On
I landbook of Fnology: The Microbiology of Wlie and Vindications
celyl and 2>bujncdlo
Ibc other hand. Ihe lactic fermentation of sugars
(tactic disease) forms im — I laclK acid. Lac Ik bac-
teria have transformed subslraics other thai malic
*■ id. wbci in—) luclK acid concentrations exceed
200 to 300 mg/l.
Yeasts also niakc use of pyruvic acid to form
accloin. diacctyl and 2.3-but»cdiol (Figure 2.17).
This process begins wlih Ihe condensation of a
pyruvate molecule aid active ate (aldehyde bound
to thiamine pyrophosphate, leading to the forma-
tion of a-acclolaclk acid The oxidative decar-
boxylation of n-acctolaclk acid produces diacetyl.
Accloin Is produced by either the non-oxidative
decarboxylation of n-acctolactk acid or the reduc-
tion of diacctyl The rcduclwi of accloii leads k>
the formation of 2.3-butiKdiol: Ibis last reaction
Is reversible.
From the start of alcoholic fermentation. yeasts
produce diacctyl. which Pi rapidly reduced k>
accloin aid 23-buGiicdiol. This reduction tikes
place in the days that follow the end of alcoholic
fcrmcntilioa. when wines are conserved on the
ycasl btomass (dc Revel el ill.. 1996). Accloin and
especially d lace lyl are stroag-smclliug compounds
which evoke a butlcrv aroma. Above a certain
concentration, they have a negative effect on wine
aroma. The concentration of wines that have nol
undergone uudolactK fcrmcntiuoa is too low (a
few milligrams per liter for diacctyl) to have
an olfaciivc Influence. On the olhcr hand, lactic
hacicrla can degrade cilric acid to produce much
higher quantities of these carboayl compounds
than yeasts (Section 5J.2).
Finally, yeasts condense accllc acid (in the
form of acetyl CoA) and pyruvic acid to pro-
duce citramalK acid (0-300 mg/ll aid dimcthyl-
glyceric acid (O-OO nigfl) (Figure 2181- These
compounds have little organoleptic incidence.
ii,c — c — o
Vie. 218. i.iii'ia«l> acid and (b)dimelhyfclyc«
Biochemistry of Alcoholic Fcmicniation and Metabolic Pathways of Wine Yraso
KN
I'ifi J. 1". DccoaiuMdnaaf n
i .kiting alcoholic fern
2.3.6 Degradation or Malic Acid
by Yeast
Saechartmytes cerevtsuie partially degrades musl
malic acid (IO-25'i> during alcoholic fermenfa-
uoi Different strains degrade varying amonnLs
of Ibis acid, and degradation Is niotc signifi-
cant when tic pH is kiw. Alcoholic fcmicilalion
is ihc principal pathway degrading malic acid.
The pyruvic acid resulting from this transforma-
tion is dccarboxylalcd into cihanal. which is then
reduced b> cihanol. The malic enzyme is respon-
sible for the transformation of italic acid into
pyruvic acid (Figure 2.19) This oxidative decar-
boxylation requires NAD' (Fuck and Radlcr.
1972). This makulcoholic fermentation lowers
wine acidity significantly more than atalolacuc
fcmicilalion.
Scfu&aacclHvtmiyces differs from wine ycaso.
The alcoholic fcraicnlaiion of malic acid is com-
plete in yeas is of Ihts genus, which possess an
active malalc lianspon system lln S. cereviaae.
malic acid pciciralcs Ihc cell by simple diflu-
sioa.l Ycl ai prescnl no alicuipn to use Schizi'SiK'-
chanmwes in wiicniakiig have been successful
IPcytaudrtrt/.. 1964: Carre if of., 1983). Fits! of
all. Ihc i m plan Li In in of these >c;ists in the prcs-
ence of S ceivvisiae is difficult in a non-sKrilixd
must Secondly, their optimum growth lempcra-
lurc (30'C) higher than for 5. cermriae, imposes
warmer fcraicnlaiion conditions. Sometimes, the
higher lempcralnre adverser)' affects the oiganolcp-
IKal quality of wine Finally, some grape varieties
fermented by Sclvfuicelttimtncei do not express
their variclal aromas The acidic Gros Nlanscng
variety produces a very fruily wine when correctly
vinificd wiih S. cerensUie. hut has no varietal
aroma when fcraicnlcd by Schizotactlwmnntes.
To resolve Ihese problems, some researchers have
used non-prol iterating populations of Schizpstic-
chtmmxces enclosed in alginate balls. These pop-
ulations degrade Ihc malic acid in wines hav-
ing already completed Ihciralcoholic fcraicnlaiion
(Magyar and Paiyid. 1989. Taillandicr aid Sircha-
iano. 1991)). Although no organolcplkal defect is
found in ihese wines, ihc Kchniques have nol yet
been developed for practical use.
Today, molecular biology permits aiolhcr strat-
egy for making use of lie ability of Sehizt'SiK-
chiavmycts u fcraicil malic acid. Il i ousels of
inicgraiing Sclnzmticchiirtmiyces mafcilc pcnKasc
gcucs and Ihc malic cn/yme (mac I and mac 2)
ii ihc 5. eetefisiiie genome (Van Vuuren if of.,
19%). The technological interest of a wiic yeast
genetically modified in this manner is not yet clear,
nor arc the risks of ib> proliferation ii
nature.
2.4 METABOLISM OF NITROC.'KN
COMPOUNDS
The nilrogen requircmcnLs of wine yeasts and
Ihe mitogen supply in grape musts are dis-
cussed ii Chapter .1 (Section 3.4.2) The fol-
lowing section covers the general mechanisms
of assimilalion. biosynthesis, aid degradation of
amino acids in ycasfe. The consequences of Ihese
metabolisms, which occur during alcoholic fer-
nienution and affeel Ihc production of higher alco-
hols and their associated esicrs in wine, are also
discussed.
Handbook or Etiology: The Microbiology of Wive and VnufkatkMS
2.4.1 Amino Ackl Synthesis Path"
The ammonium km and amino acids found in
grape musl supply the ycasl with nitrogen. The
ycasi eai aLso synthesize most of lie amino
acids necessary for constructing it prolcins. II
fixes an ammonium ion on a caibon skeleton
dciivcd from Inc mcubolism of sugars. Tic yeast
■scs Inc same icactional pathways as all organ-
Rms. Glutauialc and glntamlnc play an imporunl
role in thR process (Cooper. 1982; Magasanik.
1992).
TllcNADP , glnlanlalcdcbydrogcnasc(NADP , -
CDH). product of the GDH I gene, produces glnla-
malc (Figure 2.20) from an ammonium ion and an
a-kcioglutiralc molecule. The killer R an interme-
diary product of Inc citric acid cycle. The ycasi
aLso possesses an NAD' glucimalc dehydroge-
nase (NAD'-GDHl. pioducl of the GDH2 gcic.
This dehydrogenase is involved in the oxidative
calabolism of glutauialc. It ptoduc
reaction of Ihc picccdcnt. libciuling the a
Km used in the synthesis of glntauiinc. NAIJP'-
GDH activity is al its maximnm when Ihc ycasl
Is cultivated on a medium containing exclusively
ammonium as lis source of nitrogo The NAIV-
GDH activity, however, is at its highest level
when the principal source of nilrogcn is glula-
matc Glnlaminc synthetase (GS) prodnccs glu-
■amine from glulamalc and ammonium This ami-
nation requires the hydrolysis of an AIT- mokxuk
(Figure 2.21).
Through transamination reactions, glutamale
then serves as an amino group donor in Ihc bio-
syn thesis ofdifferent amino acids. Pyridoxal phos-
phate is the transaminase cofactor (Figure 2.22); ii
is derived from pyrkloxiK(vitimin Bit.
The carbon skeleton of amino acids originates
from glycolysis intermediary products (pyruvate.
3-phosp*oglyccialc. pbosphocnolpyruvalc). the
Fi); 291. lacorpoMion of (be »»mo«i
INADP-GDP)
i- !■.'... :lu limn: ..iii. : red hy NADP -.'I. .I.iii, .11. ..■.Ii;- ..!■.,%: -.,
*NHj C II
Fig 221. AmablioB of C>IU^*i into pluUimlnc by fhiUrcmc v. E*hclnc ((JSt
Biochemistry of Akoholic Fcraicnialion anil Mctabolk Pathways of Wine YcasR
"o — p — ati,c
a—r—o *V
lift 122. IVrkloxj] pkmplmc (I'll'i imI pyrkkixiatnc pki%f hate I PMP.i
citric acid cycle ta-kcloglatarak. oxaloacctatc) or
Ihc pentose phosphate cycle Irlbasc 5-phosphak.
crythrosc 4- phosphate). Some of these iraclions
arc very simple, such as ihc formation of aspartate
or alanine by transamination of glntamaK into
oxakmcctalc or p> mate:
oxaloacctalc + glulauuuc ■
aspartate + a-kcugluurate (2.51
pyruvate + g la Lunate ■
alanine + a-kctoglutaratc (2.6)
Other biosyutuclk pathways aic more complex.
hut still occur in ycasK as in the rest of the living
world The amino acids can he classified into six
hiosynlhcuc families depending on their nainic and
their carbon pirciisor (Figure 223):
\?
|'r r w'-"^| — *
Hj^Sr*
I. In addition loglulamalc andglutuninc. proline
and aigininc ate formed from a-kdoglatanMc.
2 Asparag inc. mcthKiniac. lysine, threonine and
tsolcuclnc are derived from aspariakr. which
is issued from oxakmcclat. ATP can acti-
vale methionine to form 5-adenosylmclhionine.
which can be dcncthylaKd to form S-
adcaosylbomocysicinc. the bydrotysRof whkh
liberates adenine k> produce homocysteine.
3. Pyruvate Is the starting point for the synthesis
of alanine, valine and leucine.
4. 3-Phosphoglyccratc leads to the formation
of serine aid glycine. The condensation of
homocysteine and serine paidnccs cystathion-
ine, a precursor of cysteine.
VV, 2.21 Gcncal bHn.v«kcsn jMihnay* of amino
nchk
5. The imidazole cycle of i tsiidliic R formed from
ribosc 5- phosphate and adenine of ATP
6. The amino acids possessing an aromaiK
cycle (tyrosine, phenylalanine, irylophan) are
derived from crythrosc 4-phosphalc and phos-
phocBolpyruvatc. These two compounds arc
in K rated larks of the pentose cyck and gly-
colysis, rcspcctively. Their condcnsalioi forms
snlkimaic The condensation of this compound
with another molecule of phospbocnolpyruvaic
produces chorismaic. a prccntsor of aromalk
amino acids.
Handbook or Etiology: The Microbiology of Wine and Vitrifications
2.4.2 Assimilation Mcchai
Ammonium and A
"In pcuciralk
Idio Ihc yeast
pfolei
Acids
mi no iK ids
clivatcs numcious membrane
rpcrmcascs(Scelion 132).
sporlei
' has al least two specific i
wn transports iDubois and Grcison. l979J.Thcir
activity R inhibited by several amino acids, in a
■on-competitive manner
Two distinct categories ol* transporters ensure
amino acid transport:
1 A general amino acid permease (GAP) trans-
ports all or the amino acids. The ammonium ioa
inhibiLsand represses the GAP. The GAP Incre-
fonr oil)' appears to be active during the second
half of fermentation . when the must no longer
contains ammonium It acLs as a nitrogen
scavenger* lowaids amino acids (Caitvvrigbl
el tit . 1989).
2 S ceievisiiie aLso has many specific amino
acid permeases (at least 1 1 1 Each one ensures
the transport of one or more amino acids.
In Contrast to GAP. the ammonium ion docs
not limit their activity. Erom the beginning of
the yeast log phase during the first suges of
fermentation, these iransporvis ensure the rapid
assimilaim of mnst amino acids.
Gluumuilc and glitaminccrossiDads of amino acid
synthesis, arc not the only amino acids rapidly
assimilated Most of the amino acids arc practically
depleted from Ihc must by the lime the first 30 g of
sagar have been fermented. Alanine and arginine
arc the principal amino acids found in must. Yeasts
make nse of these two compounds and ammonium
slightly afKr Ihc depletion of other amino acids.
Furthermore, yeasts massively assimilate arginine
only after the disappearance of ammonium from
the inedinm Sometimes, yeasts do not completely
consume y-amiaobu lyric acid. Ycas&do nol utilise
proline daring fermentation, although il is one of
the principal amino acids found in must
During fermentation. ycasK assimilate between
1 and 2 g/l of amino acids. Towards the end of
fcnncutilion.ycash excrete significant bit variable
amount of different amino acids. Finally, at the
end of alcoholic fcrmcnuiion. a few handled
milligrams of amino acids per liter remain; proline
generally represent half.
Contrary to must hexoscs that penclralc the
cell by facilitated ditfnsioa. ammonium and amino
acids requite active transport. Their concentration
in the cell is generally higher than in the external
medium The permease involved couples Ihc trans-
port of an amino acid molecule (or ammoninm ionl
with Ihc transport of a hydrogen ion. The hydro-
gen ion moves in Ihc direction of Ihc concentration
gradient: the concentration of prouns in Ihc must
is higher than in Ihc cytoplasm The amino acid
and the proton are linked to Ihc same transpofl
protein and penclralc Ihc cell simultaneously . This
concerted transport of two subsGinccs in Ihc same
direction is called sympuri (Eigure 2.24). Obvi-
ously, the prolan thai penetrates the cell must then
be exported to avoid acidification of the cytoplasm.
This movement is made against the coaccalra-
lion gradient and requires energy. The membrane
ATPasc ensures the excretion of the hydrogen ion
across the plasm ic membrane, acting as a pro-
Ion pump.
Biochemistry of Alcoholic Fcraicniation and Metabolic Pathways of Wine Yeasts
>i
E Hanoi strongly limits amino acid transport II
modifies the composition and Ihc properties of
lie phospholipids of the pLisniit membrane. The
membrane becomes nunc permeable. The H~ ions
of Ihc median massively pcnclraic the interior
of Ihc cell by simple difl'tsioa The membrane
ATPasc mnsi incic;isc irs operation h> control Ihc
intracellular p!l. As soon as this task monopolizes
Ihc ATPasc. Ihc syniporl of Ihc amino acids no
longer f ime (ions. In olhcr worts, al Ihc beginning
of fermentation, and for as long as ihc cihanol
conecn iralion in ihc musi is low. yeasts can rapidly
assimilate amino acids aid coaccnlralc ihcm in
Ihc vac notes for lalcr use. according u ihcir
biosynthesis needs.
14.3 Calabolk
The
i of An
> Acids
is essential for Ihc synthesis
of amino acids necessary for building piolcins.
bul >easts cannot always rind siflicicnt quanlilics
in their environment K'rtun.itcly . ihcy can obtain
fn>m available amino acids thnwgb
lions.
The most common pathway is Ihc transfer of
a-amino group, originating from one of many
different amino acids, onto a-kcioglataric acid
to form glntamaK. Aminotransferases or transam-
inases catalyze Ibis reaction, whose prosthetic
giunp is pyridoxal phosphate I PI. Pi Glutamatc is
then dcaminalcd by oxidative pathways to form
NHt* (Figure 235). These Iwo rcactwns can be
summarized as follows:
a-amino acid + NAD r + H.O
o-kclonicacid+NH4 , + NADH + H , (2.7)
During transamination, pyndoial phosphate is
temporarily transformed into pyridoxamiac phos-
phaK (PMP). The PI.P aktchydK group is linked lo
a lysine residue .--amino group on the active sin? of
Ihc aminotransferase lo form an intermediary pn-d-
nct (E-PLP) (Figure 2.26) The a-amino gronp of
KIH 225. Oiidwiv
<AD'*Mp
anl £<kil.iailc <khyiln>|KiUi
f-ifi 220. Mode Of ii.iii.ii uf pviidoxal pb» r h*IC il'I.I'i ib I a (Ml
pnxlutt between 1*1.1* J ml jmiaPlrjcAfcciM DtthC nroio-i .iv ill UlSslQ
i taadbook of Etiology: The Microbiology of Wine anil Vnificatkws
Fift 227. Dciainariin i>l tunc b, * dchvdcuiii
I'ili 128. i-o(B.ili.>Bof hnihcr jkohob I'niRi
Ibc amino acid substrate of ihc transamination dis-
places lie lysine residue (--amino g ronp linked h>
PLP. The cleavage of ihis in termed iary product lib-
cralcs PMP and kcuuK acid, corresponding to the
amino acid -.uI--u.il PMP can in tnrn react with
another kctonic acid to furnish a second amino acid
and icgencralc pyridoxal phosphate. The partial
reactions can be written in the following manner;
amino acid I + E-PLP r
kctonic acid I + E-PMP (2.8)
kctonic acid 2+ E-PMP
amino acid 2 + E-PLP (2.9|
the balance sheet for which is:
amino acid I + kctonic acid 2 ■
kctonic acid I + amino acid 2 (2.10)
Some amino acids, snch as serine and threonine,
possess a hydroxy! grotp on their (• carbon.
The)* can be directly dcaminakd by dehydration.
A dehydratase catalyses this reaction, producing
the corresponding kctonic acid and ammonium
(Eigne 227).
2. -1.4 Formation of Higher Alcohols
and Esters
Yeasts can cxciclc kctonic acids originating fn>m
the dcaminalion of amino acids only after their
decarboxylation into aklchyde and reduction into
alcohol (Figure 2.28). This mechanism, known as
the Ehrlich rcaclion. explains in pan the formation
of higher alcohols in wine. Table 2.4 lists the
principal higher alcohols and their corresponding
amino acids, possible precursors of these alcohoK
Several experiments clearly indicate, however,
that Ihc degradation of amino acids Is not the only
pathway for loaning higher alcohols in wine In
fact, certain ones, snch as propan- l-ol and bntirt-
l-ol. do not have amino acid prcciirsors Moreover,
certain mitan&dcficicnlin Ihc synthesis ofspccilic
amino acids do not produce the corresponding
higher alcohol, even if Ihc amino acid is present
in ihc cnllare medium. There is no relationship
between Ihc amonni of amino acids in must and the
amount of corresponding higher alcohols in wine
Higher alcohol pruduclion by yeasts appears io
be linked not only to Ihc catabolism of amino acids
hni also to Ibeir synthesis via the corresponding
kctonic acids. These acids ate derived ftoni the
metabolism of sugars. Eor example, pmpan-1-ol
has no corresponding amino acid It isdcn\cd from
o-kclobutytale which can be formed fn>m pyrovaic
and acetyl coenzyme A. a-Kckviocapfoalc Is a
precursor of isoamylic alcohol and an inicimcdiary
product in Ibc synthesis of leucine. It too can be
piudnccd from a-acctolaclaic. which is derived
from pyruvate. Most higher alcohols in wine can
also be formed by the mcEtbolism of glncosc
without the involvement of amino acids
BMichciiisliy of Alcoholic RimcnLitloii and Metabolic Painw-ays of Wine Yrasfc
Tabic 14. The principal ulcuhoh found ill ■.ioc iixl ItKir ,imii» at id pre.
76
Handbook of Enology: The Microbiology of Wine anil Vindications
Tbc physiotogKal function of higher alcohol
production by yeasts Pi nol clear. It may be a
simple waste of sugars, a detox ideation process of
the intracellular medium, or a means of regulating
the metabolism of amino ackls.
With the exception of phcnylclhanol. which
has a rose-like fragrance, higher alcohols smell
bad. Most, sach as isoamylic alcohol, have heavy
solvent-like odois. Methanol is a peculiar alcohol
because it contains a sulfnr atom. Its cooked-
cabbage odor has the lowest perception threshold
(1.2 mg/l). h can be responsible fot the most
persistent and dKigrceablc olfactory flaws of
redaction, especially in while wines In general, the
winemakcr should avoid excessive higher alcohol
odors. Fortunately, their organolcptical impact Is
limited at their usual concentrations in wine, bnt
it depends on the overall aromatic intensity of
the wine. Excessive yickls and rain at the end of
maturation can dilute the mnsi. in which case the
wine will have a low aromatic intensity and the
heavy, common character of higher alcohols can
be prononnccd.
The wincmaking parameters that increase higher
alcohol production by yeasts arc well known:
high pH. elevated fermentation temperature, and
aeration In red wincmaking. the extraction of
pomace constituent and the concern for rapid
and complete fermen rations impose aeration and
elevated temperatures, and in this case higher
alcohol production by yeast cannot be limited
In white wincmaking. a fermentation temperature
between 20 and 22 'C limie* the formation of higher
alcohols.
Ammonium and amino acid deficiencies in
mast lead to an increased formation of higher
alcohols In these conditions, the yeast appears b>
recuperate all of the animated nitrogen available
by transamination. It abandons the auascd carbon
skeleton in the form of higher alcohols. Racking
white must also limits the production of higher
alcohols (Chapter 131.
The nature of the yeast (species, strain)
responsible for fermentation also affects the
production of higher alcohols. Certain species,
snch as llnmemilti imomiilti. have long been
known lo produce a lot. especially in acrobiosis
(Gnymon etal.. 1961). Yet production by
wine yeasts is limited, even in spontaneous
fermentation More recently, various researchers
have shown that most S. btiyama teximmsiit
produce considerably more phenykrlhanol than
Finally, higher alcohol production
> depends on the strain. A limited
higher alcohol production (with the exception of
pncnytclhanol) should be among selection criteria
for wine yeast*.
Due to their esterase activities, yeasts form var-
ious esters (a few milligrams per liter). The most
important acetates of higher alcohols arc rsoamyl
acetate (rxinana aroma) and pncnylelhyl acctaic
(rose aroma). Althongb they are not linked to nitro-
gen metabolism, ethyl esters of medium-chain fatty
Kids arc also involved They arc formed by the
condensation of acetyl coenzyme A. These esters
have more interesting aromas than the others. Hex-
anoale has a Dower)' and (miry aroma reminiscent
of green apples. Ethyl decanoatc has a soap-like
odor. In white wincmaking. the production of these
esters can be increased by lowering the fermenta-
tion temperature and increasing must clarification.
Ccrciinycmt strains (7 IB (produce large quantities
of these compounds, which contribute to the fer-
mentation aroma of young wines. They arc rapidly
bydrolycd during their first year in bottle and have
no long-term influence oh the aromatic character
of while wines.
REFERENCES
Alcumlic II. Nguyen Via l.ii* I. Fcullbt M. »*1
ClnrpcMierC.. 1994. An: Fr. OjioI.. caniee m»
illiqun. 140. 11-20.
Hclv M..Ruuhli A.*»dDubourdku D..2O01.J. ftonv.
B».rt.jj. 90 (b). 507.
Bpmki I.T.. 1993. Ycuta-Mciabobm of mpm. in
\Viie ivicrobiology mil biotri'lmolo^. f. 55-75.
G-H- Fleet.. Hamood Academic Puhlnbcr.. Chur.
Swfexiaiad.
1 -i . niK-iy A. ..1.1 Alder L, 1992. Adi-. Microbiol. Ph>s-
foL.33, 115-212.
Bkrndut II . Dopiln $.. SiiiM Pm F. *ad SabUyiulfcj,
IM., 21X1 2. l-n formttion o~ itrides niA.ti/i pia les In-
ures, dms If* Siipoiiuiv heeriiiiitNiiiii'tnioIiipe
09-10 Jam 2f»7.'lNSA. 1NRA. MoMfcllicr. Faux.
Carre E.. i . ■ n-1 . ixiitadc S. . r.l Bcarand A.. I'- I
Ohio. Wpie Hi, 17.43-53.
BKxrhciiltfiy of Alcoholic Rimcniatioii anil Metabolic Pauways of Wine Yeasts
Canuripta CI'.. Rose A H . Ciklcib.nt I and Kccnan
M.H J., 1989. Sokac lanspoit. In The Yciats. vol. 3.
p. 5-5S. \ if !■!..%:■. 1 V ff.mv.ii. Acadcmlt Pnrss.
Loodon.
Caubei R.. Guerin B. and Guerin M.. 1988. An*.
Microbiol.. 149. 324-329.
Cooper TG. 1982. Nhmpen mclabolnm la Stnftoh
ramyret ceretisiiie. in The moleculig biolog, of
r.'n- ■i-i.v Sice lummy ccs: metitiwlivti mil gene
expression p. 39-99. J.N. Slmihein. E.W. Jones. J.R.
Bioach. Cold Spring llaibnr Laboniiorj. Ne» Yoik.
Ciabuce H.G.. 1929. Biorhem. J.. 23. 536-545.
lit Revel CLanvaud-rlinel A. and Beoiand A.. 1990.
Elude des composes dicaibonylcs au inun de* fci-
mcnuiions jIiooIIiuk el malukiciknK. In (Eaoto-
gie °S. S 1 Simposiaitt inieriuiinniJ it 'oitologie .
p. 32l-325.A!li>nvaud.Tct:ci Doe.Pauv
DcllinlC. and CervcUi F.. 1992. Mffc Enol. Sri.. 40.
142-150.
Dubois E. and Gnrmon M.. 1979. Malemltt -id gen-
etiil genetics . 175.67-76.
Dubounlicu D.. 1982. Reckoihes su. let. polysaccha-
rides sccicic* pat Batrytis cineren (bra U bate dc
rabin. Tltete Daciant es Sciences, L'aivcraiic dc
Bordcaui II.
Futk E. and Radler F.. 1972, AVrftii; fir Mitrobiologie .
87. .J" 104
Cuvmon J.F., Ingraham I.L. and On.cH EA., 1901.
Anil. Biocheiu. Biopbyy. 95. 163-168.
Kuhimoio M.. 1994. J. of Fermeituiion mil Bin. engi-
neering. 77. 4. 432-435.
ljfon-l.iiu.iiv.ulc $.. 1983. Whk »*l Brandy, in flio-
terltnolagy. 83- 103 H J. Rehm ci G. Reed. Wrliu
Chemie.Weinhcim.
Lavignc V.. 1990. Recherche sur lei compotes totalis
formes pit In leitire <ni coins tie In tinificition ei
de teleiiige ilet tins blmct leex. These dc Doctoral.
UbrmU dc Bonleaux II.
Mapaunik B.. 1992. Regutaikm of -iiogci unliolbn.
In Tlie molmitr biology of the iivio Sixxhirotvyces.
Caie expression. p.283-JI7. E.W. tone*. J.R.
Pringle.J.R. 8f«uh..CoUSpriiigHaiborLaboraioiy.
New York.
Magyar I. mil ftinyik 1 . 1989. Am. J. Oenol. Uric. 40.
233-240.
XitoKul \.. 19W. Rettocrttocs stir r i4ciaific.tio}i geiieti-
que iles leturei ile *>nifici&ion. Applici
ones. These dc Dnciocu dc I'L'nlv
demi
[I.
Navann-Avino J.P.. Posad R.. Mia Iks V.I. Benin
RM.and Senano R.. 1999. Ye.ist. 15.929-842.
NeubcigC.. 1946. Aim. Re*. Biorliem.. 15.435.
Oura E.. 1977. Proves* Bioiheia. . 12. 19-21.
Pcyaaud E., Domcivu. S. and Boidntn A.. Lafon-
Lafouicade S.. GuimbcrteauG.. 1904. ArdiSv. fur
Miltobiologie.iS. 150-105.
Remit P.. Andricu E. and Dcquin S.. 2000. Appl.
Eniiron. Microbiol.. 00. 3151-3159.
Rfccnrau-Cayon J-. Peynaud P_and lafoun-adc S.. 1952.
CR.ArtKl.Se. 214.478.
Rl>cnrau-Ga)»n P.. Ul«>a-U»nun:adc S., Dubour
dku D..Lucmaiei V. and Lame P.. 1979. C.R.Acad.
Sc.289 D.441.
Sliver L.. 1992. Mciaholnmc: tanccpti a <uc d'en-
xmblc. In UibioehimiedelMber! Sirycr. p. 315-328.
Plammariun. Paris.
TailUndktP. Slietabno P.. 1990. Dcgiadaiiun dc
I acidc maltpic par ScUizosifchnoxi-cei. in Art«-
iiliiei imologiques fl". C. R. rfu 4' Symposium inter-
nitiaui d'osnotogie. Bordems /9S9. P. Rbcreau-
Gayon.A. Lonvaud. Ihinnd-Bonlai . Pans.
Van Vuuien H.J.J.. Viljucn M..Gn*lerl. VoKc henk H..
Bauer F. and Subdcn R.E.. 1990. Gencm analyse
of ihc Sifiizoutriititoittirci pomoe mabic perme-
ases. Marl and malic cn/vmc. M.vll. genes and ihcir
cvpiessKin in SorHueoivyces cereiiiiir. in fEaato-
gie 9£ S' Symposium haeritiliomi iftriiologic.
p. 195-197. A. Lonvaud. Trek a Doc. Pint.
Vcnlhuyn C Posima E.. Schclfce. W.A. and Van Dijkcn
J.P.. 1990. / Gen. Micro. 136. 359-403.
Conditions of Yeast Development
3 1 Introduction
3 2 Monitoring UBd controlling fermentations
3 3 Yeast growth cyckr and fermentation kinetics
3.4 NlMilli.Ti ii\|U.iYllk'Iils
3.5 Fermentation activators
3 6 Inhibition of Ihc fermentation
3.7 Physkochcmical factors affecting yeast growth aid fem
38 Stuck fermentations
oration kinetics
3.1 INTRODUCTION
Crape ninsi is a highly fermentable mcdiim in
which ycasfi find the accessary sabstanccs to
ensure (heir vitil functions Carbohyd rales (glu-
cose and fructose) air nscd as carbon and energy
sources— the ycasK deriving ethanol. Bthanol
gives wines their principal character. Organic acids
(lariaric aid malic acid) and mineral salb (phos-
phate, sulfalc. chloride, potassium, calcium and
magnesium) ensure a suitable pH. Nitrogen com-
pounds exist )■ several forms; ammonia, amino
acids, polypeptides and proteins. Grape must also
con tuns substances serving as growth (vitamins)
and survival factors. Olhcr grape consiliums
(phenolic compounds, aromas) contribute *< wine
character, but do not play an essential role in fer-
mentation kinetics
In general, with an adequate inoculation (It/'
cells/ml), fermentation is easily initialed in grape
must It is complete, if the initial carbohydrate
concentration is not excessive, but different fac-
tors can disrupt yeast growth and fermentation
kinetics Some factors have a chemical nature
and correspond with either nutritional dclkicn-
cies or the presence of inhibitors formed dur-
ing fermentation (clhaual. fatly acids, etc.). Oth-
ers have a paysKochcmical nature --for example.
I '.v ■. .*.„.,.-■ ..
P. K-i-MUm
Handbook or Etiology: The Microbiology of Wine and Vinifie.itions
oxygenation, temperature and must clarification.
Final))'. Ici»cn ration difficulties can lead (o ihc
development of u •desired microorganisms They
can antagonize Ihc desired wincmakingycaslstrain.
The successful completion of fermentation de-
pends oa all of these factors. A perfect mastery of
fermentation is one of the primary responsibilities
of an cuokigisl. who must use Ihc necessary means
lo avoid microbial deviations and lead Ihc fermen-
GHioa to ifc completion— the comptcic depletion
of sugars in dry wines Slack fermentations arc a
serious problem. They arc not only often difficult
to restart but can also lead lo baclcrial spoilage
such as lac Ik disease, in sugar- containing media.
These issues have been discussed in connection
with practical wincmaking applications (Ribc'rcau-
Gayot eltil . 1975a. 1976). Thcotclical informa-
tion concerning yeast physiology can be found in
mocc fundamental works iFlccL 1992).
3.2 MONITORING AND
CONTROLLING
FERMENTATIONS
3.2,1 Counting Yeasts
Different methods (described elsewhere) pcrniil the
industrial charactcriration of ycasl strains involved
in ft mien union. Tracking the yeast population
according lo fermentation kinetics can be useful.
Lafon-Lafomcade aid Joycnx (1979) described
enumeration and identification techniques for dif-
ferent micnxitganisms in must and wine tyeast.
acetic and lactic bacteria).
After the appropriale dilution of fcrmcniing
must, the tool number of ycasl cells can be esti-
mated under Ihc microscope, using a Malassc/ cell.
After calibration, this determination can also be
made by measuring the oplic density of Ihc fer-
mentation medium al 620 in. This measurement
permits a yeast cell count by interpretation of the
medium's cloudiness, provoked by ycasK
The total cells counted in this manner include
both dead' yeast and live' yeast .Tic two must be
diffcrcn tilted in cnology. In fact, counting viable
mKroorgan isms* is preferable When placed in a
suitiblc. solid nutritive nKdium. vEiblc cells ate
capable of developing and forming a microscopic
cluster. vRiblc to the naked eye. called a colony.
The number of vtablc ycasl cellscan bcdcKtmincd
by counting Ihc colonics formed on this medium
after 3-4 days. The culture medium is prepared
by adding I ml of correctly diluted yeast solution
lo 5 ml of nutritive medium. Tins mixture is
transferred in lis liquid form al 40'C into a Petri
disk: it solidities as it cools. The nutritive nKdium
consists ol equal volumes of a 20 g/1 agar solution
and grape must (1 80 g sugar/land .1.2 pH) diluted
Id half its initial concentration. The avetagc of two
population count* having between 100 and 300
colonics is calculated
Viable ycasl populations can also he estimated
directly by counting under Ihc microscope using
specific coloration or c pi fluoresce nee techniques.
Viable populations can also be determined with
ATP measurements using biolumincsccncc. Bouix
("/ •;! ( 1997) proposed Ihc use of immunofluores-
cence lo detect baclcrial contaminations during
wincmaking.
Microbiological control Is useful for research
wort, but these control methods are relatively long
and difficult, for this reason, fermentations are
genctully followed by more simple methods at the
winery.
3.2.2 Monitoring Fermentation
Kinetics
Wine make is must closely monitor wine fermenta-
tion in each tank of the winery Thiscloscsupcrvi-
skm allows them w observe transformations, antic-
ipate ibeir evolution, and act quickly if necessary.
They should effectuate both fermentation and tem-
perature controls daily (Figure 3.1).
Fermentation kinetics can be tracked by measur-
ing Inc amount of sugar consumed, alcohol formed,
or carbon dioxide released, but the measurement
of Ihc mass per unit volume (density i is a simpler-
method for tracking its evolution. Mass per unit
volume constitutes an approximate measure of the
amount of sugar contained in Ihc grape must.
Since a relationship exists between Ihc amount
of alcohol produced during fermentation and the
Conditions or Ycasl Development
81
<*W
Fig XI. ILvjmpIt of dairy ferae Hal kin monaorimi in
l»o lank* (iml.il uuiu dent-iy = 1.0851. (I) Normal
fermentation curve: alter u laic icy pcr»iil. fermentation
iuiiam. accclciam anil lien ikivis be In it Mopping
on ihc lOih day at . <km.ky below 0.995. "ben all
Uigur n (crmcnicd. (II) FcrmcMaliD* curve Iciiliu In
a iIikI Ic (menial hid: leimcaiaiain uora on ihc llih
day at a dcasiy of 1X105: unlcrmcmcd uuur fcaaiav
Fcnncutation ifctni early enough lo take preventive
;-!■■■. (IlllEvoinbn of Ike temperature tied wine
fermentation): arrow* Indkaic tooling
iiilial concentration of sui'.ir in the mum. mum
density can directly give an approximate potcnllal
alcohol The density and polenta! akohol aic
generally indKaKd on the stem or the hydrometer
approximately 17 g of sugar produces I'i akohol
in volumc<Scc!ion 10.32-Tabk 10.6). Expressing
potential akohol is without doubt the besl solution.
During fermentation, sugar depletion and clhanol
fomalion result in decreased density A hydrom-
eter is nscd to Monitor nasi density Samples
arc Liken Iron the middk of the tank by using
Ike lasting lauccl. Before faking a sample, the
fauccl should be cleared by letting a few ccn-
ti liters flow The density* is then corrected accord-
ing u must temperature No other conversions or
inlcrprctaiKMis arc necessary. Plotting the points
in the form of a graph permits the winenutker
h> evaluate fcmicntalion kinetics The regularity
of Ihc fermentation can also be evaluated More
importantly, fermentation diflkullics. which lead
lo stuck fermentations, can be anticipated. Dnc lo
Ihc heterogeneity of fermentation kinetics in a ted
wine making tank (the fermentation Is most active
under Ihc pomace cap), homogenizing the lank is
recommended before biking samples.
3.2.3 Taking the Temperature
The daily monitoring of tank temperature during
fermentation is indispensable, but this measurement
must be taken properly. In red wincmaking . in par-
ticular, the tank temperature is never homogeneous
The temperature is highest in the pomace cap and
(invest at the bottom of Ihc tin k. In Ihc first hours of
fermentation, abrupt temperature increases occur in
the pomace and art sometimes very localized Asa
result. Ihc must temperature against the tank lining
Is always less than in the center of the Link The tem-
perature taken in these conditions, even after prop-
erly ckaring the lasting lancet. Is not representative
of the entire tank The temperature should he taken
after a pa mping-ovcr. which homogenizes tank tem-
perature. In this manner. Ihc average temperature
can be obtained, bul Ihc maximum temperature, also
important. remains unknown.
The mist temperature can be taken with a dial
thermometer having a 1 5 m probe. This effective
method can measure must Kmpcralurc directly in
different areas, especially jnsl below the pomace
cap in ihc hottest part of the lank. This /one
has the most signiflcant fermentation activity. The
temperature can also be taken by thcrmockcirk
probes judiciously placed in each tank The probes
arc linked to a measurement system in the winery
laboratory. With this system . ihc wincmaker can
verify the temperature of the Links at any moment
Certain temperature control systems automatically
regulate Link temperature when the temperature
reaches certain value.
3.2.4 Fermentation Control Systems
Various automated systems simplify Ihc monitor-
ing of fermentation and make it more rigorous
These sy sic ins can also an tomatkally heat and cool
the must according to temperature
82
Handbook or Etiology: The Microbiology of Wine anil Vindications
Sonic of these systems can be vciy sophisticated.
For example, when the (cnperaluir exceeds ihc
set limit, the apparatus initially homogenizes ihe
■i mi In ihc lank by pumping. If ihc temperature is
Mill too high, ihc refrigeration nnii of ihe system
cools Ihc nasi Owing 10 Ihe seasonal character of
'.'■ j m- iii.it. iii!.- . some wincmakcismust make do will
manual conliol systems Automation can. however,
be filly juMilicd. when it permit, greater precision
in wincmaking For example, a temperature gra-
dicnl can be produced in this manner during red
wincmaking. At Ihc beginning, a moderate lempcr-
alntc 1 18-20'C) favotsccll giowlh and vitality; at
the end. a higher temperature (>30'C) facilitates
ihc extraction of pomace constituents
New approaches to automated systems could be
further dcvckipcd ( Flan/y. 1998). For the moment
at least, they influence temperature, modulating
it according to fermentation kinetics. In addition
to on-line' temperature control, a system shonld
be developed 10 monitor fermentation kinetics.
Various methods have been tested: measurement
of carbon dioxide given off. the decrease in
weight, and Ihc decrease in density (measured as
the difference in pressure between the top and
the bottom of the tank). Measurement of gas
released seems to be the most reliable method (by
weighing in Ihc laboratory or by asiug a domestic
gas conntcr for large capacity tinks: El Halaoni
el ill.. 19871. This process assumes thai Ihe tanks
arc completely airtight. The method, however, is
not particularly recommended, especially in red
wincmaking where pampiag-ovcr is indispensable.
Sablayrolks el at. (1940) stated that automated
systems should modulate lempcralnic in order to
control fermentation kinetics better and limit the
asc of cooling systems
AuiomaKd control systems shonld be linked to
fermentation speed and therefore yeast .utility, a
parameter certainly as important as the tempera-
tare When Ihc speed of CO? production execedsa
previously established limit. Ihc obtained temper-
ature shonld be maintained As soon as the speed
decreases. Ihc apparatus should let ihc temperature
rise in order to revive Ihe fermentation. This opera-
tion would permit Ihe fermentation to be completed
more quickly. Temperature modulation should be
related to must fennen lability Simultaneously let-
ting the tcmpcralarc increase would result in a
decrease in Ihc tola! energy demand (Table 3.1).
Of course, the apparatus shonld maintain the tem-
perature within compatible etiological limits.
Another approach consists of creating a model of
the alcoholic fermentation process Different calcu-
lations concerning lime, lempcralnic. and alcohol
and sugar concentrations air used lo predict fer-
mentation behavior — especially Ihe risk of a stuck
fermentation (Bovcc el ul.. 1990). In this way. the
need and moment of a certain operation (especially
temperature control) could be anticipated
Finally, these control system riles mast be
adapted lo Ihc particular needs of each tank in
tents of <pian titrable data and Ihc cnokrgist s
experience. A highly advance antoataled system
optimizing alcoholic fermentations in wincmaking
would have lomakc use of artificial intelligence lo
take into account Ihc enologist's own experience
(Grciicrfifl/.. 1990).
3.2.5 Avoiding Foam Formation
During fermentiuOa. foam can be formed as a
bon dioxide is released This can result in tl
tank overflowing. To avoid Ihe problem, finks a
r '""*""
UothcrB.il
17*0
U« hernial
Tcapcwuic-
eunr Milled
I7"C-22"C
Dueuiuaof fcrmcniMbn <h>
203
171
181
\ti\imu- nlC if CO..'1'kl
0.09
1.12
0.72
Total friporilic uaa* needed (kcal/l)
NUlUDUai fcitlorili.- uni .Icmniiil (bal/IA)
182
0.174
10. 1
0257
10
0.179
Conditions or Yeast Dcvclopnicil
-' mi nuns tilled h) only hall their capacity. This
constraint is not acccpctblc Paclors that influence
loan) formation include musl nitrogen composition
(especially piotcic concentration I. fcmicHalion
lempcraiurc. and the nature of the yeas! strain.
Allcmpcs lo creale It rmen union conditions (Tot
example, eliminating proteins by using bentouite)
capable of limiting this phenomenon have nol ted
k> suusfaclory results
For this reason, some AmetKan wineries have
adopted ihc use of products thai increase surface
tension This process reduces foam formation and
stability Two auti-foaming agents arc gaining
popularity: dimethyl polysiloxanc and a mixlarc of
oleic acid mono- and diglyccridc They are used al
a concentration of less than 10 mg/l and do not
leave a residue in wine, especially after filtration.
Due to their efficiency, red wine tanks can be
rilled K>75 -Hffi capacity and white wine baits to
85-90* These products arc not mic. The Office
International dc laVigncctdn Vin recommends the
exclusive use of the mixture of oleic acid mono-
.ni'.l diglyccridc.
I'i<; 32, Yeast pin»ih cycle ,m.l fcracnatkin Unci-
« t>f papc *"« conuinini* hiph «n»r conceal in-
■ ban <120 g/lKliifon-IafiiuKfldc. 1983). <I) Total yew
popubiuD ill) Viable jean populitun (III l IV untitled
auBnr
3.3 YEAST GROWTH CYCLE AND
FERMENTATION KINETICS
In an unsulfilcd and non- inoculated mast, con-
tamination ye: cis can begin to develop within a
few hours of filling the tank. ApKulatcd ycasLs
{Kloefhera. Himseniuspora) arc Ihc most frequen-
tly encountered. Aerobic yeasts also develop ( Can-
iliiki. Picliiii. Htinsemdtil. pioducing acetic acid
and ethyl acclak: RretUuumytes and its character-
istic animal-like odors arc rare in must. Althoagh
such yeasts can be relatively resistant to sulfur
dioxide (Fleet. 1992). salfiling folkiwed by inoc-
ulating with a selected strain of Sactharmtyees
cerevisiae constitute, in practice, an effective
means of avoiding contamination (Section 35.4)
In general. S cerevisiae inoculated at H ■" celLs/
ml. cither nam rally or by a sclcclcd strain inocu-
lation, induces grape mast fermentation.
The yeast growth cycle and grape mast fermen-
tation kinclKs arc depicted in Figure 3.2 iLafon-
Lafourcadc. 1983). In order k> acccntualc ccnain
phenomena, the figure concerns a must con tuning
particularly high sugar concentrations, which can-
nol be completely fermented. Analysis of this
figure prompt! the following remarks
1. The growth cycle has three principal phases: a
limited growth phase (2-5 days) increases the
population lo between lt> and ID* cells/ml. a
qnasi-sEilionary phase follows and lasK about
8 days: finally, the death phase progrcssivcly
reduces the viable population lo 10* cells/ml.
The linal phase can last for several weeks
2. During Ihis particularly long cycle, growth is
limited lo four or live gencrauons
3. The slopping of growth is not the result of a
disappearance ofcncigy nnlricus
4. The duration of these different phases Is nol
equal. The death phase, in particular, is Ihrcc lo
four times longer than the growth phase
5. Fermentation kinetics arc directly linked lo
the growth cycle The fermentation speed Is
al let maximum and practically constant for
Handbook or Enology: The Microbiology of Wine anil Vindications
a Utile over 10 days. This tlnie period corre-
sponds wiib Ihc lirst two phases of the growth
cycle. The Icrnicn union speed then progres-
sively slows but If in icn union nevertheless Lists
several weeks. Al this stage, the ycasl popula-
tion Is in the sarviv.il phase I ; inally. Ihc snip-
ping of fcrmcntilioa is not simply the rcsull of
insufficient yeaslgmwih Thcmctibolicaclivity
of non-prolifcraling celLs can aba be iibibilcd.
The cessation of metabolic activity has been
interpreted as Ihc depletion of cellular ATP and
the accu mi Union of clhaaol ii the cell — most
likely due to transport difficulties across the mem-
braies because of cellular sterol depletion The
cell enzymatic systems slid function during this
survival phase but the intracellular sugar concen-
tration decreases gradually (Larue el at., 1982).
These phenomena have several technological
consequences for a limited concentration in .sugar
(less than 200 g/l). fermentation occurs during the
first two phases of the cycle. II tikes place rapidly
and most olfcn without a pniMcm. On the other
hand, if their is an elevated amount of sugar, a
population in lis death phase carries out Ihc List
part of fcrmcuQIiOB In this case, its mclabolic
activity continues to decrease throughout the pro-
cess The total transformation of sugar inlo alcohol
depends on the survival capacity of thus population;
it is as important as )■ Ihc initial growth phase
Excessive temperatures and sugar concciilral ions
can provoke sluggish or stuck Ic mien unions
Nutritional deficiencies and inhibition phenomena
cai also be involved. All of them have cither
chemical or pbysicochcmKal origins. Fermentation
kinetics can be ameliorated by different methods
which influence these phenomena Early action
appears to increase their effectiveness: ycasLs in
their growth phase and in a medium containing
Utile ethanol arc more icccpUve to externa! stimuli.
The wincmakcr should anticipate fcrmcntilion
difficulties: Ihc possible operations arc much less
cffcclivc after they occur.
Moicovcr.cc tnin operations, intended to activate
fcrmcutiUoa. affect yeast growth and improve
fermentation kinetics at its beginning bit do not
always affect ycasl survival or Ihc final stages of
fcrmcntilioa. at least in musts with high sugar
m f
//
'/./
///
/
Yvf, XX Ex»»plc of u ibcoitiKi
■mm fcrmcM.it kin I S t mii.il sjj.i concnt*. S t : Hjgai
ciioich m (be cm! of irnNUibai. 11) Control: fcf-
mc mat kin Mopi. leaving unfcrmcntol uq3.ii |S. ( - \,)
|II| A.i iv j* ion ji ihc inhalant of fcuntnuiNm. bul the
mm plciton nl Icracmni km In riumn luv iny high *u£ni
conceal ai ion* u, •m Improved. (Ill) Activation naing
on -nxl population lim-iiih i*l uirviv.il: fcrnicnlMhin
n complete
concentrations (the most dillicult to ferment) In-
creasing must temperature is a classic example of
an operation thai increases Ihc Icrmcntition speed
at Ihc beginning bul leads lo sluck fcrmcilalions
Isec Sectin 3.7.1, Figure38). In other cases.
Ihc activation of the fermentation influences both
growth and survival; the duration of fermentation is
prolonged. Eiguie 3 J gives an example: activation
(curved) apparent)' improved the fermentation
kinetics of musts with relatively low sugar levels
as compared to Ihc control (curve I). However, in a
high-sugar must, an activator that did not enhance
survival was unable to prevent sluck renncntaliou.
In Ihc case of curve III. where both survival
and growth factors were added, fermentation was
completed smoolhly. even in a high-sugar must.
3.4 NUTRITION REQUIREMENTS
3.4.1 Carbon Supply
In grape must, yeast* find glucose and fruc-
tose—sources of carbon and energy The total
Conditions or Yeast Development
sugar concentration n must is between 170 and
220 g/l. correspond ing u wines bcrvvcci 10 and
li'.i vol clhanol alfcr fermentation The amount of
dissolved sugar can be even higher in grapes for
Ihc production of sweel wines — np lo 350 g/l in
Sautcrncsmusls The must sugar concentration can
influence Ihc selection of Inc yeasl strains, ensuring
fermentation (Fleet. 1992).
fxrrntcniation is slow in a medium con tuning a
few grants of sugar per liner, lis speed increases
in musts which have 15-20 g/l and remains stable
until about 2(1) g/l Above this concentration, fer-
mentation slows In fact, alcohol prodnction can
be lower in a must containing 300 g/l than in
another con tuning only 200 g/l. Prom 600 to 650 g
of sugar per liter. Ihc conccnlralcd grape must
becomes practically unfermen table. The presence
of sugar, as well as akohol. contributes to ihc sta-
bility of fortified wine.
Thus, an elevated amount i>f sugar hinders yeast
growth and decreases the maximum population.
Consequently, fermentation stows even before the
prodnction of a significant quantity of clhanol —
which normally has an antiseptic effect (Sec-
tion 3.6.1).
Fermentation slows down in the same way when
the high sugar concentration is due u the addition
of sugar (chapiali/atron ) or concentrated must, or
the elimination of waicr by reverse osmosis or
vacuum evaporation (Section 155.1). Trie effect is
exacerbated if sugar is added when fermentation
is already well advanced and alcohol has started
lo inhibit yeast development, although intervening
at this stage has the advantage of avoiding
overheating the must When sugar Is added, it
is advisable to wait until the second day after
fermentation start*, i c the end of Ihc yeast growth
phase. In these conditions, ihc population reaches
a higher value, because it grows in a medium
with a relatively low sugar concentration Next, the
addition of sugar in a medium containing yeasl in
full activity increases the fcniientition capacity of
the cells and therefore the transformation of sugar.
Of course, a refrigeration system is necessary
to compensate for Ihc corresponding temperature
c of the must.
3.4.2 Nitrogcm Supply
Hcnschkc and Jiranck ( 1992) analyzed many theo-
retical works on this subject in detail. The research
for these different works was carried out under a
large range of conditions and so the results were
not always applicable to the wincmaking condi-
tions analyzed by Ribc'rcau-Cayon el ill. 1 1975a).
Grape must contains a relatively high concen-
tration of nitrogen compounds (0.1 to I g of solu-
ble nitrogen per likr). although only representing
about a quarter of toctl berry nitrogen These con-
stituents include the ammonium cation (3- )Vfi of
total nitrogen), amino acids 125-30'ii. polypep-
tides (25 --«« ) and proteins (5-10** ). The grape
nitrogen concentration depends on variety, tool-
slock, environment and growing conditions—
especially mitogen fertilization. It decreases when
rot develops on Ihc grapes or Ihc vines suffer
from drought conditions. Water stress, however,
is generally a positive factor in quality red wine
production. Planting cover crop in the vineyard
lo control yields also reduces Ihc grapes nitrogen
lcvclsThccffccrsarcvariablc.es. 118 mg/l nitro-
gen in grapes from Ihc control plot and 46 mg/l
nitrogen for Ihc plot with cover crop in one case
and 354 and 2l0ntg/l nitrogen, respectively, in
another The nitrogen content of overripe grapes
may increase due lo conccnlralion of the juice.
In dry while wincmaking. juice extraction meth-
ods influence the amino group compound and pro-
tein conccnlralion in must Stow pressing and skin
maceration, which favor Ihc extraction of skin con-
stituents, increase their conccnlralion (Dnboutdicu
rial.. 1986).
Yeasts find ihc nitrogen supply necessary for
Ihcir growth in grape musl. The ammonium cation
is easily assimilakrd and can satisfy yeasl nitro-
gen needs, in particular, for the synthesis of amino
acids Polypeptides and proteins do not patlicr-
palc in .V cererisilie growth, since this yeasl cannot
hydroly /c these substances S. cerevisiae docs not
need amino acids as part of ils nitrogen supply,
since it is capable of synthesizing them individu-
ally, hut their addition stimulates yeasts more than
amnion lic al nitrogen A mixture of amino acids
and animoniacal nitrogen is an even more cffcclivc
i landbook of Hnology: The Microbiology of Win* anil Vindications
stimulant Ycasfc use amino acids according lo
three mechanisms (Hcnschkc and Jiranck. 1992):
1. Difccl integration without transformation into
proteins.
2. Decomposition of ihc amino cmip. which is
"--.■i! for Ihc biosynthesis of different amino
constituents The corresponding carbon mole-
cule is exacted Sich a icaciroi is one of the
pathways of higher alcohol formation present
R-CHNHj-COOH + HjO
R-CHjOH + CO, + NH,
Yeasts are probably capable of obtaining ammo-
niacal nitn^cn from amino acids through other
pathways
3 The amino acid molecule can be tscd as
a source of carbon in metabolic reactions.
The yciist simultaneously iccupcralcs the cor-
responding ammoniacal nitrogen
The assimilation of different amino acids de-
pends on the functioning of transport systems and
the regulation of metabolic systems. Several studies
have been published on this subject (Castor 1953.
Rrhdicau-Cayon and Peyiaud. 1966). Due to the
diversity of mist composition, the results are not
Klcnlical The assimilation of amino acids by yeasts
docs not always improve growth. Tic most easily
assimilated amino acids arc not necessarily the most
significant in cell composition . bnt arc instead the
most easily transformed by yeasts. Yeasts have
difficulty assimilating argininc when it is the only
amino acid in the environment, but argininc iscasily
assimilated when famished in a mixture. Yeasts do
not use it when ammonium is present.
To avoid the difficulty of precisely defining
grape must composition. Hcnschkc and liranck
( 1992) carried out their experiment! using a well-
known incdinm model Their results have given
researchers a new understanding of this subject
Although complex mixtures of ammonium sails
and amino acids ate more effective for promot-
ing yeast growth and fermentation speed, ammo-
nium salts ate used almost exclusively b> it
nitrogen concentrations in must, for t
simplicity. Positive results have been obtained in
laboratory tsts bnl thci reflectiveness is less spec-
tacular in practical conditions Moreover, the addi-
tion of assimitiblc nitrogen is not always sufficient
for resolving difficult final stages of fermentation,
although it accelerates fermentation in the early
stages
Fora long time, diammoiinm phosphate was Ihc
exclusive form of ammonium salts used. The phos-
phate ion involved in glucidk metabolic reactions
was also thought to favor fermentation In real-
ity. Ihc must is suflicicnlly rich in Ihc phosphate
Km (incidentally participating in the iron cassc of
while wines) aid for this reason il is preferable to
nsc diammonium sulfate EU regulations authorize
Ihc addition of 30 g of one of Ihc these two sails
per hectoliter, corresponding to 63 mg/l nitmgen.
In the USA. Ihc limit is set at 95 g of diammo-
nium phospbak: per hechditcr. In Australia, its
addition must not lead to a concentration of inor-
ganic phosphate gtcarcr than 40 g/hl. The standard
dose is between lOaad 20 g/hl (Nov thai 100 g of
diammonium phosphate or sulfate con Grins approx-
imately 27 mg of ammonium and 73 mg of phos-
phate or sulfate ions). The addition of this form
nitrogen to must increases acidity, dnc to Ihc con-
tribution of the anion. For 10 g of diammonium
sail per hcctolikr. Ihc must acidity can increase by
0J5g/l(in H!SQi)or052g/Uin tartaric acid).
The initial concentration of ammonium cations
and amino acids in Ihc must is one of Ihc most
important clement in determining the need lor
supplements When the NH, ' concentration is less
than 25 mg/l. nitrogen addition is necessary It is
useful for concentrations « be between 25 aid
50 mg/l. Above Ibis concentration, supplementing
has no adverse cffccK. Il Pi. however, unlikely that
an add r lion will activate Ihc fermentation. If the
values are expressed in free amino nitrogen (FAN),
delectable by mnhydnii. between 70 and 140 mg/l
arc necessary w have a complete fermentation of
musts containing between 160 and 250 g of sugar
per liter (Hcnschkc and Jiranck. 1992).
Bcly el ill. ( 1990) dcKraiined thai addiig mito-
gen was effective if the available nitrogen content
• S'tU + amino acKls. except proline) )■ Ihc must
Conditions or Yeast Development
87
was below 130 mg/l. bill was unnecessary UBd
coukl even be harmful al initial concentrations of
200-350 mg/l. Aeriy's (1996) formal index pro-
vides a simple estimate of ihc fire amino acid and
ammonium cation conienl A formal index of I
corresponds k> 14 mg of amino nitrogen per liter.
According to Loren/jni( 1996). the addition ofnilro-
gen in Swiss varietal musts is indispensable if the
index is less than 10. and is recommended if the
index is between 10 and 14. The Ibmiol method
provides a quick, simple assessment of available
nitrogen deficiency by assaying Nil,* and free
amino acids, except proline It shoaM be more
widely used m monitor ripeness and fermentation.
However, the nitrogen requirements of 5 ceirvisiiie
vary from one strain to another Jalien el til. (2000)
icccntly proposed a Ksl for comparing yeast nitro-
gen requirements, estimated dnring the stationary
phase of alcoholic fermentation This test measures
Ihc quantity of nitrogen reqiired to maintain a con-
stant fermentation rale during this stationary phase.
Nitrogen requirements varied byafactorof2 among
Ihc 26 wincmaking yeast strains tested An assess-
ment of Ihc nitrogen requirement is certainly an
important criterion in selecting yeast strains to use
in nitrogen- deficient mists.
For example, in a study of musts from Bor-
deaux vineyards in the 1996 to 2000 vintages
(Table 3.2). Masncul ei ,il (2000) found nitrogen
levels of 36-270 mg/l in while must*, with defi-
ciencies in 22'i of the samples (nitrogen concen-
trations under 140 mg/l). In reds, levels ranged
from 46 to 354 mg/l. with deficiencies in 49»;
in roses, levels ranged from 42 to 294 mg/l. with
deficiencies in GO*, while SW of botryli/cd musts
were niirogcn-dcficicni
Chonci (2000) analyycd Cabernet Sauvignon
musts in 1997 and found significant variations in
nitrogen levels, from 95 to 218 mg/l
Finally, different nitrogen concentrations have
also been found in individual plots within the
same vineyard, c.g 25-45 mg/l in vines with less
vegetative growth and 152-294 mg/l in grapes
from more vigorous vines.
All these analytical findings show the cxlcnt
and frequency of nitrogen deficiencies, which arc
much more common than was generally thought
in the past, perhaps due to changes in vineyard
management techniques It was generally accepted
previously that nitrogen concentrations in musts
from northerly vineyards in the northern hemi-
sphere (temperate, oceanic climate) were sulli-
cicnlly high.
Adding nitrogen io musts containing insufficient
levels is extremely useful in achieving good
fcrmcntilion kinclKs. In some cases of severe
nitrogen deficiency, it may even be opportune lo
add as much as 40 g/l of ammonium sulfate, which
would require a change to EU legislation, as the
current limit is 30 g/1. corresponding lo 63 mg/l
of mitogen. Some observations suggest thai an
excessive increase in nitrogen content may haw
a negative effect on fcrmcnialion kinetics, so il is
advisable to modulate nitrogen additions according
lo Ihc natural level in the mist and ensure that the
lotal never exceeds 200 mg/l
Adding excessive amounts of nilrogen may also
result in Ihc presence of non-assimilated icsidual
nilrogen al the end of fermentation Although
there are no specific data on this issue, residua!
nilrogen may have a negative impact on a wine s
microbiological stability. An excess of ammoniacal
Tabic 32 Available ailnipcn conical :MI. and ftcc arnlwi acid* cxficucd In
mg/l) In annU fmm Bnidemn vincvanb <I090- 1999 vim-pes) determined by inc
tormol method (Mas oeof a -i.. 20011)
Maximum value
Mean
Siambnldcvuikii
Dcliricnt rnuM* i r
1) <MQ mg/1
1 Limlkxk or Etiology: The Microbiology of Wine anil Vitrifications
nitrogen cun also lead (o a modifKation of ihc
aromatic characters of wine. Since Ihc yeast bo
kwger needs n> deaurinale amino acids, u forms
less secondary products (higher alcohols and Ihcir
esters). This modifies wine aroma, cspcciall)' white
wines. Finally, the nitrogen supply affect* elbyl
carbamate production. This undesirable coBslitucni
has carcinogenic properties and R continued by
legislation.
The must sugar concentration abo affects the
impact of Ihc nitrogen supply on fermentation
badics. especially the successful completion of
fermentation For modctalc concentrations of sagar
(less than 200 g/1). the addibon of nitrogen
increases Ihc biomass of yeast formed and in con-
sequence the fermentation speed. Ihe fermentation
R completed a few days in advance For high
concentrations of sagar. the fcrmcntilioa is accel-
erated al the beginning with trspect K) the con-
trol sample but. as Ihc fcrnicn laiion conbines. the
gap between the control sample aid the supplc-
■Knlcd sample decreases. Finally, their fermenta-
tions spontaneously slop with similar quantities of
residual sugar remaining. Curve II in Fignre 3.3
depicts the effect of supplemental nitrogen (or
other activator effect ton a must with a high sagar
concentration, having a normal nitrogen concen-
tration. On the other hand, if fermentation slug-
gishness is due lo a nitrogen deficiency. Ihc addi-
tion of ammonium salts manifestly stimulates it
(Curve III. Figure 3 J). Stack fermentations can
sometimes be avoalcd in this manner
Other factors affect Ihc assimilation of nitrogen
daring fermentation. Yeasts have strain-specific
capabilities. Henschkc and Jiranck ( 1992) reported
that different 5. cererisiiie strains fermenting giape
mast assimilated quantities of nitrogen varying
from 329 to 451 mg/l al 15 5 'C and from 392 to
473 mg/l at 20'C. These last figures also show,
among other things, that temperature increases
nitrogen assimilation. Julici rial. (2000) com-
pared Ihc nitrogen and oxygen requirements of
several yeast strains used in wincmaking.
Oxygen, however, has the most effect on the
assimilation of nitrogen. Yeasts have long been
known to use considerably more nitrogen in
the presence of oxygen (Ribcreau-Gayon el al..
1975a). It has been observed that yeasts fcrmcnbng
in ihc complete absence of oxygen assimilate
2(1) nig of nitrogen per liter. When they develop in
the presence of oxygen, their assimilation inc teases
lo 300 mg/l. In acrobiosis. they can assimilate up
to 735 mg/l without a proportional increase in cel-
lular multiplication.
The impacl of oxygen on fermentation kinetics,
irrespective of any addition of NH^ '. is appar-
ently complex and dependent on several factors
tSabktyrollcs el rrf . 1996a and 1996b). as well as
lac type of must (sugar content and possible mito-
gen deficiency). It Is accepted thai adding nitrogen
accelerates fermentation, resulting in faster com-
pletion. It is. however, more difficult to identify
the conditions under which adding nitrogen can
prolong sugar convention by the yeasts and pre-
vent fermentation from becoming stuck, al least in
musts rich in sagar
In an experiment earned out by Ro«s el ill.
1 1988). using a must containing 222 g/l of sugar
with a normal nitrogen content 135 mg/l Nll 4
corresponding lo approximately 200 mg/l nitro-
gen), fermentation stopped prematurely in the
absence of air. Adding NH,* (0.15 or 050 g/l
(NHjbSO,) initially accelerated fermentation but
did not increase Ihc amount of sugar fermented.
With aeration on the .I 1- day. fermentation was
faster and all the sugar was fermented Adding
NTU r did noi improve fermentation kinetics, but.
on the contrary, after an initial acceleration, ycasl
activity stopped when 9 g/lofsugarwasstill unfer-
mented Of coin, these experimental results must
be interpreted in the light of the specific coadi-
tioas( sugar and nitrogen content of the must! The
results would not necessarily have been Ihe same
under different conditions, particularly if there had
been a significant nitrogen deficiency in the must.
In any case, this experiment shows quite clearly
thai adding mitogen docs not necessarily elimi-
nate problems wilb Ihc end of fermentation Fur-
ther experiments using niirogcn-dcficicm must are
required to identify a possible improvement
The timing of (he addition of ammonium
salts appeals to be important. Ribcreau-Gayon
f.<<:l. (1975a) had suggested their addition in
must before the initiation of fermentation. Yeasts
Conditions of Yeast Development
89
react best li> stimuli during ihc growth phase
ii a medium containing link clbanol. They
witnessed an assimilation of amnion iacal nitrogen
supplement (lOOmg/l) varying between 100 and
5(Yi when Ihc addition was niadc before Ihc
initiation of fermentation or on ihc foonh day.
Enhanced nitrogen assimilation did nol necessarily
incica.se the yeasts' fcmicntilion potcniial. This
Fxpkuns why adding nitrogen has no significant
impact on accckraling a sluggish final stage in
fermentation anil Is even less effective in resulting
a stuck fcmicntilion.
Sablayrollcs end i hMu and 1996b) reported
slightly* diffcrenl findings. Accoiding to these
authors, nitrogen supplements were niosl effective
in mid- fermentation, together with aeration. This
combined operation had more impact on fermen-
tation ki hc lies than aeration alone and provided an
optimum solution for avoiding ptcmalurcly siuck
fermentation (Sablayrollcs anil Bkitcyron. 2001 ).
In conclusion, supplementing masK with natu-
rally tow nitrogen kvcls (S, ald £. 140 mg/l) with
nitrogen sails is likely to improve fermentation
kinetics, wiih varying effects on yeast growth
and sagar conversion For maximnm effective-
ness, total nitrogen after supplementation should
nol exceed 200 mg/l. Some experimental lindings
indicate that fermentation may slow down follow-
ing Ihc addition of excessive amounts of nitrogen.
If Ihc must alicady bad a sufficiently high nitro-
gen content, further supplementation was likely
to cause an initial acceleration in fermentation,
bnl the effect wore off gradually. Adding nitro-
gen cannot be expected to remedy problems in the
final stages of fermentation (high-sugar musts or
strictly anaerobic conditions) It is. however, true
that nitrogen deficiencies loM vines or vineyards
with caver crop) have not been given sulficicnl
consideration in the past, and that completion of
fcmicntilion is facilitated in these cases by adding
ammonium salts Total nitrogen in must should be
analyzed in vat before Ihc skirt of fermentation as
a matter of course, together with sugar and acidity
levels.
Adding oxyg en at thestirtof fermentation (Sec-
tion 3.72) when the yeast population is in the
growth phase is still the most effective way of
accelerating fermentation and preventing prema-
ture stoppages. Opinions diverge on the correct
lime to add ammonium sails, varying from the
beginning of fermentation to halfway through In
my case, nitrogen supplements .ire more effec-
tive at accekrating fermentation than preventing
it from becoming stuck with unwanted residual
sngar.
3.4.3 Mineral Requirements
The ycasls that Pasteur cultivated in the following
medium proliferated well: water. II") ml. sugar.
100 g: ammonium tartrate. 1 g: ashes of 10 g of
yeast Yeast ashes supply the yeast with all of
its required minerals Dry yeast contiins 5- \lf.i
mineral matter, whose average composition (in
pcrccntigc weight of ashes) is as follows:
K 2 o
2.1-48
Nil*' ■
O.06-2.2
r.i: '
1 .0-4.5
V- ■
3.7-8.5
FczOi
O.06-7.3
|-,1 !-
45-59
v:,
04-6.3
5* ';
0-1.8
CI
O.03- 1.0
Other minerals not listed above arc present in trace
amounts: Al. Br. Cr.Cu. Pb. Mn. Ag. Sr. Ti. Sn.
Zn. etc. These arc called trace ckments. Not all
of them are indispensable but some arc essential
en /vine constituents.
The precise function of only a lew minerals
is known. Grape mnst contiins. both qualitatively
and quantitatively, a sufficient mineral supply to
ensure yeast development.
3.5 FERMENTATION ACTIVATORS
3.5.1 (Jrovvfh Factors
Growth factors affect cellular multiplication and
activity, even in small concentrations. They are
indispensable to microorganisms and a deficiency
in these substtnees disturbs the metabolism
Microorganisms behave differently in relation lo
90
i taadbonk of Etiology: The Microbiology of Wine anil Vmificalions
growth facloiv Some can lolally or partially syn-
thesi/c litem, others cannoi anil mnst Had them in
ibcir environment.
The subsumes ihal arc growth factors fur
microoigantsms arc also inmairy vitamins (or
higher oiganisats II-lgnre3.41 Tbcy aic essen-
tial components or coenzymes and aic involved
in mciabolic reactions Grape must has an ample
supply of growth factors (Table 3 3) bul alco-
holic It iincn union alters io vitamin composition.
For example. Ibiaminc disappear almost entirely:
yeast, arc capable orconsuming greatcramonnlsof
thiamine (600-800 P g/I) thai Ibc mast contains:
hm yeasts form riboflavin. The concentration of
nKotinamKIc icmains conslait in red wines and
masts, but only <**i remains in white wines. Pan-
tothenic acid, pyridoxinc and biolin ;uc used by
yeasts and then released: their concentration?, aic
nearly identical In niusis and wines. %icsoinosib>l
Is practically unuached.
Although musts contain saflkicnt amounts of
growth factors lo cnsaic yeast development and
alcoholic fermentation, natural concentrations do
not necessarily correspond with optimal concen-
trations, l-or this reason. supplementing mast with
certain growth factors Is recommended
c^c — ctii — cn^m
Hit CH(CII9.C
(> C C ClljQtt
ItdClli C CHDM — CO — Ntt — CI!,—
PviUniK
Fifi Xi. Ye»M grouth fact on,
Conditions or Yeast Development
Tabic XX thiH
ccmraiiomOu.lt ■.
n «f.. 1975*)
in and i
■«I
mm pniuth iMiottrun-
kiks, 1 ' Ribcicau-Cavon
pc mm
u,
Who
Viumim (ic
Whin Re*
Thuaioc I00-4SO 2-58 103-215
Rfcalavln 1 oil S 111 047-19
Pantothenic OJ-IJ 055-1.2 0.I3-OA8
acid
PynloiiK 0.16-0.5 0.12-0.07 0.13-068
Ni.oiio.««ic 0.08-26 044- IJ 0.79-1.7
BkHla 1 3-4 J I-3A 06-46
MaoMMkol 380-710 220-730 290-334
lmg/1)
Cutubmiac 0-0.10 004-0.10
Choline 19-39 19-27 20-43
A deficiency In pantothenic acid causes ihc ycasl
to ;iieunibUilc acetic ;icid bul ll I;moI been pfoven
thai (he (unauthorized) addition ol pantothenic acid
Id a fermenting ninM lowers the wine's volatile
. ■-'j-:j'.> "i. mating from yeast The production by
yeasts of abnormally high levebt of volatile acidity
is probably due to the musts deficiencies ii certain
lipris These deficiencies ate nx>st likely linked to
deficiencies in paitothcnic acid, which is involved
in the formation of acetyl coenzyme A. responsible
for fatly acid and lipid synthesis.
The supplementing of biolin and especially thi-
amine improved the mast le mien union kinetics in
n nine rons experiments. An addition of 03 nig of
Ihiaminc per liter can increase the viable popu-
lalion by _>(f>. the Icnncn union of sugar is also
quicker. These result, although regularly observed
in laboratory experiments, arc not always obtained
under practical conditions. The natural concentra-
tion of thiamine may or may not be a limiting
factor of fermentation kinetics, depending on the
nature of the grape and on maturation conditions
The addition of Ihiaminc Is legal in several
countries (EU. at a dose ol 50 mg/hl) bul il is rarely
used lo accelerate fermentation in wincmaking.
It effectively decreases signifkail kctouic acid
concentrations by decarboxylation (pyruvic and a-
kctogluciiic acid). Large quantities of these acids
bind u sulfur dioxide in botrytized sweet wines
I Sec lion 8.42).
3.5.2 Survival Factors
The Mica of survival factors Is derived from the
interpret! lion of the mode of action of sterols
and certain king-chain fairy acids on yeast activity
and fermentation kinetics. The first works on
Ibis subject (Andrcascn and Slier. 1953: Brcchot
elal.. 1971) were analyzed by Ribc'rcau-Cayon
el til. (1975a). The growth factor activity of
crgoslcrol in complete anacrobtosst Is optimal
at a concentration of 7 mg/1; It is solu bilized
with Twecn SO. For example, in a must with a
high sugar concentration 1260 g/1). S. cerevuiae
fcraicnts in complete anacrobiosis 175 g of sugar
per liter in ID days in the control sample and
258 g/1 ii the presence of 5 mg/1 of crgoslcrol In
acrobiosis. on Ihc other hand, a slight inhibition
of the fcrmcntutioi Is observed when crgoslcrol is
added . The au Ihors cone I uded that these sterols are
indispensable to ycasR in complete anacn>biosIs.
because they cannot be synthesized in these
conditions. Sterols arc necessary for ensuring
cell membrane permeability. In the presence of
oxygen, yeasts ait capable of producing sterols In
anacrobiosis. crgoslcrol is In sonic ways an oxygen
substitute for yeasts
Other sterols and long-chain fatty acids skate
most of the properties of crgoslcrol Some an con-
stituents of grape bloom and cuticular wax. such as
oleanolic acid — especially when associated with
oleic acid (Figure 33). These constituents explain
Ihc results of past experiments, indicating an accel-
eration of the fermentation speed of grape must in
complete anacrobiosis when grape skins and seeds
wen; added in suspension.
Laicr works (Larnc el of., 1980; Lafot-Lafour-
cadc. 1983) showed that the action mechanism of
sterols is In fact more complex. These authors
confirmed the growth factor action In a strictly
anaerobic fermentation: the maximum population
increases. They also witnessed Ihc inhibitory cflcct
of sterols on a fcrmcii Eilkn with permanent aera-
tion Neither of these two conditions correspond
exactly in xviucmaklng conditions
In Ihc winery, laige- volume fcmicntiDons arc
certainly anaerobic, but Ihc must Is aerated during
extraction and inoculakd with a ycasl scutcr
which was pit-cultivated Ii acrobiosCs: the ycasn
i taadbonk of finology: The Microbiology of Wive anil Vitrifications
a™uaoi cmjiuo
'Miknliml <'irtli(<l
Kift AS. Siiumu
e MCM>Mb and litt. iciU f Uylnp .
II VMM [■'.-.>". 1 /.
air therefore well equipped in stroK Both
commercial active diy yeasts and indigenous
yeasts, which develop oi the surface of nialcrEil
In contact wilh Ihe harvest, inilially develop in
acrobiosis. In ihc.se conditions, ihe addition of
crgosKrol or olcaiolic acid docs nol increase ihe
maximum population. The fermentation speed Is
also nol affecled during Ihe first 10 days. Yel the
yeast cells well equipped in sterols niainlain Iheir
fcrmcuttlkm activity for a longer Iiik. At the end
of fermentation, they will have degraded a larger
amount of sugar than noa-supplcmcntcd cells
(Table J.4). The Krm survival factor has been
proposed for this action thai does not correspond
with an increase in growth. The evolution of yeasl
populations during fermentation in the presence of
sterols Is represented in Figure 3.6. the incidence
of the Kmpcraturc R also indicated.
The notion of survival factors complements
the notion of growth factors. They arc espe-
cially interesting in the case of difficult fcrmcnla-
tions — for example, musts containing high sugar
concentrations Of course, the direct addition of
sterols to tanks in fermentation should not be
considered. Wincmaking can. however, be ori-
entated towards methods which promote sterol
synthesis Moreover, their cxrstcncc in the solid
parts of the grape suoaki be token into account:
crashed red grapes ferment bclKrthan while grape
mnsts because of solids contact during fermenta-
tion Ii addition, the elimination of sterols dur-
ing the excessive clarification of white grape
Conditions or Ycasl Development
Tabic 14. Sterol c
DKCMniDir
m ynu
ilni;-- 1 akrohulk fcimcnui km uf pra
(Li
ucnJ.. 1981)1
Coal* win
Co lull i* wo* ion
Amoohkii*.
C
-E +OA
C
+E
+OA
Djy 2
KcimcMcd ui(ar(py'll
Sterol* (*i nl'diy weight)
Vublc eclh ( HrVal)
Day 5
Feime«ed*ig4i(g/I)
Sterol* rinfdiy vciphl)
VUhk nib ( KrVal)
Day 9
i'cnncucil uig4i(g/l)
SlcroKl'i afdiy weight)
VUhk cclH ( 1"°.— It
Kndof teracnuitkin
I'cnncucil Nig4i(g/I)
SicroUCi oldiy weight)
VUbk celH ( loVal)
of Mcrob. un vea« suivival during
oi riiflcica Icapeatoics I La to o-
IDContial. (II) Phs, eig-wte «>l
muslcan rcsnll in extremely dilficill fermentations
(Section 3.7.3). Thiscotccpt can abo explain p;r%t
experiments which show increased fermentation
speeds with (he addition of groand grape skins
and seeds.
3.5.3 Other Fermentation Activators
R ibdrcan-Gayon end. (1975a) examined o(hcr
fcraicitalion ac iivau>rs. These activators generally
help (he ycasl m make belter isc of iibsi nitrogen
Incidentally. Ihe same phenomenon Is observed
each linic ihai (he fermentation Is accelerated by
(be picscncc of air or by Ihe addition of ycasl
exuact nutriments or suimv.iI faclors (Table 33).
Hydroly/cd ycasl cxIracR are rich in assintilaMc
nilrogen. survival faclors and mineral sails They
haw oficn been used (al high concentrations, up
to 4 g/1) lo accclcrat? It mentation in ihe food
industry. Some can impait foreign odors and tisles
Among Ihe aciivalor form las proposed
(Ribcrcau-Gayon etiil.. 1975a). 200 mg/l of the
following mixture may faciliEile It men union in
musts with vitamin and nilrogen deficiencies:
Tabic 3.5.
InKlcdf". .
Handbook of Enotogy: The Microbiology of Wive anil Vindications
l.ffcii tif ail i oil Mir. ii 1 1 I. aim oa giapc mie.1 nimytn auiaubllta ijcbpcil (mm
ml Kuntec. WHS. hy Camaiclli. 1989. tied by Hciochkc and liomck. 1992)
Aim.
ftffaHC
AdditniB.
utilintba
(mgof KAN/I)
Number of
eelk
(xlO'/l)
Itmfi.
(go
100
mill h)
Si rope n
Nirogen
100 g of diam mon iu m sulfate. 250 nig of thiamine.
230 nig of calcium pantothenakr. and 2 nig of
btotin.
OUtcr product!, extracted from fnngi. arc also
alcoholic fermentation activators. Some have bcci
com mere rali zed in the past One of the most
effective is prepared from a culture medium of
Aspergillus niger. II modifies the conccntratioi of
secondary products by promoting ihc glyccropyrti-
vK fermentation of sugar. A yeast activator is also
obtained from Ihc mycelium of BtXrytis cineien.
These activators arc nol aulhori/cd by vilKullural
legislation, at least in the EU.
In while wincmaking. suspended solids activate
fcrmcuttlloa (Section 3.73). Certain conslltaenis.
probably Mentis aid fairy acids, arc involved la
this phenomenon (RibCrcau-Cayon etitl.. 1975b).
Although these substances arc lot wry soluble,
yeasts arc capable of using them to improve
fermentation kinetics. They probably acl in con-
junction with other factors, such as oxygenation
and possibly uilrogcn additions Yeast hulls have a
similar cffccl independent of their ability to elim-
inate inhibition (Section 3.6.2).
As with nitrogen or growth factor supplemen-
tation. rcmcnUition Is not activated to the same
degree in the winery as in the laboratory Oxygena-
tion, yeast sUirtcr preparation and the fermentation
■Kdium play an essential role. In addition to the
mist's possible nutritional deficiencies.
3.5.4 Adding Yeast Starter
Wine makers have always been intc rested in
improving fcraicnUition kinetics and wine quality
by inoculating with activated yeast suiter. This
practice has certainly become mi tic widespread
since relatively economical, casy-lo-tsc Dried
Activated Yeast (DAY) became available on the
market. DAY arc simply reactivated in water or a
mixture of equal volumes water and must at a tem-
perature of .15— K)C There arc around a hundred
commercial yeast preparations on the market, aid
each one should be prepared for use according lo
the manufacturers instructions. DAY also makes
it possible to eliminate apiculatcd yeasts and select
strains with a high sngar-aleohol conversion rate.
Together with other winegrowing practices, the use
of DAY has contributed to Ihc general increase in
average alcohol con lent in wines and lo Ihc cor-
responding decrease in the need for chapLili/aimii
(adding sugar) in certain regions. On the contrary.
)■ some situations, there is an interest in using
yeast strains with a lower conversion rale. This
mainly concerns hot areas where there may be a
high sugar level in Ihc grape flesh although the
other ripeness indicators (skin) have nol reached
optimum levels In this case, it is necessary lo
delay the harvest, wilh the risk of obtaining very
high-sugar musts that arc difficult lo ferment in
order to produce high-alcohol wines
An initial inoculum of I"* cells/nil is generally
considered necessary lo obctin good fermentation
kinetics. In view of the current constraints on while
wincmaking. this initial level is rarely achieved:
so the use of ycasl starter has become practically
compulsory. In red wincmaking. there may be
insuflicicnt inoculum in the first few vats tilled, but
grape and must handling operations in the winery
Conditions or Yeast Dcvclopnicil
95
rapidly result in proliferation of ihc yeasts The
nsc of DAY is mainly justified in Ihc Him vat..
Luici vats cither require no seiner ai all. or DAY
cun be replaced will 2-5'* must from a vat where
fermentation is going well.
There has. however, been interest for sonic linic
in Ihc possibility of improving a wine's quality fry
selecting the appropriate sttuin for fermentation. It
is certainly Ime that ihc composition of grapes and
other natural facloiMc i> .lerraa )arc the main ele-
ment of the specific characteristics recognized as
Ihc basis of quality, especially in the concept of
itppellmion <f uridine amiri'Aee. It is aLso true that
positive results have been ob tuned, especially with
while wines (Section 13.7). Several strains capa-
ble of fermenting musts wiih tow iirbidily without
producing excessive volatile acidity have been iso-
lated Strains have also been Mien tilled thai do not
produce vinyl pneuofe. with their unptcasant chem-
ical odor and other undesirable characteristics, due
to high levels of fermentation esters These neutral-
ize varietal aromas and can only be recommended
for wines made from non-aromatic varieties h is
clear lhal using yeast starter Is a good way of
avoiding these types of defects and. consequently',
making the most of ihc grapes* intrinsic quality.
Another example is Ihc development of anaerobic
yeasts likely h> produce elbyl acetate as soon as
vab arc filled wilh non-sulfilcd grapes or must.
Reports in the litciaturc indicate that it Is possible
k> avoid these defects by using appropriate yeast
strains after the grapes/must have been sullied.
Today, there ts increasing interest in select-
ing yeast strains capable of enhancing the vari-
etal aromas of various grape varieties by releasing
variable quantities of odoriferous molecules from
their odorless precursors Research has focused on
Muscat varieties and. above all. Sauvignon Blanc
■Section 13.7.2). Although different ycasi strains
have varying impacts on wine aromas, it is impos-
sible to say thai the use of yeast starter leads to Ihc
development of a uniform character that depends
mainly on the grapes' composition in terms of
aroma precursors.
In the case of red wines, it has been reponcd
lhai Ihc use of specific yeast starters has an impact
on color inknsilv and the aromatic character of
some grape varieties. These effects may occur
during fermentation itself or result from the
autolysis of dead yeast cells, which justifies
■he practice of aging wine on Ihc lees. These
observations, however, require a more detailed
theoretical investigation.
A dose of 10* nils. nl yeast starter is generally
recommended, which corresponds to 10-20 g/hl
of DAY As the yeast population in strongly fer-
menting must is of Ihc order of 10" cells/ml. a \'t
inoculum is theoretically snfticicnl. but ¥- is more
commonly used, or even 5'i . to offset any potential
difficulties, l:\peniiienis carried out wilh higher
doses of yeast starter (20-25 g/hl of DAY) indi-
cated thai there was a lower risk of fermentation
becoming slugged towards the end. but some off-
aromas conld be produced. In any case. Ihc most
important selection criteria for wincmaking yeast
starlets are trnperature resistance and Ihc ability lo
complete fermentation in high-sugar musts These
properties are characteristic of yeasts formerly
known as Stiec Htn nances btnams (Section 18.4)
When Ihc yeast starter is added, it is important
lo avoid antagonism with other strains naturally
present in the must Antagonistic reactions may
reduce the fermentation rat and contribute to
causing stuck fermentation (Section 3H.I). Rh
inoculation wilh DAY lo be successful. Ihc yeast
starter must be more abundant and more active
lhan the indigenous yeasts, which must be inhibited
by proper hygiene, sufficiently low temperatures,
and appropriate use of sulfite.
3.6 INHIBITION OF THE
FERMENTATION
This section covers the phenomenon of inhibition
in grape must fermentation A laigcnumbcrof sub-
stances exist that may hinder yeast multiplication:
chemical antiseptics and antibiotics and fungicides
(Ribcrcau-GayoD el ill.. 1975a). Inhibitors used for
Ihc conservation of wines (in particular sulfur dkiv
idci arc described in Chapter, 8 and 9.
3.6.1 Inhibition by Ethanol
Hfhanol produced by fermentation slows the assim-
ilation of nitrogen and paraly /es the yeast. Blhanol
i landbook or Etiology: The Microbiology of Wive anil Vindications
mcnuiioiUU limited 4e.ohkir.Bai 25 C) iRlicmu-Gayon c <>i..
Alcohol
Delay in
Ycau
Alcohol
Alcohol
Kcslhol
NiuopcD
Glycerol
addition
Iniotion of
pofublion
tonic n
formed
uaair
luimilaicd
(mmol/l)
(S vol.)
ferment* ion
(lOVmll
(r, vol.)
<'i vol.)
(g/l)
(■gfll
•H>
1 day
80
140
Ufl
2
252
5T
+2
2 d*y*
n-
ISA
130
6
233
OS
+*
i nuyn
62
182
122
IS
194
7?
. 10
12 d*>*
30
ion
60
125
81
m
act* by modifying cell active Iran-spoil systems
iiim Ihc membrane (Hcnschkc and Jiranck.
1992). The quantity of alcohol necessary lo block
fcrnicn tilion depends on many factors, including
ycasl strain, krmpcraturc and aeration.
The presence of clhaBol al Ihc lime of inocula-
tion prolongs Ihc la lent phase and reduces cellular
■liUiplicabon. An elevated fcmpcraturc increases
this inhibitory action This effect of cthanol on
yeast v row Hi and fermentation speed occurs even
al low concentrations from the Mart of fcrmcnla-
tion. The difficulty of restarting a sluck fementa-
tion is. therefore. understandable.
The experiment In TaNe 3 6 shows lie cfTect of
the addition of alcohol to grape must. It slows the
initiation of fermentation and limit* the assimila-
tion of nitrogen and Ihc formation of alcohol. Ycl
thcycaslscanconiinac their activity up to a higher
alcohol content, as long as the inhibitory action
of cthanol Is nol excessive In this experiment,
the variation of Ihc glycerol concentration repre-
sent* significant metabolic modification As seen
in Section 3.4.1. elhanol intensities Ibe inhibitory
effect of an elevated sugar concentration in must.
3.6,2 Inhibition by Fermentation
By- Products: Ihc Use
of Yeast Hulk
Past observations have indicated the potability of
the formation of substances other than cthanol. dur-
ing fermentation, having an inhibitory action on
yeasl Gcicix rt «/. (1983) confirmed Ihishypolhc-
sRI Figure 3.7) Synthetic fermentation media con-
tuning variable concentrations of cthanol were
Fnjt 3.7. Evohfion of Saxhtramum eonfaar pop-
ububn In fermcnlimi media co«aial*a dilfcicM alco-
hol concern M ion* (A =1.7» vol.: B =7.0S vol.:
C=9JS vol.. obtained by fctmcMaibn or akohol
.i.kli. i.-.i-. : .< Jenei* n ,*.. 1983). N, =cdl count m lime
I; N, = ccll count al staa (approximately I" ccllv/ml).
II) non-fermented -cd«i A and It <ll) no o- fermented
medium C. (Hippie-fermented medhim A. (IV) pic-
fcimcntcd medium B |V| ptc-fcimcmcd medium C
inoculalcd with S. ceiwisiiie . A first scries con-
sisted of non- fermented and entirely synthetic
media. A second scries consisted of pre- (cnuented
media The cthanol in the second scries came from
a fermentation stopped by double ccntrifugalion in
Conditions or Yeast Development
''■
the g rowtb. htuion.li}' and death phases or ihc pop-
ulation growth cycle.
The composition or Ihc non-fcrmcnicd and pic-
fcnncikrd media was us follows:
A: alcohol content = 1.7* vol : sugar = 160 g/1
B: ukohol content = 7.(X* vol.; sugar =65 g/l
C: alcohol content =9.5% vol.: sugar =23 g/l
Figure 3 7 shows lhal ye;r%ts grow in all Ihc aon
prc-fcrmcntcd media. Ii Ihc pic-fcniicnfcd media,
on ihc other hand, growth ts oily possible in
medium A. which has a low alcohol concentration
Population decline is significant in prc-fcrmcnicd
medium C. which has an elevated alcohol content.
According to this experiment, fcnncnttliot cre-
ates othcrsubstanccs besides c I hanol which inhibit
ycasl growth and alcoholic fcmicntillon. A com-
plementary cxpennieni indicated thai these sub-
stances are climinaKd bycharcoal. confirming pasl
observations. For a stink fcrmcnGition. charcoal
helps to restart ycasl activity by removing ycasl
metabolism products from wine
Research inio the impact of various fermentation
by-product on yeast demonstrated the inhibiting
ei'feci of C 6 . C h . and C l0 short-chain tatty acids
found in wine at concentrations of a few mil-
ligrams per liter. They atlcct cell membrane per-
meability and hinder exchanges between the inside
of ihc cell and the fermenting mediam. When fcr-
mcucilion slops, the yeast cn/yuuilK systems still
function, but the sugais can no longer penetrate
Ihc cell to be metabolized (Larue etal., 1982).
Salmon el of. ( 1993) confiriKd that loss of activity
ofS. cerevisitir incnologiealcoinlitioss w:r* linked
Id inhibition of the transport of sagar. Fermentation
is inhibited by these (",.C h and C,„ saluralcd fatly
acids — hcxanoic (caproK). oclaaoic (caprylic) and
dccaioic tcapricl acids. Other unsaturated, king-
chain fatty acids (Cut), however, are activator* in
certain conditions: oleic acid, one double bond: and
linoleic acid, two double bonds (Section 35 21
The Kim fatty acid' used in both rases can lead
to con fusion
The preceding facts lead » the use of yeast hulls
in wine making They air currently the most effec-
tive fermenEtlkw activators known foe wincmak-
ing (Lafon-Lafomcade el ul . 1984). Ycasl hulLs
eliminaie the inhibition of the fermentation by Ik-
ing the toxic fatty acids (Lafon-Lafouicadc el al.,
19841 The permeability of the cellular mem-
branes is ic-esEiblishcd in this manner Muno/
and Inglcdcw (19891 confirmed the toxic effect of
C*. C H aid C|| fatty acids and the activation of
fermentation by different varieties of yeast halls.
According to these authors, in addition to their
pmpenics of adsorption of fatty acRts. ycasl hulls
contribute sterols and unsaturated, long-chain fatly
acids to the mediam These constituents arc consid-
ered to be 'oxygen substitutes or survival factors
(Section 3.5.2). Whatever their mode of action,
ycasl hulls arc universally recognized as fermen-
tation activdiors. Table 3 7 gives an example of
Ihc activation of the fermentation of a grape must
containing high sugar concentrations The numbcis
show the superiority of ycasl hulls with respect to
ammonium salts for the activation of the fermen-
tation. During the final Stage of fermentation, the
Addition uf
van bulk
Addhbaof
(NH,),SO.
(02 s/I)
Supir icimcntnl (g.'l)
Total populainaUCT cclK/al)
Viable pupuht»n ( I" " cclb/ml)
Handbook or Etiology: The Microbiology of Wine anil Vindications
lotil cell population has ioi greatly Increased but
the viable yeast population is clearly more signif-
Kail In ihc presence of ycasl bulls. Tbis charac-
teristic survival factor effect docs noi exist when
oily ammonium salts aic added. The addition of
ycasl hulls oa the fifth day following the Initiation
of fermentation, af let the growth phase, has a note
pronounced effect on population snnlv.il than an
addition before fermentation.
Yeast hulls have proven to be effective in
ntasK that are difficult to ferment: for example,
those containing high sugar concentrations or
containing pesticide residues. Yeasts are also moic
temperature rcsiscinl in their presence (Table 3.8).
They may be used, although less effectively, in
cases of stuck fcrmcuutioi (Section 38 J).
Yeast hulls must be perfectly purified to avoid
an organoleptics! impact on wines. The industrial
preparation of yeast extract results in pronounced
odors or tastes in the product and these must be
removed from the envelopes before use in wine.
Moreover, if the halls arc not sufficiently purified,
a souring (due lo the presence of residual lipids)
may occur during storage in certain conditions.
This souring leads k> an unfavorable development
In the wine's organoleptic characters. Such circum-
stances have incited excessive criticism concerning
the use of yeast hulls
The involvement of yeast hulls in fermentation
processes is also accompanied by variation in
the concentration of secondary products (higher
alcohols, fatty acids and their esters). As a result,
wine aromas and tastes can be modified All
operations thai affect fermentation kinetics affect
the wine— temperature, oxygenation, addition of
ammonium salo. ck— and yeast hulls have no
nunc of an impact on the fermentation than these
other factors, and certainly less than Kmpcraturc.
for example. Whatever Ike case, yeast bulls should
be used with prudence for the fermentation of
wines having a simple structure, such as certain
dry white wines. In this case, their effect can be
more significant
3.6.3 Inhibition from Different Origins
Souk vine treatment spray residues teg Folpcl)
arc well known to Inhibit fermentation. Sulfur-
and chloride- based compounds arc the most harm-
ful to yeasts Inoculation with fresh yeast once
the Inhibiting residue has broken down Is gen-
erally sufficient to reactivate fermentation in the
must (sec Hal/idiniilriou ft ill.. 1997). However,
certain difficult final stages of fermentation can be
attributed u the presence of these residues The
minimum lime between the List application of a
product and the harvest date indicated by the man-
ufacturer is not always sufficient.
Elevated concentrations of tannins and colored
matter found in certain varieties of red wines can
binder yeast activity They bind to Ihc cell wall
by a kind of tanning process. The effect of these
substances Is not clear some activate fermentation,
while others inhibit It
Carbon dioxide produced by fermentation is
known to have an inhibitory effect. This occurs
during fermentations under pressure (sparkling
wines). A slight internal pressure in the tank Is
sufficient to slow the fermentation, and above
7 bats fermentation becomes Impossible. In nor-
mal winemaking conditions, carbon dioxide is
released freely and exercises bo Inhibition on the
fermentation.
The difference in femen lability of various
grapes and musts Is linked with many poorly
controlled factors In the same way that specific
Tabic J.8. Slimultfbn of icd wn
(minimum IcnncMai ■>■ Icmpcnriui
n ill.. I9S4)
* fcimcnuik
c of M"C mi
in by the *kl*bn ol
tiaoi ob div "Htifor
vcan hi lb.
i-Ufouwsdc
Rnkhul
iMipin during lennca
uiba <gd)
Day i
l>... 8 l).v. 9
Day 10
Cotf ml
Addition of vcaM bulb (0 J i?'l.
194
200
15
15 2
IS
Conditions or Yeast Dcvclopnicil
activators nisi exist, ibc presence of natural
inhibitors )■ grapes has been considered. Their
interaction would affect ilium fcrmcntabillty.
Molt specifically, the preliminary development
of yeasts. Uiclic bacteria or BiAryiit einerea can
binder ihe alcoholic fermentation process In the
case of alcoholic fcmienGilion difficulties. lxKicrl.il
growth and malolactic fermentation exacerbate
these difficulties. Stuck fermentations often result
t Sections 3.8.1; 6.4.1).
Among these caascs of fermentation difftcal-
IKs. the involvement of Batryris tineieu has been
the most sladicd. Must derived from parasitized
grapes (noble or gray rot) is more difficult to fer-
ment than must originating from healthy grapes
Past works, summarized by Ribcrcau-Gayon el id.
(1975a). identified a sutastincc with antibiotic
propcrtics; the authors named it bolryticin. Sul-
liting and piolongcd heating at I20'C destroy
this substance Btuanol at -i '. can precipitin:
it. Subsequent work showed that this fungistaiK
substance is an either partially or completely
man nose- based neutral polysaccharide (Ribercaa-
Gayon ei ill.. 1979). The phyutoxic propenics of
such substances arc known and this polysaccharRlc
affects fcriKnlalion kinetics It is also the cause
of certain metabolic deviations induced by Botiy-
lis cinerea — 'm particular an increase in the pro-
duction of glycerol and acetic acid (Section 23.4;
Volume 2. Section 3.7.2).
3.7 I'HYSICOCHEMICAL FACTORS
AFFECTING YEAST GROWTH
AND FERMENTATION
KINETICS
3.7.1 EtTccI of Tempera lure
Alcoholic fermentalKii. depicted by the following
chemical cqaation:
C„Hi?0, -
-2C2H5OH + 2CO2.
liberates 40 kcal of free energy per molecule
YcasLs use part of this energy to ensure their
vital functions, in particular their growth and
multiplication, and to form two AT? mokcaks
from a sugar molecule These ATP mokcuks have
a high energy potential:
2ADP4-2HiP04 2ATP4-2HiO
It takes 7 .1 kcal of cncigy to form one ATP
molctnlc. The difference (40- 146 =25.4 kcal)
is non-u till red cncigy that is dissipated in the form
of beat, causing the fermentation tanks to heat.
This estimate of non-ullli/cd energy Is open 0>
discussion. In sllaations where part of the ATP
formed is not needed by the yeast, it is hydroly/cd
by the corresponding enzymes. Yet 25 kcal corre-
sponds fairly well with past thermodynamic mea-
sures of dissipated heat by the fermentation of one
molecule of sagar
The fermentation of must containing ISO g (one
molecule) of sagar per liter therefore liberates
25 kcal in the form of heat This liberation of heat
theoretically could raise the temperature from 20 to
45'C. Such an increase In Krmpcrature woald kill
the yeast. Fortunately, this increase Is the hypo-
thetical case of an explosive, insttnlancous fer-
mentation or a fermentation in a fully insulated
tank In reality, fermentation lakes place over sev-
eral days During this time, the calorics produced
arc dissipated by several phenomena: by being
entrained with the large quantity of carbon dMixMIe
released during fermentation: by cooling resulting
from the evaporation of water and alcohol: and by
exchanges across the tank wall.
Temperature increases in fcrmcnlcrs depend on
several factors.
1. The must sugar concentration, which deter-
mines the amount of calories liberated.
2. The initial must Krmpcrature.
3. The fermentation speed, which depends on must
composition (nitrogen-based sahstanccs) and
yeast inoculation condltKins Operations such
as aeration, chaptali ration and inoculation will
increase the fermentation speed, limit the dis-
sipation of calorics and increase the maximum
temperature Reciprocally, not crashing the red
harvest in carbonic maceration (Section 12.9.1)
will slow fermentation kinetics and lower the
maximum temperature.
100
Handbook of Etiology: The Microbiology of Wine anil Vinifhralions
4 Tank dimensions. When the volume i
the surface of the w:iiis and Ihcir ihcraiic
exchange capacity decrease when the same pro-
portion* arc main tuned.
5. Tank material The global thermic exchange
coefficient (K . expressed in cal/Vnr for each
degree difference in temperature) is from 0.7
to 0.78 for a cokicIc wall. 10 cm thick, from
1.46 to 1.49 foraS cm wooden wall. and from
5 .34 lo 32.0 fora 3 mm slainlcssslccl wall. The
coefficient varies the most in the case of stain-
less sKcl. A stainless steel lank is sensitive to
the external conditions (lempcralnrc. aeration)
in which it is placed.
6 Aeration and cellar lempcralnrc The vcntilinon
of the winery limit, fermenting tank tempera-
tures by dissipating heat.
The maximum tank temperature is related to all
of these factors by complex fcivvs and R difficult
to predict Depending on the circumstances, the
maximum lempcralnrc can be compatible with icd
wincntaking. In this case, a maximum temperature
between 25 and 30 C ensures sufficient extraction
of phcnolK compounds from the solid part, during
maceration. In other cases, refrigeration is neces-
sary to avoid exceeding the maximum temperature
limit. Refrigeration is always necessary foe white
wines: their fermentation must be carried oul at
aroand 20 'C lo retain their aromas.
Current refrigeration methods include circulat-
ing cold water orothcrcold fluid through the dou-
ble lining of metallic ranks ot through a temper-
ature exchanger submerged in the rank In ccrttin
cases, spraying Ihc exterior of a mcullK tink can
be saffkicnt The mast can also be scnl throng a
tubalar exchange!* cooled by circulating water.
iLsclf rcfrigcralcd by an air exchanger.
Temperature has an impact on yeast develop-
ment and fermentation kinetics. According lo Heel
and Heard (1992). temperature can affect indige-
nous yeast ecology. The authors suggest thai dif-
ferent strains arc more or less adapted lo dif-
ferent temperatures, ranging from 10 to 30 C.
More precisely, the growth rale varies for each
strain according to temperature— for instance.
wilh Kloechcra iipiailait/ and S. cererisine The
possible cnological consequences of this phe-
nomenon merit further research.
Numerous overlapping faciois make it difficult
to anticipate the impact of temperature on fer-
mentation kinetics. Ough (1964. 1966) developed
equations for the estimation of the impact of tem-
perature on fermentation kinetics as a function of
numerous parameters Bordeaux cnologisls dedi-
cated much research to this subject, summarized
by Ribcrcan-Gayoi end. ( 1975a). Temperature
profoundly affects yeast respiratory and femenra-
lioH intensity (Tabic 3.9): the fcrmcnuiion inten-
sity doubles for every 10'C temperature increase.
It is at its maximum al 35 C and begins to decrease
at 41) C". These numbers show the imporuncc of
the fcrntcnutioa lempcralnrc. The fermentation of
sagar is twice as fasl at 30'C as at 20'C. and
for each temperature increase of IT Ihc ycasl
transforms U'f.i more sagar in the same elapsed
lime. The optimal fermentation temperature varies
according to the ycasl species
Temperature influences fermentation kinetics
The alcohol yield is generally lower at elevated
temperatures, in which case some of the alcohol
may be entrained with Ihc intense release of car-
bon dioxide Additionally, most of Ihc secondary
products of glyce ropy rn vie fermentation arc found
in greater concentrations. Fatly acids, higher alco-
hols and their esters arc the mosi affected: their
formation Is al its maximum at aboal 20"C and
then progressively diminishes Low fermentation
Tabic J. 'I. Avenge IcrmcntMiun inil mpinilory Imcn-
nih (mm' of O, conumed or of CO, irk^cd/p ol
dry ynsis/bour) of virknn Si.nftiroraire'A ctrriitoiv
%pccfc* ucconlin|j lo icmpcniuic (RlKicnu-Ga^oa
n it.. 1975a)
is*c
2ff°C
2S*C
n ■:
40*C
Re* ■ Iraion Fcrmc Halloa
Conditions or Yeast DcvclopnicK
101
k'tnpcratnrcs arc jnstHicd when these pmdncts aic
i k -ii -j i.l In white ivincmaking
In addition to Its inflacucc on yeast activity,
k'tnpcniinrc affects fermentation speed and limits
Between 15 and 35'C. the duration of the latent
phase and Ihe delay before the InilEilion of
fcmicitition become skoilcr as the lempcralnrc
increases. Simultaneously, ycasl consumption of
nilrogen increases (Section 3A2).
For example, a \z rape mast with a liniilcd sugar
concentration (less than 200 g/l) tikes several
weeks kilcnncnlal 10*C. 15 daysai20'Cand3 lo
4 days al 30'C For musts with higher sugar con-
ccnlraliOBs. the fcniKnlation becomes nunc limited
as Ihe lenipcralarc incicases. in faci. fcmicitilion
can stop, leaving non-fcrmcukd sugar
Table 3.10 concerns a must from Saatcrncs
containing more than 300 g ofsagar per liter The
same phenomenon occurred in Mullcr-Thurgaa s
experiment in ISS4 IciKd in RibCrcau-Cayon
eiul.. 1975a). He fcrmciied the same mist with
increasing concentrations of sugar (Table 3.11).
The initial sugar concentration of mnsl and
excessive lempcralnrcs limit ethanol production.
Other factors, sach as Ihe amount of oxygen
Tabic 111
A
nfcol lb ratal bo
*i vol.) weeding io
Icimc ill 11 1
"
■ pcratun:
(Miiiic
[ 1...IJ--...1 IK84)
supn
Potent!.
Aknbol pmduced ul
4lcoh.ll
(p/n
fi vol.
9*C
I8*C 27*C »°C
127
7.2
7fl
6.9 09 4.2
217
124
HE
II A 94 4.8
KB
17.3
9.9
9.1 7.7 S.I
present, can limit or slop Ihe fermentation a(
rclalivcly low alcohol concentrations (11—12%
vol) and tcmpcralurcs (less than 30'C>. An
excessively high lempcralnrc (25-30'C). daring
the ycasl multiplication phase affect* Ihcir viability
and favors stuck fermentations. The Impact of
icmpcraiaic on fcrmcnlation kinetics also varies
from one yeast strain lo another
These fact* arc important for winery practices
and show the dill lenity of determining a maximum
acceptable temperature limit
The impact of Kmpcraturc on fermentation Is
depicted in Figure 38 The latency "ate decreases
and the Initial fermentation speed increases as the
Icmpcraiaic rises. The risks and severity ofastuck
fcrmcntilion also incieasc with Knipcraiarc Of
coarse, if the iaitEU sugar conccnlralion had been
lower in this example. Ihe fermentation would
have been complete at 35'C. On the other band, a
higher sugar concentration would have resulted in
a stuck fermen tiiion even at 25 'C. This shows that
fcrmcnlation speed increases as the temperature
rises but thai the fcrmcntilion is also increasingly
limited.
Tnhlc3.ll)
Km
cnuiHm mill.
■in ipeed and limit*
KWWBjagl
tcmpcratuie IRfrcic
u-Gavonrt.rf. 1975a)
[tripe rati!
Inuutkin of
Ak.ihol.ni.cnt
B
fcnCMilllin
atMiacd.fr vol)
1
I
10"C
K da)*
lb.2
15 '<
b .la)*
as
20"C
4 da)*
ISJ
-
2S°C
3 da)*
us
u i
30 hour*
10.2
3ST
24 hour*
bD
a
!■'< 3.8. Influence of temperature on (crmcaMliua
»pccd iad limit (So ainiul Mipir concert Mba). Al
25"C. fcrrtcntalbn n ihnrcr. but complete. Al 3trC.
and especially 35*C, it n mure rapid, kit Mop* at Ici-
memed *upar concent nit kin* 5, and i,. mpccilvcly.
bcfawSa
Handbook of Etiology: The Microbiology of Wine and Vmific.uions
An abrupt temperature change during fermenta-
lion can lead to ihcnnH shock. This phenomenon
rs different final ihc notion of shock or slrcss
lhal is used in microbiology (Section 625). Ii
certain wineries, while wine fermentation tanks
arc situated ouUoors due to space limitations.
These wines, fermenting al modcrak: temperatures
(20"C). have dilficnlty withstanding Ihc abrupt
Kmpcraturc v. n--.it ions between day and night
when autumn cold Mist arrives. The fermentation
progressively slows and finally slops. A second
InoculalKM is not effective. If these wines aic
transferred to .1 tank at a constant temperature
before Ihc fermentation has completely slopped
and after a consequent inoculation, fermentation
will be completed. Laboratory tcsK confirm Hurl
an abrupt temperature change, in one direction or
the other, affects yeast activity. This phenomenon
mcnis more in-depth study.
The data in Table 3.12 arc taken from a
fctboraloiy experiment At 12 C. the fermentation
is slow but complete At I9"C. Ihc fermentation is
«l nicker and sugar Itunsfomiation is also complete.
II the fermentation begins al I9'C and ts abruptly
lowered to 12 C. it stops, leaving ion-degraded
sugar The same is true for a fermentation whose
temperature abruptly increases from 12 to 19 C.
If thermic shock ex'curs during the final stage of
fermentation (after the fermentation of 120 g of
TuhfcJ.ll Ellen of tcmpeoiuic tariaibiu. (thermal
ih«l>)ongnipc ami ler-enuiKMUiuc-'Wi.. 1987)
ferment* .on
Duratnn »f
Kcikfcul uipar
temperature
ferraemaibn
CDKCMMb*
(days)
41 end of
ferment it kin
(gfl)
I2C
91
<2
12-C.iia inferred
*i 19 "C .derm
formation of 40
B
UlfUI.'l
56
27
I9"C
%
<2
19Cimmferrcd
M I2T tnerm
formalin* of 40
e
iUfitlll
21
108
sugar per liter, for example), its effect is less
significant.
To recapitulate, an increase in temperature accel-
erates the fermentation but also adversely affects
its limit. A stuck fermentation can also occur if
other limiting factors add their effecfe {richness in
sugar, anacrobiosis). For this reason, there is no
fixed Icmpcraiuic limit above which fermentation
Is no longer possible. In red winemaking. Ihc fer-
mentation Icmpcraiuic should never exceed 30 l C:
above Ibis temperature, the risks are certain. Of
course, this docs not mean that a fermentation can-
not be complete at temperatures al or above 35 "C.
The yeast is most sensitive 10 temperature al the
beginning of Ik development: it is more icsistini
during the final stage of fermentation. For this
icason.in red winemaking. the fermentation should
begin al I8-20T and be allowed to increase
progressively lo 32 : C or even a little higher.
The higher final temperature favors maceration
phenomena. An excessively high temperature al
the initiation of fermentation (around 30"C) can
icsult in a difficult final stage of fermentation
3.7.2 Influence of Oxygen— Effect
of Must Aeration
Ycasls use enctgy derived fiom the degradation of
sugars This degradation is carried out by either the
tcspiralory or the fermentation pathway. In grape
must, due Hi Ihc catabolic repression of respiration
exerted by musl glucose in S. cererisioe. sugar
degradation is carried out exclusively by alcoholic
fermentation.
Vcasl respirator)' capacity Is put lo good use In
enology for the production of (lor wines. In Ibis
case, ycasls oxidize cfhanol into aklchydc in dry
wines. Oxidative yeasts can also develop during
winemaking: they oxidize cfhanol into carbon
dioxide and arc considcicd to he spoilage ycasls.
Pasteur spoke of must fermentation as a type of
life without air* Yeast development and fermen-
tation have long been known to be Impossible in
Ihc complete absence of oxygen (Ribcieau-Gayon
el ill.. 1951 ). The complete absence of oxygen sup-
poses Ihc fermentation of a musl devoid of oxygen
in complete anacrobiosis The musl would also
Conditions or Yeast Development
103
have lo be inoculated by a yeast-starter cultivated
ii (he absence of oxygen. I\perimcntal conditions
in complete anacrobiosis arc difficult lo maintain.
For this reason . some authors might have thought
Uat oxygen was not absolutely necessary (or fcr-
mentation kinetics.
On the contrary, oxygen has a considerable
impact 01 Ike fermentation kinetics of wine The
addition of oxygen is probably the most effective
method available 10 tic wiicmakcr for controlling
must fermentation for this reason, the terms
complete anacrobiosis* . scmi-anacrobiosis or
limited aerobiosis* arc sometimes nsed to explain
the anioint of oxygen added during fermentation.
In laboratory experiment, samples arc scaled
with a sterile wad which permits the controlled
introduction of oxygen and ensures an exchange
in both directions between the interior and exterior
environment. Complete anacrobiosis is obtained
by obturating the opening of the samples with a
fermentation kxrk
In the winery, open tanks leaving the wine
in contact with air permit a permanent aeration.
They arc not recommended, because of the risk
of bacterial dcvck>pmcnt — closed lanksaic prefer-
able but a controlled amount of oxygen should be
added lo the fermenting wine in these tanks dur-
ing pumping-ovcr. for example. This technique has
been wktcly nsed in icd wincmaking for many
years. The fermenting must Is easily saturated
in oxygen (6-8 ntg/ll Pumping-ovcr. however, is
less used in white wincmaking becanse of fears
of oxidating the must and modifying the aromas
In fact, litis fear has not been confirmed in prac-
tice, since the yeast can absorb a huge amount of
oxygen during fcrmcutilioa. The aromas of musl
before fermentation and in while wine separated
! i. ■(■! is Ices after fermentaton arc susceptible lo
oxidation, bui the aromas of musl during fermen-
tation arc probably less affected by aeration.
Aeration accelerates fermentation and as a
result increases the demand for nitrogen-containing
nutrient. Oxygen favors the synthesis of sterols
and unsaturated fatly acids, improving the cell
membrane permeability and consequently glncMlc
penetration The addition of oxygen has an effect
similar lo the addition of sterols, which arc
consKlcrcd to be oxygen substitutes.
The data in Table 3.13. taken from an experi-
ment carried out many years ago in the labora-
tory of Ribcrcan-Gayoi (Riblirau-Gayon eiul..
1951). show the effects of controlled aeration
on the fermentation kinetics of a relatively high-
sugar mast. In complete anacrobiosis. fermentation
skips on (he 14th day. leaving 75 g of sugar per
liter. On the 21' day. the yeast population was
5 x 10' cells/ml In limited aerobiosis. the fermen-
tation is coaiptclc with a yeast population twice as
great. Moreover. Ihc iniliation of fermentation and
its initial speed arc greater in the presence of oxy-
gen. Of con isc. if the initial sugar concentration
had been lower in Ihc experiment in Table 3.13.
the fcmicntilion would have been complete in both
cases, although slower in anacrobiosis A higher
sugar concentration wouM have led to a stick fer-
men titioa in both cases before Ihc complete deple-
tion of sugar.
In wincmaking. permanent aeration is rarely
possible, bnt momentary aerations arc a suitable
replacement The data in Table 3.14 arc also
Tabic 1 11
1. Evoknioa of ihc fermentation ol
ion 1270 p/l) ii wonting to ac nil ..ncc
i pope muu containing ,
(■InionMRfccicMi-Cavoii
. high iupar
rt.rf.. 1951)
Time
l.lmlol ucmbauB
U'mum iioppcnrd laiki)
AnaerobiMh.
(bubbler Muppcnd lub)
Rc>*hiil *ugaK>
<g/l)
Toul «lb
( lOVmll
1 ■ . 1 i 1. UlgaD.
Total celb
(KT/mlJ
7 .!*■.■.
U day*
21 <by*
80
10.7
140
75
75
5
I landbook of Etiology: The Microbiology of Wiae anil Vinifie.nions
l.ibk- i Ii. EflWi of nvygen aiUnion .« different mj-j«-n of gape aim Kiacnuifon
>>S.uit!ir'tiI(w;.iuininciii made on .la;. 14 of teimematkin)(Rtocreau-Cayan<i«r.. 1951)
Icimcntcil mijjji
( Hailed p
WkkjUair
WitDUa
cnvjoi.
c <-t|I i-.miI >i nw.'l tiy ■■■■ filiin a
3 (A
>( tnywii «■ Hi ri^xii u
tiken from a past experiment (Ribcrcau-Gayon
erof., 1951V They confirm Ihc difference ii
fcrmcii cibility between musts n ihc presence aid
In ihc absence of oxygen. The elicit ol oxygen oa
fcrntcntiiioa kinetics increases wiih Ihc quantity
of oxygen introduced The lining of ihc addiltoa
of oxygen appeals lo be especially important. The
acceleration of Ihc fermentation Is niosl significant
when oxygen is added on lie second day following
lbc initiation of fermentation, during lie growth
phase of Ihc yeast population. In Ibis case, the
fcrmentilKw is nearly as ill nol as) rapid as a
fcrmcnuiKM with limited permanent aeration, the
same amount of sugar is also transformed Ii fact,
it is not Ihc must thai needs to be aerated but rather
the ycasLs fermenting the mast— especially the
population in the growth phase. Other experiments
confirm yeast use of oxygen primarily during the
first stages of fermentation. They do not benefit
from an aeration when the fermentation is in its
advanced stages and the alcohol concentration Is
These obscrvauoas arc significant for prac-
tical purposes and they shoukl be Liken into
accountduring wincmaking — especially red wine-
making (Section 12.421 The risks of wiicmakitg
in open links, or permanent acrobiosis. arc known
(Section 12.5.1). Auacrobiosis in unks is recom-
mended if oxygen is added at the right moment
The acceleration of the fermentation indued by
a momentary aeration mast nevertheless be antic-
ipated This acceleration result in a more signifi-
cant heating of the wine.
The experiment cilcd in this section date back
to Ribficau-Gayon ei iH. ( 195 1 1 Even if no longer
cilcd in recent works, the results aic still valid
(Fleet. 1992). In particular. Sablayiollcs and Baric
1 1986) retained the same values for yeast oxygen
needs, i.c around 10 mg/1. they also conlirn>cd the
significant influence of oxygen aid lbc momcni
of Ik addition on fcmicitiUoa kinetics. Other
icscaich has shown thai aeration, combined with
Ihc addition of nitrogen in rnkt-fcrmentation. is
ntoic effective than aeration alone (Sablavrolks
rt.rf.. 1996a and 1996b) (Section 3.4.2). This
effect is apparently more marked in certain media
under certain fermentation coadiliois.
3.7.3 Effect of Musi Clarification
on White Crapes
Mnsi clarification before the initiation of fermen-
tation has long been known to affect the qiality
Conditions or Yeast DcvclopnicK
11 n
of while w)k (Section 13.5.1). Yeasts fermenting
clear must form ukhc higher alcohols, fairy acids
ami corresponding esters In addition, mist sus-
pended solids can imparl heavy aid disagreeable
vegetal odors The racking ol musl is fbcfcfoie
essential
Musi clarification also atlcets fermentation phe-
nomena ll eliminates some of Ihe wild yeasts,
along wilh the natural vegetal sediment. bn( inoc-
ulation compensates for ih is k*cs. and is also often
recommended k> compensate for Ihe small popu-
lation present al Ihe lime of tilling Ihe link. In
this manner, fermentation is tarried onl by selected
strains thai best express musl quality, without ihe
development of olfactory flaws
During juice striding . ccrlain conditions useful
for sedimentation (sach as low temperature and
snliitingl can promote the development of certain
strains resistant to these conditions. Of coarse,
this growth must not become a fermentation:
otherwise, it would put the sediment, back in
suspension Nevertheless, these strains can develop
preferentially during fcrmentilion even after an
active yeast inoculation (Fleet. 1992).
Clarification essentially modifies must ferment-
ability. Clear ninst is known to ferment with
more difficulty than cloudy must (though the
elimination of ycasfc is not the only rcasoa for this
fermentation difficulty, as was once thought) Yet
clarification simultaneously favois the aiomaiic
finesse of the wine. White wine quality is thought
to be enhanced by a somewhat difficult and
slow fermentation In general, all operations that
accelerate fermentation will kiwer wine quality,
and vice versa. A comptomise permitting complete
fermentation and satisfactory wine quality must
be sought.
Several researchers have studied the effect of
grape must Ices and other solid materials on
fermentation kinetics (Ongh and Croat. 1978;
Dclfini and Costa. 1993). RiWreau-Gayon el id
(1975b) evaluated the effect of several related
(actors on must fcrmcnubility the elimination
of yeast, the elimination of nutritive clement,
released by grape must sediment and a possible
support effect which would permit a greater
yeast activity, possibly by the fixation of ioxk
compounds (short-chain fatty acids). A number of
fermentations were carried out in the laboratory
under different conditions, for this experiment,
the medium was heated at 100'C for 5 mimics
to dcsln>y Ihe yeasts and ensure the solubilization
of the nutritive ck-ments likely to be involved
in fe mien tn ion In view of the destruction of
the yeasts under certain coadilKns. the medium
was systematically inocufciled. using a strain of
S. cererisiiie with good fermentation potential at a
relatively km* concentration iloVnih to assess any
possible effect of natural elimination of the yeast.
In this manner, the subsequent el feci of the natural
elimination of yeast, can be appreciated. By
computing the different samples, the contribution
of each of the three parameter to the loss of
fennel lability could be evaluated. The results of
two tests with different concentrations of sugar
are given in Table 3.15. Fcnncnubility lossdne to
Tabic 115. .ruh-..-. ol the clfccl of different I
Givon H trf.. 1975b)
t>n lcnnci<ibil« ; . km* tKfrci
1 Su
ic of (cnmcMahiliv law.
(nihil
Mc
Tri*t A
Mi|iar concentration
22D g/l>
Trot B
<inial %upit coaccalerib*
2H5 p/l)
Mc«uired on tUv 5
in ol yean* In Ice*
m of -- . - 1' f n cllcci of ICO
I" 1 ,
21%
106
Handbook of Etiology: The Microbiology of Wiae anil Vinlflcaimns
JuKc clarification appeared K> be significant The
measurements were taken on Ibe second and liflh
day or fermentation and il should be uken Into
account ihat Ibe slowing of the fcmiciialion due
» juke settling and clarification is most obvwas
al Ibis Iiik. The differences lend to become less
appaicm over nine In icsl A. the fcrmcnuiion
Is complelc even after juice settling. In test il.
juke settling result, in a stuck fcrmenuikm. In
spile or the necessary approximations made iu
earning out fit is experiment, it shows the multitude
of effects of juice scliling. Furthermore. Ibe sum
of Ihc i »d iv ►ii.il femen lability losses corresponds
fairly well with the total loss.
The nature of the scdinicnland incited on mnsl
fcrmcu lability arc rclakd lo grape origin, grape
sanitary conditions and mnsl extraction condi-
tions Lalbn-Lalbi trade el ill. 1 1980) demonstrated
the essential role of Ihc crushing and pressing
conditions of while grapes. In identical clarifica-
tion conditions, musts extracted after Ihc cncigetic
crashing of grapes ferment less well than musts
originating from pressing without crushing. This
difference in fermentation can result in a stack fer-
mentation when associated with olhcr unfavorable
conditions (elevated sagar concentrations, com-
pfclc amierobtosis. etc i Pressing wilhonl crashing
maintains Ihc Jaicc in contact wilh the skins for
a ccrttin period, and this coatacl seems to per-
mit the diffusion of grape skin slcroKls In the
same manner, pre- fermentation skin conGtct gen-
erally results in a must wilh a good tcrmctiiabil-
ity. even after careful juice settling. Dclfini rial.
(1992) noticed thai mnsl clarification eliminates
king-chain falty acids. Their elimination has been
linked u Ihc increased production of acclic acid
frequently reported in while musts wilh fcrmen-
uiion problems, parficularly Ibosc thai have been
highly clarified
Mast Ices particles and even glacidic macro-
mok-euks. making up part of the colloidal turbidity
of musls such .i- yeasl bulls, can adsorb short-
chain fatty acids <C8 and CIO) (Section ? .62)
(Ollivicr evitf. 1987) In conscqacncc. Ike level
of must clarification should be controlled for
each type of while wincmaking by measuring
mast cloudiness or turbidity, expressed in NTU
(Seclioas 135.2; 135.3). For vineyards in the
Bordeaux region, a turbidity of less than 60 NTU
can lead loscnous fermentation difficulties Above
200 NTU. the nsk of olfaclivc dcviatkms due lo
Ike presence of mast Ices is certain.
3.8 STUCK FERMENTATIONS
3.8.1 Ca
of Sluck Fermentations
Stuck fermentations kave always been a major
problem in wincmaking French cuotogkal liter-
ature has mentioned Ihcm since ihc beginning of
lac 20th century. The production of fortified wines
was definitely a response to difficult final stt'cs
of fermentation and the ensuing microbial acci-
dents, especially in countries wilh warm climates.
These wines were rapidly stabilized by the addi-
tion of pure alcohol Around the world, many of
these wines disappeared as progress in mk robin!-
ogy permitted Ike elaboration of dry wines
Stuck fcrmcntilioa conlinncs to be a much
discussed subject In some cases, a modification
of vine varieties has produced grapes thai have
high sagar concentrations. These grapes arc more
difficult lo ferment than past varieties In other
cases, wincmakers have recently realized thai
slaggish fcrmcntal ions spread over several months
arc not ideal for making wine
As red wincmaking techniques used in Bordeaux
(grapes with a relatively high sagar content and
King valtiug requiring closed vafc.i arc particularly
conducive to this problem, il has been studied here
for many years. In Ihc 1950s in-depth research
in Bordeaux resulted in important discoveries
concerning temperature rcgulalion and aeration.
The ubiquity of sluck fcmcnlations in other
viticulinral regions led lo new research confirming
past work
The slowing of fermentation can be monitored
by tracking the mass per nnil volume If a density
below 1.005 decreases by only 0.001 or 0.002
per day. a sluck fermentation can be anticipated
before the complete depletion of sagar From
past experience. Ihc consequences of a stuck
fermentation arc not loo sennas if it occurs wilh al
least 15 g of sugar per liter and a moderate alcohol
Conditions or Yeast Development
Hi-
con lent ( < 12% vol.). In this case, ihc restarting of
fermentation docs not pose any major problem.
On ihc oihcr hand, with a sugar concentration of
less than 10 g/l. It is olfcn very diliicoli to rcstarl
stack fcnncatuM>ns. particularly when ntalolaclK
fermentation is initialed.
A stuck fermentation can rcsull from a panic-
nlat cause. For example, an excessive must sugar
concentration makes a complete fermentation im-
possible: in this case, only a sweel wine can be
made. Stuck fermentations can be expected in the
case of excessive temperatures, and this type of
stick fermentation is generally the icsultofscvcr.il
causes The effects are cumulative, although some-
times individually v. I ihout consequence Wine-mak-
ers do not always undcistaud possible cumulative
risks and the precautions that mast be Liken
To summarize. Ihc following factors can be
involved in stuck fermentations
1 . The must sugar concentration has an inhibitory
cffccl which compounds Ihc uxicily of the
alcohol formed. The addition of sugar to
must IcbapeUiraiKan). when it is loo laic,
requires ycasls to puisne their metabolic activ-
ity although already hindered by Ihc alcohol
fo rated.
2. An excessive temperature results from the ini-
tial temperature, the quantity of sugar fer-
mented, and the type of tank used (dimensions
and materia)). All operations thai accelerate the
transformation speed of sugar increase the max-
imum temperature The temperature becomes a
limiting factor at about 30'C. The effect is more
pronounced when Ihc temperature is elevated in
the early stages of fermentation. Normally, the
fermentation should begin al a moderate tem-
perature (20°C).
i. Conversely, too low of an initial temperature
can Until ycasl growth and lead to an insuf-
ficient yeasl population. Al moderate tempera-
tures, ycasft have difficulty supporting extreme
kmpcraturc changes I thermic shocks).
4. Complete anacrobiosis docs not permit satisfac-
tory yeast activity (growth and survival! Aera-
tion increases fermentation speed. It must lake
place in the early stages of fermentation, during
the population growth phase Oxygen substi-
tutes snch as steroids and long-chain fatry acids
can also improve fermentation kinetics.
5. Ycasl activity can be aflcckd by nntrilional
deficiencies: nitrogen compounds, growth fac-
tors, and possibly minerals Combined additions
of oxygen and ammoniacal nitrogen appear
to be particularly cffcclivc. These nilrogen
dellcicncKs probably occur in specific silua-
twns thai we are bow capable of predicting
The effectiveness of the addition of nutritive
elements, observed in laboratory work, should
be interpreted with respect to other fermenta-
tion conditions (sugar concentration and aera-
tion). Certain grape growing conditions, such
as nydnc stress. oH vines, and cover cropping
vineyards to decrease vine vigor, can lead lo
less fermentable musts Under these conditions,
stack fermentation is probably due lo nutrient
deficicncKs(nilrogcn). which probably require
further investigation
6. Metabolic byproducts (Co. CS and CIO sata-
rakd fatty acids) inhibit yeast growth, intensi-
fying alcohol toxicity
7. Anli-fungal substances can be present in
mast— pcslicidc residues used to protect the
vine or compounds produced by Bonyiis
einerea in rollcn grapes.
8. In while wincmaking. must extraction condi-
tions have a signilkani influence: grape crash-
ing, conditions of juke draining, pressing of
the crushed grapes, and especially the level of
mast clarification (juice settling) These oper-
ations may result in the excessive elimination
of steroids, which act as survival factors for
the yeast
In Ihc acidity range of the must, a high acidity
does nol seem to favor fermentation, bat an ele-
vated pH can make the consequences of a stuck
fermentation mach more scnoas A low pH com-
bines with the effect of sultlling lo inhibit bac-
terial growth. In this case, antagonistic phenom-
ena between backria and yeasts diminish and the
108
Handbook of Ending y: The Microbiology of Wiie anil Vinlfications
fcimcntilKHi H unite steady Another unfortunate
consequence of low pH is thai il promotes the for-
mation of volatile acidify by ihc yeast.
In addition to chemical and physKochcmical
causes of Stack 1'crmcnlations. microbial pbeno-
■vena also aic involved. First of all. ihc quantity
of the initial ycasi Inoculum can be insufficient
(Section 35.4) If. foe example, must tempera-
ture is exaggeratedly low Antagonistic phenom-
ena between different yeast strains can also occur,
and the killer factor (Section 1.7) explains Ibis
fairly widespread antagonism Fermentation can
be rapid In some tanks while being slower in
others, and strain identification techniques have
shown thai fermentation Is carried out almost
exclusively by one strain in the first case, whereas
several strains fcmicni ihc mast in the second
case (Section 1.10.2). These antagonistic phenom-
ena can affect an inoculation. In certain conditions
(for example, a significant natural inocalam Ii full
activity), inoculating with dry commercial yeast
leads toa slower fermentation than not inoculating.
Yeast strains must therefore be sclccied according
to the type of wine being made, ensuring that they
ate mote active and numerous than the Indigenous
yeasts Tic accessary conditions for controlling
fermenuiKin include: cleanliness: Inhibiting nat-
aral yeasts saffkicatly carry by maintaining low
temperatures: sulliting appropriately: and inoculat-
ing with an active yeast sorter as soon as the Gink
is filled to ensure its raptd implantation
Antagonistic phenomena between yeasts and
lactic acid bacteria can also cause fermentation
difficulties (Section 6.4 I), especially in red wine-
making (Section 12.4.3). TV Initial sulliting of
the grapes must temporarily inhibit the bacteria
while at the same lime permitting yeast devel-
opment and sugar fermentation. Bacteria do not
develop as king as yeast activity Is sufficient, bnl if
alcoholic fermentation slows forsomc reason, bac-
teria can begin to grow— especially If the Initial
salfiting was insufficient This bacterial develop-
ment aggravates yeast difllculDcs and inc teases the
risks of a premature, stuck alcoholic fermentation.
The bacterial risk R an additional justification
for icd grape sulfitlng (5 g/hl) before fermen-
Etlton (Section 8.7.4). The addition of lysozyme
(200-300 mg/l). extracted from egg whites, has
been suggested lo reinforce the Inhibitory effect
of sulfa ring on bacteria in difficnll fermentations
(Section 952). In addition, the Inoculation of lac-
tic acid bacteria (Qenoctvctu iieni) bcfoic alco-
holic fermentation Id activate a subsequent mal-
olaclK fermentation is not recommended: in the
case of difficult alcoholic fcmicncilions. this opera-
tion Increases the risk of bacterial spoilage t Section
382) Yet this Is sdndard practice in some vine-
yards. The relationship between this practice and
an increase in volatile acidity should be considered.
For a long time, difltcalt final stages of fermen-
tation and slack fermentations were a real prob-
lem during red wlncmaklng Temperature control
systems and the general praclKc of pumping-over
wilb aeration limited these incident. While in lists.
however, have become increasingly diflkull to fer-
ment because of excessive clarification and mech-
anized must extraction conditions. This excessive
clarification removes must lonslilacnts essential
for a complete fermentation. In while wlncmak-
lng . the mast is often not aerated Id avoid oxi-
dation, yet a lack of aeration during fermentation
also contributes to fermentation difficulties. Today,
controlled aeration of white musts Is recommended
during fermentation as Ihc CO? being released pro-
tects them from oxidation
Human error is another factor that certainly has
an impact on stuck fermentations, allhoagb it is
difficult lo prove II Is not nnnsual to find wineries
where stuck fermcncilions occur with some reg-
ularity, as thoagh there was a specific, technical
cause thai could be Idcntilicd and corrccKd. then
disappear completely following a change in wine-
maker.
3.8.2 Consequences of Stuck
Fermentations
Residual sugar Is not acceptable in dry while aid
most red wines. A stack fcmienEilrnn therefore
requires Ihc restarting of yeast activity is a
bosllle medium Evidently, if Ihc alcohol content
Is already elevated (lf'» vol), the chances of
restarting the fermentation arc slim
The risk of bacterial spoilage is Ihc principal
danger of a stack fermentation.
Conditions or Yeast DcvclopnicK
Fig 1ft Ellen oldiflcicM microbial phemj
i.(Kihcrc.iiK^vo 1999)
Figure 3 9 schematizes Ike involvement of differ-
cm microbial phenomena, (red wincmaking is
specifically represented since bi.iIoI.kIii feruienti-
lion is taken intoaccountHRibcrcau-Gayon. 1999).
The various stages of fcrmcutilion arc understood.
YcasLs tcniteni sugars, anil yeast activity should
stop oily when all of (he sagar moke ales have
been consumed. The Luetic acid txiclcria (ben assert
themselves, exclusively decompiling malic acid
molecules in a process called maktlaclic fermen-
tation. IT yeast activity slops bcfoic the complete
depletion of mast sagar. bacteria can develop.
I ■ .'.'.-■ i al development depends on several faclois.
including (be initial sulfiliig of the grapes and the
possible addition of lyso/ymc (Section 95.2) The
inoculation of makihictic fermentation baclcria in
Ihc must also promotes their development. Acetic
acid is formed when lactic bacteria, mainly hcicro-
fermentative Oenacocaa^are picscnt in a iKdinm
contiining sugar In these situations, the vokilik:
acidity can rapidly increase to ■■acceptable levels
e'en if there Is a relatively small rcsidual sagar con-
centration In tact, baclcria form acetic acid from
sugar after thcirgnnvih phase, during which malic
acid is assimilated In consequence, ia the case of a
slack alcoholic fermentation, the wincmakcr can k:i
Ihc makiktctic fcrmcnuitoa continue anlil its cotv
plction before inhibiting the bacteria
The undc islanding of Ihc processes re pre-
sented in Figure 3 9 constituted .in unquestionable
progress in wine m)crobiok>gy. As a result, certain
operations were initialed lo control Ihc alcoholic
fermentation and avoid stuck fermentations. The
volatile acidity of top- tanked red wines decreased
substantially, with a corresponding impiuvcmcnlin
quality. In the 193% in Ihc Bordeaux region, the
volatile acidity of these wines was often I.Og/l
(expressed in H>S0 4 ) or 12g/l (expressed in
acetic acid). The content has been decreased half
Ibis value and today s higher figures are dac to
various problems during fermentation and storage,
which can and must be avoided
3.8.3 Action in Case of a Stuck
Fcrntcn ta t ion
Many stuck fementations result from winemaking
errors. Moreover, systematic stuck fermentations
have been observed in certain wineries year after
year They disappear without any apparent reason
at the same time thai the wincmakcr changes. More
often than not. the necessary operations are known
but not carried oat properly. In red winemak-
ing. stack fermentations often resalt from exces-
sive temperatures at the initiation of fermentation
and a poor control of tink tcmpcralarc daring
110
Handbook of Etiology: The Microbiology of Wine anil Vindications
fcrmcntition. Insufficient dissolution of oxygen
dimit' pnmpiag-ovcr can also contribute. In white
wine nuking, excessive musi clarification, temper-
ature varEitioftsand ihc absence of air contribnic to
fcrmcntition problems. Of course, a higher sagar
concentration in ihc mnsl increases Ihc risks, but
in certain situations there Is no satisfactory expla-
nation for suck fermentations
A density thai remains stable daring 24 or
48 hours confirms a slack fermentation In this
case, different procedures exist u resent the fer-
mentation while avoiding bacterial spoilage
Restarting a fermentation is often a difficult
operation. Elrsl of all. this medium is rich in alco-
hol and poor In sagar— it is not conducive to the
development of a second fermentation. In addition,
the ycasK arc cxhanslcd from the litsl fermenta-
tion and they tract poorly to Ihc different sllm-
all employed They bcnclil nunc from dlfTcrcnl
operations sach as nitrogen supplcmcn union and
oxygenation at Ihc beginning of their development,
when the median contains high sagar concentra-
tions and docs not contain cthanol. Thcrcfoic. an
operation after a stuck fermentation cannot com-
pensate for a winemaking error All operations
benefkial to the fermentation should be employed
from the stut of the winemaking process to avoid
stuck fermentations The prevention of stuck fcr-
■Kntatlonslscsscnllal to winemaking and It shoald
tike into account all of the recommendations pre-
vnasly stated.
In spit of all precautions, a stuck fermentation
nay still occur In this case, white wines mnsl
be treated differently from reds which undergo
malolaclK fermentation. At the lime of the stuck
fermentation, the red wine tank contains mnsl
and pomace rich )■ batterer. The wine should be
drained rapidly, even if the skin aud seed mac-
eration Is not complete Draining eliminates part
of the baclcrial contamination and introduces oxy-
gen, which favors the rcstirting of fcrmcntition
and decreases the temperature. The wine can be
salliled at the same time, to inblbil baclcrial devel-
opment. In some cases, the fermentation restarts
spontaneously.
Even if the stack fermentation results from the
com hi nation of several clc incntuy causes, each has
lb own effect on the case of restuting the fermen-
tation. Excessive temperatures destroy yeasts bnl
do not make the medium nn fermentable, as docs
fermentation in complete .macrobiosis
If the fermentation docs not restart on its
own. an inoculation with active yeast is required.
At present, commercial dry ycasK are inactive
in media containing more that S-'/l vol. of
alcohol, due to manufacturing conditions. In the
future, industrially prepared yeast capable of
developing in a medium contiinlag alcohol would
be desirable. Baclcrti with this properly have now
been dcvckipcd for makflactic fermentation.
An active ycasl starter must be prepared using
Ihc slack fermentation medium adjasKd fc> Vi
vo! alcohol and 15 g of sugar per liter. 3 g of SOi
per hectoliter is also added. The active dry yeasts
arc added at a concentration of 20g/hl. Their
growth at 20 C requires several days and It ismon-
luted by measuring the density or measuring the
sagars When all of the sugar has been consumed.
Ihc yeast*, arc al Ihc peak of their growth phase.
This yeast starter, rich in activated yeasts and no
longer contain ing sagar. Is Inoculated into the stuck
fermentation medium at a concentration of 5 - Nfi
Several days arc required for the complete exhaus-
tion the last few remaining grams of sugar II is a
king and painstaking operation. The volume of the
ycasl stirrer can also be progressively increased
by adding larger and larger quantities of the stuck
fermentation wiuc to It
In choosing a ycasl strain, the yeasts should
certainly be icststinl to cthanol Yeasts cotJaKr-
ciali/cxl under the name S. Iniynmit could be rec-
ommended but they seem to have a propensity
to form volatile acidity iu these conditions. Com-
mercially available S. eerewisiae yeasts (formerly
5. ''v i. ■ '"i i. known for their resFtance to cthanol
aud low probability of producing volatile acidity,
arc recommended for this purpose
Effectively. Ihc volatile acidity of Ihc wine tends
to increase during Ihc restuting of a stuck fer-
mentation This generally occurs when the yeasts
encounter unfavorable conditions. Certain ycasl
strains arc nx>rc predisposed to forming it than oth-
ers The addition of 50 mg of pantothenic acid per
hectoliter (not authorized by III: legislation) not
Conditions or Yeast DcvclopnicK
only can Inula its formation bat also can contribute
hi ihc disappearance of an excess of acclK acid.
The rmpcraturc lor restarting the fermentation
must he considered. A slightly elevated temper-
ature favors cellular multiplication but Ihc anti-
septic properties ofcthanol increase with temper-
ature. Tie risk of an increase in volatile acidity
also seems K> he in Unction of temperature For
these reasons, the restarting of ihc fermentation
should be carried out at a temperature between 20
and 25°C.
taunting activated yeasts in the winery can aLso
he used to restart a stuck fermentation. If there is a
large volume of fresh harvest available at Ihc right
moment, the tank with Ihc stuck fermentation can
be diowncd with if This operation, however, is in
conflict with the legitimaK desire to select cuvces
The practice of adding 5-20*f of a medium
in tall fermentation to a stopped tink should
be carried out with prudence Active yeasts arc
certain ly added bit sugar is too. In this situation,
the fermentation has sometimes been observed to
restart and then stop again, leaving about the saute
amount of sugar that existed before the operation.
The Ices of a tank that has normally completed its
fermentation can also be used as a yeast stirrer to
restart a stuck fermentation. Supplemental sugar
is not inliuliccd into the medium, but yeast
in their death phase are no longer very active
The correct restarting of a fermentation requires
the introduction of active yeasts in this alcoholic
medium without introducing supplemental sugar.
The preparation of a yeast stirrer nsiig dry yeast
gives Ihc most satisfactory mulls.
In while xvincmakiug. at least when malolaclk
fermentation is not sought. Ihc wine with a stuck
fermentation shonld be lightly salfitcd lo piotccl
against bacterial development. The fermentation
can then be restarted using a ycasl starrer prepared
accotding to Ihc preceding instructions
Many possible adjuvants helping to restart a
slack fermentation have been proposed The addi-
tion of ammonium salts does not r.ase any coantcr-
indKaliOBs.bul no appreciable improvement of the
second fermentation has been observed. The addi-
tion of ammonium sulfate should be limited !■>
5 g/hl due to Ihc limited use of nitrogen by ycasfe.
Mash- pasteurization (heating between 72 and
76'C for 20 seconds) seems to be effective. It
impiovcs Ihc Ic rare n lability of wines with stuck
fermentation (Dubernet. 1994). This operation is
valid for red. rose and dry while wines and
should be carried oul before inoculating Its heating
effect can be likened to the effect observed dur-
ing Ihcrmo-vinificalion (Section 123.3). Ii spite
of the destruction of yeasts, the beared musts
ferment especially well The effect, of this pro-
cess merit further slady bat several explanations
can be proposed: fcrmcntilion by a sole strain
avoiding microbial antagonisms, addirion of nutri-
tive elements due to yeast lysis; elimination of
toxic substances: and modilkation of the colloidal
structure
Active charcoal has also been used for a
long time « reactivate fermentations! 10-20 g/hl)
Such an addition is hardly conceivable in red
wines, but its effectiveness for stuck fermentations
in while wines is recognized. Il works by eliminat-
ing yeast inhibiuts (fatty acids) (Section 3.621.
The addition of yeast halls is certainly the most
effective way of restarting a slnck fcrmcntilion.
although less so than in preventing fermentation
from Mopping in the first place. (Section 3.62)
They can be added tu the yeast starter prepa-
ration or directly lo Ihc medium with Ihc stuck
fermentation.
In result* of an experiment given in Table 3.16.
Ihc first fermentation of a must initially containing
250 g of sagar per liter slops at 67 g of non-
fcrmcnicd sugar per liter The second fcrmcntilion
is conducted after an inoculation at 10* cells of
5. cererisiiM' per milliliter, without the addition
of yeast hulLs in the control sample and with an
addition of 05 g/l in Ihc lest sample 24 hours later
This addition permits a complete fcrmcntilion
Tabic) 10. Rcuurtinp Icrmcnmion Ulici
nana Mini, ferine mi kin) hy aiUkioa uf ;
lUtoo-Uti.nnidert.jF-. IWU>
KJM hull.
Rokhul iu(4i(
Mgl)
D*y 9 I>jv 10
Day 30
Control 57 36
-SO p/hlofycnu hulk S3 23
IJ
HamUxxik of Isitology: The Microbiology of Wiac anil Vinifkralions
1 36 days, which is aot passible ii ihc control Chune X. <!0D0> Cattiibtttim
i Fttuile dei I
A massive addition of ycasl hills combined with
an inoculation or active yeast can result ii olfactivc
modifications or light wines sack as whites ami
roses Doses between 20 and 30 g/hl (maximum)
arc therefore recommended
In while as well as red wincmaking. Ihc restart-
ing of a stuck fcniKnIation should be closely mon-
itored, especially by measuring volatile acidity to
ensure that the alcoholic fermentation is pure. The
smallest increase in volatile acidity represents a
bacterial contamination, which should absolutely
be avoided. A judicious sultiling should prevent
con Crmi nation; without a doubt, it slows the fer-
mentation. Yet if the doses are adapted to the
situation (3-5 g/bl). Ihc fcrnicntilioa will not be
delinitivcly com promised, it will restart after inoc-
ulating It masi also prevent all bacterial develop-
ment befoic the complete depletion of sugars, even
though its addition can make malolactic Icrmcnia-
tion more difficult.
Tanks with slack fermentations must be resulted
as soon as possible. In the middle of winter,
this operation can become impossible and in
these situations it is preferable u wail until Ihc
following spring, when fermentation may restart
spontaneously.
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101.
Andrcatcn A ,\ j -I SiKrTI.lt (195.1)7 Cell. Camp.
PkjBoL.il, 73.
Bclv M .. SabUyiollo, IM. ..i-l Banc P. (1990) /
Ferment. Biaeng. . 70. 246.
Bouli M.. BtBMinC. Cbarpentkr M.. Lcvcau I.V..
IXuerfieB. (1997) J. kit. Sri. Hjw tin. 31 (1).
II.
Bovee I.P.. Blnuia I. Marion J.M. aad Sinrkiiano P.
( 1990) In ,\nu,jiic> IJi,n'o K iuuei 90 (lit* P. Rheieau-
' ;.-.. ui, .ml A. ! ...it. .ikI : Dumd. Pair,, r; 281-280.
Brecnol P.. Chauvci J . Dupuy P.. Cmimn M aad
RabMu U. (1971) CR And. Sri.. 272.890.
CaatarclliC. (1989) In ltiff>«fti*>/i>*.i Applictaion In
Setn.tge Produilion (E* C. Camaicll) and G.
Lumen). Ehevkr Applkd Science*. London.
Cauor J.G.B. 1 1953) J. Fond Ret . 18. 146.
botileliix etude tie Finiuciur de\ deficit! Iitiirii/ue.i
motlere'i et de I'tJimeiiUl'wn en <epte mr le poieinlel
itramtiiqtte iles rtiwit de tiiit linijirii i*i. mmntgum
blot.: Tbcie de doctoral. UmychM Vkior Scpalcn
Bordcaw 2.
DclflniC. CoMa A. (1993) Aw. J. Kiwi. \UU:. 44 (I).
8ft.
DclHni C. Contcrao L.. Giacna □., Cocao C. Kj-. au-
la I. and Banli L. 1 1992) Uric. Etiot. Sri.. 47. 09.
Dubcroet M. ( 1994) Im Mffte. Dec.. 52.
Dubourdku I) OllivkrC. and Boidam J-V (1980)
Cmm. Hffie Wh.20(I).53.
El Haboui N.. Rkqic D. and Corrku.G. 11987) Sri.
Mm.. 7. 241.
Fbaiy C. ( 1998) Otjiologle. Fondetnentt Srieiuifiitues
et TeilmpJoffques . lavonkr. TcchaUfic A dmumen*
latba. Pari..
Fleet G.ll . ( 1992) lUw Mioobioltiit} itttl Binteeloinl-
agj. Harwood Academic Publnhcn. Chur. Swi-
/eriand.
Fleet G.II . awl Heard G.M. ( 1992) In Vine Micmbinl-
agj oxi Sintechnnlogt (Ed. G.M. Fleet). Haruood
Acadcmk Pobloaerj,. Chur. S=.«rUnd. pp. 27-S4.
Gcacix C.Lalbn-Lnfourcadc SandRfeeieau-Gajon P.
( 1983) CR. Art*!. Sri.. Serle 111. 290. 943.
• im«i P.. Feuilblcy P. and Sabbyiolle* J.M. ( 1990)
In Aeim/itesEnt>ioffqiieitlt9.iEikP. Rlvcicau-Gayon
and A. Loavaud). Duaod. Pari*, pp. 521-526.
GuiaundG. H9S9lOfouped'e.ipemierlinnliigieilu rid.
Office Inernaibml de b VBjdc et du Via. Parr..
Hatridimuriou E.. Daniel P.. Bed rand A. and Duhouc
dicu D. 119971/ aa>. Sri. Ugne Vfn. II (1). 51.
Hcawhkc P.A. aad Jlranck V. ( 1992) la Wuw Mitrnbi-
olnx-'iid Bitxechnologx (Ed. G.ll. Heel). Haruood
Acadcaar PublnaccCaur. S»nwrland. pp. 77- 169.
lagledcu W.M.aod Kunlxc R.( 1985) Ait. I. total. Ui.,
36.65.
ii I xii A.. RouMaa I.E.. Dubu 1.. and Sabbyrollo J.M.
(2000) Am. J. HK. (I.TI..I.. 51 (3). 215.
lafon-ljfourcade S. (1983) Wine and Branh'. In
Sioteelmnlnxt. Vol. S (Ed* H J. Rehm andG. Read):
Vcrbg Chenic. Wcinhciai. pp. 81- 163.
Lafon-Lafnunadc S.and Jnycut A.(1979)Cofur. Mgie
H/i. 13(41.295.
Ufon-UKHiitadc S. Uaje F. Bnfehot P. and Ra>c-
reau-Ga>on P. ( 1977) CR Actrl. Sri. 284. 1939.
Ufon-Ufimrcadc S.. EUbuunlieu D.. Hadjlnktdaou D.
and Rtoctcau-Giivoa P. (1980) Ctttn. Mffie UH,
14 (2). 127.
Latbn-Lafcmrcadc S.Gencit C.andRi>cicau-Ga>on P.
1 1984) Appl. Emir. MimtbinL.il (6). 1246.
lame F.. Ufoa-UfouKadc S. and Ri>cieau-Ga>on P.
tmoi Appl. Emir. Mitmbinl.. 39 (41.808.
lanx F.. lafoa-Ufouwade 5. and Ri>cicau-Gayon P.
( 1982) CR Attil. Sri.. SrlriV III. 294. 587.
Ctwdiliots or Yeast Development
b« F., Cdiinki M i*l Rfccirau-Gayon P. (1987)
In dHftpte-ienilu det ixthiiet de irciuvdics (/**W-
/9S6J de I'hOiiui •rEnolo&e. LWitrnV 4e Bor-
riaau tf .
LorcnaaiF. (1990) fciiif Svi'm* Arbaric. Hartic.
28(3). 109.
Ma»*cul I.. Mural Ml... Cboac X. and Dubounlieu I).
(2000>ifii.249.4l.
MullccTauipu M.U8B4)Rcpo«cdb;, Ribcreau-Gayoa
el ill. 1 1975a).
MuaozE and loplcdcw W.M. (1989) A-> J firo/.
Hi«\.40(l).0l.
OMh (crC. SloncMicci T . Eaiuc F. and DubounUoi D.
1 1987) Cowl if jw Ha, 2 1 . 59.
Ougn.C.S.(l964)AMi. / £>«/. H/i<\. 15(4). 107.
Ougfc C.S.I 1900) /VwJ t™* Htfc, 17 (11.20 anil 74.
OughC.S. and Giuai M.L. (1978) AppS. Emir. Micro-
bial. . 35. $81.
IV Icnlo N.M. 1 1988 ) Group* iTe.iperis tfthnafog/e du
int. Office Imc notional dc la Vjpne el du Via. Pare.
Rfcercau-Gayon J. and Peynaud E. i 1900) Aw. Ahfrff.
-Vim. 20. 1.
RlVicau-Gavon I.. Pcyuaud E. and Lafounadc S.
1 195 1 ) Aid. A*W. /Vim. 08. 14 1 .
Rfccrcau-Gayon J.. Peynaud E. Rfccorau-Gayon P. and
Sudani P. ( 1975a) Seism ei Techniques du ifii .
Vol 2: Cinneret des Vfnn Miiuneinn du naiiu.
leiures a bixieiles. Duood. Pain.
Rfccnrau-Cayon P. ( 1999) J. Mr S.v. W*w Vfcr. 33 ( I ).
39.
Rfccorau-Gayon P.. Ufon-Ulixinr.uk S. and Bcrtiand
A .(l975b)Cow.. Hjbw Hit. 2. 117.
Rfccicau-Gayon J.. Peynaud E.. Rfccicau-Cayon P. and
Sudoud P. |I970) Srimif.i el Tetimiquei da ifn.
Vol. 3: Vftt/fi<*iifHwi.Y Tniinjoriwiians da tin. IXinod.
Rticicau-Gayon P.. Lafon-LatbuKade S.. Duhouidku
D. liAiium V.aad Unit F.(I979) CR Actil. St . .
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Sabbyiolle* J.M. and Bane P. (1980) &-i'. AKbl,6, 177
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Sabbyiollc* J.M . Dubon C. Mao£)m* C Roma*
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377.
Sabbyiollc* J.M . Salman J.M. and Banc P. (1990b)
Prof/ei agr'tc. ti/ic. 111. 339.
Salaon J.M.. VinccM <>.. Mauricio Jr.. Bcly M. and
Banc P. ( 1993) Aiu. J. Eiiol. iff iV.. 44 (I). 58.
Lactic Acid Bacteria
4 I The different components of lie baclcria cell
4.2 Taxonomy
4J Identification of lactic acid bacteria
Luetic ackl bacteria aic present in all grape
nmsis and wines Depending on the Mage of
lie wiucmaking process, environmental conditions
dctcmiinc Ibcir ability 10 in In ply When ihcy
develop. Ihcy metabolize numerous substrates.
Laclic .11. 'I bacteria therefore play an importint
n'le in the transformation of grape musl iiio wine.
Their impact on wine qnalily depends nol only on
environ menu I faclois acting at Inc cellular level
but also on lie selection of the best adapted species
and straits or bacteria.
All the strains have a similar cellular oiganim-
uon. bnl their physiological differences accounl for
Ihcir specific characteristics and varying impact on
wine qui i ty They aic clarified according to their
morphological, genetic, and biochemical traits
4.1 THE DIFFERENT
COMPONENTS OF THE
BACTERIA CELL
Bacteria arc procaryolic cells with an extremely
simple otganiAition. They can be distinguished
from cucaryolcs(to which yeast belong) by their
small si/c aid a tack of a nuclear membrane
delimiting a nucleus.
It is impossible u distinguish between sach dif-
ferent bacteria as Escherichia caii and OeaiKoc-
ciu oeni ( 0. oen. formerly known as Leucoimslir
oenos or L aema) by simple microscopic exami-
nation. In facl. the stricture of all bacteria is very
simitar. It can be divided into three principal clc-
mcn&tr-igure 4.1):
£ n*- U.iaVuhri »i
Handbook of Enotogy: The Microbiology of Wine anil VinifitaiMws
I'it 4.1. Utile acid lawn* Uobicil (a wine under
i Kian)ap ckilnm •kmnopt (DcpjRCBcn ilc
Mkntwopk Kktuoniquc. I'mvcniiy of Elonlmu II
{>) Photograph of Urtobitrillus phtiutunt cell* na».-
■ (una. La maud. 1975): c = cyiapkum: pm = pb»au
-Cmhi.K,w =.cll vv,ill:s =«pluin ; « = m»i»oaic:
• = ■Kkw.. (hi PniMopaph of teuconnttoe anwi
(OmocmviiJUrtu I (Manning cleciiun mkimtopc).
4.1.1 ThcCdl Wall
The cell wall of G nun- positive baclcria. such as
lactic acid txicicrM. is essentially composed of a
pcpiiclojf lycan thai is mly found in procaryoics
(Fnjnic 4.2). This polymer wraps Ike bacterial
cell wiih a kind of mesbwork made up of
polysaccharKlic chains linked by peptides. The
oses arc glucose derivatives: jV-accIylmaramlc
acid and ,V-accty (glucosamine (Figure 4.2). They
alUrrnaie along ihccilire length of the chain, linked
by fi-typc (1-4) glycosidic bonds lhal can be
hydrolycd by lysozymc or mulaiolyslftc.
A chain of fonr amino acids is linked lo
mi ram k acid: L-alamnc. D-akinincand D-glutamK
ackl .iic in majority. A peptide bond links the
tclrapc|Midc of another polysaccharide chain lo the
third amino acid (Figure 4.3). The peptidic chains
vary depending on Ihc species of the bacteria.
The sequence of (heir amino acids can be used
in taxonomy.
The cell walls of lactic acid bacteria, like those
of nearly all Gram-positive baclcria. also con tun
• Cellular envelopes, including Ihc cell wall and
the membrane. The cell r< delimited by (he
cytoplasmic membrane doubled towards (he
exterior by Ihc cell wall. Between Ihc cell wall
and Ihc membrane, (he periplasms space Is a
moic or less fluid gel wherein prolcins move
a bo »t.
• The cytoplasm.
• The nucleus.
t
Pq*UKt>
*
Pq*UKt>
I'ifi 4.2 Pol>w
Kb/can
Lactic AcHl Bacteria
ir
ribilol phosphate or glycerol phosphate polymers
caltcd tcichoK acids Pbosphodicstcr linkages can
fix amino acids and oscs to these chains Glycerol
Ixtscd Icicboic acids contain a glycoliptd by which
they altach themselves lo the external layer of
the plasniK membrane. They pass through Ihc
prptidoglj'can anil air al Ihc sutfacc of Ihc cell
wall acting as ihc antigenic sites or bacteria The
proportion of pcptidoglycans and Icicboic acids
varies depending on the species and afco the phase
of the cell development cycle. Tcichoic acids can
represent up to 5(K* of the weight of the cell wall.
The cell wall is rigid and gives the cell its
form: round for cocci, elongated for bacilli. It
prrmibt the cell to irsw very high in Kraal osmotic
pressures ( up lo 20 bars). The culture of cells in the
presence of penicillin, inhibiting the synthesis of
Ihc cell wall, leads lo the formation of protoplast:
they are only viable in isoRmK media. Similariy.
lyso/ymc hydroly/cs the glycosidic linkages of
peptidoglycan. provoking the bursting of the cell
in a hypotonic mediant.
Water, mineral ions, substrates and metabolic
prod k- is diffuse freely across the cell wall Al
this level, proteases also release amino acids from
proteins and peptides which are nscd for cellular
mctabotem.
Observations under the clcclron microscope
have also proven the existence of a protein layer on
the cell wall surface (S- layer) in several laclK acid
bacteria species. The study of this S-laycr ii wine
bacteria has not jet been attempted. Hnallj . the
accumulation of polysaccharides piled upon these
proteins can form a more or less distinct capsule
Id thickness varies according lo environmental
conditions. In cnokigy. Pettioeoceus tkminmais
gives the best example of this phenomenon. In
certain conditions, strains of this species syn thesi/e
significant quantities of polysaccharides which
make the wine viscons ( lupiucss). These cells arc
easily recognized under an optical microscope by
Ihc refringent halo that surrounds them.
4.1.2 The Plasnik- Membrane
The membrane is situated against the cell
wall, delimiting a pcriplasmic space, Folds arc
sometimes visible in the interior of ihc cell: Ihcsc
The membrane of lactic acid bacteria has the
classic stricture of all biological membranes:
a lipid bilaycr creating a central hydrophobic
oik (Chapter). Figure 1.6). The proteins arc
more or less tightly joined to It. Among them,
the n yd insoluble proteins are only lixed lo the
surface by ionic or hydrogen bonds (periph-
eral proKrins. i(¥i of Ihc proteins) The oth-
ers are lodged In Ihc membrane by hydrophobic
bonds (integral proteins) The peripheral prolcins
have a certain mobility in the pcriplasmic space
between the pcptidoglycan and the membrane.
whereas the integral proteins are almost immo-
bile. Some protrndc from the membrane while
others only appear on the surface. Hydropho-
bic bonds between aliphatic lipid* and protein
chains create the framework of the membrane The
high number of these bonds ensures the solid-
ity of this stratum-, but there arc no covalcnt
bonds and so Ihc framework created remains lluKl
The biochemical functions ensured by the mem-
brane depend on this llaHlity. i.e. lipid -protein
interactions The structure can be destroyed by
organic solvent! and detergents. II is also dis-
torted by wine components. Finally, on the sur-
face, the hydrophilie parts of the lipids and the
ion i/cd group* of the prolcins cstaMcdi ionic bonds
between themselves
Membranous lipids represent nearly all (95-
W< ) bacKria cell lipids. They essentially include
Handbook of Hnology: The Microbiology of Wine anil VinHlc.uions
tl— C— O— CHi
iwMiiliil HhJliii.ltil n tulpiytiiai
a i ii
o ciij— o— r— o— cn,-cn-ctipn
n,N-cn
I AtytOt^tulUyliMnol
II— C — O-CHj
MiC-O-C-R
It*" -O — t — O-Crtj — C — CHi— O-P — O-Clli
•V"S"««''?">"
ViH 4.4. Chemical fonm.be of*.
icatmnc phmfhullfHk
phi*>pholipids anil glyeolipkls. Phospholipids air
■km abundant they consist of a glycerol molecule
which has a primary alcohol function anil a sec-
ondary alcohol function cstcrificd by Tally acids.
The other primary Unction Is cslcrilied by phos-
phoric acid, which Is cstcritied by glycerol, form-
ing phosphatidyl glycerol. Lactic acid bacKria
also con tun diphosphatidyl glycerol (cardiolipid).
amino cslers of phosphatidyl glycerol with ala-
nine I OeiiociKCUS ««) and lysine {UiciohacB-
tits ptaMunmH (l-tguic 4.4). Bacteria phospholipid
concentrations vary according to growth stage and
cultural conditions
Glyeo lipids— generally glycosides of dlglyc-
cmlcs — arc formed byglycostdk bonds between a
mono or disaccharidc (glncosc. fructose, galactose,
rhamnosc) and the primary alcohol function of a
djglycefidc (Figure 45).
,P« ^i_,'o-c
iijc-o-ca-tt
l''ifi 4.5 liirmuU of* ulycolipkl
Lactic Ac Id Bacteria
Fatty ik Vis possess a long hydrocarbon chain
ami a Kraiinal carboxylic acid friction. These
molecules air charactcri/cd by tbc length or their
chain. Ihcir level of unsaiuralion. the cis or trans
conformation of the double bonds, and t for Gram-
positive) the istt oninH-iui ramification:
CHj— (CHj),— CH 2 — COOH
CHi— (CH2)-— CHj — CH— CHj— COOH
H»C— CH— CH2-
HjC— CHj— CH-
CHi — (CHj)-— CHj — CH =CH — COOH
In bacteria. most faiiy acids have 14 to 20 car-
bon atoms and arc saturated or noto- unsaturated.
Lactic acid bacteria afco contain a characteristic
eye lop ro panic acid: laclobacillK ackl (rii-11.12-
meihylcne-ocnxlccanoic). TaHe 4. 1 lists ihc prin-
cipal fairy acids of lactic acid bacteria found in
wine— notably Oeaococaa iieia (Louvaud-Funcl
and Dcscns. J 990). Malonyl CoA and acclale con-
dense to form tally acids wilh an even number
of carbon atoms. For an odd number of carbon
atoms. lady acids arc synthesized by the conden-
sation of malonyl CoA and propionate Anaerobic
bacteria synthesize unsaturated acids by the action
of a dchydralasc on hydroxydccanoaic which is
formed by the addition of a malonyl unit on an
octunoic acid molecule
The following reactions arc given very schemat-
ically:
The progressive elongation of this acid leads
to tbc formation of ivi-v-accenic acid Ids), a
precursor of lactobacillic acid tt",., > In this List
step, the double bond of Ine unsaturated acid (tbc
precursor) is methylated to form the corresponding
cyclopropuuic acid. Tie fait)* acid composition of
Ibc bacteria lipids varies during the physiological
cycle and is also strongly influenced by several
environmental factors.
Finally, besides polar lipids, the bacteria mem-
branes contain ncnlral lipids, analogous to sterols
in cucaryotcs. These IrilcrpcuK and pcnGicyclic
molecules air called bapanoids They ate formed
by thccyclizaiion of squalcnc in an anaerobic pro-
cess They have not been clearly identified in lactic
acid bacteria
Thc membrane is even more vital to bacteria
than Ihc cell wall Numerous proteins in the mem-
brane ensure essential enzymatic functions such as
substrate and metabolic product transfers and the
Toblc4.l. Principal fattj ackU of luctk-
Mvrbtk
acU
laodccanoic
CI4fl
Palnik
icid
hetaefceanok
ClOfl
Pilmitilclc icid
iv>9- hexadeca noic
C10:l A 9
Stearic i
ii.il
ociadccaaoic
CIBfl
Okie at
id
.v.-9-ocwdecjnok
: 18:1 i 9
.ii- Vac.
HydnMI
l.actoba
emc ackl
ciculk acid*
u iliac- ackl'
.v>ll-.Kia.lccai»k
<y><M0-mclhvknc .
.v>l 1-12- met byk*c
wa.kcunok
< 18:1 .', II
C19cyc-"»
C 19 eye- II
Jl«*cii
■cacrtmuai
it a cyckfi.fianiL' cycle —
CH-CII —
120
Handbook of Etiology: The Microbiology o( Wine anil Vindications
ATPasc system. Lactic acid bacteria do nor have
a respiratory system. The selective permeability
ensured by ihc membrane creates a transmem-
brane electrochemical prolan gradient between ihc
inside and outside of ihc cell This difference gen-
crates, e lec troc bent real energy nscd in the synthe-
sis of ATP. Moreover, the membrane maintuins
an optimum cellular pH for the functioning of
numerous reactions of the cellular metabolism. It
constilntes a barrier whose optimal functioning
is guaranteed by the fluidity The fluidity dcicr-
mincs the specific activiry of the proteins accord-
ing to the lipidic environment, bat this fluidity
mist be cottrollcd for Ihc membrane *> remain
an effective barrier between the cytoplasm and
the environment Hiring Ihc cell growth cycle
and in response *> multiple external parameters
sneh as temperature. pH and Ike presence of toxic
subsumes tclkanol). the cell manages to mod-
ify membrane composition to adapt to and resist
environment)) effects. Tic physical properties of
the membrane are maintained at least as king
as the stress factor remains within certain lim-
its. The mccnaueims pnt into play act together
on Ihc sane properties. They affect the aver-
age length and Ihc unsaluralKm. ramification and
eye )i /at ion level of fatly acid chains, the propor-
tion of neutral and polar lipids and the quan-
tity of proteins In this way. from the growth
phase until the stationary phase, m-vacccnic acid
diminishes greatly to ihc point where it rep-
resents less lhan KM of the total fatty acids,
whereas laclobacillic acid attains a proportion of
55% in Oenoeocais oem. Lactobacillus planianmi
and Pcibococcus tkannasus (Lonvaud-Funcl and
Descns. 1990).
The effect of temperature on membrane com-
position is one of the most understand effect.
At low temperatures. Ihc fatty acid unsaluration
rale increases as does the proportion of acids with
ramified chains. At the same lime, the length of
Ihc chains decreases In this manner, palmitic acid
(On) increases and m-vacccnic and laclobacillic
acid decrease in Oenacoccus vein and Lactobacil-
lus plaitianm when Ihc temperature of ihc cul-
ture increases from 25 to 30'C. The introduction
of a methyl group, the formation of a propauic
cycle, has the same effect on Ihc physical prop-
erties of bacteria as a double bond The inverse
phenomena occur when the culture temperature is
higher The unsaturated fatty acids are less abun-
dant. Neutral lipids also participate in cell adap-
tation io Ihc medium by increasing membrane
viscosity.
The presence of clhanol in the medium provokes
significant modifications in membrane straclurc. II
exerts a dclcrgcnt elf eel by intercalating in the
hydrophobic mac of the membrane, whose polarity
increases as a result The fluidity is increased and
the proteins arc denatured. In general, an increase
in the unsatu rated .saturated tally acid ratio is
observed In Oenoeocais oem. this ratio increases
from 0.4 to 2.1. when bacteria arc cultivated
in Ihc presence of ft clhanol. The results are
ihc same for the species Utciobacillus hilf,ariUi
whose straits, like Oenacoccus arm. arc capable
of growing belter than other species in an alcoholic
medium (Descns. 1989).
The membrane proteins also participate in cell
response to an environmental change. The stress
proteins in microorganisms are becoming better
known Their synthesis is increased, for example,
by tempera (ire. acidify or the concentration in
ethanol. Certain proteins also change when the
cell enters the stationary phase Several families
of these proteins have been constituted and the
specific functions of some of them have been
Ktcntiflcd. Their ovcicxprcssioi in Ihc cell is
related to a belter resntancc to stress factors.
Their induction by heal shock protects the cell
not only against the toxic effect of heal but also
against Ihc effect of other factors, such as ethanol
and acidity. In ccitain wine lactic acid baclcria.
especially Oenacoccus oem. the proteins exist bnl
their role is not known Their synthesis is increased
when wine is added to Ihcir culture medium or
when the cells are directly inoculated inlo wine.
The concentrations found in Oenacoccus oeui have
been found Io be up to five limes higher than in
other species. IGarbuy. 19941 Among these, two
proteins have been identified and coded by the Ontr
A and L'ls H genes (Bourdincaud etal., 2003a.
2003b).
Lactic AcMI Bactcrii
[21
41.3 The Cytoplasm
The cytoplasm contains the main elements for cell
opcmiion: in/ymy nuclear niaicrEil awl somc-
Umcs reserve subsEinccs. The cnlirc metabolism—
bolh degradation rcaclions (calabolisni) aid syn-
thesis reactions (unabolisai) — Is carried out in a
prog mined manner according to exchanges with
the external environment, to produce Ihc energy
necessary tor cell growth.
Coded by the genome, the cytoplasmic pro-
teins aic always the same for any given bacte-
rial strain, but for some of them their level of
expression varies with cullaral conditions Stress
proteins, produced by draslic changes li condi-
IKibs. have also been identified One of those
produced in 0. iieni. IjiIS. has been particularly
studied (Dclmas el ill.. 2001). The clccltophorctie
profile of the soluble proteins of the cell can there-
fore be ascd as an Identification method by com-
parison with established strains.
Cytoplasmic granulations can be revealed by
specilic coloration techniques. They arc insoluble
reserve sabstanccs of an organic nalnrc: polymers
of glucose or of the polyester of 0-hydroxyburyrK
acid. These reserve sabstanccs accumulate in Ihc
event of a nitrogen deficiency, when a source of
carbon is still present. lucUstonsofvDlulln (a poly-
mer of Insoluble, inorganic phosphak) arc char-
acteristic in laclic acid bacteria, especially certain
species of the genus of strictly homofcrmen la-
live IjKlobacilhu. Volilln comprises a phosphak:
reserve available for Ihc synthesis of phosphoiy-
tiled molecules snch as nucleic acids.
Under Ihc transmission electron mlcaiscopc. the
interior of the bacterial cell appears granular. This
is due to the nbosomes. which arc essential players
in protein synthesis They ensure, along with the
t-RNA. Ihc translation of the generic code The
ribosomes consist of two parts characterized by
their sedimentation speed, expressed In Svcdbcrg
values IS). These two sub-units are different in
size: 30 S and 50 S in procaryotcs. The assembled
ribosonic has a sedimentation constant of 70 S.
The 30 S snb-unit contains a 16 S ribosomal RNA
molecule 1 1542 nucleotides) and 21 different pro-
tein molecules. Its molccilar mass is 900 KDa.
The larger 50 5 snb-unit contiins two ribosomal
RNA molccnlcs. a 23 S and a 5 5 molecule (2904
and 120 nucleotides, respectively). It also con-
tains 35 proteins and has a molecular mass of
1600 KDa. During protein synthesis at Ihc transla-
tion step, the sib-units |al first separated) reassem-
ble. The genes encoding proteins and ribosomal
RNA arc known for the fuctcriu Escherichia aAi
They arc organized In operons. ensuring the con-
trol of Ihc synthesis of ribosomal components The
opcron of genes encoding the rRNA have the fol-
lowing structure:
The nucleotide sequences of these genes, espe-
cially loose of 1RNAI6S. arc known lor many
species and identified in gene banks Sequence
comparison forms the basis of molecular identi-
fication methods.
4.1.4 The Nucleus and Generic
Material
The bacteria nucleus consists of a single circular
chromosome of doable stranded DNA suspended
in Ihc cytoplasm wlthoat any separation. (Ls si/c
varies depending on Ihc species In LKtuixteillm
p/wiiiman. its length is about 2400 kb. It Is
much smaller in Oeaococeus oeiu (about 1400 kb)
and PetSacoceui pentataceia (1200 kb> (Daniel.
1993) The chromosome carries the essential
genetic information of a cell.
Other more or less vital tunc lions arc determined
by plasm ids These small, circular DNA molccnlcs
arc completely Independent of the chromosome
They vary In si/c and namber depending on the
species aid strain of bacteria In Oenacoccus
i>ein. ptasmtds of 2 to 40 kb arc often identified
and one of them has been sequenced (Frcmaux
el ill.. 1994). So far. no function of cnological or
physiological interest has been attributed to them
In general. Ihc plasmkts determine functions such
as the fermentation ofccrialu sugars. Ihc hydrolysis
of proteins, resistance to phages, antibiotics, heavy
metals, etc
i2:
Handbook of Fnology: The Microbiology of Wine and Vindications
In wine luetic acid backria or Ihc specks Pe<Uo-
coccus ctmmosiis. a plasmid has been identified as
a determinant of pulysaccharidic synthesis. Strains
ili.it n'ii tu- il arc responsible lor ropiness in wines
( Lonvaud-FoKl fid/.. 1993) This plasnrid has
been entirely sequenced. Unas Ihrcc coding regions.
obc ofwhKh Is probably responsible for synthesiz-
ing the ex polysaccharide I Section 5 44>;isii proba-
bly codes foraglucosy I transferase (Walling. 2003).
Characteristically, ptasmkts arc relatively unstable
from one gencialioa to Ihc next, but some, in Oeiiei-
coccus cieni maaifest an immense stability. Others
ate easily lost in the absence of cnviionnicn til pres-
sures Coning alive plasaiRlscan naturally transfer
from one strain to another, though this properly has
■ever been demonstrated for the lactic acid backria
of wine. A strain s ptasmklK profile can therefore
wiiy.
4.1.5 Multiplication of Bacteria
All backria multiply by binary division (Figure
4 1 1. A cell gives two completely identical daugh-
kr cells. Multiplication supposes, on the one hand,
division of nuclear material, and on Ihc other
hand, syn thesis for Ihc construction of new cellular
envelopes aid cytoplasmic elements, in particular
ribosomes aid en /vines.
The genetic makrial is transmitted al'Jer the
duplication of the chromosomal DNA and the
potentially existing plasmids. DNA replication,
according to the semi- conservative mechanism,
leads in Ihc formation of rvvo molecules that are
identical to the parental chromosome or plasntid.
The replication occurs almost during the entire
cellular cyckr at Ihc mesosomes When il is
finished, the scission of the cytoplasm begins.
A septum Is formal in the middle of the cell as a
result of the synthesis of portions of the membrane
and the cell wall It separates the mother cell little
by little into two daughkr cells. The genetic mate-
rial and Ihc other cellular components arc simul-
taneously distributed between them. Finally, when
the septum is completely formed, the two daugh-
kr cells separate Cell and nucleus division ate
not synchronous: replication Is quicker Moreover,
a replication cycle can start before cell division is
com pic led. For this reason, backria cells in their
active growth phase contain more than one chro-
mosome per cell During division, plasmids (much
smaller than Ihc chromosome! arc not always cor-
iCCtrydEuriblkd between the cells alter their repli-
cation, hence their instability over generations.
4.2 TAXONOMY
The objective of tixonomy is to identify, describe
and class microorganisms C tassllication is made
according lo several hicrarchkal levels. For bacte-
ria. Ihc highest level corresponds with their clas-
sification among procaryotcs. The lowest level is
species. In a specks of backrium. strains grouped
logethcr share a number of identical charackrs
These characters radically differentiate them from
other sixains
Lactic acid backria belong u the Gram- positive
group, based on color ksts (Section 43.2). The
primary product of their metabolism of glucose Is
lactic acid.
4.2.1 Phcnotypic Taxonomy, Molecular
Taxonomy and Phytogeny
Phenotypcs include morphological. physiokogKal.
biochemical and immunological cbarackrs as a
whok and ihc composition of ccrttin cellular
components. Certain phenorypic charackrs appear
kt vaiy in a given strain— for example, the
assimilation of certain sugars. Certain strains
having different phenotypcs bnl belonging lo the
same species are atypical strains.
Progress in molecular biology provides new
classification criteria based on genome analysis
Molecular taxonomy consist of classifying bac-
kria accoiding lo similarities in their genome.
Diverse methods exist, permuting several levels of
classification.
A first level lakes into account the percentage
of guanine and cyiosinc bases in the DNA — the
il ' + ■■'*'» wilh respect lo Ihc total number. Two
strains arc not necessarily related because they
have the same (G+CJ». In fact, the base
composition docs not give any indication of the
DNA sequence Among Gram- positives, lactic acid
backria belong to the phylum CUankliiaii. The
Lactic AcMI BackrrU
123
Tabic 4.2.
according 1
Sub-divUbn of
Gam-po*aivc htticria
(G-Cfi
w.
>W*
I'll, hi m
Cloeritliam
ArtiitPrv jm rt
UttnOtt-iUus
Lmcoitoiaoc
Wnnrflu
( "i IVT-'i-i I..1 !.,
MierabtictftiuBi
Propiimibacitriui
Em,
fiimiftii In
{G + C)'* of Uis phylum rs less lhan 5(W. The
AcliamyceWs whose (G + C)% is greater lhan
SCi iiclude other bacteria thai arc aLso important
n> ike food and beverage induslrics (Table 4.2).
The CUaiiiilhan branch ctMstso of three groups:
the liisl includes the Un'iiiixKilhis. Pednicocaa.
leiicowsitK. OentKocaa. and WvaWffl genera:
the second. Streptococcus ami Lictococcus:
and the third. Carnabtwteriunt. Mi&ikocciu and
Enierocixcia (Gasser el «/.. 1994).
Dicks ei id. (1995) proposed a new species.
OeiKKuccm oeat. for bacteria previously known
as LeuconiistiK oeixn, currently the only species
ii the OtntKticciu genus. This proposition was
based on the phylogcactic distance of 0. oeni with
icspccl lo other lactic acid bacleria.
The homology of genomic DNA permits the
definition of haclerial species by their nucleotide
sequence. The homology Is measured by the
icassociation percentage between strands of DNA
from the strain u he classed and a type strain
of the species The strands air isolated by DNA
ileu am rat Hin Two bacterial strains belong lo the
same species if the hybrid! ration percentage is
greater than Kfi . For a lesser value, the strains
arc part of the sane genns. on the condition thai
the hybridization remains measurable
Nucleotide sequence companion can be carried
oul on portions of the genome rather than the
cutircgcuomc. The chosen portions correspond with
essential functions, common to the strains to he
compared. The genes encoding Ihc synthesis of
nhosomes. particularly for the ribosomal RNA (a
conserved molecule), arc a noteworthy example
Several types of analysis are possible In one of
them, the 16 S RNA is affected by the action of a
restriction endonuetease The liny oligonucleotide
fragments smaller lhan 20 bp arc separated by
electrophoresis and then sequenced, permitting the
creation of a data hunk Sequences specific ft) groups
of bacleria can be Rlcntiticd in this manner. The
similarity coefficient between strains can also be
defined.
Another type of analysts consist of sequencing
the 16 S. 23 S. and 5 S RNA The 16 S RNA
sequence can provide the most interesting indi-
cations. Il contains conserved rones and variable
rones: Ihc comptireoi of conserved rones is valid
for distantly related bacleria: variable /one com-
parison can be used on closely related bacleria
Sequencing of the 16 S rRNA divides lactic ucid
bacleria into ihrcc pbylogcnclic groups:
1. The gioup LacmbttcHlus delbrueckii contains
this species and other slriclly homofermcntallvc
lactohacilli.
2. The group Leuconmtoc is divided into two sub-
groups: one containing L ptmmeseiUemides
and hclcmfcmientillve lacbihacllli: Ihc other
containing LeucomsUK sensu sm'cio. «j which
OenKOCCia tieni beings (although Individual-
ized).
3. The gioup hicinbtxillus auei—PettitKoccits
Is a more heterogeneous group since It com-
prises strictly and facultilivcly hclcrofcrmcn-
talive species and strictly homofermcntallvc
species.
Grouping by means of 16 S rRNA sequences is
based on phylogcnctic rckilionships between bac-
leria. It docs not support Ihc grouping realized by
using phototypes, snch as morphokigy and physi-
ology. Al present, then-lore. It is difficult k> class
lactic acid bacteria if referring lo both ihc pheno-
lypc and Ihc genome. Physiological and biochem-
ical criteria remain useful, but the contribution
of molecular taxonomy Is considerable and seems
more absolute since it Is directly rckiicd *i ihc
gciclic heritage of a strain
Handbook of Etiology: The Microbiology of Wiie anil Vindications
4.2.2 Classification of Wine Lactic
Acid Bacteria. Description
of Genera
The lactic acid bacterid or grape must and wine
belong lo the genera Un'tubttcilliis. LeucimotUK.
Oemieoccus aid Pediitcoeciu. Besides their mor-
phology In coccal or rod-like tonus, the homofcr-
mcnlalivc or hctcrotcmicitalivc character is a
deciding (actor in their classification Homofcr-
mcnlativc bacteria produce more than 85'* tac-
tic acid from glicosc. HcKrofcrmcnuiivc baclcria
produce carbon dioxRIc. ethanol aid acetic acHl )■
addition to lactic acid.
Among the cocci. Ihc baclcria from the gems
PediociKciis an: homofcrmenters aid those from
ihc gcicra leucimosioc aid Oenocaccus aic
hclcrolcrnicnlativc.rhc loclobacilllcan present the
two behaviors They arc divided Into thicc groups:
• Gimp I: strict homoferiKntcrs (this group has
never been Klentllicd in wine).
• Grotp II: facultative bcKrofcrmcntcrs.
• Gronp III: strict bete role rate liters
The strictly homofcrmcitilivc lactobacilli do not
ferment pentose, and form two molccilcs of
tactic acid from one molecule of glucose by the
Embdci-Mcycthoff pathway.
In facultative hclcrofcmicilcrs (Group II). one
glKOse molecule, as in the case of Group I. leads
10 two molecules of taciic acid, but the pentoses
arc fcrmcilcd iiu lactic and acetic acNl by the hct-
erofcrmcitativc pentose phosphate pathway The
strictly hcKiofcrmcncillvc bacteria ii Group III do
not possess the fructose 1 ^-diphosphate aldolase
that R characteristic of the Embdcu-Mcycrhoff
pathway. They ferment glucose into CO), lactic
and acetic acid, and ethanol by Ihc pentose phos-
phate pathway, and pentose into lactic and acetic
acid in the same manner as bacteria from Group II.
Table 4 3 lists the taciic acRI bacteria most often
encountered in grape must and wine. Oenococeus
iieni is known for ensuring ntakilaciic fcrmcnla-
lioi in the great majority of cases. So far. the
strictly homolcruicntativc tactobacilli of Group I
have not been isotatcd ii must or wIk. The species
arc Iherefoie divided into facultative and strut
hctc tolerate tilers for lactobacilli and into bomofcr-
mcntcrs(Peifi<"i"«rn«)aid bctciofcmcntcfslto'-
eotuatoc) for cocci It is likely that this classi-
fication will he modified— on one hand due to
anticipated progress in the Nlcntificaliou of new
species ii wine, and at the other haid die lo even-
tual reclassiricatiots of lactobacilli )■ the gimps
described above.
No lactic acid bacteria possess cytochrome The
catalasc activity is gcncially assumed not lo exist,
but several species of bacteria { Lactobacillus.
PetUnciKcus and LeiKiHtositK) cai synihcsi/e a
manganesc-dcpendcil. non-hemic pscndocalafcisc.
A hemic catalasc activity has been identified in
many strains.
The following is a general description of three
genera of lactic acid bacteria in wine.
Tjbk-4.J. I.ai tiiihcn
Facukatrvi
: bncmlcrmcait
b Lattobo
rilla* aaei
iMftfctcili
iCnmp II
)
Uniobix
dlla \ plinitaatv
Slnct hoc
mteraieiuen
rillat twin
(Grnup II
1)
Uxtabc
Paliara
att*\liilg.r>lil
cm*tfnm>vw
Httmolcm
icMcn
Cocci
Palinca
m* **,**«*
Leucoimanc <miu ( Ottwcaccus otai)
Hacmfcn
IinvIK meirtiieroidtt
1U1M1V .-.'i'Ai'.'I.'i i'i.'.M
Laclic AcMI Bactcrii
Genus teuconoxtoc Oeiiacocciis
• Nou- mobile, non-sporulaling. spherical or slig ta-
lly elongated celt, assembled Id pairs or
small chains: diameter 03-0.7 jim. a length
0.7 12 pm
• Facultative anacrobiosts.
• Cbcmo-organolroph: rcqnircsa rich medium and
fermentable sugars.
• Optimum growth icmpcraliic 20-30"C.
• Mcttbolic product* of glucose: CO;, laclic acid
aid cthanol.
• Argininc R metabolized by ccrciln strains of
OeiioctKcus oew\ whereas other Leuconostoc
species respond negatively to Ihis lest.
• (G + C)*i from 38 lo-l-H
• No leichoic acid
Genus Pediococais
• Non-mobile, non-sporulaling. sometimes tso-
btcd. spherical ( never cktngakrd) cells: diameter
1-2 pit: division in rvvo righl-anglcd planes
which leads to Ihc formation of Ktrads — no
chains.
• Facultative auacrobiosis
• Chcmo-organotropfc: icqnircsa rich medium and
fermentable sugars.
• Mctibolic product, of glucose: di. or i. laclic
acid, no O0 2 .
• (G + C)'i from 34 lo 42*.
• No leichoic acid
Genus- Lactobacillus
• Non-mobile, non-spornlating. regular elongated
cells. 05-1.2 pm by 1.0- 10 p.m. oficn long
rod-like forms. Some arc wry small (nearly the
smtc dimensions .is /i-i-riirnvi/'v Assembled in
pairs or in variably si/cd chains
• Pacnltalive anacrobiosis.
• C hemo- organotropy. : requircsa rich medinmand
fermentable sugars.
■ Fermentative metabolism: at least half of the
products of the metabolism of glucose is laclK
acid. The homofcmicitilive metabolism leads
lo this sole molecule. The hcKrofcrmcntativc
metabolism also produces acetic acid, cthanol
aidCCv
. (G + Cfi from 36 lo 47».
• Many species contain leichoic acid in the cell
wall"
4.3 IDENTIFICATION OF LACTIC
ACID BACTERIA
4.3.1 I .^ IH'l.il Pi ilKI|,I.S
Since Ihc beginning of microbiology, the iden-
tification of bacteria has been based on their
phciolypic characters (Section 43.2). Besides by
its morphology, which gives link- information, a
strain is identified essentially by the substrates and
products of ifs metabolism When more discrimi-
nating analytical methods appeared, the chemical
composition of mKroorgau cutis (fairy acids and
proteins. Section 4.38) abo participated in their
identification.
More recently, and in a spectacular manner,
the tools of molecular biology have made the
identification even more precise at the genus and
species level and even within the sane species
For a long time, lactic acid bacteria of wine
were identified by their phenotypes. Now. with
DNA analysis, more reliable results aic obtained
(Scctioos4.3.3-4J.6).
Identification by phenotypic analysts of clones
isolated in wine often poses two kinds of problems
First, these clones arc difficult to multiply in
laboratory conditions Numerous sub-cultures arc
needed to obtain a sufficient biomass to carry out
all of the ursts For the same reasons. Ihc response
to biochemical Ksts in the API tcse. t Sec lion 4.3.2)
can be ambiguous. The change of color of the
indicator is not distinct if the strain docs not
multiply sufficiently in Ihc microtube. Second, the
phenotypic character, such as the assimilation of a
126
Handbook of Fnokigy: The Microbiology of Wine anil Vindications
snbstralc of the formation of a panic ifcir product,
represents Ihc mull or a mctibolic chain ihai
depends on Ihc entirety of cell enzymatic activity.
For a pbenorype lo be positive, all of (he genes
encoding Ihc en Allies of Ihc particular chain must
be expressed: ihc en ■■> mes musl also be functional.
The indnciion or repression of enzyme synthesis
as well as inhibitions dnc lo certain medium
conditions can Ihcrcfoic modify a phcnolype.
The DNA coti position of strains is stiiclly
specific. Il is not influenced by cnlturc conditions.
Il can. however, undergo punctual mutitioas
over generations. Al the laboratory culture scale,
these inn unions do km significantly affcel the
i! c iion iii- DNA characteristics Strain klentiricatkHi
by genomic analysis Ihcrcfoic appears to be the
■>ost reliable approach. Several types of analysis
cxisl which permit diffcicnl levels of identification:
strain, species, genus.
The general principle consists of looking for
similarities between the DNA of the unidcntilicd
strain and the DNA of the reference strain There
arc several methods based on various bwls aid
propenies of Ihc DNA molecule. The study of
restriction polymorphism Is bused on Ihc specific
action of rcslriction enzymes Hybridization Is
bused on the ability of single-strand DNA chains
K> reassemble in double-strand chains. Combining
these two methods and varied uses of each method
considerably broaden the possibilities of analysis
Finally, polymerization chain reaction (PCR)
permits Ihc amplification of portions of ihc genome
delimited by markers. These markets arc primers
(oligonucleotides) which musl hybridize with the
DNA matrix for amplification »» skin. Depending
oa the printers chosen, the elcctrophorclK profile
of Ihc amplicon obkrincd can. pcrniil difl'cicnl
k-vcK of classifkalion within Ihc genus or the
species.
4.3.2 Phcnutypk- Analysis
Phcnolypic analysis encompasses morphology, the
assimilalion of diverse sibsiralcs and Ihc nalurc of
metabolic product.
Morpholog nal observations can be made with
fresh cells but Ihcy arc more prccisc with fixed
preparations. Microscopic observation can be cou-
pled wilh Ihc Cram coloration lest, which is used
to verify lhal hacicria arc Gram- positive. Alter the
hacieria arc placed oa the slide and diicd by the
ftinieol a Hunsrn burner. Ihc preparation Is dipped
first in a violet colorant, then in an alcohol-acetone
soln lion, and Dually in a rose colorant The cell
wall of G rani- positive bacteria is not altered by
Ihc organic solvent: these bucKrria renin the viofcl
coloration. Convene ly. G tarn- negative bacteria arc
lose colored Cell form, whether coccal or rod- like.
Is easy to identify, as is cell arrangement (pairs,
iclrads. small chains).
Secondly, the homofcrmenutive or bctciofcr-
men tilrvc character isdclcrniiucd. The unidcntilicd
strain Is cultivated In a medium with glncose as
the energy source. After cell growth, the metabolic
piodnclsaie characterized and measured A release
of CO; manifests the hetciufcmicntative character
of the strain. This result is regularly confirmed by
measuring acetic acid and ethanol concentrations.
Their presence is also pioof of a hcicrolcrmcnui-
livc metabolism Conversely, the exclusive forma-
tion of tic tic acid attests lo a homofermcntilivc
character. In culture conditions, facultative hct-
eiTifcnncntallve bacilli (for example. Lactobucillits
plaiittmotif have a homofcrmcutitlvc metabolism
wilh rcspect la glncose.
During the same test, it Is iilcrcsting lo deter-
mine the optical nature of lactic acid formed from
glncose. This analysis makes use of an en zymatK
process. The two stereoisomers of lactic acid: (i.
and d) arc analyzed separately This form of anal-
ysis is particularly adapted to the identification of
bctcrofcrmcnuilivc cocci {Oenococeus item. Leu-
eontatoc nieseittenntles). which only fom the i>
isomer, and of Itictabiicillus casei. which only
forms i .-lactic acid.
These initial investigations permit baclerlal
Idcntilkalion al the genus level: Lactobacillus by
morphology, aid PetSacoccus and LeucontuUK
by morphology and determination of their
homofcrmcnlativc or bckrofcnncntaiivc chancier
Classification al Ihc species level makes tse of
the analysis of the fermen tnioa profiles of a large
number of sugais.
Lactic AchI Bacteria
12"
For this lypc of analysis. Ihc API 50 CM.
idcutiticalioa sysicm (Bio-Mcricux) is commonly
used In this system, the classic Ksfc thai were
once performed Id test lubes ate miniaturi/cd The
on identified strain is inoculated Id »» a nutritive
medium that contains all of Ihc nitrogen- based
nutrients, vilauiins and salts necessary for its
growth. Different carbohydrate energy somes arc
represented In each niktutubc of the sysicm. In
this manner. 49 substances arc tested, including
nexuses, penuscs. dlsaccharides. etc. An IndKaur
in Ihc culture medium, which changes color,
facilitates Ihc reading of results Fermentation in
a mKiotubc acKIUics ihc medium. provoking the
indicator lo change color.
Tu carry ou i the API KSI.O.I ml off* ictc rial sus-
pension is dcposilcd in each of the SO microlnbcs
The lubes aic seated with a drop of paraffin lo
ensure anacrobiosis Generally. Ihc sysicm is incu-
bated at 15 'C for 24 hours Tubes in which Ihc
cokir changes from blue *) yellow IndKale pus-
ilivc characters. In this manner. Ihc fermentation
profile of the examined strain can be established
(Figure 46)
This method iswclladapKd for the identification
of numerous lactic acid bacteria, but il musl be
carried out very carefully with bacteria isolated
in wine Experience has shown lhal Ihc strain
should undergo several successive sub-cultures
in the standard laboratory medium beforehand
lift 4.0. BkKbcakalwbwra
robin (API 50 CHL gallery) of t°» bclK acid ba«ci
iik
Handbook or finology: The Microbiology of Witc anil VnifkatkMS
This preparation is cxscbIi.iI Tot obtaining profile
.stihiliiy . which Is indispensable before referring to
ibc Klcnlilicaiion key In any case, a strain cainol
be identified solely on Ihesc tesulLs. The method's
disc tint mating powcis aic mi sufficient and the
similarity in profiles of Ijicitibtia'llus plamantm
and tsruciHUisKK mexeiHermiles dcmonstralcs litis
point All of the other phenotypes previously
described should also be lakcn into coasidc ration
at the same liiK.ThcsccharacKrisasawholcmakc
the determination of ;i species possible without too
ninth ambiguity, by referring w Ber&ey's Manual
if Detentiimiire Bacierinlogy (19B6).
Tables 4.4 and 43 siBiiiari/c the pheno-
lypK characlcrs used to determine genus aid
species. Most of the strains for each species have
Tabic 4.4. Dcicm
kin uf the peiu* «f Utile acid buct
tabled in oil
Eb«paicdcclb (bacilli)
Cell iiRtn^cmcn
Small cbaiM.
HctcintcimcnlJiii-
Ijtiohtrliltii
(Onwi)
rau)
Tubk-4.5. Dacrmim
Bixierialng). 1980)
lb.:
if nine Uclic
acid hjacrii
i spec«\
andninukr
npccfc
* IB"*-/' Mtmtti of Swnutic
Spnfe r
«■"
e Oknm Ci»i
xlme Cihxn
Ml MM
™ *'" w
xyhw
: r.Miiliw liidKack) uirat
Imtuivih Mima <Mii<li>tiw
ft wpiiuit
1 , ptHiivc: . iirjathc: \ v
ptiXa ( .mi
Lactic AchI BacKiEi
[29
fermentative profiles which correspond with those
listed in Tabic 45. Nevertheless, besides differ-
ences that can be introduced by ihc prc-cullurc
of ihc strain befote ihc lest. Ihc niorc pronounced
iniraspcclnc variability of ccittin characters musl
be tiken inio account For example. Leueonottae
lin particular OenvctKi'M oenii was long though!
■Dl lo possess the argininc hydrolysR character, bul
recent studies have proven that numerous strains
of Oenocaccus iieiu possess all of the necessary
enzymatic equipnieni » hydroly /c argininc. lead-
ing to Ihc prodnction of cilrullinc. ornithine and
carbamyl phosphate (Lin etal., 1994; Makaga.
1994). The hydrolysis activity depends on envi-
ronmental conditions which determine not only the
enzymatic activity but also its synthesis. Further-
more. Ihc arc operon. conttinlng genes coding
for the various enzymes in the metabolic path-
way, has been idcntilicd and sequenced in strains
of 0. mm (Tonon el of., 2001).
The use of fermentative prolilcsasan Mlcntifica-
iKm method should therefore tv standardized Bac-
El il characters can then be expressed in the most
reproducible manner possible Finally, these KsLs
place baclcrEt in optimal growth and metabolic
conditions— they give no indication of their true
metabolism in wine. The fermentation of a car-
bohydrate foind in the API SOCHL system can
be tocilly impossible In wine: its nutritional condi-
tions are far from those in the synthetic medium.
Conversely, a substrate that cannot be mctibolizcd
In optimal conditions can be metabolized in wine
becanse of the totally different metabolic regula-
tions in pfcty.
4.3.3 Extraction and Visualization
of DNA for Genomic Study
Before analysis. Ihc entire genomic DNA of
txiclciia musl be separated from the lipids, glucidcs
and proteins constituting the cell. The extraction
protocols for lactic ackl bacteria include all of
Ihc stages of cell lysis, dcprolclnlzation and DNA
precipitation. There arc slight differences between
Ihc protocols ILonvaud-Futcl el id . 1989). Lysis
of Gram-positive cells is obtained by the action
of lysozymc. The pcptidoglycans arc hydrolyzcd
10 form proupfctsts. which arc submitted ta the
action of SDS — a powerful detergent that destroys
Ihc membranes and liberates the cellular contents
The addition of phenol, most often mixed with
chloroform and isoaniylK alcohol, precipitates the
proteins The organic and aqueous phases arc
separated by ccntrifngation The denatured proteins
assemble logclhcr at the Interphase. The lower
phenolic phase contains the lipids and proteins: Ihc
upper phase contains Ihc dissolved DNA
The phenol is climinaKd from this phase by suc-
cessive extractions with a mixture of chloroform,
alcohol, and isixunylk alcohol. The DNA Is pre-
cipitated by cthanol in the presence of salts. It
can be stored at -20'C after being dissolved in
a buffer
E Ice irophorcsR Is Ihc most popular, simple and
reliable technique for analyzing DNA extract. At
an alkaline pH. the DNA phosphate groups arc
Ionized. The molecules placed In an electric field
therefore migrate towards the anode. In a viscous
gel (most often agarose), the elccirophorctic mobil-
ity depends on the size and conformation of the
molecules. The smaller the linear molecules arc.
Ihc faslcr Ihcy mlgralc. Circular molecules of equal
size are less mobile than linear molecules Plas-
mlds. for example, exest in circular and colled cir-
cular form. The size of linear DNA is easily calcu-
lated from the migration distance of DNA weight
markers There is an inverse relationship between
the mobility and the logarithm of the molecule
size. Molecules separated by electrophoresis arc
revealed by an cthidium bromide bused coloration
This compound isan analogue of an aromatic base:
11 intercalates In the DNA and fluoresces orange
under ultraviolet light
4.3.4 Identification Based on
Restriction Fragment Length
Polymorphwn
This method consists of hydroly zing the DNA with
the help of restriction enzymes. These enzymes
produce different sized fragments which arc sepa-
rated by cIccUophorcsBt. The clcctropborclic pro-
file differs depending on the strain. The enzymes.
i.;i>
Handbook of Etiology: The Microbiology of Wine anil Vindications
Id l. m. act on specific sites, recognizing palin
dromic sequences of generally lour io scvei
nucleotides on Ihc two DNA strands
The firo Rl ciiAiR-. for example, hydrolyze
DNA accoiding in Ibe following schema:
C-3'
S'-G |AA1
3'-C TT AA| G-5'
Depending on the sequence recognized, ihc
number of sites cui on Ihc polynucleotide varies,
bui ii is always Klcnili.il for a given enzyme aid
■■cleolktc. On Ihc one baMl. different fragments
varying in size and imbcr can be obtained
fion ihc same DNA by using a variety of
restriction cn/ymes available. On the other hand,
the icstriclion of different DNA by the same
enzyme leads to different sized moments
The rcstrictm product) arc analyzed by elec-
trophoresis. The characteristic profiles ate obtained
after revclalioa by coloration Considering the
nimherand sequence of the nucleotides, enzymes
that statistically cut the DNA too often will pro-
duce complex prollles that are difficult to study. If
tbc number of cnt sites Is very low. the profiles
arc simpler bnl the length of the fragments pro-
duced requites the use of pulse- field clccirophorc-
sr In oidcr to separate them This technique is very
reliable and well adapted for the identification of
yeasts but remains very difficult to use for bacteria
(Daniel. I993>.
Restriction polymorphism is not relevant for the
Rlcnlilicaiiou of bacterid species. No profile type
exists for OeiuKocais oem. for example, nor for
each of the other species that arc of interest in
wincmaking. This method seems belter adapted to
the differentiation ofstiains of the same species. It
Is thus easy to identify strains of 0. aeni. following
hydrolysis of their DNA by the Noll enzyme with
rare restriction sites This method is used to mon-
itor development of inoculated selected strains,
used during w incmaking to promote malotactic fer-
mentation The restriction profile of the biomass
collected in wine during malolactK fermentation
Is computed with that of the selected strain that
was added (Gindrcau el of., 1997; 2003).
4.3.5 Identification by Specific Probe
DNA/DNA Hybridization
Hybridization is a tcchn(t|K often used in molecu-
lar genetics and it is very well adapted for the iden-
lification of species and even strains. The technique
Is based on the ability of double-strand DNA to
separate, rcvcisibly. into two single sltands. in cer-
tain conditions that destroy their hydrogen bonds.
Along with the ionic force of the medium, the
temperature is Ihc determining parameter of DNA
dcnalnraUOa. A temperature increase provokes the
separation of the two strands, which teassociale
when the temperature diminishes once again.
In favorable environmental conditions, the chains
can rcassociatc if Ihc nucleotide sequences present
arc complementary For example, the oligonu-
cleotides of the following sequences recombinc: 5'-
ATGCAATTGGCC-and 3'-TACGTTAACCGG-.
A hybridization cotsws of two single strands,
each coaling from different cells, rcassociating due
to their complementary sequences. This property is
used for iden miration and strains arc considered
to belong to the same species if they have a ~l(ft
homology of their genomic DNA sequence.
The DNA of a reference strain is needed to
identify a sliain by DNA hybridization. One of
the two fragments r must often Ihc reference DNA)
constitutes the isotopic probe or the chemically
derived base analogue labeled probe A probe is
a single-strand DNA fragment that combines with
the complementary sequence of the target DNA.
These operations arc schematized in Figure 4.7.
The target DNA (the DNA of Ihc unidentified
strain! Is fixed and denatured on a nylon
membrane The membrane is then placed in a
hybridization buffer without probes During this
pre- hybridization step, all of the non-sprcllk
sites on Ihc nylon are saturated by a mixture of
macromolcculcs These molecules have no affinity
for Ihc probe. The hybridization takes place in the
same physKochcmical conditions after the addition
of the marked DNA probe. Al Ihc end of this
step, the single strands of the DNA probe will
have strongly combined with the complementary
tatget DNA. but also more weakly wilh DNA
having less similar sequences RcvclatNw is used Io
localize the tiigct DNA that truly corresponds with
Lactic Ackl BatKiEi
/ 7
/ a a ti u u & f\
"O^J
* 1
7
/
ISJJ'IJ Si
I'lfi 4.7. SvhcniJiH' ilia|!iiB t>l ibc gcncal prim if 1c fur (he iikailk-Mioii uf belie iicitl bacicrn uihijj *fc<.ih<
DNA/DNA pn*c
hyhnds of strains or ihc same species. Therefore, purlkipaic In the specificity uid ibc sensitivity of
Uis Mcp is performed .ilfcr the diminution of this method Tic icvcfclltoa process makes use
Ihc piobc aid or ibc DNA strauls that present or autoradiography fot the Lsolopic pn>hcs and
little homology Successive washes with ionic Immiiocn/ymatic reactions Toe the non-tsofcipK
bnfrersof decreasing force air vciy imponaiiland piubcs bksi often used.
132
Handbook of Enotogy: The Microbiology of Wine anil Vwifications
Depending on ihc problem w be resolved, ihc
pcobc is prepared from cither the enilic DNA or
a specific DNA fragment. In the first case, the
species of an ■■known strain can be identified.
In Ihc second case, strains possessing a specific
gene and in consequence a characteristic functional
property can It identified.
Most tactic acid baclcriaor wine can be identified
al the species level in this manner The lirsl
working probe was developed for the species
OtntKOCtus •"'".'. It was created by using the
ucd DNA of different strains taken as references
(Lonvaud-Funcl Hal., 19(9). The DNA probes
of oem do not hybridize with the genomic
DNA of other species; the inverse is also true.
The presence of the O. tieni species of bacterium
can he identified even in a mixture containing
other bacteria Subsequently, this method also
proved to be well adapted for other species fond
in grape must and wine (Table 46) (Lonvaud-
Funcl etitl.. 1991b). However, the similarity of
the /- l/ilgiinbi and /. brevis species necessitated
the development of a more specific probe targeting
L /Bfjrmir (Sofcicrrt rtf.. 1999).
By hybridizing Ihc probe directly with the
DNA of bacteria colonics, this method becomes
considerably more inlcrcsting than the previous
Kchniqnc of hybridizing Ihc probe with the DNA
extracted from the strain and then placed on
a membrane. In the new tcchnK|K. the nylon
membrane is placed on Ihc surface of a Petri dish
after the development of colonies. It is then treated
successively in diffcrenl buffers and reagents
which provoke Ihc lysis of Ihc bacteria and the
immobilization of the DNA on the membrane. The
prchybridi/alion steps follow; hybridization and
washes arc then carried out In these conditions, a
mixed population of laclK acid bacteria can easily
be studied. In fact, afleran initial hybridization of
the membrane with a given probe, dehybridizalion
and then rehybridizauon with a second probe
permit Ihc localization of clones belonging k>
another species. At least five different species
can successively be detected in a mixture wilh
this system (Figure 4S) (Lonvaud-Funcl ei irf .
1991a). Thanks lo Ibis method, by preparing
probes representing the eight species most often
encountered in etiology, the dynamics of each of
Ihc species were studied during winemaking for
Ihc first lime.
DNA/DNA hybridization is also an excellent
lool for identifying strains thai differ in phcnoiypc
bni belong to Ihc same species The difference
rests on a metabolic fanction which depends on
the presence of one or more enzymes and therefore
ihc presence of the corresponding genes In this
case, the probe is prepared from a DNA fragment
representing all or pari of the gene
Al present in cnology. two particular cases arc
analyzed in this manner: strains of PetSococeus
ikaiuu'Siis. responsible for ropiness disease, and
strains which produce histamine, notably 0. oeni.
Preliminary stndics have shown that P tkmmmis
strains capable of synthesizing Ihc ropy wine
polysaccharKIc possess a supplementary plasmid.
contrary u> normal strains. The ropy character
is linked lo the presence of this plasmid. A
frag men I was cloned in £ i ■■"> and now constitutes
Ihc base material for preparing Ihc probe. In
this manner, colony hybridization permits the
idc limitation of ropy' clones even when mixed
wilh other Pedtocaccus clones or other species of
bacteria. This method is routinely used lo identify
this undesirable population in Ihc microflora of
wines at fhe end of winemaking and during aging
iLonvaud-Funel <•/«/.. 1993).
The other cloned specific probe is prepared from
a gene fragment of hisiidinc decarboxylase. This
enzyme catalyzes the decarboxylation of hisiidinc
into histamine. The hybridization of a bacterium
with this probe, to more than half of the gene
length, signifies that Ihc strain possesses the gene
(Le Jcune rtrtf.. 1995). The presence of these
strains in wine most likely increases the histamine
concentration. Daring winemaking. and also aging,
these strains in specific can be counted by colony
hybridization.
4.3.6 Identification by Polymerization
Chain Reaction (PCR)
PCR consisb of using polymerization lo amplify
one or more DNA fragment, localed by specific
sequences. The obtained product exists in sufficient
Lactic AcMl Batlcru
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Handbook of Etiology: The Microbiology of Wine anil Vinificalions
l-'ifc 4.K Spccitc ls,i i. acid baclcra pofubiina counts
by hyhridimian tin colonic* uUh reference DNA
probe*. U) h>brkli/aiioa on tobtkt ofcukurcd Own-
cdcom MiT. (b) detection of Lhilp:fdii colonic*
In a mix of 5 ijnii (" oetti. X. rvnrrnnvuifri.
/'. IJUTKM.'Jl. /_ pitl/Ul um . L. l/ilgiriJii (. I'cslll
obuincd afier 4 ukccmivc bybrkliaitoiu. ami deny-
bridinrrkim a(h probe* (mm fouroi net *pccic*
quantities ki be easily repeated by electrophoresis.
This method includes an enzymatic reaction fot
the synthesis of DNA which requires primers and
a Icniplulc. The polymerase copies the DNA target
stilting from the primer al 3' towaidsS'. The PCR
technique KHCs two oligonucleotide primers, cho-
sen foe their complementary sequences: each one
is complementary to a single strand of the DNA
tugcl Synthesis is carried out between the two
primers by a polymerase The cxtcnsional product
of one of (he primers serves as a template for Ibc
other alter dcna(u ration. The repetition of cycles
comprising primer annealing, extension reactions
and dcnatiralion leads lo an accumulation of iden-
tical neosyuthesi/cd molecules, flanked by the cho-
sen oligonucleotides (Chapter I. Fignre 1.21).
For the three steps of the amplification to be suc-
cessful and to avoid complicated manipulations of
the sample during multiplication, a temperature-
resistant cn/yiK* is necessary The use of the Taq
polymerase icsolvccl this problem: it is ihcrmorc-
stsEinl and functions at elevated lempcralures up
<ol2'C The cycles are therefore repealed, gener-
ally 30 to 40 times. A large quantity of specific
DNA fragments determined by the primers are
produced in this manner Theoretically. 2" target
fragments are obtained after n cycles The auu-
malic equipment currently in nsc permits the dif-
ferent parameters of the three steps of each cycle
to he programmed. From a single copy of a DNA
fragment a sufficient number of copies can be
obtained in older lo view cosily after cfhidinm
bromide coloration. The products of PCR ate ana-
lyzed by cIcctiophofcsfA. If a fluorescent band Is
revealed with the expected sine, this means that the
DNA matrix contained the sequence identified by
the primers Thus, within a genome with approxi-
mately two million nucleotides, like thai of Ortui-
coccus tx-iu. it is possible in determine whether a
gene or gene fragment several hundred nucleotides
long is present or not.
The specificity of PCR is based on the level
of hybridisation between Ihc oligonucleotides and
the template It therefore depends on the primer
sequence and length, the ionic force of the
medium, ihc temperature and the concentration of
Mg" ions. The choice of primers can Ijc very
precise when ihc sequence of the regions bordering
the /one u be amplified arc known. The use of
random primers also gives valuable results, when
Ihc buctcrEi genome is totally unknown.
The primary vuluc of PCR Is it. sensitivity.
The presence of a gene in a small number of
cells can generate a quantity of DNA which
is easily analy/ablc by gel electrophoresis. In
cnokrgy. PCR is also used to identify the two
problems previously described, through Ihc use
Lactic AchI BucKrii
lis
of ropy' strain and histamine- prod Being strum-
specific probes Tbc amplication reaction R very
specific: Ihc size of Ihc amplified fragment Is veri-
fied afcr electrophoresis These undesirable strains
of bacteria ait therefore detected in a mixture of
olbet bacteria, even if they ate few in number
PCR ampliricalkHi using a try ion of the histidine
decarboxylase gene makes it possible lo idcilify
baclcria likely to produce histamine, irrespective
of the species (Colon end.. 1998) Sequencing the
plasm id of baefcria Ihat produced glucanc led lo
Ihe identification of Ihc itps gene responsible for
Ihis effect Primers were selected for PCR delcc-
lioi of these baclcria directly in wine (Gindrcan
end.. 2001). It is now also possible lo detect
lactic baclcria thai break down glycerol lo pro-
duce acrolein (Claissc and Lonvaad-f-uncl. 20011
as well as those ihat decaitoxylale tyrosine lo form
PCR will soon have another application in our
domain for Ihc differentiation of strains of Ihc same
species. Random primers arc used for the moment
In Ibis case, the (tactions amplify several mes
of the backrium genome. After electrophoresis.
Ihe amplifKalion products inrush a profile thai
can be characteristic of the strain The difficully
lies in linding Ihe primers. The best adapted ones
for recognizing strains must give a profile for
each strain in a reproducible manner. Among Ihc
lactic bacteria in wine, this process has only been
applied lo strains of O oeni The main application
is in monitoring selected bacteria for malolaclic
fermentation.
PCR P. a useful tool, especially die lo ils
great sensitivity and speed. This method comple-
ments the colony hybridization method by spe-
cific probes, and the tvvo methods pcrniil the
early detection (PCR) and quantification (specific
probes) of bacterial strains thai alter wine. How-
ever, in Ihe near future, the more recently devel-
oped quantitative PCR in real lintc is likely lo
provide quantitative data with all the accuracy and
speed of PCR. In the future, other methods of
genome analysis will probably permit Ihc klcntitt-
catioa of species ksscommon lo wine with greater
certainly These species ait ink-resting because
of their inctibolism or their itsistincc to wine
stabilization processes. Ribotyping. for example,
consols of hybridizing Ihc genomic DNA with a
probe prepared from the DNA encoding ribaso-
mal genes Before this process. Ihe genomic DNA
musi first be submilicd to the action of restriction
enzymes and undergo separation by electrophore-
sis This method, which permits ihc analysis of
simplified hybridization patterns, has been used lo
slady a few strains of /. hHf,imhi and L foievts
Profile types permit the classification of strains inlo
two species thai truly correspond with the habit-
ual phenomena described (Lc Jcnnc and Lonvatd-
Fnncl. 1994).
4.3.7 Identification by Fatty Acid
and Protein Composition
Besides Ihcir phcuotypK characteristics, the pro-
tein and Tally acid composition of baclcria is also
dcKimincd by the mass of information in Ihe
genome and can. therefore, be used for identifi-
cation purposes. In both cases, these components
result from a succession of genetically determined
syntheses. Differences in the tally acid and prolcin
composition therefore reflect differences between
strains They can possibly even lead to idcntilica-
n. ■■■ of genns and species.
The lolal Tally acids are dosed in the form
of cslcrsaffcr saponification The analysis makes
use of gas phase chromatography (Ro«s. 1993)
Even if this analysis Is reliable, ii musi be
used with caution for identification, in fact,
several slndics have clcaity proven thai, for a
given luetic bacterium, the same fatty acids art
always represented, but their proportion varies
significantly according lo Ihe cellular cycle phase
and even more so the pnysicocbcmKa! growth
conditions. Modifications essentially concern the
level of saturation and Ihc length of the carbon
chains. Moreover, for a given species, strains art
capable of synthesizing very long-chain fatry acids
(more than 20 carbon aloms) to adapt togrowih in
an alcoholic environment iDescns. 1989: Kalmar.
1995).
Badcr&i can therefore only be identified by their
composition in lolal fatly acids when the culture
of the cells to be analyzed is standardized. Even
136
Handbook of Etiology: The Microbiology of Wiac anil Vinificitions
if Ibis method docs not seen easy to use. il
aieri is being mentioned In lie genus Pettiac/KCiu.
it was used to characterize Ihrcc groaps ia
wbicb six specks arc classified. P dimiiu'sta aad
P pentosaceiu. encountered ia cnology. belotg
to two of ibese groups Tbc authors of Ibis
work (Uchlda and Magi. 1972) observed thai
culture age and environmental conditions modify
the pmpoitions of tally ackls withoul affecting the
separation of the groups
Bacteria cell proteins constitute another level of
genomic expression. The amino acid scqacncc of
proteins is Ibe resaltol.idln.-it Iraaslalion of gcaes.
Il is therefore normal h» distinguish baclcria from
one another by Ihc proteins thai they contain. The
primary siruclarc determines atotccak: mobility
in an clcclropfcoiclic gel in conditions where the
secondary, tertiary aad quaternary structures air
denatured This identification BKlhod therefore
involves subjecting Ibe lolal cell coukrnts of
budena to electrophoresis. After slaining. the
protein profiles arc coaipuied cither vpually or
by coil paler- assisted analysis The clcclrophorctic
profiles arc reproducible. They arc slaadardi/ed by
marker", which arc required fc> compare several gels.
According u Kerslcrs (1935). prolein pn>lilcs
of strains arc identical when iheir UNA presents
a homology greater lhan 9W : they are very sim-
ilar up u TCf . Strains can therefore be idcali-
ficd in this manner at the species level. Ncvcrlhc-
less, as with the method asing fairy acids, all of
the following conditions mnsi be rigorously sian-
dardi/ed: baclcria cullnrc conditions; Ihc moment
of sampliag. cxtractiOB. and clccUophorctic proto-
col. Recently. Lie tic acid baclcria spoiling torn lied
wines have been discovered in this manner O oeni
(Dicks el <#.. 1993). aad diverse species of tactt-
bucilli (L luipmtit. L fmctmrnita. L eaUimmks
and /. "hill— the last three being rare) (Coulo aad
Hogg. 1994).
REFERENCES
Bounlincaud ).P.. Nchmc B.. True I. und Loimut
ftim:l A..f20aji.) Appl. Fjiiiron. Microbiol.. 09.2512.
Bounlincaud I.P.. Nchmc R.. Tcur I. und ljin.aud-
Juncl A . (2003b) fa . / Food Microbiol. 92. 1-14.
Chine O. aad Umvuud-fiincl A. 12001) J. Food Prai. .
64. 813.
Cocoa E.. RolaaCC.. Bed rand A. und Lonvautl-
Fuael A. ( 19981 A™. / Fitol. ItSc . 49. 199.
Cuurn JA.and ll.>j.-j T • h>«4. .'. Appl. &■**-»'.. 70.
487.
Daakl P. 1 1993) These dc Doctor*. Unlverske dc
Naatca.
Dclmu* ('.. 1'ieiit 1 . CukIickv I" . Divtc* C. aad
Guioi J. (2001) J. Mol. Microbial. Biotcdmol. .
1, 001.
DcscnsC. (1989) These dc Doctoral. UmvcBit dc
Boakau* II.
Dicks 1 M I" ..Dclbglb F.aad Collins M.D.(I99S)*».
/. Sytfnw BociaioL.iS. 395.
Dick. L.M.T.. ImfcxrPA. und AupB.ivnO.PJi.
(1995)/ Appl. Bmitrwl . 79.43.
Frcmau* C. Aajk M. and Lonvuud-Fuael A. (1993)
PJaandifi-.3D.2I2.
Gartxiy 5. (1994) These dc Docloai. Univenae dc
Boidcau. II.
Guscr F. M»«cl M.C.. Tabu R. und Chumpomrr M
I I99il i* U* BaeHriet Lnctieaei ud* H.dc Ruis.ua
und P.M. tu*ici I- lnricu. Uria^c. Fiance.
Gindreau E., Joyou A., dc Revel G . Clui.sc O. and
1-onvjud-fiinelA. 11997) J. *«. Sri. Mjyie Mil.
31. 197.
Gindreau E., Khclm H.. dc Revel G . Be. rand A. aad
Lonvaud-ruaclA. (20031 J. bi. Sri. M>»- Mb,
37.51.
Gindreau E., Wullinp E. aad Innvuud-FUoel A. (2001)
/. Appl. MkrabioL. 90. 535.
KalmnrZ. (1995) These dc Docioial. Uaivcndc dc
Bonleau. II.
Kcnrcis K. ( 1985) la Coinp^ier-<atit/eil Bi&eriii S\s-
leiaaics (ed* M.Goodfcllow und D£. Minakin).
Academic Pku. London.
U Jcune C. (1994) These dc Doelocu. Unnciatc dc
Bo«fcam II.
U JcuncC. and Lnnvuud-Kincl A. (1994) Food
Microbiol.. II. 195.
U Jeunc C. Unv.ud-Funel A. Ten Brink B.. Hubn
H. und Van dcr Vossen I.M-B.M. (1995) / Appl.
Microbiol.. 78. 316-320.
Liu SQ-. Piichuid G.G.. Kuidmun M J.aad Pikinc G J.
1 1994) Amer. J. FjioI. »iic..45. 235.
Innvuvid-Funcl A. (1986) Tbcsc dc Dociom ca
Sciences, rjnivcisac dc Boafcam II.
Innvuvkl-Hincl A.. Bacuu S" und Fremain C. (1989)
So. A'/m..9.S33.
l«nvuud-Kincl A. aad Desens C. (1990) Sri. Af.m.. 10.
817.
lnnvuvat-Hioel A.Jovem A.aad Lcdoui 0.(l99lu)/
Appl. BiKieriol.. 71.501.
l«Dvuud-fuaclA..Fn:muuiC..BheauN.undJi>>xui A.
( 199 lb) Food Microbiol.. 8. 215-222.
Laclic Ac hi Buckriu
lnnvaud-Fuael A.. OUIIkmi Y. and Imcm A. (1993)
J. Appl. Bucteriol.. 74.41.
MiUjsi E, (1994) lli fie tie Doctorit. Vnv.cn.ki dc
Rein.
Rojib N.. Giihjy S.. Deiuymllc* M. >*d Unvaud-
Miacl A. ( 1993) l*fi. »p/>A Microbiol.. 17. 126.
Sohici D.. Coutoo J. ltd 1-onnud-fiiacl A. ( 1999) ha.
J. Sy*. Bucirrlol.. A9. 1075.
ToaoaT.. Bounliacaud J.P. and Lonvwd-KiKl A.
(200 1 > Rei. Microbiol. . 152. 653.
I'.-biih K. and Miwi K. ( 1972) J. Geii. Appl. Microbiol..
18. 109.
Walliag E. (2003) Tfcoe Duciom. UnhcnU dc Bor-
dean.
Metabolism of Lactic Acid Bacteria
5 1 Gene lull lies— a review
5 2 Mctabolnni of sagais by kiciK acid bacteria
5 .' Metabolism of ibe principal organic acids of wine
5 4 Olher transformations likely (o occur ■■ wincmaking
5 5 Effect of ihc mcEibolism of lactic acid bacteria on wine compositio
qialiry
5.1 GENERALITIES— A REVIEW
Metabolism represents the biochemical tractions
of degradation and synthesis carried onl by (he
ruclcriacell during multiplication. Caiabolic reac-
tions piovidc energy, transforming subslralcs from
Ike environment or reserve substances of ihc
cell, anabolic reactions guarantee cellular synthe-
sis from environmental substrates and intermediary
cata holism products
Lactic acid bacteria are cacmotrophic: ihey lind
ihc energy required tor their entire metabolism from
Ihc oxidation of chemical compounds. The oxida-
lioi of sahstraKs represents ihc lora of electrons
that must be accepted by another molecule, which is
reduced. Most oxidations, simultaneously liberal:
protons and electrons. The transport of these two
particles to the final acceptor can activate a chain
of successive oxidation -rcdnc lions.
Thus the biological oxidation of a substrate
is always coupled with ihc reduction of another
molecule. In the following oxidation-redaction
reaction the oxidized substance is noted as
DH. and the final election and pioton acceptor
be A:
DHi »D + 2H' + 2c~+cnciBy <&■>
A + 2H' + 2e" » AH 2 <S2)
The overall reaction is:
DHj + A r AH 2 + D + cn«By. (5.3)
140
Handboak of Etiology: The Microbiology of Wiie anil Vinilicaiions
Tbc nature of ibc final election acceptor A
determines Ibc type of metabolism: fermentative
or respiratory. The presence of oxygen also distin-
guishes aerobic aid anaerobic microorganisms.
In acrobiosis. the electrons and protons are trans-
ported to oxygen, which Is most often reduced
lo water. This process is called aerobic respira-
tion. The transport system consist* of a group
of cytochromes The proton flux creates a pro-
ioi motive forte, which permits the synthesis of
ATP molecules. The conservation of the oxidation
energy is ensured by the synthesis of the pyrophos-
phate boad of ATP. This bond generates energy
when it is bydrolyzcd. This system does not exist
in Lie ik acid bacteria, although some species can
syilbcsirc cytochromes from precursors.
Some lactic acid bacterid rcdncc oxygen from
ibc environment by forming hydrogen peroxide
according lo the following reaction:
Oj + 2c"+2H' -
-H2O1
(S.4)
Hydrogen peroxide nasi be eliminated since it is
toxK. Cells that arc not capable of eliminating il
cannot develop in the presence of oxygen: they
arc strict anacmbes. Depending on their behavior
with respect to oxygen, lactic acid bacteria arc
classed as strict anaerobes, faculuiivc anacmbes.
mKroucrophilcs or acro*olcrunts. The distinction
between these different categories is often difficult
10 establish for a given strain.
Most lactic acid bacteria tolerate the presence
of oxygen but do not use it in cncigy- producing
mechanisms Depending on the species, they use
different pathways to eliminate the toxic peroxide,
activating peroxidases which nsc NADH as a
reducer: a supcioxidc dLsmntasc. a pseudo catalasc
and soniclinics Mn J ' ions (Dcsma/caud and
Roissan. 1994). To date, this subject has not been
specifically studied for species .~ > all .1 in wine.
If the final election and proton acceptor isa min-
eral ion (sulfate, nitrate), the microorganism func-
tions in aiacrobiosis. but a rcspiraUiy mechanism
R siill involved. This process is called anaerobic
respiration.
In aiaciobiosis. the reduced molecule can also
be an endogenic substincc— one of Ibc products
of metabolism This is the case witb fermentation
In lactic acid bacteria. Ibis molecule is pyruvate.
Il Is rcdnccd into lactate in Ibc reaction which
. li. 11.11. i.'ii/.-s lactic fcmicitilion:
pyravatc+NADH + H r lactate + NAD'
<5.5)
Contrary lo the rcoxidalion of the coenzyme by
tbc respiratory chain, this rcactKw is not cncigy
producing.
Other kinds of reactions can lead to ATP
synthesis. They occur in acrobiosisoranacrolnoMs
During these reactions. Ibc oxidation of the
substrate accompanies the creation of an cncigy-
rich bond between Ibc oxidized carbon and a
phosphate molecule:
XHj+|Pi|-
-P + 2H'+2c- I* 6 )
The cncigy of tbc cslcrphospho
stored in a pyrophosphate bond:
X - P + ADP -
- X + ATP (5.7)
In Ibis manner the pbosphoenolpyruvalc and the
ace ty (phosphate can transfer their phosphate group
lo tbc ADP in the same type of icaciion. Tbc two
intermediary molecules in the catabolisai of sugar
aie Ibeicforc very imporlant from an energetic
viewpoint
5.2 METABOLISM OF SUGARS
BY LACTIC ACID BACTERIA
Tbc oxidation of sugars constitutes the principal
cncigy- prod ucing pathway. This cncigy is essential
for baclcrial growth. In laclK acid bacteria,
fcmicitilion is the pathway for the assimilation
of sugars For a given species, the type of
sugar fermented and environmental conditions (the
presence of clcclion acceptors. pH. etc.) modify
lie cncigy yield and the nature of the tiial
products
The cytoplasmic membrane is an effective bar-
rier separating the external environment from the
cellular cytoplasm. Although pcimcable lo water,
salts aid low molecular weight molecules, it is
impermeable to many organic substances Various
Metabolism of Lactic Acid Bacteria
141
worts describe Ihe different active sugar trans-
port systems in lactic acid bacteria. 1 hey arc
for the bum put ATP dependent and activalc
enzymatic systems — sometimes complex. These
systems arc specific (o Ihe sugars being trans-
ported Hclcrofermcnbitivc baclcrta. parliculatly
Ihe species lhal interest cnoktglsts. have nol been
slad led ■■ depth, bat Ihe existence of active trans-
port systems using ATP-dcpcndcnt permeases is
highly probable.
Lactic acid bacteria of Ihe genera iiKlifbaeilha.
leiK'munliK and Petbtroccus assimilate sugars by
eilher a homofcmcnlaiive or bcicrofcrmcntaiivc
pathway. Among the cocci. Pediocvceus baefc-
ria are homofcmicitabvc. while UtKoaosme and
OeiUKnecm arc hclcrofcrmcntalivc. In laclobacilli.
bctcrofcrmcntcrs and homofcrmcnicrs arc distin-
guished accoiding to the pulhway used for hexose
degradation. Pentoses, when degraded, arc metab-
olized by be tcrofc mentation
5.2.1 Horn ofcrmenta the Metabolism
of Hcxoscs
Homofc men tali vc bacteria transform nearly all of
Ihe hexoscs thai they use. especially glucose, inlo
lactic acid Depending on Ihe species, cither the
i. or d lactic Isomer is foimed (see Chapter 41
The homo fc rate nlativc pathway or Ihe Embdct-
Mcycihof pathway includes a first phase confin-
ing all of the fractions of glycolysis lhat lead from
hexose to pyruvate During this stige. the oxida-
tion reaction tikes place generating the reduced
coenzyme NADH + H'. This pathway is used by
numerous cells l*or aerobic organisms, this path-
way is followed by Ihe citric acid or Kichs cycle
In lactic acid bacteria, the reaction of the
second phase characterizes lactic fermentation. The
reduced coenzyme is oxidized inu NAD' during
the reduction of pyruvate into lactate
The reactions of glycolysis are llsKd in
Figure 5.1 In the first stage, the glucokinase
phosphorylatcs glucose into glucose 6-P (glucose
6- phosphate). This molecule then undergoes an
■some riza lion u become fructose 6-P. Another
phosphorylation leads to the formation of fructose
1 ,6-di phosphate. At Ibis stage, the two mosl
important reactions have already occurred. They
activate the kinases which require bivalcnl ions
IMg* '.Mi 1 ') and use an ATP molecule each
lime. One of them, the pb(^phofructokin;tsc. an
alloslciic enzyme con in tiled by ATP. determines
Ihe speed of glycolysis.
The fructose 1.6-dlphosphalc Is then split inlo
two molecules of Irioscphosphak:. This reaction
is caialy/cd by aldolase, a key enzyme of the
glycolytic pathway Homofcraicntativc bacteria
present a high fructose 1 .6-diphosphalc aldolase
activity The products of this reaction are glycer-
aldcbyde 3-P and dibydtoxyaccmne-P.
Only glyceraldcbyde 3-P pursues the trans-
formation pathway The dibydroxyaccionc-P is
rapidly isomcrized into glyccraklchydc 3-P. In
reality. Ihe equilibrium between these two
molecules favors dihydroxyacclone-P. bnl It is
continually reversed, since the glyccraldcuydc .'-
P is eliminated by the reaction which follows
In the next stige. energy production processes
begin The glyceraldcbyde 3-P is oxidized inlo
1 3-diphosphoglyccraK. A phosphorylation from
inorganic phosphate accompanies the oxidation.
The NAD' coenzyme is reduced to NADH +
H ". These reactions permit the synthesis of an
acyl- phosphate bond— a high energy potential
bond. During the hydrolysis of this bond, the reac-
tion immediately following recnperaKs Ihe energy
by the synthesis of an ATP molecule.
The 1 J-dlphosphoglyicnitc is transformed inlo
3-P glyccialc. This molecule undergoes a rear-
rangement: its phosphate group passes from posi-
tion 3 to position 2. cstcrifylng in Ibis manner the
secondary alcohol function of the glyccialc. An
Internal dehydration of the molecule then occurs
The Important reaction which follows generates an
enolptosphaKr. a high energy potential molecule,
called phosphocnolpynivatc. Finally, this energy
is used for Ihe synthesis of ATP from ADP in a
reaction which forms pyruvate
Prom Ihe moment when Ihe Iriosc molecules arc
utilized, the second part of glycolysis comprises
Ihe most important energy- producing phases. Two
reactions ensure the synthesis of ATP for each
of the glyccialdchydc-P molecules coming from
hexose. The lotal reaction energy from the
Handbook of Etiology: The Microbiology of Wine anil Vhiiialiwa
I niciou' f-|t ifftiBc
. ?«H<« rfiHhiflar
FiK SI. Metabolic pith* ay of (flu..
transformation of a glucose molecule is therefore
ihc synlhcsls of two ATP molecules and incttlcn-
tilly Ihc reduction of NAD'.
For each hexose molecule assimilated, ihc
cell requires an NAD 1 molecule. The cell must
therefore make use of a system lhal maintains an
acceptable NAD' level. Lactic acid bacieriu use
the pyruvate formed by glycolysis as an electron
acceptor D oxidi/e NADH. This character defines
lactic fermentation. In general. bacKria thcrcfoit
transform a hexose molecule into two lactate
molecules by Ihc homolaclic pathway.
Metabolism ol Lac lie Acid Bacteria
5.2.2 Hclcrofcrmcnlativi
of He
Bacteria using the hctcrofcrmcnmivc pathway
transform nexuses principally but not exclusively
into lactate Tic tuber molecules produced by
Ibis mcftbolism air essentially CO?, acclatc and
nil. mi ■!. Ibis is Ihc pcnlosc phosphate pathway.
Alter being transported inin (be cell, a glncoki-
nasc phcaphcuylatcs Ibc glucose ink) glncosc 6-P
(glucose 6-phosptalc>. I Is destination is completely
different from ihc glucose 6-P of ihc homofcr-
men trine pathway. Two oxidation reactions occur
sncccsslvcly: the tirsi leads to glncoualc 6-P. the
second, accompanied by a decarboxylation, forms
ribulose S-P (Figure 5.2>. In each of these reac-
tions, a molecule of the coenzyme NAD' or
NADP' us rednccd. TV ribulose 5-P is then
epimcrircd into xylulose 5-P.
The xylulose 5-P phosphokclolasc is Ihc key
cn/ymc of this pathway: it catalyzes the cleavage
ol* the pcntnkise 5-P molecule into acctyl-P
c— c— c— c— c— cn*3— ^
A~...
144
Handbook of finology: The Microbiology of Wine and Vwifiiaiions
anil glyccraktchydc J- P. His reaction requires
phosphaK. The glyccraldchydc 3-P Is metabolized
inio laciic acid by following the same pathway
as in ihc homol'crmcntativc pathway. The acetyl-
P has two possible destinations, depending on
environmental conditions. This molecule can be
successively reduced inloclhaialand then cthanol.
In which case the molecules of lie coenzyme
NADH + H * or NADPH + H * . formed during the
two oxidation reaction of hexose at lie beginning
of the he krrofcrmen (alive pathway, arc rcoxidi/cd.
This rcoxidation Is esseniial for regenerating the
coenzymes necessary for the assimilation of sugar.
In ccrtiin conditions, when the cell makes
use of other coenzyme rcoxidalion systems, the
acclaK kinase cataly/cs a reaction thai leads to Ihc
formation of acetate from acctyl-P. This reaction
simultaneously recuperates the bond energy of
the P groip of acctyl-P by the synthesis of
an ATP molecule. In this case, the coenzyme
rcoxklalion systems activate NADH or NADPH
oxidases, when the cells arc in acrobtose or
redaction reactions such as the transformation of
fructose inu mannitol. When acctyl-P leads to the
formation of acctilc. there Is a definite energetic
advantage. A supplementary ATP molecule Is
formed for each hexose molecule transformed.
The final quantity of glucose metabolism prod-
nets (presence of cthanol and acetate) from beero
fcrmcnuiivc bacteria demonstrates thai this path-
way is nearly always used. Vet the use of this
pathway varies more or less depending on the
degree of acrauon and the presence of other proton
and electron acceptors. In this way. bacKria of the
genus Leucimi'sioc preferentially produce lactate
and cthanol In a slightly aerated environment and.
on the contrary, lactile and acctite in an aerated
environment. Changes in conditions therefore not
only influence ihc nature of the products formed
but also the energy yield and thus growth.
Hctcrofcrmcnutlvc bacteria produce acetic acid
from hexoscs. but regulation mechanisms modify
production. In anaerobic conditions, the NADH
oxidase cannot regenerate NAD Glucose pref-
erentially leads to the formation of laciic acid
and cthanol. When NADH can be rcoxidi/cd by
another process, the amount of cthanol formed
decreases, resulting in an Increase in acetic acid.
This occurs In aerobic conditions or in the presence
of another substance that can be reduced Homo-
lactic bacteria ferment glucose almost exclusively
Into laciic acid In an anaerobic environment with
a limited glucose concentration, homolcrmcntativc
Ixicieria such as UicliiitKilhis etisei form less tur-
tle acid: the primary products can become acetic
acid, formic acid and cthanol The change is linked
to the regulation of the i-I.DII by frucUsc 1.6-
di phosphate The change is less obvious w hen the
homo fermentative species possess the two LDH
types, i_ and t> FDP does not regulate the d-LDH
5.2.3 Metabolism of Pentoses
Ccrtiin strains of Ijitttitoticillus. PeitkKOcnu at
LeucimotttK ferment pentoses sach as ribosc. ara-
binosc and xylose, whether they arc homofcr-
men tcrs or hclcrofcrmcniers. according to the same
schema (Figure 5J). The pentoses arc pfecspho-
rylanrd by reactions activating kinases and using
ATP. Specific isomcrascs Ihcn lead to the forma-
tion of ihc xylulose 5-1' molecule. The following
reactions ate described in the nctciDfcrmencilivc
pathway for glucose assimilation. In spite of glyc-
eraldchydc 3-P having the same laic in this case.
acctyl-P exclusively leads to Ihc formation of the
acetate molecule, generating an ATP molecule In
this manner. In fact, a reduced coenzyme molecule
is nol available to reduce acctyl-P into cthanol. The
pathway furnishes two ATP molecules for each
pcniasc molecule fermented. This pathway there-
fore has a greater yield than the fcrmcntition of a
hexose by Ihc pentose phosphate pathway.
The study of the homofcrmcncitivc and Itcicro-
fcmicntativc metabolic pathways of sugars there-
fore permits Ihc prediction of the nature of the
producLs formed. Pentoses arc always al Ihc origin
of ace Ik acid and of course lactic acid production.
5.3 METABOLISM OF THE
PRINCIPAL ORGANIC
ACIDS OF WINE
Bacteria essentially degrade two organic acids of
wine: malic and citric acid Other acids can of
Metabolism or Unlit Ac Ml B actcria
■>n puthuny by kicik ickl
course be deg raded bat arc or less interest in ecol-
ogy — with ihc exception of tannic acid, winch
has rarely been studied. Since the initial research
of lactic acid bacteria and their role in wincmaking.
malic acid has been the foens or a large number
of studies. Yet the degradation of citric acid aLso
plays ;in imporcinl role in wincmaking. The major-
ity of bacterial species ptepondciunt in wine after
alcoholic fcmicntalion degrade these two acids.
This degradation is evidently the source of many
oiganolcptical changes noted after their develop-
ment. Ihc cnologist ntay consider the transforma-
tion of malic acid to be the most imporcinl phe-
nomenon of the matobctic fermentation phase, but
other transformations, of citric acid in particular,
should also be taken into account
5.3.1 Transform at ion of Malic Acid
In the case of non- proliferating cclfc in a laboratory
medium and during wincmaking. lactic acid bacte-
ria of wine transform l- malic acid exclusively into
i.- lac tic acid. Scifeit (1901) esQblisked Ihc reac-
tion of the malolactic transformation accotding to
Inc following eg nation:
italic acid lactic add + COj. (5.8)
Hi is equation was confirmed when the stereoiso-
mers could be separately determined for each of
Inc two acids
This reaction therefore involves a decarboxy-
lation without an intermediary product capable
of following another mcttbolic pathway Several
authors have reported that certain bacterial sttains
form other molecules from malic acid, suggest-
ing in this manner Ihc existence of other reactions
Even if their existence cannot be ruled out. ni.[). -
lactic transformation Is the only reaction that exisLs
in the lactic acid bacteria involved in wincmaking.
AliAide and Simon (1973) Mndred the stereo-
chemistry of this transfo in ration Enzymatic meth-
ods were used to determine the specific quantities
of the stereoisomers In addition, the fermenta-
tion of radkxiciivcly labeled glucose and malic
acid permitted the study of their products The
hckrnifcrmcncttivc cocci lOenacoceia). abundant
or cxclnsivc during xvincmaking. were found to
present several properties. They form exclusively
D-laclic acid from glucose (Chapter 4) and exclu-
sively i-Lictic acid from i.- malic acid (Figure 5.4).
This observation suggests that the transforma-
lion of malic acid docs not pass by the intermediary
of pyruvic acid. Pcynaud (1968) concluded that
the sabsirate was decarboxylatcd directly A lot of
E'iK 54. Equabn uf ihc
14r.
Handbook of Fnology: The Microbiology of Wine anil Vwifications
research was carried obi lo elucidate ibis mccha-
■isai. k naturally leads lo ihc examination of ihc
enzymatic aspect of this transformation.
Al thai lime oily the motile dcbydrogcu;ise
<MDH)and ihc malic enzymes were, known lobe
capable of fixing aid catalyzing a reaction whose
sabstralc is r -malic ac«l These Iwo enzymes were
described in numcious vegetal aid animal cells
and in diverse microorganisms. They catalyze ihe
following reactions
MDH: L-maUitc
oxaloacctalc4-NADH + H*4-NAD*
malic enzyme: r.-malalc+ NAD r =
pyruvate -|- CO< + NADH + H " (S10)
Since oxaloacctatc is easily dccarhoxylalcd into
pyruvate aid CO;. Ibese two reactions lead K>
the formation of pyruvic from i-maLitc Since
Ihc linal product of the maktlociic iron slonnot ion
in wine is L-laclK acid. MDH or Ihc malic
enzyme wcwld be associated to an LDH catalyzing
the red lie lion of pyruvate ink) 1 -lactate in Ibis
metabolic pathway At least fin wine bacteria. Ibis
concept is not acceptable since Ibese bacteria only
possess a d-LDH. Malic acid would only lead in
the fonualioa of i> lactic acid.
Therefore, the hypothesis of Ihe existence of
an enzyme catalyzing Ihc direct dccarboxytalion
of 1.- malic acid into L-laclK acid was made. The
enzyme, called Ihc malolaciic enzyme, was iso-
tiicd for ihc first time in Lactatxtcilha planktrum
(Lonvaud. 1975: Scbutzand Radlcr. 1974). From
accllular bacterial extracts and thanks to sicccs-
sivc purification stages, the authors obtiincd puri-
fied fractions responding lo the funciiona! criteria
of the malolaciic enzyme i-MalK acid is trans-
formed sloichionKlrically inio 1.- lactic acid. These
fractions do not bavc an LDH activity.
Al leas! in /- oiescnleiiH'iles and L iiemn
{0. oeni). (he mulolactk enzyme is inducible. Cul-
tivated without malic acid during numerous gen-
erations, the cells conserve a very small residual
activity. They regain ihcir maximum activity as
soon as malic acid is added 1 1 g/1 or mote). The
presence of fermentable sugars I hexose or pentose)
also favors in activity.
Some lime later, the same enzyme was purilicd
in other strains and species of lactic acid bacte-
ria, notably in strains of /. plmHiawi. I. nnirima.
L ntestntenriiles. 0. item and L laciis. The phys-
ical characteristics and kinetics of all of the
described malolaciic enzymes are Ihc same The
enzyme is a dimcric or Klramctic protein formed
by the association of a 60 kDa polypeptide. The
pH, of ihe enzyme is 4 ..15. It functions only in
Ihc presence of the NAD 1 cofacKx and biva-
lent ions. Mn' r being the most effective, and
uses a sequential mechanism. The Mn Jl and the
NAD* fix themselves on the prolcin before the
L-malatc. Al the optimum pH. Ihc Michaclis con-
stants arc 2 x 10" 1 vt for malatc aid 4 x 10"' m
for NAD. The optimum pH of the eizymalK
reaction is 5.9 At this pH. Ihe kinetics are
MKhaclian At a pH far from ihe optimum pH.
it is sigmoidal— demonstrating a positive cooper-
ative mechanism which signifies a growing affin-
ity for Ihc malatc. Hontopolymcric enzymes shore
this characteristic: the binding of the fiisi sahstratc
molccalc on the first promoter transmits a defor-
mation, increasing Ihc aflinity of the others. This
coopcralivencss permits an inciease in the effec-
tiveness of the system in unfavorable conditions.
Evidently, in wincmakiig. bacteria are in far from
optimal conditions (Lonvaud-Funcl and Strusscrdc
Saad. 1982).
The carboxylic acids of wine — succinic, citric
and t-lartaric acid — arc competitive inhibiurs
with the following respective inhibition constants
8 x ID 1 m. I x 10- m aid 0.1 u. L-Laclk acid,
a product of the reaction, is an inefficient, non-
competitive inhibitor whose inhibition consimi of
01 vt indtcaKs a weak aftiiity
Although thiscnzymcnbccoming bciicrknown.
a question still remains unanswered: what is the
real tote of NAD* in the oxidation -reduction
exchange? The indispensable coenzyme of the
icactkin is not involved, al Icasl in a conventional
The malolaciic enzyme purified from Laciacac~
iu hietis. a tactic bacterium of milk origin, has
Metabolism of Lactic Acid Bacteria
14"
cxaclly ihc same charackrislics as Uc cnological
strain en/ymc. It was used \a study the stricture
of Ike gene The protoitcr N-Krmina) cad was
sequenced on 20 amino acids (IBMC Laboiutoiy.
University or Bordeaux II). Tic corresponding
nuckolidc sequences were deduced from ihc live
litsl and live last amino acids of litis portion or the
protein (Dcnayrolles el ill.. 1994). These oligouu-
clcolidc sequences were used as primers in a PCR
amplilicalioa reaction with bacterid DNA as tem-
plates In this manner, a 60-nnckolidc fragment
was isolated aid used to produce a piobc. permit-
ling (he identification of the malolaclic gene in Ihc
bacterial chromosome This fragment, and progres-
sively the entire gene, was sequenced. Ansanay
el al. 1 1993) obeiiicd the same result by another
method, starling wilh Ihc same purified en/ymc
preparation.
The nucleotide sequence encoding the malotac-
IK en/ymc is therefore known and shows a strong
resemblance to Ihc malic en/ymc. The binding
slics of the coenzyme on the protein have also
been located IFtgiic55). Finally, after having
been inserted into a vector, this gene was trans-
ferred into E raft and also into laboratory strains
of S cerevisiae ycasl: the gene was expressed in
Ihcsc conditions (Ansanay el til . 1993: Dcnay-
iolles el al.. 1995).
In Ihc talc 1980s, the program aimed at devel-
oping a malolaclK yeast" capable of carrying onl
the malolaclic transformation during alcoholic fer-
mentation was supported by wincmakcrs in France
and abroad. The first stage coasiskd of cloning
Ihc gene of the malolaclic en /vine and express-
ing it ii an yeast. Unforlnnaicly. il very rapidly
became obvious that the system was limited by the
fact that malic acid entered Ihc yeast. To overcome
this problem. Ihc Skllcnbosch Icam (South Africa)
decided to clone the malalc permease gene from
Sehi&isatchiBomyces pmihe. another yeast fonnd
in wine (GroMcr rtitf.. 1995). Having demon-
strated thai a ycasl could be transformed by a
vector hearing the gene coding for permease and
another hearing the malolaclic enzyme, the same
Icam inserted these two genes into a yeast chro-
mosome to stabilise Ihc desired genetic data. A
ycasl strain wilh malolaclic activity now exist*.
can be produced on an industrial scale, and has
shown a certain level of performance (Van Vnurcn
and Hnsnik. 2003) There is no question of carry-
ing out full malolaclic fermentation as this yeast
docs not exhibit all the other bacterial activities
involved in enhancing the gustatory qualities of
wine. Il may be useful as an organic agent for
dcacRlifying wines when the wincmakcr wishes
to preserve the characteristic aromas revealed by
yeast. However, this ycasl isagcnctKally modified
microorganism and. as such, is far from gaining
universal acceptance
The production of the en/ymc for direct use in
wines R of no use. since this protein Pi rapidly
inhibited by diverse sibstanccs in wine— acids,
alcohol aid polyphenols. The malolaclic reaction
takes place at Ihc interior of Ihc backrium in a
medium protected from inhibitors by Ihc bacterial
membrane. The degradation rale of malic acid is
limited by its transport speed in the interior of the
cell Although the optimal pH for enzyme activity
is around 6.0. il is around 3.0 to 33 for whole cells
of 0. item. At this pH. malic acid penetrates more
easily into the backrium than al higher pHs.
The inhibitory action of tartaric and saccilk
acid is even stronger on whole cells than on
prokins. Citric acid al a concentration of OS g/l.
normally not reached in wine, only slows cellular
activity by around $'f (Lonvand-l-uncl and SUas-
serdc Saad. 1982).
Finally, among Ihc questions raised as early as
the period of initial research on malolaclK fcrmci-
lation. the physiological role of malic acid remains
to be interpreted. The addition of malK acid il a
culture mcdiim of lactic acid backru simultane-
ously increases the yield and the growth rate A
partial explanation of this observation was discov-
ered only recently. The malolaclK reaction inclf
is iol very excigonK. yet it indirectly constitutes
a real energy soiree for the cell Poolman 1 1993)
demonstrated that, following the decarboxylation
reaction, the increase of the internal pH (whKh
imposes an influx of protons), the uptake of malK
acid and the efflux of laclK acid combine to creak
a proton motor force, permitting the conservation
of energy via the membrane ATPasc.
Hamllxxik of linokigy: The Microbiology <"( Wiie anil Vinifkalions
dp 55. NiKkoiklE softcntc of ibe DNA fag mem onylnp Ihc nul<
icijiKaio. ended bv ihn liagmca.. Ccnaln pnvcic man panKuhriv well
is-1 ma Ik carymc* lave been uailc dined. A piMcnital faneiba h» bee« specified for*
Metabolism of Lactic Acid Bacteria
149
5.3.2 Metabolism of Citric Acid
Ccrtiiii Lie tic acid bacteria (hctcrofcrnKntilivc
cocci and honiofcrmcntativc bacilli) degrade cit-
ric acid. Anions ihc species found in wine.
/- planmnm. I- aaa, 0. veto and /. mesemer-
irides lupidly nsc curie acid Strains of Ihc genus
fetbiroccia aid of Ihc species L Inlgtmtii and
L b/rvii cannot.
In cerbiin dairy industry baclcria. Ihc lack of
utilization of citric acid is linked li> Ihc kiss of the
plasmid encoding Ihc cilratc permease, c-tcntial
for Ihc uptake of Ihc acid In hiclcria isolated in
wine. Ihc citrate permease may exist but its role is
inconsequential, since at Ihc pH of wine, the non-
dissociated sntstrafc diffuses across ihc membrane
without needing Ihc permease The species and
ihc strains that do nol degrade cilric acid arc
therefore at Icasl deficient in Ihc lirslcn/ymcof ihc
metabolic pathway: the citrate lyase. This enzyme
was studied in wine Lie lie acid bacteria and
more particularly in a strain of L aiesfiHemiiles
(Wcinzon. 1985).
Within baclcria. cilric acid is split in*) an
oxaloacetic molecule and an acctitc notccilc
by the lyase (Figure 5.6) The largest quantities
of this cn/yntc are synthesized in low sugar
conccilration media containing cilric acid Glucose
acts as a repressor. The protein is active in an
acctylatcd form. The inactive dcacctylatd form
can be rcacctylatcd in vin> by the cilrale lyase
ligasc wilh acetyl CoA or acetate and ATP. This
Kifc 5.0. Mtuhulic pjihwiy lore
mi dtp a dub a tv. belle acid battel
150
Handbook or Enology: The Microbiology of Wine anil Vitrifications
first degradation stage leads to Ihc romialion of
an acelaic molecule for each molecule of ihc
substrate.
Oxatoncctaic is ihci dccarboxytiilcd into pyru-
vale in Oenococais. ihc most impunanl bacteria )■
etiology. In certain /.fti(rf*ii7//ni. ileal also lead to
a partial formation of succinate and formate Pyru-
vate is Ihc source ofacetoin compounds: diacctyl.
accloin and 2 J-butancdiol. Tie first is particularly
imporumt oiganolcptically. It is the vcty aromatic
molecule thai gives bnilcr its smell. The olfac-
tive intensity of accloin and buunedKil. which ate
derived from diacclyl by reduction of the ketonic
functions. Is much lower (Figure 5.6).
Two diacclyl synthesis pathways have been pro-
posed. In one. diacctyl results from the reac-
tion of acetyl CoA with cthanal-TPP (active
acctaMchydc). catalysed by a dEiccryl synthetase,
which has never been isolated The other path-
way supposes lhat from two pyruvate molecules,
a -ace to lac tale synthclasc produces o-acctolaclale
which is then dccarboxylatcd iiu acetoin. The
diaccryl R derived from it by -nidation This is
an aerobic pathway
In addition *> accloinic suhsEinccs. the pyruvate
molecules coming from citrate have other destina-
tions First of all. if Ihc coenzyme NADH. pro-
dnccd by olhcr pathways, is available, il leads io
the formation of lactate Next comes Ihc decar-
boxylation of pyruvate and then a reduction pro-
dnccscthanol. Finally, the pyruvate derived from
cilraK panic i pales in the synlhesls of fait)* acids
and lipids via acetyl CoA. The radioactivity of
labeled citrate supplied *i the bacteria B incor-
porated into the cellular malcrtal. In thct pathway,
part of the acetyl CoA can also gcncmlc acctaic
molecules (Figure 5.6).
The citric acid metabolism pioducts arc Iherc-
fore very divcise. Whatever the conditions, moit
than a molecule of acclK acid is surely formed
from asabstraK molecule. The production of oth-
ers is hugely dclcrmined by factors influenced by
growth conditions In limited glucose concentra-
tion conditions, a low pH and the presence of
growth inhibitors, citric acid preferentially leads
u Ihc formation of accloinic substances. In fact,
due to conditions, pyruvate is orientated neither
towards the synthesis of ccllnlar material, since
growth Is difficull. nor towards tactile and cthanol.
becansc of a tack of reduced coenzymes. The acc-
loinic sahstancc synthesis pathway is considered
m be a detoxification process of the cell. In order
h> mainlain id intracellular pH. il mnsl eliminate
pyruvate. Conversely, when growth Is easy, pyru-
vate is utilized by fatty acid synthesis pathways:
acetic acid is produced li larger quantities, la a
laboratory medium. OenoctKcut forms more than
two acetic acid molecules from one citric acid
molecule al a pi I of 4K and only 12 molecules
ata pH of 4.1. Conversely, the production of acc-
loinic molecules is four times higher lLoavaud-
Fuacl el erf. 1984).
It Is therefore not surprising that some wines
contain more than lOatg of diacctyl per liter. Ycl
it has been dclcrmined that several milligrams of
diaccryl per liter (2-3 mg/l for while wines and
about 5 mg/l for red wines) contribute favorably
to Ihc bouquet. Above these concentrations, the
bnlttiyarouu.distinc try perceived, diminishes wine
qualify. Malotactic fermentation conditions and
the quantity of citric acid degraded (from 0.2
Mi 0.3 g/l) and also without doubt the species of
ii zv/ir involved determines the quantity of diacctyl
produced. Several other reactions contribute to
Ik final concentration. First of all. ycase also
synthesize diacctyl during alcoholic fermentation by
a completely different pathway linked to the mcta-
holisai of amino acids Diacctyl is then reduced
into accloin by the diacctyl reductase, an cn/ymc
present in ycasK and lactic acid bacteria. The
diacctyl concentration attains two maxima in this
manner oae during alcoholic fermentation, the olhcr
during the degradation ofcitric acid by bacteria. Il
diminishes between the two fermentations andal the
final stage of bacterial activity Malntiining wine on
yeast and bacteria Ices al the cad of fcrmcatabon
ensures this reduction and also determines the
final diacclyl level (Dc Revel errrf. 1989). Finally,
sulfur dioxide further diminishes its concentration
by combining with the ketonic functions
Citric acid is always mcubolizcd during fer-
mentation because in nearly every case the species
O m-iu is involved. Is degradation begins at the
same time as malic acid degradation, but it is
Metabolism of Laclic Acid B acicria
151
much slower — si> much so ibal at the end (if
iii.il' 'It- in fcraicnlation. citric acid ■■: mi i.-m., its
Dp lo 0.15 I'll of sometimes cvcd more It rcprc-
scms an addilional cncigy source In fuel. ATP
Is formed from acctyl-P. derived from pyruvate,
which Ls diicclcd towards Ihc productioii of cell
compose iiis In the presence of residual solars
(glucose or fractosc) degraded by the betcrofet-
mentalivc pathway, part of the pyruvate derived
froii citric acid acts as an electro* aid proton
acceptor. Part of foe . i-l-i- :- ! ■ originating fn>m sug-
ars leads lo the formation of acetate Id produciig
ATP. Id Ibis manner the presence of citrK acid
it a wine favors bacterial growth and survival,
notably in the presence of residual sugars. This
metabolism therefore participates, along with the
malic acid metabolism, in the microbiological sta-
bilization of wine by eliminating energy soirees
(Loivaud-Fiicl. 1986).
5.3.3 Metabolism of Tartaric Acid
Wine laclic acid bacteria can degrade tartaric
acid, but this metabolism differs from malic and
citric acid metabolisms It B a veritable bacterial
spoilage. Pastcir described it )■ the last century
and named il iiwiie disease. It R dangerous since
the disappearance of tartaric acid, ai essential
acid in wine, lowers the fixed acidity and is
accompanied by an increase In the volatile acidity.
The degradation cai be total or partial, dcpciding
on the level of bacterial devclopmcit. bnl il always
lowers » lie qnality.
This spoilage Ls rare slice the strains capable of
degrading tartaric acid seem u be relatively few
ii number. Stidlcs carried oul on this subject in
the 1960s and 1970s showed thai this properly
Is not linked u a particular bacterial species.
Strains hcloigiig to diffcrcil species have been
Isolated by various authors, but they are hum often
laciobacilli. Amoig them. Radler aid Yannlssis
( 1972) found lour strains of /. ptanttmm and one
strain of L Nevis having this trail oul of Ine 78
strains examined Peynaud (1967) discovered 30
or so straits capable of partially or totally nslig
tartiric acid in a study earned oil on more than 700
strains Tie scarcity of this property constitutes in
sum the Hist prolix tion against this disease.
Since a high pH Is always propitious lo the
multiplication of a larger numbcrof bacteria, higher
acidify wines arc less affected Moreover, these
bacteria are sensitive to SOj. Therefore, respecting
the current rules of hygiene ii the vvincccllar aid in
wine should be sufficient*) avoid this problem
Pew studies exist cxamiiiig the metabolic path-
ways of Ihc trausformalioi of tartaric acid The
only resulbi cxisllig describe different pathways
for L pliintiinm aid L Kit-vis I Radler and Yai-
nissis. 1972). Prom a molecule of tartaric ackl.
L planmrttii produccs05 molccnlcsof acetic acid.
0.3 of succinic acKI and 1 J of CO. L hreris forms
0.7 molecules of acetic acid. 0.3 of succinic acid and
1.3 of CO! (FigureSJ).
5.4 OTHER TRANSFORMATIONS
LIKELY TO OCCUR IN
WINEMAKING
5.4.1 Degradation or Glycerol
Glycerol Is one of the principal components of
wine, both in its concentration (5-8 g/l) and in
Its contribution to taste. Yeasts form glycerol by
glyec ropy ru vie fcrmcitalion at Ihc bcgiuniDg of
fennci la i ion The degradation of glycerol harms
wine quality, partly because of the decrease ii its
coKCilraliou and partly because of the resulting
products of ihc metabolism.
Certain back' rial strains produce bitterness in
wine— a fact known since the time of Pasteur
Laclic acid bacteria make use of a glycerol
dehydratase lo transform glycerol iiu ^-hydroxy-
propioialdchydc (Figure S8). This molecule Is the
precursor of acrolein, which Is formed in wine by
ncaliig. or slowly duriig aging. The combination
of wine tannins and acrolein, or its precursor, uncs
a bitter taslc
1. lie ukim?. Ihlsspoilagc is nol widespread, due
lo the rarity of siralis capable of degrading glyc-
erol by this pathway. No single species of hacleria
Ls responsible for degrading glycerol In wine. !. ti-
tle research has been devoted to this problem, bul
strains of two species of bacteria. Ltettibacilliis
hilgitnUi and IjictobaeitlustUolivoraits. have been
Isolated from wine following degradation of the
Handbook of Etiology: The Microbiology of Wine anil Vhiitialiwa
Fift 57. Tana.*
ubolnm by kirtk
a<R*llc>ii(Ui Vtni
glycerol (Clalssc. 2002). A key cn/ymc. glycerol
dehydrogenase, has been sindicd in several strains.
In strains of L fiteris and /- Inwliiieii. St nut/
and KiHikr ( l'J84> dcnionslratcd ihc degradation
of glycerol by the glycerol dehydratase lo IJ-
propanedkd via r>- hydroxypropiomddchydc when
the medium also contained glucose or fructose.
NADH ior NADPH). produced b>' Ihc fcr»cnra-
tionof sagar. reduces aldehyde lo I J- propanediol.
As described earlier, iirs co- metabolism leads to a
dcviaini of acctyl-P derived front sugar towards
the production of additional ATP and acetate. II
tbcrcforc facilitales baclcrial growth.
Some strains degrade glycerol in wine by
the glycerol dehydratase pathway, while others
also use Ihc 3-P-glyccrol dehydrogenase path-
way. The genes coding the enzymes for the lirsl
pathway have been studied They ait organized
ii a set including a totil of 13 genes, prob-
ably all necessary for the funcuoiing of glyc-
erol dehydratase, which consists of three pro-
tein MibueiLs and propane- 1 J-diol-dcbydrogenasc.
leading, finally . n> propane- 1 J-diol. L bHf,ar<Ui
and L ttivlrritram arc organized in the same
way. as arc strains of L ailliiktitles isolated
from cider affected by acrolein spoilage (Gorga
Metabolism of Unlit Acid Bacteria
Kip, 5.8. '.il.itml ifcrjuul.ii i.-o puih.i ,,. « lv. lyokicid hMlcria ( RaScRau-Gtivt
el al.. 20021 Oligonucleotide prims for detecting
these luck'iLi nave been selected from the gene
sequence coding for one of the u lycc ml dehy-
dratase subunits (Ctaissc and Lonvand-l-nncl.
2001). Degradation of glycerol results not only
in the production of hydroxy- 3- propioualdchydc.
lit precursor of acrolein, bnl also, by metabolic
coupling, to an increase in volatile acidity pro-
duced from the i -lactic acid in the wine. Tic other
pathway consists of glyecrokinase phospborylat-
ing the glycerol and 3-P-glyccrol dehydrogenase
resitting in 3-P-dchydroxyacclonc. This iiolccilc
ciiitrs into glycolysis reactions by oxidaitoa into
d (hydroxy ace tone- P which result in the formation
of pyruvate. 71k Ileal produces of this pathway arc
those previously described from pyruvate degrada-
tion, notably acctK acid and accloinic sabstanccs.
The quantity of products varies depending on envi-
ronmental conditions, in panic liar the amount i'f
fermentable sugar and aeration
In particular, large amounts of Lactic acid arc
formed, increasing the wines total and fixed
acidity and causing its pH m drop Acidification
was as nil? h as 08 g/l (expressed in tartaric acid)
in wines where the pH dropped by 0.25 to OJ0.
Accloinic molecules may also be formed from
pyruvate The fate of the pyruvate molecules is
probably, as usual, determined by the availability
of NADH/NAD coenzymes. ic. the intracellular
redox conditio* If NADH is available, lactate
is produced If not. the pyruvate is eliminated
in the form of accloinic compounds. Interactions
between the mcttbolic pathways and the bacteria's
environment arc the decisive factor. In any case,
the presence of bacteria capable of degrading
glycerol is a risk to the extent that, even if it is
not totally mclabolind. its metabolic by-prodicts
may spoil the wine to varying degrees.
5.4.2 Dccarboxj hi I i f HisluliiK-
Hisildinc. an amino acid in wine, r dccarboxylatcd
into histamine, whose toxicity, although low. is
additive to the toxicity of other biogenic amines
I tyrant inc. phenyl ethytaminc. pntrescinc and cada-
vcrinc) (Figure 5.9) Tyrosine is decarboxylatcd to
form tyraminc by a similar reaction.
In general, biogenic amines— histamine in partic-
ular—arc more abundant in wines after malolactK
fermentation. This explains the results presented in
various works: red wines appeared to be richer in
amines than while wines Some researchers (Ac ray.
1985) also proved that histtminc is formed mainly
at the end of matobctic fcrmcutatton. and even later
Handbook of Etiology: The Microbiology o( Wive anil Vindications
C=C— Ctlj— C — V
C=C-CII,-CtI,-N
t'ii; SA llniallnc dccubtHyhtiM icKiba
I mi" .1 long lime. many atlcmpK were made lo isolate
bacteria capable of dccarboxylaling histidinc. and
ibcy led lo ihc conclusion ihal only contamination
strains belonging w the genus Peifiocrtrw.* had this
. ■ ."' '|v i i j According lo sonic authors, (he presence
of hisominc indicated a lack of vvincmaking skill
and hygiene in the winery.
Yci this phenomenon occirs even during ihc
ntalolaclK lc rate n rations of wines whose micro-
flora Is almosl exclusively " txni. This facl
conmidicicd the above results An in-depth study
of the microflora of wines rich In histamine
finally ted in the isolation of O ivni strains
lhal pioduce histamine fioni bislkline (Lonvaud-
Funcl and Joycnx. 1994) In laboratory media, the
Initiation of hisLimine by these strains increases
as Ihc growth conditions become less favoraHc:
Ihc absence of other sibstrafcs lin particular,
sigar) al a low pH and in Ihc presence of
clhanol The histamine concentration also, of
.' m iv. depends on the hislidine concentration.
The addition of yeast Ices, which progressively
liberate amino acids and peptides, increases the
concentration in the mcdiim Bacteria that also
possess peptidases find a significant hislidine
soiree there It is. therefore, not surprising lhal
Ihc htstiminc concentration increases toward the
end and even after fermentation, as long as Lactic
acid bacteria are present. A precise study of the
hislidine carboxylase activity of a strain of oein
shows that even non-viable cells conserve their
activity for a very long lime in wine
This enzyme has been fully purified. Its char-
acteristics and properties arc similar lo those of
Ihc en nine purified from a Ijiciobaeilliis of non-
etiological origin The protein comprises two dif-
ferent submit, grouped in a type [ojllt hcxamcr.
The activity is optimal at a pH of 4.5: il is the same
for whole cells. The en zymc is synthesized in the
form of an inactive precirsor D II is then acti-
vated by the scission of Tl into a and p Like Ihc
malolaclic reaction, the decarboxylation of hisli-
dine does nol directly generate cellular energy, bul
the strains of 0. ivia ihal ate capable of using il
profit by iK presence in the medium and glow mote
quickly than strains lhal do nol have tins capa-
biliry. The cneigctK advantage is explained fas in
/- builineii) according lo the process described for
inalolaclic fermentation by Poolman 11993). The
exchange between hislidine and histamine al Ihc
membrane level creates a prolan gradient and a
proton native force, generating ATP.
The strains of " iieiu lhal use hislidine. there-
fore, have an additional advanttgc which can
be a deciding factor after wincmaking when the
medium is poor in nutrients Their survival can be
facilitated in this manner.
Nol all ft tteiri strains possess Ihc hislidine
decarboxylase; in fact, there ate only a few. The
detection of these particular strains was made pos-
sible by the use of molecular loots. DNA amplifi-
catioa by PCR. by using the appropriate oligonu-
cleotide primers, permits the identification of these
particular strains in a mixture. The labeling of
a gene fragment, or a whole gene, that encodes
Ihc enzyme supplies Ihc specilic probe. Thus, the
detection and enumeration of strains capable of
forming histamine no longer present a problem.
For example. Ihc analysis of 251) samples of Bor-
deaux wines showed that ncariy 5fM contained
sic h strains. This method can be extended lo other
bacleria. since it is specific only to the presence of
the gene and not Ihc bacterial species (Lc Jcunc
rtnT., 1995: Colon. 1996).
Unlil now. Ihc only strains isolated from wine
capable of piodicing lyraminc were identified
as /- brevis and L /irfjjrovfi/ (Moicno-Arribas
etui.. 2<XX». The tyrosine decarboxylase enzyme
was studied in a particilarty active strain of
Metabolism of Lactic Acid B acicria
/. .".'■:■ i', 1 ' . ihcn i- m". .. ii.-.i :mnl ii ( Moreno- Arribas
ami Louvaud-l-ancl.20DI) As Ibc aim was lo pro-
duce dclcclion tools specific lo Ibcsc strains, ibc
protein was sequenced to find Ibc coding sequence
of Ihc gene and. filially, ihc oligonucleotide
primers required forPCR amplification (Lucas and
Lonvaud-Funcl. 2002; Lamlctc el al., 2003).
In future. w»'1s for ihc rapid, accnialc iden-
lificalion of strains producing biogenic amines
will enable wlneniakcrs to assess Ihc risks and
also u cany out stndics of ihc ecology or these
strains, rurticuLirly Ihcir distribution and coudi-
aons encouraging Ihcir presence in si
ofArginin
5.4.3 Mctabolni
Among grape must amino acids, aiginine is ihc
most rapidly and completely consamed at the
beginning of alcoholic km ten union It is Ihcn
secreted by the yeast and liberated during autolysis
The use of this amino acid by backrria during
fermentation was not closely studied until recently,
notably as part of research on the origins of ethyl
carbamate (Malaga. 1994: Liu el ill.. 1994).
The microoiganisms employ several metabolic
pathways for using aiginine. In laclK acid bacte-
ria. Ihc most widely used Is Ibc aiginine dcimi-
nasc pathway. The enzyme of ihis first step cat-
alyzes ihc dcaminalion of aiginine into ciirullinc
(Figure 5 10) Next, ornithine trauscarbamy Use and
carbamate kinase lead to the formation of ornithine.
COj.ummoaiamand the synthesis of ATP. Strictly
he icrofcrn tenia live laciobacilli were long consid-
ered to be Ihc only ones capable of these trans-
formations. HctciofcrmcuGilivc lactobucilli wcic
long considered to be the only ones capable of
effecting these transformations. This is Ihc case
of L lalgtuttii. which has a highly active aiginine
metabolism thai providesa high-level cncigy sou ire
(Tonon and Lonvaud-Funcl. 2002)
However, some studies also identified this path-
way in optional!)' hctcrofcrmcnlaiivc laciobacilli,
such as Lactobacillus plantanon (Arena eial.,
1999). Intcrcsiingly. Oenoeocaa oeni {0 new)
was classified in Sergey's reference manual for the
identification of bacteria as being among those lhal
do not hydroly/c aiginine Rcccniworks of authors
cited earlier have, however, definitively con firmed
that certain strains of ibc species O. oem are capa-
ble of degrading aiginine by Ihc aiginine deiminasc
pathway The frequency of this character is not cur-
rently known.
The synthesis of three cn/ymes— aiginine deim-
inasc. ornithine Iranscarbantylasc and carbamate
kinase — is induced by the presence of aiginine in
the medium A strain of O oeni studied b\ i.nn-' (if
( 1994) transforms aiginine sloKbiometrically into a
mole of ornithine and two moles of ammonium. In
O oeni. the genes that code forthccnTymcsof this
mccibolK pathway arc organized in an opcron on the
chromosome including air A. It. and C coding for
aig inlncdclniinasc. ornithine Iranscarbantylasc. and
carbamaK kinase. preceded by a regulation gene, air
R . and followed by two genes coding for transport
proKins. arc Dl and tar D2 (Tonon elal., 2001;
Divolrt«/..2003).
In a collection of " oeni. Ibc strains do not
systematically have this complete region of ihc
chromosome Of course, only those that have the
com pie le region are capable of assimilating aiginine
via ibis pathway.
A small quantity of ciirullinc is still libcrakd in
Ihc medium (up lo l ■' ■ >. it Is not taken up in the
156
Handbook of linokigy: The Microbiology of Wine anil VinUkraUons
catabolism of ii,-iii as opposed to lactobacilli.
This mulcenk is ibe source of clhyl carbamate,
whose presence corresponds with ihc degradation
of arg ininc by wine bacteria.
However, quantities formed are considerably
lower than those originating from Ihc urea released
by yeasts during alcoholic It- mentation There is
■ii need Id worry about the development of strains
of " mm that degrade arginine during makHaciic
fcrmcntiiMia. Il b. however, advisable to prevent
these strains, as well as he krofcrmc Native lacio-
bacilli. from proliferating after malolaclic fcrmcu-
tttioa. It is atlvtviblc not to risk Ihc formation of
these ptccirsors of ethyl carbamate
As Is Ihc case in decarboxylation reactions
involving malic acid, bisbdiac. and tyrosine.
degrading arginine provides ycasVt wilh additional
energy resources The ncl energy gain via the
arginine pathway consists of a molccnk: of ATP
produced from carbauiyl-P.
Nevertheless, as wilh strains thai dccarboxylatc
hislidine. strains ihai degrade argiiiic have an
advantage over inner strains The net energy gain
is an ATP molecule produced from carbamyl-P.
In tuclobacilli. il has been demonstrated thai the
uptake of arginine B coupled wilh Ihc excretion of
ornithine by an anlipott system, therefore it docs
noi require energy Al least in lactobacilli. arginine
stimulates growth. The same effect has been
de ii tons! rated in O if ni (Toaon and Lonvaud-
Fauci. 2000).
5.4.4 Synthesis of Exocdlular
Po lysa cc ha rides
The symhesis of exoccllular polysaccharides by
tic tic acid bacicria R a very widespread char-
acter /- mesenteiiriiles and Slrepmcvccta naitans
produce g Incase homopolymcis sach as dcxlran
and glucan: fntckisc bomopolymcrs (levans) and
■ctcropolymcis are also synthesized. Dextran of
/. itKseniemiiles is the best known, as much for
is different structures and ic biosynthesis its for
its varions applications.
The produclioi of cxoccllnlar polysaccharides
increases viscosiry and it can be measured or eval-
uated visually by the ropycbaraclcroflhc medium.
Exoccllular polysaccharides ate of interest n> the
milk industry and for industrial and medical appli-
cations, bnl much less so in cnotogy. They give
rise to ropincss and Ihc gmltw disease, studied by
Paslcur. A lot less rare than immte and ivneramie.
this spoilage has incited new research.
In the literature, increased viscosity in cidcrsand
beers is attributed lo diffcrcnl ticlic acid bactcrEi
species. notably P it/mtimnu and/, breris — which
are also found in wine (Williamson. 1959; Beech
and Carr. 1977). Lathi 1 1957) established that the
symbiosis between luetic ackl bacteria producing
polysaccharides and acetic bacteria accckrulcs the
increase in viscosiry of Ihc medium.
In Ihc early 1980s. Ibis spoilage reappeared wilh
increasing frequency. Isolations carried ont since
then have demonstrated Ihc involvement of the
species I' tkmtiHxaa. This facl docs not exclude
Ihc possible participation of other species, bul
they are generally in much smaller proportions.
Polysaccharide production was studied both by
measuring medium viscosity and by determining
polysaccharide concentrations. For a given wine,
the viscosity increase corresponds directly to the
production of the polysiccbaridc.
Il is not simply a matter of measuring viscosity:
it is the visual aspect of Ihc wine that Is the deter-
mining criterion forcharactcri/ing this problem. For
example, a wine wilh a viscosity of 1 .617 ccitntoke
test) and a polysaccharide concentration of 95 mg/l
is not ropy, as opposed lo a wine with a viscos-
ity of 1 .615 and a polysaccharide concentration of
30Qmg/l. Many other medium factors con tribute to
wine viscosity, notably Ihc alcoholic content.
Compared wilh non-ropy strains of P. (Avrrnjiw.
the ropy strains are distinguished by Ihc existence
of a sort of rcfringent capsule around the cell,
clearly visible under the microscope. The colonics
formed on a solid medium arc ;Uso easily identified
by the formation of a thick liber when picked with
a platinum fiber. Al Ihc physiological level, the
ropy strains demonstrate Ihc occasional ability lo
adapt to giowth in wine. They develop wilh the
same case whatever the alcoholic content, even
greater than 12'* Their growth rate is hardly
reduced at a pll of 35 compared with a pH of
45. and is not much affected by sulfur dK>xKtc.
Metabolism of Lactic Acid Bacteria
157
> loinial .11 pH 3.7 with a free
SO? cokci (ration of 30 iii g/l ( l.omaud- Funci and
Joycux. 19R8).
Ropy P ikmintmu strains increase wiie viscosity
when they multiply in a medium containing g lucosc
The disc. inc is clear!) visible when ihe population
exceeds III' units forming colony (UFCWnil Wine
sugars such ;ts fructose or pentoses do mi pcraiil
Ihe synthesis of Ihe polysaccharide. It is a glucan. a
glucose honiopolyiKr with a structure comprising
afi 1-3 chain on which a glucose nnil is attached in
fi 1-2 every twounibULtaubcrcsirf «/.. 1990):
-fefc-glc]-,
I
gle
The particular structure of Ills glican docs nol
permit an enzymatic treatment of affected wines
with currently known enzymes.
In wine, lie polysaccharide is therefore formed
from residual glucose. Sevciul do/en milligrams
per lilcr suffice lo increase Ihe viscosily. The
spoilage can occur in ihe tunkalihcendof femiei-
tilion. but itost problems arc posed by spoilage in
Ihe bottle— mainly a few months aflcr botlliig. In-
depih studies have shown lhat Ibcsc strains adopt
physiological forms that ensure not only their sur-
vival but also their growth. Furthermore, glucan
production is greater in a nuiricut-poor medium,
such as wine Km the same amount of bacte-
rial biomass. two lo three 3 times more glucan
Is formed in a medium containing 0.1 g of glu-
cose per liter than in a medium with 2 g/l Simi-
larly, for Ihe same glucose concentration, the pro-
duction Is gieatcr in a nitrogen-deficient medium.
Independent of the survival and gtuwlh rale, the
strain physkikigy in extreme media directs their
metabolism towards the synthesis of glucan.
Laboratory studies of several strains have shown
the great instability of the ropy phenorype. Strains
transferred lo a medium without cthanol rapidly
lose this property This result led lo the comparison
of ropy strains and their mutants. The presence of
a plasmid distinguishes the strains. On this is a
small plasmid with 53 kbp: three coding genes
have been identified by homology, one probably
for rcplicasc. the second fora mobilization protein.
and the third for a glucosyl transferase. This
■bird gene is probably the key lo is properly
of synthesizing exopolysaccharide (Walling el <»..
2001). Knowledge of this plasmid has made
il possible to develop tools for delecting these
strains, cither by bybrRli&ition wilb a probe or by
PCR (Lonvand- Funci ei al., 1993. Gindrcau el al..
2001 ) They make il possible lo identify and count
ropy*' strains in a heterogeneous population of
wine bacteria, including non-ropy P ikmwsiis
or other species
II spoilage occurs al Ihe winery or a warehouse,
ihe lirsi precaution is evidently the disinfection of
all of the tanks and winery material lo avoid future
con tami natrons. In general, a ropy wine docs nol
prcsentanyothcrorganolepiii.il delects and ilean
therefore be commercialized after the appropriate
treatments The viscosity can generally be lowered
by Ihe mechanical action of shaking the wine
Snlfiting at a minimum of 30 mg of free SO?
per liter and progressive nitrations np h> a slerik
miration ensure Ihe preparation of the wine for a
risk-free re-bottling. Heal treatment of the wine.
J nst before boliling. Is another a reliable solution
for these fragile wines.
5.5 EFFECT OF THE METABOLISM
OF LACTIC ACID BACTERIA
ON WINE COMPOSITION AND
QUALITY
Unless the appropriate inhibitory Ircaimcu fs arc
applied. laclK acid bacteria are part of the normal
microflora of all while aid red wines. From the
start to the end of fermentation, and even during
aging and storage. Ihcy alienate between succes-
sive growth and regression periods depending on
Ihe species aid Ihe strains. All multiplication or
survival involves a metabolism thai Is perhaps very
active or. oi the contrary, hardly perceptible and
even impossible to detect with current analytical
methods Substrates arc transformed and conse-
qucilly organoleptic characters arc modified. Sonic
metabolic activities are favorable and others arc
without consequence, while some are totally detri-
mental lo wine quality (Volume 2. Section 8.3).
158
Handbook of Etiology: The Microbiology of Wive anil Vinific.uions
The main substrates for wine buclciia known
k" dale ate simple molecules: sugars and organic
acids Although their transformation is not enr-
iciily verified, oihcr more complex wiac com-
ponents, such as phenolic compounds, axomaiic
compounds or aroma precursors, piescnl in small
quantities, arc withonl donbl panially mctaboliTcd.
The rcpcicnssion of these minor transformations
on organoleptic characters can be (depending on
the molecules concerned) at least as important as
the principal tractions.
The only substrate always metabolized by the
same pathway by all species of wine bacteria is
■ •malic acid. Cellular activity Is nodulated by the
presence of other compounds acting on the trans-
port level or on the enzyme activity. The growth of
tactic acid bacteria in wine is sought after because
of this activity: indeed, it is the only activity truly
dcsiitd. It permits the softening of wine provoked
by dcacidrficalion and by the replacement of malic
acid with lactic acid, a compound with a less
aggressive flavor.
Bacteria degrade must and wine sugars with
a different affinity depending on the species and
perhaps even the strain. Hcxoscs arc fermented
into i.- or o-lactk acid, or a mixture of the two
fonts, depending on the species. In general, bac-
Krial development occurs after yeast development.
Therefore, the lactic acid formed from sugars is
in negligible quantities compared with the amount
coming from malic acid. Several bacterial species
produce D-laciK acid but it is the exclusive form
for he (croft rate ntatrvc cocci, and thus " <*•/». the
most importtnl bacterium to cnology Among the
sugar fermentation products of O <*■«/. acetic acid
Is significant because of its contribution to the
volatile acidity of wines Like o-lacllc acid, it
Is produced in small quantities as long as the
bacteria do not ferment loo muh rcsKlaal sugar.
An increase in volatile acidity can therefore be
attributed lo lactic acid bacteria, if an abnormal
amount i -0 ! g/l) of i>lactic acid is simultane-
ously formed. In this case. O. aeni fermented a
significant quantity of sugars (a few grams per
liter). This sitaation is called lactic disease
Acetic acid is also one of the unavoidable
metabolic products of citric acid, produced by
homo fermentative lach>bacilli and especially by
he tc rote rn tentative cocci. The fermentation of a
few hundred milligrams of sugars per liter increa-
ses the volatile acidity during malolactic fermen-
tation. Although carried oat on a small quantity of
the substrate, the degradation of citric acid Is cer-
tainly important oa accoanl of the prodaclion of
diaceryl.
Diacctyl. like the other a-dicarboaylalcd com-
pounds in wine, glyoxal. mclhylgl><>xal. and pen-
tancdione. produced partly by the metabolism of
lactic bacteria, arc highly reactive. Reactions, in
particular those with cysteine in wine, produce net-
crocyclcs sach as thiamlc. described as smelling
of popcorn, toast, and hazelnuts, and Ihiophcne.
and fur.ui. with ammas of coffee and burnt rubber
(Marchaudf?<i'..2000).
Methionine and cysteine arc metabolized into
volatile snlfur compounds. The oent species
is particularly active in converting cysteine into
hydrogen sulflde and 2-salfaayl clhaaol. and
methionine into dimethyl disalrkle. 3-(mclha-
salfaayl) propanol. 3-imclhasulfanyl) propan-1-ol
and 3-(mcthasalfaayl) propionic acid. The most
interesting of these compounds from a sensory
point of view is 3-(mcthasnlfanyl) propionic acid,
with its canny, red-berry fruit nuances <Pripts-
Nicotau. 20021.
As for the other known metabolisms of lactic
acid bacteria in wine, they all participate in one
way or another in the spoilage of the wine. The
degradation of essential wine components such as
Ltituic acid and glycerol ink) volatile acidity and
bi IK r- tasting subsEinccs. respectively, completely
destroys the organoleptic quality of the wine. The
metabolism of amino acids (argininc. hislidinc.
etc I docs noi affect taste, but at a kixicological
level it creates a problem by increasing the con-
centrations of biogenic amine and ethyl carbomale
precursors in the wine. All things considered, ropi-
ness seems to be the most widespread and spectac-
ular disease, but even if it causes economic loss,
the damages can be limited since the spoiled wine
can be treated and commercial) *d.
In contrast k> the metabolisms of malic acid.
sugars and citric acid, these last transformations
arc carried out by certain strains belonging to
Metabolism of Lac lie Acid Bacteria
normally inoffensive species Bacterial spoilage
can no longer he aliribuled *> a specific bacterial
species, as in Ifce past Certain strains of O. tiem
font biogenic amines, and olhcr strains fonii
cilnillinc— precursor of ethyl carbomaie.
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Wllliaasnn I) Ji(I9S9)/ A/»p/. A*»mo/.. 22. 392-402.
Lactic Acid Bacteria Development in Wine
6 1 Laclic acid Ixicicrla niirilKii in wiic
62 Phystcochcmical factors of bacterial growih
6 J Evolution of belle acid bacteria microflora during fermentation and
aging, aid influence on wine composition
64 Microbial interactions during witcmakiig
65 Bacteriophages
6.1 LACTIC ACID BACTERIA
NUTRITION IN WINE
Like all microorganisms, laclic bacteria cells nul-
liply when conditions arc favorable: presence of
nutritional factors, absence or toxic factors, and
adequate temperature. All or tbc principal rcac-
liois of its metabolism arc directed towards the
biosynthesis of cellular components: nucleic acids
for the transmission of genetic heritage, carbo-
hydrates, lipids, structure proteins and of course
biologically active proteins. To ensure these syn-
theses, the cell must first find the necessary chem-
ical elements in the medium: carbon, nitrogen and
minerals — in nsablc forms. Since all of these syn-
thesis reactions arc endcrgonic. the medium must
also supply ntokccitcs capable of liberating the
necessary cncigy. Most of the energy P* supplied
by the assimilation of varions substrates In addi-
tion, the cell receives cncigy from sophisticated
systems which activate electron and proton trans-
port phenomena. Although these systems cannot
ensue the totality of cell growth, they contribute
to it very actively in ccrtiin cases, particularly
when the cells arc in nutritionally limited condi-
tions
6.1.1 Energy Sources
Most of the cncigy comes from the assimilation of
numerous organic substrates, sigars. amino acids
and otganic acids Laclic acid bacteria arc encmo-
orgunolrophK oiganisms The oxidation of these
r. r*,. ..i •„ <■,
K-:
I landbook of linology: The Microbiology of Wine anil Vmifications
sihsinilcs ts principally reprcsennrd by ihc fermen-
-iii. ■ ii of sugais. H ctcrofc mien tat ivc anil nomofcr-
mentative laclK acid bacteria degrade nexuses aad
pentoses. At different stages of their metabolism,
cxcigonic reactions pcmiil the slocking of energy
in ATP molecules (Section 521 The oxidation of
sugars Is always coupled with the reduction of
atn/yn's In a necrobiosis, the laclK fcratcnta-
tion process is responsible for their reoxidalion.
In the metabolism of other substrates, the libera-
tion of energy by reactions can be accompanied
by the synthesis of ATP: this is the case with the
degradation of citrk acid and arginine.
The energetic importance of the proton motive
force created al the membrane level has been
demonstrated in several lactic acid bacteria species.
The bacterial membrane in fact has a dual role:
on the one hand, it is a barrier opposing Ihc free
diffusion of component of the medium and ihc
cytoplasm, on Ihc other hand, il is the site of
proton and election exchange. The proton motive
force has two components: a difference in electric
potential (negative inside) and a proton gradient (of
pH). Maintaining a proton motive force requires
an H'-ATPasc of ihc membrane, which functions
rcvcrsibly. An influx of piotons leads to ihc
synthesis of ATP; conversely, the cfllnx of protons
consumes energy. In lactic acid bacteria, the
efflux of lactate from the metabolism Is associated
wilh Ihc efflux of two protons (symporl). In this
manner, the efflux of prolons does not require
cncigy.
During malofciclic fermentation, the use of
malatc produces a sufficient proton motive force
for the synthesis of ATP. The influx of negatively
chaiged malatc into Ihc cell Is coupled with ihc
efflux of neutral lactate, a difference in potential
B created, furthermore. Ihc decarboxylation pro-
vokes the alkalini ration of Ihc cytoplasm and this
increases the pH gradient. All of this leads u ihc
creation of the pioton motive force. The energy
indirectly furnished by Ihc malolactic transforma-
tion ts ihcicforc conserved This same paxess
explains the energy gain by ihc decarboxylation
of histidinc and tyrosine. The hislklinc/hlslaminc
exchange, accompanied by the transfer of a
negative charge, and the decarboxylation traction
provoke the alkaliuicition of the inicmal environ-
ment, ensuring ihc conservation of energy as in Ihc
previous case (Poolman. 1993).
6,1,2 Nutrients. Vitamins
and Trace Elements
Apart f mm water) the most important component).
cells draw carbon, nitrogen and mineral elements
such as phosphorus and snlfur from their environ-
ment. These substances enter inu ihc composition
of cellular componenLs.
Carbon essentially conies from sugais and
sometimes organic acids. Glucose and frucusc
are Ihc most represented sugars in wine after
alcoholic fermentation ta few hundred milligrams
per liter). Mannosc. galactose pentoses tarabinosc.
xylose, ribosc). rbamnosc and a few dlsaccharidcs
are also present in small concentrations (a do/en
milligrams of each per liter). The sugar degradation
capacity depends on the bacterial species and (for
example, for glucose) on environmental factors.
Oenocaccta iieni degrades fructose more easily
than glucose. Its presence in a mixture with glucose
is bencricial to growth lis reduction into mannitol
regenerates coenzyme molecules necessary for ihc
oxidation of glucose. Through a lack of reduced
coenzymes, acctylphosphatc does not lead to the
formation of ctnanol. bui rather to acetic acid
and ATP.
The cncigy obtained by ihc fermentation of
residual sugars largely suffices to ensure ihc
necessary growth for successfully starling and
completing malolactic fermentation. According to
Radlcr (1967). less than I g of glucose per liter
covets the needs of the bacicria to form ihc
hioniav. necessary for malolactic fermentation. In
fact, much less than I g of glucose suffices, since
other sugars in the medium are also used. The
available sugais no! only cone directly from grape
must but probably also from Ihc hydrolysis of some
of iR components, notably polysaccharides.
Amino acids and sometimes peptides supply
lactic acid bacteria with their assimilable nitrogen.
Amino acid requirements vary with respect lo the
species and even Ihc strain These acids can he
strictly indispensable or simply grow th activators.
Lactic Ac id Bacteria Development n Wine
!'..:
Accordiig id Rittfrcai-Gayoa rial. (1975). the
following amino acids arc necessary as a whole
or in pin. depending on the strain Ala. Are. Cys,
Glu. His. Leu. Phc. Ser. Trp. Tyr aid Val. Cocci
have stricter demands than bacilli. The result, of
auxotrophic slndicsarc. however, diflkull to obtain
and interpret. In a more recent study oa " arm.
Fremanx 1 1990) demonstrated Ihclr autotroph)* for
lie. Lcn aid Vol. The synihcsts pathways Tor these
acids have enzymes in common for the production
of aromatic acids I Pic. Trp. Tyr), derived from the
same precursor, chorismic acid, and for Are. His.
Strand Met New observations suggest that His Is
a stimulant and not an essential
Although these data remain very imprecise,
an amino ackl deficiency does not appear to he
responsible for growth difficulties of lactic acid
bacteria in wine. Temporary deficiencies can be
noted at the beginning of alcoholic fermentation
during the rapid yeast multiplication phase, but at
the end this is no longer the case The metabolism
and then the autolysis of yeasts release a large vari-
ety and quantity of amino acids into the environ-
ment. Tie culture of OenociKaa and luctobacit-
ha in a synthetic laboratory medium shows that all
of the amino acids of the medium can be consumed
during growth. In wine. certain amino acids dimin-
ish while others increase in concentration, probably
became of the simultaneous hydrolysis of peptides
or proteins. In addition, the ammonium concentra-
tion increases following the dcaminalion reaction
tRibcrcau-Gayon eial.. 1975). Amino acids arc
essentially used for protein synthesis. Depending
on the strain, some can be catabolizcd and serve as
enctgy sources i are in inc. heuidinc. and tyrosine).
Among nitrogen compounds, piiric and pyrin-
idn bases play an important role in activating
growth. In this case, the needs lor adenine, gua-
nine, aracilc. thymine and thymidine are also
dependent on the strain. They are not always
essential.
Minerals snch as Mg ?r . Mn ! \ K~. and Na*
arc necessary. The lirst two arc often used as
key cizymc cofactors of the mecibolism t kinases,
malolactic enzyme). The following trace elements
arc involved in the nutrition of fctclococci: Or * .
Fc". Mo 4 ' aid Sc 4 '. Yet the role of these metal
ions is not yet established for wine tactic acid
bacteria.
Vitamins arc coenzymes or coenzyme precur-
sors. Lactic acid bacterid are incapable of syn-
thesizing B -group vitamins, in particular nicotinic
acid, thiamin, hiobn and pantothenic acid. Aglyco-
sy led derivative of pantothenic acid was Klcntificd
in grape JiKc. it had been initially purified from
tomato juice (Tomato Juice Factor: Amain i. 1975).
Finally, among the important chemical elements,
phosphorus plays a primordial role in lactic acid
bacteria, as in all cells, in the composition of
nucleic acids, phospholipids and in the stocking
of energy in the form of ATP.
All of the minerals and vitamins cited, as well
as carbon sabsiratcs and nitrogen nutriments, arc
found in sufficient quantities in wine. Only in
exceptional cases, are developmental difficulties of
bacteria alter alcoholic fermentation likely to be
due lo nutritional deficiencies. A simple experi-
ment suffices to prove this statement a favorable
modification of one of the physicochcmical factors
that will be studied later I temperature, pill usu-
ally permits the multiplication of the population.
Independent of these physicochcmical factors, the
absence of growth must be considered to be caused
by inhibitors
6.2 PHYSCOCHEMICAL FACTORS
OF BACTERIAL GROWTH
Fonr parameters very distinctly determine the
growth rale of lactic ackl bacteria in wine: pH.
temperature, alcohol content aid SO. concentra-
tion. Other factors arc also in play but to a
lesser degree and can only be determinant in souk
conditions.
These lour essential factors have been known for
a king lime They permitted the establishment of
cnological rules' Progress in winery equipment
has made these rules progressively easier lo follow
tScction 12.7.4). Noic of these factors can be
considered independently of the others the fonr
act together as a unit. A favorable level of
one compensates an unfavorable value of one or
several others. It is also rather difficult to give
.(■4
Handbook of Etiology: The Microbiology of Wit* anil Vindications
an exact liniil Tot each of them. In this way.
hniciii tolerate higher alcohol contents and SO?
concentrations in wines with favorable pHs than in
wines with low pHs
6.2.1 Influence of pH
The variation n growth rale related to the pH
presents an optimum value aid extreme limits
Most bacleria develop bcllcr at a pH near neutral-
ity This is not the case with acidogenic bacteria
sKh as lactic acid bacleria: their acidophily per-
nio their active development in wit* at low pHs.
around .15. At pHs as low as 29-3.0. growth
remains possible bnl stow. Al Ihc upper pH lim-
its of wine (.1 7-38). it is inch quicker. Slopped
growth due to environmental acidity occurs when
the intracellular pH attains a certain limit (pH ( |.
It not only depends on Ihc environmental pH but
also on the nature of Ihc acids (McDonald et of.,
1990). In fact, the fraction of acids that freely pen-
etrate in non-dissociated form is dissociated inside
the cell, resulting in a decrease )■ pH Conse-
quently. Ihc intracellular enzyme activity is more
or less inhibited with icspccl to the optimum pH
of their activity. The pioton motive force and the
dependent transports arc also stowed, interfering
with the global metabolism of the cell and thus
multiplication The lower limit tolcraled for pH l
vanes depending on Ihc species. II is appiox-
imately 4.7 and 55. respectively, for L plan-
tiauiii and /. meseiHeroMes. according lo McDon-
ald elal (1990) At pH 35. oem maintiiis
a higher pH t than /. planttmm (Henick-Kling.
1986). The strains of this species adapt bcllcr to
acidity than other species Moreover, when culti-
vated in an acidic environment, they have a higher
pH, and thnsagrcatcrprolon motive force — linked
to Ihc higher proton gradient
Acidity adaptation mechanisms arc not known
bat actively purtKipaie in Ihc natural selection
of this species In wine. II has been established
for a toig time thai wines with iclallvely high
pHs present nol only a more abundant tactic
microflora but also a much mote varied one with
respect to acidic wines These wines arc more
mKrobtologKally fragile as some of ihc bacleria
arc spoilage tailors, and as a broader range of
substrates is metabolized. High pH facilitates the
growth of bacteria in wine, as well as promotes
their survival, nol only directly but also by
reducing the effectiveness of Tree sulfur dioxide.
Spoilage may develop several months, or even
yeais. after fermentation. The pH also has an
impact on the malolaclic activity of the entire
cell.
Besides growth, the pH aiTcco Ihc matolaclK
activity of the entire cell Although the optimum
I'll of Ihc purified cn/ynic is S.9. it is not the
same for cells. The matofctctic activity of 0. aeni
strains is optimum at a pH between 3.0 and
32 and around (**i of Its maximum activity at
pH 3 8. The usual pH range of wines, their Ion:,
corresponds well with the maximum matolaciic
activity of the bacterial cell Vet the malolaclic
fermentation rale depends on not only the activity
but also Ihc quantity or cells Finally, al usual
wine values, the pH affects both in the same
way. Consequently, when all other conditions arc
equal. malolaciK fermentation Is quicker at higher
pHs. Fix example, malolaclic fermentation lasts
I64days for a wine adjusted to pH .1.15 aid
14 days for a wine adjusted lo 3.83 (Boasbouras
and Kunkcc. 1971).
Accoidiig to Rlbciran-Gayou end. (1975). the
pH also conditions the nature of the substrains
Iraisforitcd. The authors defined Ihc threshold
pH for malic acid and sugar assimilation ll
corresponds to the lowest pH al which the
substrate is transformed aid it varies according
to Ihc strain. The threshokl pH for malic acid is
tower than the threshold pH for sugars. In the
rune between these two pHs. bacteria degrade
malic acid without fcmcnllng a large quantity
of sugars and thus without producing volatile
acidity The larger Ihc /one . the bcllcr adapted for
wincnaking is the strain The average threshold
pH of 400 be tcrofc menu live coccus strains lested
is 3 23 for malic acid and 351 Tot sugars. These
values arc respectively 3.38 aid 3 32 for Ihc 250
bctciofcrmcntalivc laclobacilli strains tested. The
presence of the latter I be if line docs nol guarantee a
nialolactic fermentation wilhoul the risk of volatile
iK id it)* production.
Luetic Ac id BactcrEi Development in Win
165
The pH Is therefore vciy important aid comes
into play at several levels: Ii the selection or the
test adapted straits: in Ike growth rate and yield:
in the nialolaclK activity; and even in the nature
of the sahstruKs transformed
The role of pH has diverse practical conse-
quences in the control of Ike malolaclic fermen-
tation. First of all. the makilactic fermentation is
initialed more easily and rapidly in press wines
than in the corresponding free ran wine. A partial
chemical dcacidification of wine may he advis-
able in the most difficult cases. It is especially
recommended in the preparation of a malolaclic
fermentation starter— used fot the inoculation of
recalcitrant wine tanks. Finally, particular atten-
tion must be paid to musts and wines with elevated
pHs. They sustain a ntorc or less anarchic bacterial
growth of a large variety of bacteria and arc thus
subject to spoilage A sensible sulfiting is the only
tool for controlling these microorganisms
6.2.2 Effect of Sulfur Dioxide
In wine, sulfur dioxide (SO;) is in equilibrium
between its free and bound forms, lis effectiveness
as a germicide and as an antioxidant is directly
linked to wine composition and pH (Section 8 J.I).
The active form, in fact. Is molecular SO; which
depends on the concentration of free SO; and
the pH To calculate it. the Sudraud and Chauvcl
11985) formula can be nscd which gives the
percentage of molecular SO; in function of the pH.
^molecular SO, = IOO/IO> HIW + I (6.1)
For example, at pH 3.2 this percentage is 3 .91'* .
His. respectively. 2.rXH and 1.01** at pH 35 and
pH Mf These numbers demonstrate the Influence
of pH. Four times more free SO; is necessary at pH
38 than at pH .1.2 toobctin the same effectiveness
The mechanism of the action of SO, was stud led
in yeasts in particular, but it Is most likely very
similar in bacteria. According to Romano and
Snz/i (1992). SO; penetrans into the cell in
molecular font by diffusion. In the cytoplasm
where the pH Is highest, it dissociates and tcacrs
with essential biological molecules: enzymes with
their dlsulfur bonds, coenzymes and vitamins The
result is cessation of growth and. finally, cell death
The inhibitory action of SO; on the nialolaclK
cn/ymc of Oenococcus is in addition lo its effect
on cellular growth.
For the same concentration of total SO, . bacteria
inhibition depends on the binding power of the
wine (Section 1 3.3.2). which In turn determines the
free SO. remaining and the pH This establishes
the amount of active molecular SO,. It is only
possible lo give an approximation of the quantity
of SO, necessary u inhibit bacterial development.
As a general rule, lactic acid bacteria have
difficulty in developing at concentrations > 100 mg
of total SO; per liter and 10 mg of free SO. per
likr. Evidently, the result is not the same at pH
3.2 and at pH 3$ Their sensitivity also varies
according to the strain Finally, for a given strain,
the sensitivity varies according u environmental
growth conditions and physiological adaptation
possi bill tics
Lafon-Lafontcadc and Peynand (1974). found
that cocci seem less resistant than lactobacilli
Thus. O. oem growth Is hindered more than
/. lui&aitlii growth, for example. The effect is also
con nee led to the strain Peitiocaena ihaimmu Is
a useful example: the n<py strains arc insensitive
to SO; doses that inhibit or kill olherstrains. Alter
2 months of bottle storage, ropy type bucteria can
maintain populations between 10* and l0*UFC/ml
in wines con tuning SO mg of free SO, per liter
tLonvaud-Finel and Joycux. 1988)
Bound SO; alsocxertsagrowth inhibitor effect.
dcmonstraKd by Fomachon t l963)(Scction 8.6.3).
Lactic acid bacteria may be capable of metaboliz-
ing the aldehyde fraction of the combination and
liberating SO,. The SO; then exerts its activity on
the cell, btt it is less effective From their tests.
Lafon-Lafouicadc aid Peynand 1 1974) concluded
that i'-. ml SO; is 5 lo 10 times less active than
free SO,. Other authors have observed thai its con-
centration in wine can easily be 5 to 10 times mote
elevated.
Technological consequences can be drawn from
these rcsnlts. When the elaborated wine must
undergo malofcrctic fermentation, it Is important lo
sulfite the grapes judiciously. The su lilting must
exert a IraBsiury Inhibitory effect on the lactic acid
166
Handbook of Etiology: The Microbiology of Wine anil Vinifications
mctera Al Ihc cod -ii.iLnlM.il. fc rare n tat i m ihc
bound SO; pctscus and can delay bacterial growth.
Obviously, sniffling ihc wine during running off is
mi recommended, except in very ■■usual cases
(Section 12.62).
6.13 Inlucwccof Kthanol
Like most ntic morgan isms, lactic acid bacteria an;
sensitive to cthanol. Generally, in laboralory con-
ditions, bacteria Isolated from wine arc inhibited
alan alcoholic strength of around S-llYi volume.
Results v.uy according n> the gents, species, and
sirarn. Ribcrcau-Gayoi etal. (1975). found thai
cocci arc altogether morc sensitive lo cthanol than
ate ladobacilli At an alcohol content of 13'* vol-
■■K. mote than 5CM of the fcictobacilli resist as
opposed loonly Wi of Ihc cocci.
Thcgiowthoft?. iv/u strains Isofciied from wine
and cultivated in the laboratory is activated al
around S-o'i volume orcthanol: it is inhibiled in
environments richer in cthanol and difficult at or
above 13-I4*i volume. The cthanol tolerance of
Laboratory strains is much less than for Ihc same
strains cultivated in wine. Bacteria that multiply
in wine adapt lo Ihc presence of cthanol but also
probably lo Ihc wine environment as a whole. In
addition lo the intrinsic strain tolerance of cthanol.
their adaptation capacity varies. II is therefore
difficult to set a limit above which laclK acid
bacteria no longer multiply.
Strains of UnUAxKilliii fruclivinwu. L brevis
and /- hUgmilii (hciciofcrmcntalivc bacilli) arc
frequently isolated from fortified wines with alco-
holic streng ths from 16 to 2fW volume. They seem
lo be naturally adapted to cthanol but lose this
adaptation after isolation. Strains of L frucit'w-
rant nevertheless remain very ulcrant of cthanol.
which has an activator ioIc in their case (Kalmar.
1995). /'. tUnituma bacteria arc not particularly
rcsisGinl to alcohol, but the ropy strains multiply al
the same rate and with the same yield in the pres-
ence or absence of 10-12'* volume of alcohol.
The adaptation phenomena arc definitely dissim-
ilar in nature In most cases, they are Ihc result
of a structural (fatty acid, phospholipid and pro-
Kin composition) and functional modilkalion of
the membrane In the case of mpy /'. ilwmism
strains, the polysaecharidic capsule possibly ;
as a supplementary protector.
6,2.4 tC fleet of Temperature
Temperature influences the growth rate of all
microorganisms. As with chemical reactions, it
accelerates biochemical reactions. Cellular activity
(resulting from all of the involved enzyme activ-
ities) and consequently growth vary with temper-
atme according to a hell curve. At the optimum
temperature, generation time Is the quickest. This
curb not only varies with the species and strains
but also with the environment in which the bacteria
multiply.
In a laboratory cullnic medium, laclic acid bac-
teria strains isolated from wine multiply between
15 and 45 "C but their optimum growth range is
berween 20 to 37"C. The optimum growth tem-
perature for O. item Is from 27 u .IOC. but it is
not Ihc same in an alcoholic medium, especially
ii wiic. The optimum temperature range is mote
limited: from 20 to liX. When the alcohol con-
ical increases u 13-14'* volume of alcohol, the
optimum temperature decreases. Growth slows as
the temperature decreases, becoming nearly impos-
sible aiuutd 14-I5°C.
The ideal temperature for laclic acid bacteria
growth (notably 0. item) and for malic acid degra-
dation in wine is around 20'C. An excessive tem-
perature of 25 U C or above always slows malolac tic
fermentation— principally by inhibiting Ihc bacte-
rial biomass. Additionally, an excessive temper-
ature increases the risk of bacterial spoilage and
increased volatile acidity In practice, thciclbrc.
maintaining a wine at 20'C is recommended. II
should nol be allowed to cool too much after alco-
holic fcrnicnutron If the temperature of the winery
decreases, the wine should be warmed
When the temperature is less than 1ST. the im-
itation of malotactic Icravcnlation is delayed and
iK duration is longer. A malolaclK fermentation
under way can continue even in a wine with a
temperature between 10 and I5 U C. In these cases.
Ihc bacterial biomass was normally constituted
under favorable conditions The cooling blocks the
mo hi plication of bacteria but docs not eliminate
Lactic Ac id Bacteria Development n Wine
them. The cellular activity, however, is slower.
Malolactic fermentttion of a wine therefore con-
linKs after ie> initiation even in the case of being
cooked, but the titration is much longer Tnc time
frame for degrading all of Ihc malic acid can range
front 5-6 days io several weeks or months.
Along with pH. tcmpcrularc Is certainly the
factor that most strongly iillKnccs ihc ntalolaclic
fcmicitibon speed of a properly vilified wine not
executively sulfilcd. This factor R also the until
easily monitored and controlled.
6.2.5 Other Factors Involved in Lactic
Acid Bacteria Activity and
Growth; Adaptation of Bacteria
to Growth in Wine
The action of phenolic compounds 01 lactic acid
bacteria growth icmains relatively ■■known Past
results have shown that polyphenols tested alone
or ii a mixture had an inhibitory effect Saraiva
(1383) noticed, on Ihc contrary, that gallK acid
stimulates yeasts and lactic acid bacteria. Con-
versely. difTcrcnl phenolic acids (coumaric. pro-
tocatccbK acid, ck.) and condensed anthocyanins
Inhibited them Enological unnins were found Io
have an antifuclcrial effect (Riblreau-Gayon el ill..
1975). The effect of phenolic compounds on lactic
acid bacteria growth remains unclear.
Nevertheless, a systematic study of several types
of molecules clearly demonstrated the inhibitory
effect of vanillic acid, seed procyanidins and oak
cllagitannins and. at Ihc same time, the stimulating
effect of gallic acid and free anthocyanins (Vivas
eitil.. 19951 These rcsnlB pertain u Q.txm
growth, bat may also be valid for other bac-
terial species By favoring growth, gallic acid
and anthocyanins activate malolactic fermentation.
Bacteria degrade these two compounds. The trans-
formation of anthocyanins seems Io activate a (!•
g 1 ncosidasc— freeing Ihc aglycon fraction and the
glucose, which is mclaboli/cd by bacteria.
Polyphenols, along with wine components as
a whole, affect bacteria Some arc favorable and
others unfavorable io bacterial growth and acliviry.
bul they play a secondary rokr compared with
Ihc other four parameters examined earlier These
elements, among many others (mosl of them
unknown), determine the malolactic femen lability
of wines.
Similarly, oxygen can influence the multiplica-
tion of lactic acid bacteria in wine bnl its effect is
nol clear. In facl. the behavior of bacterial species
present in wine can be diverse with respect to oxy-
gen They can be indifferent to lis presence, adapt
better in its absence (facultative anacrobiosis). tol-
erate oxygen at io partial pressure in air bnl be
incapable of using il (acrototcianl). or linally can
require a small oxygen concentration for optimal
growth (microacrophilcs). Furthermore. Ihc bchav-
iorofagiven statin can van with in environment
In a laboratory culture medium. growth is activated
in an inert gas atmosphere: CO- and N>.
It is therefore difficult Io specify the possible
oxygen needs of lactic acid bacteria in wine. Cur-
rent observations indicate that a limited aeration,
after running off or racking wine, can strongly
favor Ihc initiation of malolactic fermentation.
Wine is an extremely complex environment and
it is nol possible to elucidate Ihc effects of all
of Ik components on lactic acid bacteria. In any
case, this would not help the cnologist. since
these individual effects are cumulative— acting in
synergy or. on the contrary, compensating each
other. In Ihis medium, lactic acid bacteria, par-
ticularly 0. oeni. develop in extreme conditions.
Acidity and cthanol combine with other molecules
to inhibit the growth of the Isolated strains.
It has long been known lhat a statin of >>. ivni
isolated from a wine undergoing malolactic fer-
mentation, therefore capable of multiplying, then
cultivated in a laboratory medium, loses iR viabil-
ity when re- inoculated in wine. Many observations,
both in the laboratory and in Ihc winery, sug-
gest Ihc existence of adaptation phenomena that
ensure Ihc survival and growth of bacteria in these
extreme conditions. Isolated cells cultivated in a
laboratory medium with wine added have a gener-
ally higher tolerance Io low pH. SO;, cthanol and
wine than isolated cells cultivated in the absence
of wine (Tabic 6.1).
The plasmic membrane probably participates
actively in these adaptation phenomena, which
have been shown to exist in 0. tvni and olhcr
Handbook of Etiology: The Microbiology of Wme anil Vniftatioas
Tubk-ri.t.
inoailtfb
Influcnc
* IB ItJ Wl
of OJIUIC -C.t«l«
iac(Cmb»y. 19941
onth
c popuUiiua HI 1','mh of four a
""" ""
in* (A. B.CimI lit .titer
Hac
Strain*
fdhjm)
A
B C
D
M
MW
M
MW M
MW
M MW
ill
<I0*
|(l'
1. 10*
2x 10*
bacterial species. Tbc first adaptation mechanism
M be discovered was modification of tbc fatty acid
com pusi lion of ihc membrane All stress by ihc
medium (addition of clnauol or wine, a temperature
change, etc ) capable of provoking a niodillcatioa
of membrane fluidity, and thus membrane tuition,
is compensated by an adjustment of the length and
■■saturation level of the fatty acid chains. Fat all
species studied (O. itm. Pettiocixeus dammvnu.
L plantiBiBH. L liilgiailu urul Lfnu'litoiwisi. the
presence of ctbanol in tbc mcdiim. for example,
greatly increases the proportion of unsaturated
fatty acids (Descns. I9B9: Garbay el ill . 1995:
Kalmar. 1995).
A second phenomenon, quantitatively and qual-
itatively sign! ft: ant. concerns membrane pro
Kins Their concentration increases following a
shock — whether physical (cold or heat) or chem-
ical (acidity, addition of ethanol. fatty acids ur
wine) (Tabic 6.2). In thrs manner, as with all liv-
ing cells. O. oem and the other kictic acid bac-
teria react to a shock by inducing the syntbe-
se> of shock proteins". These proteins participate
in Ihc reaction of the cell against environmen-
tal slicss (Garbay and Lonvaud-Fnicl. 1996). The
LolS protein is induced in O.oeni by heat, ethanol.
and acidity. It is associated with the membrane
and maintains in integrity, following induction by
a change in fluidity (Guzai el til.. 1997; Dclmas
el of., 2001).
Lactic acid bacteria arc extremely exacting in
their development in laboratory media. Contrary
u all expectations, these microorganisms dcvck>p
spontaneously in wine. Their development is due
Tubk-Cl lotocnccof ditlcrew type* of
pmlcia co ate or alio* of ihc O. ••ml plum
KJduv. 1994)
Count
Hewing tt> 37 C
Hewing tti 42 C
Hewing i« SO-'C
lacubminn Ifi cllnnol
Isouhniion Ifi crlanol -t buy
i.i.i.%
to their complex group of adaptation phenom-
ena—notably the induction of proteins whose
functions remain unknown.
6.3 EVOLUTION OF LACTIC ACID
BACTERIA MICROFLORA
DURING FERMENTATION AND
AGING, AND INFLUENCE ON
WINE COMPOSITION
6.3.1 Evolution of the Total Lactic
Acid Bacteria Population
In the production of wines requiring nudolactkr fer-
mentation, the bacterial microflora passes throng h
several phases (Figure 6 1). During the first days
of fcrmenutlon.assoonaslbc tanks arc filled, they
arc present in very variable quantities— most often
firm 10* to If* 1 UFC/tal. The extent of the pop-
ulation depends on climatic conditions during the
Lactic Ami BaclcrEt Development ii Win
[W
Fig 0.1. Eiulufon of lactic acid bacicm fopiUliu*
ill 1 1 w alcoholic Jikl mibkiclk- fcimcminn
Lfsi days of maturation Ii Is generally lower when
Ihc conditions an: pn>p)tioas for hcallhy grapes
anil in these situations a sialic bacterial colony
can be impossible to Isolate on Ihc berry. How-
ever, during Ihc dive inc operations from hancsl
to filling the tmk. Ibe ninsl is inocuktlcd very
rapidly— probably by Ihc equipment. During the
harvest period Ihc bacteria, like the yeasts, pro-
gressively colon i/e the winery. In general. Ihc lasl
Links rilled prcscil Ihc highest populations.
During the lirsi days of alcoholic fermentation,
the bacteria and yeasts multiply. The killer, better
adapted to grape mast, rapidly invade Ihc medium
with elevated populations During this time. Ihc
bacteria multiply bnl their growth remains limited,
with a naximnm population of ID 4 to 10- LTC/ml
To a huge extent their behavior at this time depends
on Ihc pH of the medium and the grape sulfiting
level.
Normally, the doses of SO? added (about 5 g/M)
at pi Is between 3.2 aid 3.4 do not prevent
their growlh. but simply limit it Then, from the
most active phase of alcoholic fermentation lo the
depletion of sugars. Ihc Ixtclcria rapidly regress to
It) 1 to I0» LTC/ml. This level also depends on
environments conditions ipH and SO.).
lidlowing akoholit fermentation. Ihc bacterial
population remains in a latent phase for a varying
period, which can last several months when the
pH.cthanoland lempcralurc parameters aical their
lower limits Usually, this phase Lists oily for a
few days, and in certain cases, il docs sol occural
all. In the most frequently encountered situation,
the multiplication phase takes place after Ihc wine
has been run off.
One microorganism follows the other the yeasts
first and then Ihc lactic acid bacteria. These arc
ideal wincmaking conditions, in which all of
the fermentable sugars arc depleted before the
bacteria invade the medium. In the opposite case,
the bacteria multiply actively towards Ihc end of
alcoholic fermentation: they ferment sugar-, using
the hclerofcmicitalivc pathway and increase the
volatile acidity of the wine.
The growth phase lasts for several days and
raises the population to around I0 1 UPC/ml of
more Evidently, its duration also depends on
Ihc composition of the ami nun The subsequent
stationary phase also varies The bacteria then
begin Ihc decline phase. As soon as the malic
acid is completely transformed, sulliling Is used
to eliminate the baclcrEi as quickly as possible
The matotactic fermentation phase beginsdnring
Ihc growth phase, as soon as the total population
exceeds 10 7 UPC/ml. It continues and is completed
during Ihc stationary phase, or sometimes at the
beginning of the death phase Ii very favorable
conditions with a limited concentration of malic
acid, malolaclic fermentations arc often completed
even befoic the end of Ihc growlh phase. The
optimum population in these cases exceeds 10 s
L'K'/ml As soon as a sufficient biomass is formed,
malic acid Is degraded The malolaclic acid
Kit lend activity is always present but depends on
various conditions, especially the lempcralurc. The
transformation of * g of malic acid per liter can
lake more time lhan 4g/l if the population level
attuned Is lower.
If wine Is nol sullitcd after malolactic fermen-
tation, bacteria coalinnc lo survive for months.
Carre (19K2) observed a small decrease from 10 ?
L'FC/iil lo ID 5 UFCYiil after 6 months of conser-
vation in a wine stored al I9 ; C with a pH of .1.9
and an cthanol volume of 1 1 25'i. Sulliling imme-
diately after Ihc end of malolaclic fermentation is
intended to accelerate this death phase No signif-
icant viable population should be left in the wine
Even if they can no longer multiply very actively.
170
Handbook of linoUigy: The Microbiology °f Wine anil Vinificalions
eclbt tan metabolize diverse substrates to ensure
their survival. These transformations have tot all
been explained bnl they increase ihe mIics con-
centrations of undesirable substances from a sen-
sory or hcallh standpoint (biogenic amines, elbyl
carbamate, etc.).
Sulfiling al ihe end of secondary fermentation
is piuciiced k> adjust Ihe free SO. concentration
u 30-40 iiit 1 '] Al Ibis conccnlralion. nearly all
of Ihe Lie tic ackl bacicrEi disappear within a few
days (<I-I0 UFC/ml). The results also depend
on the composition of the medium (Figure 6.2).
Addilionally. numerous observations have shown
thai the Lactic population is maintained more easily
in the barrel than in the tank During 18 months of
barrel aging, a decrease from only 10* UFC/ml
w 10* UFC/lnl was nolcd in spile of a free SOj
concentration of between 20 and 30 mg/l. The kisl
fining realized with egg whiles effectively helps k>
eliminate baclcria.
In fact. Ihe drop in the bacterial population
assessed by counting Ihe colonies developed on
a nutrient medium does nol apparently provide an
accurate representation of the situation after suk
filing. Counting Ihe bacteria by cpillut
shows that part of Ihe population retains *
so,,-, I)
Fifi fti lnBuc*.« of muteeutar SO, concern raibn on
berk- icil h4cicria uiivhal- (Innvaud-luncl. unpuh-
Inhcil rtuili.v) Ni = viable hucicru population 20 day*
after MiiniiKfj; \. = initial baclcria populainn. ■
wiocA:(A)*incB;<«) n iaeC
metabolic activity, although the cells are inca-
pable of multiplying in a nnlrient medium This
physiological condition is described as viable bul
non-cultivable" (Millet and Lonvaud-Fuicl. 2000)
(Seclion 632).
According to Colon (1996). histamine may be
accumulated in wines by this type of cell, which
still has a high histidinc decarboxylase activity.
Il is. therefore, possible thai baclcria may be
responsible for other transformations in wine
constituents, even aflcr sn I filing and during aging.
These effects are not necessarily entirely negative.
6.3.2 Viable but Non-cultivable
Bacteria
Viable baclcria arc usually counted as bacterial
colonics, on Ihe principle that baclcria placed on
a nutrieni gel will multiply. Aflcr an incubation
period, ihe resulting population becomes so latge
it is visible to the naked eye and is. thus, easily
counted. Counting by cpifluorcsccncc is based on
a completely different principle. Bacteria cells on
a microscope slide or filler membrane are placed
in contact with a substrate that is transformed
by passing through each cell. The most common
sabsiraK is a fluorescence cskr nydrolyied by an
esterase in Ihe cell, which makes the bacterial
content fluoresce under UV light All cells thai
fluoresce under these conditions are considered
viable as hydrolysis of the ester indicates the
existence of enzyme activity.
Both methods give the same results for bacte-
rial suspensions in Ihe growth phase. When they
move into the stationary, and then the decline
phase, ihe difference between ihe result increases.
While counting by cpiflnorcsccncc shows a slight
decrease in ihe number of cells, there is a sharp
drop in the number of colonics visiMe. This dif-
ference may be explained by the fact thai part of
Ihe population of fluorescent cells is still biologi-
cally active but is incapable of Ihe metabolic and
physiological functions necessary for mulliplica-
Ini They arc described as viable, non-cultivable"
(VNCl cells Table 6J shows the lactic baclcria
count afrcr su I filing a wine
As expected, sulliling eliminated the viable land
rcvivablc) population. However, there were still
Lactic Ami Bacteria Development ii VVir
171
Tabic ft. A l.;,in. i-.i,t.-iij population collided by Cfi-
Huomccncc (celh in It and by voWe cobnln iVlC.
rr. .1 .11..! miIIii.ii„- ■ Milkl ,. n. 1 I . >D' .u.-l . n.l 2001))
Fax SO r C
■pj)
Kf.fluoiocencc
Colonic*
30
50
(44±0)x 10'
(44 ± 5) x 10*
<1
large numbers of VNC cells (Millet and Lonvaud-
Fnncl. 2000).
This phenomenon is not exclusive Id lactic bac-
Icrtu. bul certainly applies u many other microor-
g; iii .-in- ll is easily demonstrated for acetic bac-
Icrit in wiucmakiug As soon as they arc deprived
of oxygen, the diffemec between Ike viable and
VNC populations increases rapidly, (ben disap-
pear complclcly as soon as Ihe wine is act-
atcd (Millet and Lonvaud-Funcl. 20IXI). The one
experiments showed lha( yeast and bacteria in
VNC state decrease in sis and sonic of them
may pass through tillers intended u eliminale
them.
The exKnl of Ibis phenomenon aid its impor-
tince in winemaking have yet lo be assessed. Fur-
ther research is required todcKniiinc what happens
lo VNC cells and Ibcir capacity Id recover viability,
ic ** multiply and produce colonics.
6.3.3 Evolution of Various Bacterial
Species
During fomentation, the laclic microflora evolves
not only in number bnl also in variety of species.
Carre ( 19B2) isolated bacteria on grapes before (he
harvest belonging Id the following species: UkIo-
txxilhis pitmttinoit. L hilRimlii and /_ ciaei. The
species O itni. which becomes the most signif-
icant Utter, is barely present at the beginning of
fermentation. lust after io arrival in the tank, grape
must contains a very diverse microflora, gener-
ally belonging to Ihe eight usual lacubacilli and
cocci species: L planranm. L row/. /- hilgmiHi.
L brevit. P. dtmiiuisia. P. pentotiitem. L mesen-
tenmles and O. vein) (Tabic 6.4).
All species are not always represented, or
at least cannot be identified by enrrent analyt-
ical methods, bul a natural selection has been
confirmed which tikes place progressively dur-
ing alcoholic fermentation The laclic popula-
tion regresses after reaching its optimum. At the
same time, the bomofcrmcntalivc then hckrrofcr-
mcuuiivc lactobacilli disappear, to the benefit of
O. itm. Afterwards, the homofcrntcnlativc cocci
and L mettmertiitles aba give way to 0. oenHtce
Table 6.4).
Certain species may also subsist at vciy low
residual populations— less than lOor Itf UPC/ml
Molecular methods, such as PCR and PCR-
DGGE should enhance onr knowledge of total
residual microflora. As these methods amplify
the signal specifle to a particular microorganism,
they make it possible lo identify minority species
Furthermore. PCR-DGGE reveals the presence of
unexpected microorganisms, as a region common
to all bacteria is amplified, then each species
is Klcnliricd individually (Claissc and Lonvaud-
Funcl. 2003). Some species may proliferate al a
later sttge. once fermentation is completed, if Ihe
wine is not property protected. After fermentation.
Tabic 6.4. fofutui
CabctKi Sjuvnpm
oa (Ur-r/ml) of the dilfc
n •u\t (liinvjud-FuncI n
rem belie mid b*ciei
<i.. 1991 J
a \fnK\ lining the ikoholk Icim carat ion of
]>jy Alcohol
Come*
4% vol.)
Oow.v.n-./i lanwj
not- PMSoHcni
Vli'l'I tllttttltl M I
Ltt'tfbiriltus l/tiobiirillat Umabaillat
liilgtatffi Oiei ii pliimrma
172
H ami I*** k of Enokn*y: The Microbiology of Wi»e anil Vinifkauons
some cm multiply if ibe wine Is poorly protected
In fact, species other Uian 0. ttem are most often
a-spomslWt for wine spoilage.
The spontaneous evolution or a mixture of
species corresponds with the sclcciioi of Ihosc
bcsl adapted lo wine — which is a hostile acidic
and alcoholic environment. The composition of ihc
ptasmic membrane, and Ihc various mechanisms,
lhal permit it to react lo Ihc aggressiveness of ihc
medium, seem lo influence Ihis adaptation. Cer-
tain species or strains may also differ in their
ability lo carry out these transformations Strains
of L fruciivorims adapl belter to ethanol than
/. pliiiinmBn and /- talgirtE, dnc lo a morc cfTec-
livc modification of their laity acids I unsalnralion
and chain length) ( Kalntar. 1995). Unsurprisingly,
strains of Ibis species are often idcilificd in forti-
licd wines Ciinicd by laciK disease with an alcohol
content between IS and 2<W volume.
6.3.4 Evolution of Wine Composition
in the Different Phases of
Bacterial Development
As soon as tactic acid bacteria multiply, they
inevitably modify wine composition In fact. Ihcir
growth requites the assimilation of sahstratcs
to supply Ihc cell with cncigy and carbon and
nitrogen The division of a baclcrium into two
daughter cells evidently supposes Ihc ncosyn thesis
of all of Ihc slructmal component, and molecules
having a biological activity. In wine, bacteria
transform sugars, organic acids and a mo In hide of
other components. The type of reactions and the
nalurc and concentration of these substances more
or less profoundly modify Ihc wine — improving
or. on the contrary, spoiling it.
The only bacterial intervention trnly sought
after in wincmaking is Ihc Iransfonnalion of
malic acid into lactic acid (Section 12.7.2). It
is the source of Ihc most manifest organoleptic
change, resulting from maktlaclic fcmicnlalion: the
dcacldilicatron and Ihc softening of wine Malic
acid, a dkarboxylic acid, is transformed molecule
for molecule into lacbc acid, monocarboxylk.
The k>ss of an acid function per molecule Is
intensified by Ihc replacement of an acid wilh
a particularly aggressive task; by a much softer
acid This transformation is carried out on 1 5 g/l
to 8 g/l maximum, depending on Ihc variety and
grape maturation conditions.
Bacteria do not transform all of Ihc malic
acid contained in the grape. From ihc start,
during alcoholic fermentation, yeasts metabolite a
maximum of Mfi of the malic acid. The pn>dnct.
pyruvale. then enters one of many yeast metabolic
path ways-— notably leading to ihc formation of
ethanol. This nalo-alcoholk'" fermentauon Is
catalysed al Ihc litsl stage by Ihc malic ennme.
The bacteria must develop a snffkient population
before malolactic fcrmentalkM can truly start. The
production of i_-laclk' acid Is coupled wilh the
decrease in malic acid (Figure 63).
The degradation of citric acid is aLso very
important in cnology. First of all. its disappear-
ance from Ihc medium contributes to the natural
microbiological stabilization of wine by eliminat-
ing a potentially enctgetk* substrate Additionally.
the organolcplkal impact of the products of its
metabolism, fairly well known al present, has been
proven (Section 5.3.2). (Dc Revel clal., 1996).
Diacctyl Is certainly involved and al low con-
centrations it gives wine an aromatic complexity
lhal is much appreciated. In certain kinds of wine,
tasletscvcn prefer wine that has a very pronounced
odor of this component. The degradation of citric
acid also increases volatile acidity— a maximum
of approximately 70 nig.'l (HjSO t ) . Oiganolcptical
deviations dnc lo an excess of volatile ackliry or
diacctyl coming from the degradation orcithc acid,
however, arc rare: they can have other origins.
Recently, cnologists and researchers have shown
increased intcicst in thissubfect. but opinions dif-
fer to snch a point that some currently advocate
avoiding ihc degradation of citric acid as much as
possible while others look for ways of ensuring
it. Entirely different approaches have been con-
sidered, including Ihc use of transformed bacterial
strains k> ensure or prcvcnl Ihis transformation or
even for accumulating diacctyl
In any case, citric acid is always degraded during
malol;rclic fermentation, since oem species
have all of the necessary enzyme equipment lis
transformation is nevertheless slower than malic
Lactic At hi Bacteria Dcvckipmcnt in Win
Fie; ft. J Evohabn of different wmpouo
malic Mtl: open Mir. dcmiy; filled Mjutire. cfc
.turimr IcnncMJlu
■I: open u|ihk. l.-kulk ft
acid, and several do/cn mg of tilrit acid |c 1" lilcr
often remain ul (he line of sulllliag . Yet since lactic
ackl bacteria ait noi eliminated immediately and
icniain active for several days (someiinics several
weeks), oaly liuccs of eiltlc atNl irmain in wine.
During wiacmakiag. laclic disease is a dreaded
bacterial spoilage By definition, il corresponds
■'.ul, the increase of voblilc acidily cansed by (he
be tciofc menu live fcrmcnialion of sugars. Nor-
mally, tactic ... i". bacteria multiply only aflcr the
completion of alcoholic fcrmcnialion. The rcskl-
nal sngaa— glucose, fractose and pen lose — are
in small bui sufficient concentrations K> ensure
the essential energy needs of the bacteria If bac-
terial growth occurs before the end of alcoholic
fcrmcnialion. when more than 4-5 g of reducing
sugar per lilcr remains in the wine, lactic dis-
ease can result In fact, bctcrofcrmcntailvc bacteria
(0 •vial fcrmcnl sngars not only into lac Ik acid
bul also into acetic acid. Moreover, the sludy of
metabolisms has shown that Ihc fermentation of
glucose in the presence of frnclosc. which is the
case in wine, preferentially direcrs the acetyl phos-
phate molecules towards acetic acid — fructose
being reduced inu mannitol I Section 5.22).
Lactic disease, therefore, occurs when envi-
ronmental conditions are favorable to bacte-
rial growth, even though ycasrs have nol ycl
completely fermented the sugars. This category
essentially comprises wines derived from particu-
larly ripe harvest-. In fact, the sugar concentration
is elevated, the medium is often poor in nitrogen
and the pH is high. A stuck alcoholic fermentation
(Section 3.8.2) or simply a sluggish fermentation
should be expected. These phenomena by may lead
to a rapid multiplication of lactic acid bacteria
Laclic decease is also a widespread form of bac-
terial spoilage in fortified wines These wines arc
elaborated by the addilkm of alcohol to grape must
that has been slightly (or not at all) fermented
They are generally stabilized due to their high
alcohol content Vet tactic acid bacteria, and most
often heir role rate illative tactobacilli. arc particu-
larly resistant to cthanol. They develop easily in
this very sagar-rich medium Not only is there mal-
olaclK fermentation (which is not a real problem):
there is also, laclic disease The volatile ackliry of
these wines frequently attains 1-15 g/l (H.SQ,)
This phenomenon often occurs in Ihc bottle, pro-
ducing carbon dioxide and a cloudy wine.
A poslcrkxi. the diagnosis of lactic disease is
Ixiscil on the nature of the products of ihc bacterial
metabolism (Section 14.2.3). Wines presenting an
elevated volatile acidity can also have been the
siKof acclic bacterial multiplication or a mccibolK
deviation of yeast. Vet when they produce acetic
174
Huwlbtkik of linokigy: The Microbiology of Wi»e anil Vinifkauons
acid, lactic acRI bacteria ( hclcrofcmicitcis by
dcliniUouhUso form belie acid from sugars — more
precisely, exclusively i> Lie lie for ccxxi and in-
tactic Tot kctobacilli Lafon-Lafourcadc (1933)
ilenuni.Mr.iled thai yeasts produce Utile l>lactic aid
concluded that tactic disease has occurred when the
D-laclK concentration exceeds 0.2 g/l in wine A
simple cn/yntalk dctcrmi nation of the quantity of
D-laclK acid therefore determines Ihc origin of the
wine spoilage. A limit of 0.3-O.4g/1 of i> lactic
acid per liter would seem to ensure a more reliable
diagnosis.
In wines. Ihc first step in preventing Lictic da-
ease is the proper sulliling of grapes, especially
when Hey arc very ripe The corresponding musts
arc more subject lo stuck fermentations than others
I Section 38.1 1. The wincmaker mnst react accord-
ingly and. if need be. use additives sach as nitro-
gen, vitamins and yeast hulls whose effectiveness
is clearly established. Of coarse, elemental oper-
ations, nocibly aeration and temperature control,
must also he scrupulously respected
In the funicular case of torn lied wines, studies
arc under way in propose Ihc bcsl solutions
for mic rob io logical stabilization. The hygiene of
the winery and barrels used in their produclioa
is essentia] Sulliling can resolve some of the
problems but is not authorized for certain forfifk*d
wines. Heal treatment Just before boltling is
probably also a suitable solution for these wines.
The catrbolrsm of sugars, malic acid and citric
acid arc normal occurrences during fermentation.
LaclK disease only exists if ft itei/i multiplies
prematurely. Many other transformations also
occur and some depend on ihc nature of the strain.
Malolactic fermentation has been conlirmcd to
cause chromatic changes in wines and a decrease
in their color, while stabilizing it
The AfW and Cyi sulfur-based amino acids
as well as. probably, other precursor compounds
arc converted into volatile odoriferous com-
pounds. They contribute to (he increasing com-
plexity in a wine's aromas and bouquet after
malolaclic fermentation Dc Revel el ill.. 1999.
ft ifiii produces methanethiol. dimethyl sulfide.
3 (mcihylsulfanyl) propanol-1-ol. and 3tmcthyl-
sulfanyli propkwk acid Synthetic solutions of
3( mcihylsulfanyl) propionic acid. described as hav-
ing chocolate and toasty aromas, have a perception
threshold of 50 u.g/1. Concentrations increase sig-
nilicanily after matoLictie fcrntentation. and inter-
actioa with other components of wine produces
an aroma rcminisccnl of red-berry fruit (Pripts-
Nieotiu. 2002).
After the secondary fermentation, the wine is
sal filed. Sulliling stabilizes the wine by eliminat-
ing viable bacteria and definitively blocking all
mkrobtal growth, liven then, profiling f nun a weak
salfitic protection or more often dnc to a natural
resistance, spoilage strains somclinvcs succeed in
multiplying. Wine diseases such as ropincss. rvircr-
imne and towns can he triggered (Section 5.4).
Nonviable hictic acid bacteria, or at least those
thai arc no longer capable of multiplying, can
also still modify wine composition. Some strains
produce histamine in this manner— these O. ueni
bacteria dccarboxylalc hisiidinc from Ihc must, the
metabolism and later yeast autolysis. The determi-
nation of hrstiminc concentrations has shown aa
increase in concentrations during aging. The hisii-
diic decarboxylase enzyme maintains an elevated
level of activity for several months in nonviable or
at Icasl non-cultivable cells fCown. 1996). Conse-
quently, these residual populations can be respon-
sible for other minor, unidentified transformations
of ihc wine during aging
6.4 MICROBIAL INTERACTIONS
DURING WINEMAKING
When Ihc must arrives in the tank, it contains an
extensive variety of microorganisms, fungi, ycasfr.
and lactic and acetic bacteria. Initially they come
from Ihc grape and from harvest equipment aid
then later from equipment that transports whole
and crushed grapes to the tank. From this mix-
ture, the microorganisms involved in wiacmaking
arc selected naturally — very quickly at first and
afterwards more progressively Thissclcctton lakes
place due to changes in environmental conditions
(composition, oxidation -reduction potential! and
spec ilk' antagonistic and synergistic interactions
between Ihc different microorganisms
Lactic AcHI I!. Mem Development )■ Wine
Successively or simultaneously, yeasts and bac-
Icrki interact nol only with the different types of
microorganisms (yeasts and bacteria) bnl also al
lie species and strain level. Due to Ihc great diver-
sity of microorganisms and their varying adapta-
tion ability in the medium, a multitude of interac-
tions between them can be imagined. depending on
the wine making stage. Only a few arc well known.
Sonic, on Ihc contrary, arc very difficult 10 identify
and study The ycasts/bactcrEi interactions during
fermentation seem to be the most important.
6.4.1 Interactions Between Yeasts
and Lactic Acid Bacteria
Ycasls arc well adapted to growth in grape
must Prom the lirsl days of fermentation, their
multiplication is very rapid. Lactic acid bacteria
also multiply very easily when inoculated alone in
Ihissamc environment. Yet in practical conditions,
yeasts and bacteria arc mixed: yeasts arc always
observed to dominate bacteria. The experimental
inoculation of grape must with S. cerevisiae and
diverse lactobacilli or S cerevisiae and a mixture
of 0. if ni clearly shows a behavioral difference
between the bacteria
When ■ i.L-h and tactic acid haclcria arc inocu-
lated in approximately equal concentrations (7 x
It)' UFC/ntl). ktcubacilli are completely elimi-
nated after 8 days. " oertt disappears more slowly
and subsist, al a very low concentration. If the
same must is inoculated with 10- 100 times more
bacteria, they remain viable for a longer period
bul eventually disappear— wilh ihc exception of
0. twiti This species is belter adapted than the
others to wine making
The interactions between S cerevisiae and
" vem have therefore been studied in greater
detail Grape must (220 g of sugar per liter) has
been simultaneously inoculated with both microor-
ganisms. Figure 6.4 illustrates their evolution in
must at pH 3.4. In an initial phase, corresponding
with Ihc explosive growth of ycasti. the bacte-
rial population regresses After a transitory phase,
the inverse phenomenon occurs. The yeast death
phase coincides wilh the rapid growth phase of
Ihc bacteria This evolution can be interpreted as
F«5 0-4. Evohxia
bum tn i im <<b}M
dI vcju tad bcik- m-mI bjite-
i.l popukilkiiB mixed inixuLiicil
In yrapc mini ■ I ■■ ■ .!i.-l ..r,.. . ti.. 198Kb). Gape
rauM pH=34: tanccttniikin in Mipio, =220 g/l.
(A) Ycauv. (B> bciicicld huwria
an aniagonism excrlcd by yeasts on Ihc O. aeni
population The bacteria not only do nol multi-
ply but also are partially eliminated. At this stage,
nutritional deficiencies may also be responsible in
part. Moreover. during the rapid growth period at
the beginning of fermentation, ycasls have been
proven to deplete the medium of amino acids
Argininc can be totally consumed. These deficien-
cies, hindering bacterial multiplication, arc com-
bined wilh the toxic effect, of metabolites liber-
ated by yeast.. In the first .1-4 days, the alcohol
formed cannol explain this effect. Moreover, at low
concentrations (5-6** volume.) it activates bacte-
rial growth. Other substances arc involved among
the following: fatly acids liberated by yeast*, such
as hexanoic. octinoic. decanoic and especially
dodecanoic acid (Table 65HLonvaud-i-uncle/itf .
1688a). These acids target and alter the bacterial
membrane The incubation of whole cells in the
presence of these fatly acids results in an ATP leak
and a loss of makvLu Hi activity
Handbook or Etiology: The Microbiology o( Win* and Vindications
Tabic 0.5. Influence afthc nddnioa of liay ickb n
Ihc «iabbd. term cm .it ion cue (tontc Mnribn in mil
KklU/l) (Lonvaud-Hincl (1 ,i .. 1988*1
Red wine 25 2.1 1.7 I J
Red Wme-tX,n|2J ill) 28 2.7 25 23
Whieoioe 35 24 Oft 02
Wh«c«ine-tC„(25 |iM) 45 45 45 45
As alcoholic fermentation lakes place. Ihc alco-
hol concentration increases In Ihc medium The
■cgalivc effeers of yeasl metabolism are compen-
sated in ihc end by Ihc positive ones When ihc
ycasi population enters the stationary phase. Ihc sit-
uation Is not sialic: in reality, ihc viable population
count is composed of cells that actively multiply
while others arc ly/ed. The latter cells ptuy an
important role vh-d-ris the bucicria— -they liberate
vitamins, nitrogen bases, peptides aid amino acids.
All of these components acl as growth faclois for
the bacteria.
Tberefoie. in the final stage of alcoholic fer-
mentation, yeas e. slimulatc baclctial growth. This
cffecl Is also combined with a lesser knowi phe-
nomenon corresponding to an inhibition of yeasts
by bacicria (Section .18.1) (Figure 651 More pre-
cisely, the bacteria accclc rale ihc yeast death phase
(<*>*>
tip; 0.5. Kflect of belie acid baacri* on ihc evoluioa
of the you fopubtioa liter alcoholic fcrmcnuiion
(Pnnskcvofoukn. 19SN) (■). puk ycml cukuic: (I.
■EKd cufelire (b.ucru ID* IK.'mll; (A), mlicd
aihuictbnclcra 10 : UFC/ml)
iLouvaud-Fiiel rial.. 1988b). Glicosidasc and
bacterial protease activities are certainly respon-
siMc for the hydrolysis of ihc yeasl cell wall and
lead b> Ihc lysis of ihc cnlire cell
Al Ihc end of alcoholic fermentation. bac-
teria Ihcrcforc accelerate ycasi autolysis. Their
growth is cqially stimulated by Ihc released prod-
ucts. These phenomena amplify each other and
finally lead lo a rapid decrease in yeasl activ-
ity and viability They contribute to slow or even
s(Kk alcoholic fermentations. Yet bacteria prob-
ably also produce yeasl inhibitors. In fact, grape
mist precultivaKd by buclcria (cocci or lacio-
IxK'illi i. Is less iennen table by ycasi than the con-
trol must The wines obtained conserve several
do/en grants of non- fcrnicn ted sugar per liter.
Among the species tested. ireni has the high-
est incidence (unpublished rcsilts.i. The tote of
L pJtinmrum. a species very commoa in musts,
nevcnhcless needs Ri be emphasized A strain of
iht> species inhibits not only bacteria bnl also a
laige proportion of yeasts from the genera Sixchii-
•s. Zy&taacchiimmyees aid Schizpsucthii-
t. The inhibitory sibstancc is an cxlraccl-
lilar protein that is stable bit inactivated by heal
iRamniclsbcrg aid Radlcr. 1990).
tinviroi menial conditions, in particular pH and
grape sn I tiling, play an importinl role in the cvo-
lition of these mixed ciliurcs lFignrc6.6). An
elevated pH Is favorable lo bacterial growth. Evi-
dently, the inverse is true of low pi Is Bit sultiliig
considerably limits bacterial survival and growth
al Ihc beginning of Ihc primary fermentation Its
role is essential. Yeasts shoikl be allowed to mul-
tiply Milium leaving room lor Ihc bacteria They
mist regress bit rental! in the mcdiim. all ihc
same, lo lake advailagc of Ihc ycasi death phase
and then multiply These observations illisiratc Ihc
importance of sulllliig grapes correctly. By caking
Ihc pH into account, wiiemaking incidents caused
by ihc competition between ycase* and bacte-
ria — swh as lactic disease or. on thcconlnuy. mal-
olactK fermentation d ill ie ul ties — can be avoided.
The name and quantity of peptides, polysaccha-
rides and other maciumotccilcs in wine released
by yeasts are different depending on witcmakiig
techniques and the yeasl strain As a result. Ihc
Laclic Ac id BacKrtu Development ■■ Wine
Tabic Oft Inftuence of the vcum Main an wine
midullcKc kimcalahill;, I I .on> aud-l 'unci. Hnpuhlnhcil
Fig "" Elfect of miui ill un 11
acklbiiclcru pi)fu1ai»B% ial be pic
t per Bcr (Uavaud-KuKl «<t..
malolactic fcrmcutlbility of wines obtained from
Ihc same nmM bui fermented by different ycasl
strains varies greatly Tk inhibi(oi>' contribution
of yeast fully acids, however, remains certain.
Wines (h.n arc richesi in these loxic subsemccs
arc oflcn Ihe least propitious for bacterial (level-
opine it Yet macromolcculcs. particularly polysac-
c ha rides, arc capable of adsorbing these fatty acids
and carr> T oni a veritable dcloxificaton of the
medium. Yeast autolysis, autolysis rale il.onvaud-
Funcler (i/. 1985; GniHoux-Bcnaticr and Fcuillat.
1993) and the nature of the molecules, which vary
with the sliuin. influence their liberation in the
medium.
For example, different wines were obtained
from fermentation of the same mast by eight
commercial yeast strains The difference in Ihc
duration of malolaclk fermentation wus then
compared (Tabic 6.6). The wines had similar
cthanol conkrnts and pHs Alter slcrilc liltralion
Ycui
WllH
conceal at (on*
Avenge diintiua
number
so a
Dmlcianok ackl Icimcnuiion'
(■gfl)
(■g/1)
<ifa)*l
1
8
027
12
I
in
042
925
3
3
022
035
4
21
DJ2
925
and inoculation by four different O. iiein strains,
they underwent makifciciic fcrmcnlation. Their
fcrnicn ration speeds varied from 6 to 21 days on
average Determinations of Ihc quantity of sulfur
dioxide and dcdccaioic acid showed extremely
varied concentrations, depending on Ihc wine. A
relationship between Ihe duration of the malolaclK
fermentation and the concentration of these known
bacterial inhibitory substances appears. The rote of
Ices R very important when added to a synthetic
medium containing only sugars, malic acid and
salts. Ihcy permit bacterial aciiviry and growth
6.4.2 Interactions Between Lactic
Acid Bacteria
The succession of bacterial species during alco-
holic fcrmcnlation can be cxpfciincd by a differ-
ence in the sensitivity of bacteria to interactions
with ycasCv Interactions between laclic acid bac-
teria must also exist, simultaneously. Like other
microorganisms. Ihcy can synthesize and liber-
ate substances with antimicrobial activities This
problem has been examined closely in the milk
industry, where the consequences are more serious
These substances arc simple (hydrogen peroxide,
organic acids, clc.) or more complex. Baclcriocins
arc a class of proleins whose bactericidal activity
generally has a narrow range of action. It is -(mu-
tinies even limited to Ihc same species as the pro-
ducing sliain Fundamental and applied research on
I?8
Handbook of Etiology: The Microbiology of Wiie anil Vwifications
backrnocins is at the increase and a huge range of
these sutelaiccs produced by a large variety of !ac *
lobucilli aid cocci is now known So many have
been discovered, in faci. ihal il conkl be imagined
that each strain produccsa specific bacKriocin. The
key lo proving ihcir existence rest, in finding sen-
si live slraiis.
Rammclsbcrg and Radlcr (1990). Lonvaud-
Fund and Joycux. (1993) and Strasscr dc Saad
el al. | 1996) tackled ihr> problcii for wine tactic
acid bacteria. The liisl of these works reported
the discover)' of rwo bactcriocins: brcvicin from
an L brvvis strain and caseicii from an /- ttaei
strain. The first has a large range of action
and inhibits 0. oeni and P. ikmmmis strains in
addition to /. Ixrvis Cascicin is oily active oi
L casei. Brcvicin is a small thermostable protein
(3 kDa) and is stable in a large pH range Cascicin
is less stable, with a much higher molecular
weight (40-42 kDa). The same authors observed
that a strain of /. plaiilanan has an antibacterial
activiry towards many bacterial species, including
tacubacilli and cocci, notably ft. oeni. The active
protein synthesi/cd by this strain has not yet been
rotated. Ii a /'. pentnuiteus strain. Siiasscr de
Saad el al. 1 1996) dcmonslmlcd the produclioi
of a bactericidal protein ris a ris several strains
of L liilganbi. P peatasaceiis and 0. oeni. This
buclciiocin. pioduced in latgc quantities in grape
juKc. is stable in the acidic conditions aid ethanol
concentrations of wiic.
In the same wuy. various strains he kinging to all
of the species of the FOEB (Facnltf d'Ocnologie
dc Bordcanx) collection and isolated in wine
were tested to look for possible tractions. Several
associations were clearly demonstrated lo ctcaie
reactions ii the liquid medium. The mostobviots
effects weir avoided for P. pentasaceus aid
I. planltinm. both sliongly inhibiting the growth
of ft oeni and L mesenienn'iles This inhibition
•ot only exist, in mixed cultures bnl also whci a
culture iKdium pit-fermented by these two strains
is added to 0. oeni culture medium tLoivaud-
Funcl aid Joycix. 1993) Diffcicnl experiments
have permitted the characterization of the possible
roles of hydrogen peroxide. pH and laclK acid.
For two strains, the iihibiury molecules which
accumulate in the culture medium are small
peptides, thcrmostiblc and degraded by proteases.
Their toxic effect Is oily temporary; they do not
kill the bacteria but merely lower the growth
rate and the final population. A more resistant
sab- population may develop in the end or. more
simply, these peptides are degraded by the growing
population.
In addition to the influence of yeasts and other
lactK acid bacteria, fungi and acetic bacteria
present on infected grapes also affect wine lactic
acid bacteria. The media prccnllivatcd by the
above have varying effects on lactic acid buclcria
ma I li plication with respect to the control media
tSan Romao. I9H5: Louvaud-Fnncl Hal., 1987).
Organic acids and polysaccharides accumulate in
the medium and either impede or activate bacterial
growth, but ii practice they have little effect Even
if the grapes are tainted, these metabolites remain
)■ insufficient concentrations to affect lactic acid
bacteria.
The discovery of these few active molecules—
bactcriocins or simple effectors— gives only an
indication of the true situation in wine. They are
specific not oily to genera but also to species aid
especially strains It Is therefore impossible to try
lo identify them all Nevertheless, they exist and
carry out Ihc selection of the strains observed in all
wincmaking. In the majority of cases, conditions
ensure that the undesirable strains are swept aside
during wincmaking.
6.5 BACTERIOPHAGES
Bacteriophages are viruses capable of massively
destroying cultures of sensitive bacteria) strains.
For lactic acid bacteria, bacteriophages were first
discovered in the milk and cheese industry they
provided explanations of incidents during cheese
production. Phage accidents increased in this
industry with the isc of uiK|k strain ferments.
Considerable research led to the use of mixed fer-
mentation starters, which minimized these prob-
lems. Ii the future, phagc-resistinl strains will be
developed gciciically.
The bacteriophage mist infect a bacterium in
order lo multiply. Inside the cell, il uses its
Lactic Ami Bmm Development )■ Win
own ^cnonic as code and ihc en /vine cquipitcnt
of Ike cell to ensure Ihc necessary synihcscs.
Depending on whether Ihc phage is moderate or
virulent. Ihc Multiplication cycle docs not have the
same effect on Ihc development of Ihc culture
With a moderate phage, the genome remains
inicgraled in Ihc hucml chromosome in Ihc form
of a prophage aid is replicated and transmitted
altogether normally to Ihc daughter cells. With
a viiuknl phage, ihc virus multiplies into many
copies — liberated in ihc Medium after cell lysis.
Each one of these copies then infect, another cell,
and so Ihc destruction of ihc cnllnic is massive.
In certain conditions. Ihc prophage carried by Ihc
lysogen can excise itself from the chromosome and
start another lytic cycle.
In etiology. Ihc Sur.se dc So/a Kant carried
on i ihc lirsi research on bacteriophages of laclK
acid bacicria of Ihc species O. mm i So/.n el of.,
1976; 1982). The phages were lirsi discovered
under electron microscope alter ccnlrilngaiion of
the wine (Figure 6.7). Subsequently, idcilification
was simplilicil by isolating sensitive indicator
strains. Plaques could be observed on ihc indicator
strain The phages were ihcn isolated and purified
According to ihc So//i team, abrupt stoppages
of malotactic fenncntalton are caused by a phage
attack, which destroys the total O. tteni population.
Other authors, such as Davis ei at. I 1985). Hcnick-
Kling el <tl. (1986) and Arendl el a/. (1990).
also demonstrated ihc existence of bacteriophages,
without linking them to wincmaking incident!
The DNA extracts of all of Ihc O. mm phages
hybridize together, and the marking of any of idem
furnishes a probe. By DNA/DNA hybridization,
this probe permits Ihc detection of lysogen ie strains
in a mlxiure. In this manner, we have established
thai nearly 9»i of the 0. mm strains from onr
collection, isolated during matottciic fermentation,
arc lysogcnic (Poblct and Lonvand-Funcl. 1996).
The restriclion prolilcs of isolated phages arc
nol all .■i.'iiiK.ii. which confirms ihal several
" mm strains coexist in wine during malolaclic
fermentation. Due to diverse inlcraciions and
variable phage sensilivily. these strains succeed
each other.
l-'iR <■'. Ekcimn mknotnpc phntngaph of Ornnv
cms KPil ptupn. | Photograph film Ccmic <lc Mk
*copk i:k.imn«)iK. I bi-.cimic dc Bnnluui I.)
Bacterid and phage counts in two Links during
malolaclic fermentation shewed Ihal boih popula-
tions developed in a similar way Phage popula-
tions appeared after a short lime lag. decreased as
the bacicria populations did. and reached a maxi-
mum two logarithmic imiis lower than the viable
bacteria count (Figure 68). This result is normal,
since the phage appears when bacteria multiply as
a result of the excision of the prophage.
The diversity of O. mm strains present in
wine ensures against slnck malolaclic fermenta-
tions caused by Ihc phage destruction of bacicria
None of the strains is likely to have ihc same sen-
sitivity to the phages. The elimination of one strain
by phage attick is protxiMy followed by Ihc multi-
plication of othcrslraiiis. In facl. a natural bacterial
strain rocilion can occur during wincmaking
Humllkxkk of Etiology: The Microbiology of Wmc anil VnifkalioBB
k w
H* °^
s *
^f
3 k?
f
S
Kl-
f
= ">'
S uH
/
!«■
/
w"
A Muck niatotaciic fcniKii kilion can be feared
>aly in ciccplional circumstances when Ibc phage
ind bacierial popilaiion reach the sanic number.
REFERENCES
Amatnl T. ( 1975) bi Utclic Arid Biwrieria hi Bctir-
(tjjr.v itid Food (ciU JC Can. C.V. Ciaiim? and
G.C. Whiiqa). Academic Picu. Londnn.
Awodr E. Neve II and II. nunc. W.P. (1990) -V;''
Microbiol. Bitxtduml . 34. 220-224.
BombouniG.E.and Kuubrc R.E.(I97I) /W. / £™/_
K/ic.22. 121.
Cane E.I. 19821 Tkncdc DociuM. Inuiii d Oin
I'aJvcaicdc Boidcaua II.
Cwon E. ( 1990) Thuc dc Docioeu. Fatulic d ttoc
Umvcmic dc Boidcnut II.
Cbtuc O. and U.nv.iud-Funcl A. (2003) h i
Tec cl Doc. lami
:r.<apa*iie).
nbgk.
»bgk.
aiato#c
DavB.C.R..Silveira N.F.A. and Fleet G.II(l9S5)>tpp/-
Eaiiran. Microbiol.. SO. 872-870.
Dclma* F.. PeoeF. Couchcnev F.. DiviuC. and
GurxiJ. (2001) J IfW Microbiol. Bioirdmal..
1.001.
Dc Revel O., l-onvaud-Funel A. and Bcnand A. ( 1990)
4i Qjioh>tie 95. Tec. A Doc Lavanici. fare..
De Revel G.. Maitini V. PriPR-Nkobu L. Uuvaud-
Rincl AandBemand A. (1999) J A*.i. Food Chem..
47.4003.
Dmem. C. ( 1989) TUsc dc Doctoral . Iiutiui d'CEuolo-
uie. UnivciiU dc Boolean* II
Fotnaeho. J.-C.M. ( 1903) J. Sri. Foal Affic. 14. 857.
Fie main C. I !990iThcw dc Doctoral . Inuiui d'ttnoki-
gk. Vnv.cn.ki dc Boideau- II.
Gatoy S. and Unvaud-Funcl A. (1990) J. Appl. /!...-
irriol.. 81. 013-025.
Cariny S.. Ko.O H. and 1 Dnvaud-funel A.( 199S)*V*h1
Microbiol.. 12.387-395.
GuilUu-Benntiei M. and Fcuilbl M. ( 19931 J. hi. Sri.
Mgie HH, 27. 299-311.
Guim J.. Dclma* F.. Pierre F . Jubin M.. Saayn B..
Van Bccumca I.. Carvin l.F. and Dhio, C. (1997)
Lett. Appl. Microbial.. 214. 393.
Hcakk-KliiNjT. (1980) PhD Ihcsiv Unlvcnky of
Adelaide. Auu rain.
Hcaick-Klinp T.. 1 cc T.ll . and Nichobt. D.J.D. ( 1980)
/. Appl. A*«7yo/.. 01. 525-534.
Joycm A. ( 1988) Sri. Nm/wuwu. 8. 33-49.
KalmarZ.P. (1995) Ihe*e dc Doctor*. ImlHu d a
no logic. L'nivcoitc dc Bnnkaui II.
lafon-lafourcadc S. 1 1983) b, BioiccJutolog' . Vol. 5
(eds H J. Rcnm and G. Real) Verlap-C hemic. Wcbt-
hcim.
I jIiiii-I atlHlliadc S and IV. wild E.( 1974) Com. Vi/fir
H/..8. 187.
] .- ■ ir. I'.nl In': I ' I 1986) I be* ..: ii.,,...,i •■■ v ■ii-
ca, Iiutiui .1 (J inotogic. Univcnhc dc Bonlcaw II.
lnnvaud-Funel A. and feycm A. 1 1988) Sri. AHmtws.
8. 33-49.
l«nvaud-Funcl A. and Inyan A. (1993) Food Micro-
biol.. 10.411-419.
In nvaud-K.de I A.. Zmiiuu-Bnnnamnur C. and Wcin-
mrnF. (1984) Si. AA*m..4.(H-S. IID.81-8S.
Innvaud-RincI A.. Doeo* C. and loyai* A. <I985)
Cam. Hffie Ww. 19. 229-240.
Lactic Ami BaclcrEi Dcvck>pn)cn( )■ Win
In maud- Tunc I A.. Sin Roman M.V, Joyem A. and
Chauvcl S. ( 1987) Sri. fwirioMnt*. 7. 267-274.
Uinvaud-Fuoel A..fa)«w A. and Dcm« C. < 1988a)/
Sri. Foot! Ag-icU. 183-192.
Unvaud-Funcl A.. MaKkf J.-Pl.. tojoii A. and I'm-
*kcvopouli» V i l»XSh i dim. Ujr** ifa, 22, 11-24.
InnxauU-luiicI A .. Joycw A. and l.cdou. O. ( 1991) J.
Appl. Baewiol. . 7 1 . SO I - 508 .
McDonald L.C.. nr«ln|t HI' and Hassan II .VI. ( 1990)
Appl. Emirai. Microbiol. . SO . 2 1 20 - 2 1 24 .
Milk) V. and Lo maud- tunc I A. 1 2*100 1 Let!. Appl.
Microbiol.. 3Q. 130.
Paraskcvopnuloi Y.(l9S8)Tncicdc Dncicui-lnpc'nwur.
Inviitu ri i 1 ■iiil.-pK. UahwU dc Bonlcaux II.
Puhki M and Uovaud-Funel A. (1990) fa (Eaolope
95. pp. 313-310. Tec & Doc U<obkr. Pari*.
Poolnwn B.H99HFFMS Microbiol. Re,.. 12. 125- 148.
P»ipb-NkolnuI..(2CXI2)Tne4cDocloiaidc I Universe
Vktor Scpaka. Boolean* 2.
Radfcr F ( 1907) Cam- Mg/te «w. 1. 73.
RammeMKOi M. and Radler F. < 1990) J. Appl. Micro-
faoJ..09. 177-184.
Rfccicau-Cayoa J . Pcynaud E.and Rhcieau-Gaynn P.
II97S) fa Tniied'<Eitok)$ l ie. Sciences el Teiimique*
•In \ta. vol. 2. Dunod. him.
Rontano P. ci and Suzn G. ( 1992) fa Uhte Microbi-
,.,'.>,:' '■'■-' Biotntotolot? (ed. Gil. Fkcl). Ha mood
Acadc*k- Publi.nciN.Ciiur. S»iicdand.
Sjh Romuo M.V. ( 1985) Cora.. \igne \i». 19. 109- 1 10.
Sinin R. ( 1983) Thc%c Docuuai 3cmc cycle. Inuiu
d d notogk. UbrwM dc Boidcaw II.
So.-.-i T.. Mam R. and Puulln J.M. ( 1970) F.xperientia.
32.5O8-S09.
So/dT.. GnacpiK.. dAmlco N. and Hose II. (1982)
Jfcv. Suiae Uric *D«ri<-. Hdrffc., 14. 2-8.
Slauctdc Saad A.M.. hiMcri*. S.and Manca dc Nadni
(1990) fa (Biologic 95. pp. 329-331. Tec. A Dae
Uvonici. Pirn.
SudcuidP. ci Chuivcl I. (1985) Coin,, \tpie vt». 19.
31-40.
Vivai, N.. Bclkmcic L. Unvaud-Kuncl A. and Glo-
ria Y. (1995) Jfcv.fr. Wtol.. 151.39-45.
Acetic Acid Bacteria
7.1 Principal characteristics and cytology
7.2 Classification and Rlcntiricaiiou
7 3 Principal physiological characteristics
7.4 Metabolisms
7 5 AcclK acNl bacteria development in grape must
7 6 Evolution of acclK acid bactcru during wincmaking and wii
and Die impucl on nine quality
7.1 PRINCIPAL
CHARACTERISTICS
AND CYTOLOGY
Acetic acid bacteria arc very prevalent in nalnre
and arc well adapted In growth in sagar-rKh
and alcohol-rich environment. Wine, beer and
cider arc natural habitat, of these bacteria when
production and storage conditions arc not conccdy
controlled. Their quality Is clearly lowcicd. except
in the case of certain very particular beers
Acetic acid bacteria cells generally have an
ellipsoidal or rod-like form, with dimensions of
0.6-08 pit by I -4 |im. They can be cither sin-
gle or organized in pairs or small chains. Some
arc equipped with cilia, sunoundiig the cell or at
its ends. These locomotive ofgans give the cells
a mobility that is visible under the microscope
These bacteria, like belie bacteria, do not spore-
late Their metabolism is strictly aerobic. Cellular
oxidations of sugars, cthaaol or other substrates
arc coupled with respiratory chain election inns-
port mechanisms. Oxygen is the ultimate acceptor
of electrons and proms (coming from oxidation
reactions).
The ccllufcir structure of an acetic bacterium is
simitar to that of other bacteria: a cytoplasm con-
taining genetic material (chromosome, plasmids).
ribosomes and all of the enzymatic equipment, a
plasm ic membrane and cell wall. At the structural
'- .,-. ...■..■A.. ■ ..
r r,t„ ,.i .;,. ,„
1X4
Handbook of linology: The Microbiology of Wine anil Vindications
le\cl. only the cell wall clearly (listing Irishes il
from lactic bacteria. Acetic acid bacteria arc Gram-
negative, whereas lactic bacteria arc Cram- positive
(Section 4.1.1).
The Gram coloration rcffccK a significant struc-
lural difference ol the cell wall of the two types of
bacteria. Pcptidoglycan is the principal constituent
of Gram- positive cell walls, bal ii Is much less
present in Gram- negatives. In Ihc latter, an essen-
tially lipidic external membrane is present. It is
destroyed by ethanol which acts us a solvent in
the Gram test, residing in the washing away ol
the violet dye The external membrane iscomposed
of phospholipids, lipoproteins and lipopotysaccba-
rides. Like the plasaiK membrane, it is organized
inn a lipid bilayer. a hydrophobic /ok is con-
tained between the layers. The lipopolysaccharidcs
comprise a lipidic /one integrated into the external
layer of the membrane, an oligosaccharide and a
poly sacc hand ic chain at the exterior of the mem-
brane. This chain carries the antigenic specificity of
the baclcrinm The lipoproteins join the thin pep-
tidoglycan layer to the external membrane. Bnricd
in the lipidic layers, crossing the entire membrane,
proteins called pi nines form canafe that permit
exchanges across the cell wall.
7.2 CLASSIFICATION
AND IDENTIFICATION
7.2,1 Classification
Acetic acid bacteria belong to the Actiobacier-
iiceiie family Besides Ibe previously mentioned
characters, their principal property is u oxidize
ethanol into acetic acid. Their (G + C) DNA base
composition is from 51 to 65'* They arc encmo-
organo tropic
The bacteria of ibis family arc scparakd into
two genera: Acetitixtcter and GliKontftwcier The
key distinguishing features according to Bergey's
Mamuil (Dc Ley el a) . 1984) arc as follows:
• Genus Aceiobaeler: oxidize lactic and acetic
acid into CO;: non-mobile or pciitrichous.
• Genus GtiKimotxteter: do not oxidize lactic or
acetic acid: non- mobile or polar llagclla.
Dc Ley rial. (1984) referenced a total of five
species: A. iiceli. A. liquefutcuns. A pasieuriantit
and A haiuean for the gents Aeettilxicier and
only G oAyduns for the gems Gtucmtobacter.
Later studies on acetic acid bacteria led to the
identification of new species: A <tiuzpM>pttkiis.
A. mrihiiitJkits. A. xyiimim. G. iiuiii. G. ceiiimi
(Swings. 1992) and morc recently A eumpana.
This last species is clearly separated from other
Aeetobaeter and Glutonahntter by its very low
DNA/DNA hybridization percentage of between
and 22'* (Sicvcrs el td . 1992). Tits species is
pre-eminently used in vinegar production, due to
its high ethanol and acetic acid tolerance. Ii Gin-
coaobacwr. a fourth species G fiweiaii cannot he
differentiated from G. ce/iinis by pbenotyptc com-
parison, but from iB low DNA/DNA hybridization
percentage it is very distant from il at the genetic
IcvcUSicveisrtrrf.. 1995).
The classification has been updafcd still more
recently on the bnsisof molecular phylogenK crite-
ria to include the Areuitmcier. Glucwtbaeier. Ghi-
tantMitettibticier. Actikmionns. Asai. and Kozitia
genera in the Atettibttcleriicette family of bacteria
(Yamadaef (if. 2(«>2l Bacteria in the A liqttejth
sciem. A. haiisemi. A. melhtutolinis. A .ryff nwr. A
ilialrophkut. and A europtmu species according
to Ike previous classification (Table 7.1) arc now
included in the Gluconmcelt'l'ticter genus.
Three species unaffected by recent changes
it Ihc classification. G. oxythua. A. aceti, and
A. paaeuritinus. arc the ones that arc most fre-
quently found in the course of wincmaking. as well
as. Id a lesser cxknt. Glucimtxiceiolxiclerliquefa-
xient ■MAGIucmtoacnobacierhaitseiui.'WQ three
species saccccd each other during wincmaking.
The G oxyihmt present on the grape disappears
and gives way to Aceiobaeler, which sabsisft in
wine (Lafon-Lafonrcadc and Joycnx. 1981).
7.2.2 Isolatio
ntl Identification
The isolation of acetic acid bacteria from grape
mnst or wine is carried out by culture on a solid
nutritive medium The composition of the medium
varies, depending on Ihc researcher. Nevertheless,
taking into account their nutritional demands.
Acetic Acid Bacteria
Tabic 7. 1, riiilpsl.iwin
c la cki erotic* of Aceiohaciei »pccio (Swingv 1992; Sievi
Gnra-ih on mg'k acid
Produced fmni uKkom :
5-lcit"^lutiiiiK acid
2 ^-dikctug beanie acid
Kctoak acid fiom glyccr
Gnralhoacl basal
Gnra-ihoa mcthaml
-:Pi*llvt- -: SvjMfv <l:
HI
Ihcsc bacteria only develop well on rich media
con tuning jcasl extract, amino acids and glucose
as the principal energy source Swings i 1992
described diverse media Toe isolating baclcrla
from different ecological niches. To BOtutc acclK
bacteria from wine, the same medium may be
used as lot tactic bacteria ( Ribs' rcau-Gayon el ul .
1975): 5 g/l of yeast extract. 5 g/l of amino acids
from casein. 10 g/l of glucose, and 10 ml/1 of
tomalo juice, with the pH adjusted to 45. It is
also possible *< use grape juice diluled with an
equal amonnl of waler plus 5 g/l yeast extract.
The medium is solidiricd by adding 20 g/l of
agar. To ensure thai Ihc medium supports only the
growth of acetic bacteria. 0.2 ml 0.5'.* pimaricin
and 0.1 nil 0.125'.* penicillin arc added per 10 mL
culture nvedium to eliminate yeasts and lactic
bacteria. The cilturc nasi he iicubalcxl under
aerobic conditions
After isolation, the colonics pat into pnrc
cultures art identified by a group of tests and
identification keys in Ber$ey's Manual tDc Ley
clot., 19841 The first test is Gram coloration.
Researchers also depend on the aptitude of the
strain for developing on diverse constituents and
on iCi metabolism in relation todiffcrcnlsubstralcs.
According v Swings (1992) and Sicvcrs ft ui.
11992). Table 7.1 presents Ihc identification keys
for AcetobtKtor aid Tabic 7.2 for Glucanoixxler
tSwings. 1992).
GhtconabtK'ter and Aceiobacler arc differenti-
ated by their ability to oxidize lactate: 20 g of
lactate per liter is added to the medium, already
constituted of yeast extract at 5 g/l. Acelobticler
oxidizes lactate: a ctoady zone is formed by the
precipitation of calcium aroand the colony.
Hfhanol oxidation by the two genera of bacteria
is verified by culture in a medium containing 5 g
of yeast extract per liter and 2-3'f clhanol. The
acidification of the medium Is demonstrated either
by titration or by the addition of a color-changing
indicator (brontocrcsol green)
7.3 PRINCIPAL PHYSIOLOGICAL
CHARACTERISTICS
The i". -ii! of the two genera Acelahticier and
Gtucottatxicler arc obligatory aerobic microorgan-
isms with an exclusively respiratory meubolisni
Their growth, at the expense of sa bstrakrs that they
oxidize, is therefore determined by the presence of
dissolved oxygen in the environment. All of these
species develop on the surface of IN|uRI media and
form a halo or haze, less often a cloudiness and a
deposit.
Although present in Ihc two genera. Ihc char-
acteristic metabolism of Meiobaeirr is Ihc oxi-
dation of ethanol into acetic acid with a high
transformation yield ThR is not Ihc case for
IX'.
Handbook of linology: The Microbiology of Wine and Vinificalions
Gluconobucler, which arc eharac icri /ed hy a high
oxidation activity of sugars into kelonic com-
pounds <ihls activity is low in Acettibucttr). li
s«ii. bacteria or He genus Acembocler prefer
ethanol hi glucose lor their growth, the inverse
is iruc for bacteria of ihe genus Gluctmnbacter.
In addition, eihanol tolerance is parallel. In con-
sequence. Aceiobtieier bacteria are more common
in alcoholized environments (partially fermented
must* and wines) than Glivoaobacier. which arc
■tore preseni on Ihe grape and in the must
Some Acerobacler strains form cellulose in
non-agitated culture media Ccrtiin Ghiconabacur
produce other polysaccharides tglucaus. Icvan.
etc.). which make the medium viscous
The vitamin demands are approximately iden-
tical for all acetic acid bactctti Growth Ls only
possible in environments enriched in yeast extract
and peptone, which furnish the necessary carbon
substrains In order of preference, the best sub-
strates for Aceitilxtcter are eihanol. glycerol and
tactile: for Ghteonobtieter they are mannitol. sor-
biul. glycerol, fructose and glucose Acetic acid
baclcrEi are not known lo require a specilk amino
acid Certain AcetobtKier and Glucttnobacrer arc
capable of using ammonium from its cnvisonmcnl
The optimum pH range for growth is from 5 to
6. but the majority of strains can easily multiply
in acidic environment as low as pH 35.
Although they oxidize ethanol. acetic acid bac-
teria arc not especially resistant u if On average.
GliKomHiacler do not tolciutc more than 5'i
eihanol. and few Acetiihticier develop at above
10**. Evidently, adaptation phenomena (probably
similar to those described for lactic bacteria) occur,
ensuring their eihanol tolerance in wine. Acidity
and ethanol concentration simultaneously influence
the physiology and the resistance of acclK acid
bacteria.
7.4 METABOLISMS
7.4.1 Metabolism of Sugars
The direct incomplete oxidation of sugars without
phosphorylation leads to the formation of the
corresponding ketones. The aldoses arc oxidized
into aldonic acids The aldcnydic function of
this sugar is transformed into a carboxylK acid
function. Glucose is oxidized into gluconic acid
in this manner. The glncosc oxidase catalyzes the
reaction, which Ls coupled with the reduction of
FAD. In acetic acid bacteria, electrons and protons
arc transported by the cytochrome chain to oxygen,
which Ls the final acceptor.
Bacteria of the genus Gliicimnbiit'ter in par-
ticular abo have the property of oxidizing glu-
conic acid, leading to the formation of kcu-5
gluconic, kcto-2 gluconic and dikclo-2.5 gluconic
acids (Figure 7.1). These different molecules ener-
getically bind with sulfur dioxide (Section 8.4J).
Wines made from grapes dinted by Glucanobacter
arc therefore very diflkult to conserve
Certain Aceiobtieier strains aLso form dike-
Ionic acid. Similarly, other aldoses, man nose and
galactose, lead to Ihe formation of mannonic and
gali atonic acid.
c it pit
CH,OH
cii/m
CM pit
D-GMctne
M £a*
KiUvSgMCilllC
ao.i ? fc.
Fifil
O.ldlllOB of pUKua
k »ckl by Glut
anobu-ier baclcru
Acciic Acid Bacteria
\;<-
Kcfc»cs ;uc less easily oxtdi/rd by acetic acid
bacteria. The oxidation of fruckisc can lead lo the
formation of gluconic acid aid keto-5 fructose
The carbon chain of Ihc sugar can also be
divided, mulling in Ihc accumulation of glyceric,
glycolic and succinic acid Especially for ihc
Acetobacler. the final oxklalion products of hexose
arc gluconate and kctagluconalc.
The complete oxidalioi of sugais. however,
furnishes Ihc necessary energy for bacterial
growth The hexose monophosphate pathway is ihc
meubolK pathway for the utilization of sagars In
Aceiohacter. the tricarboxylic acid cycle Is also
used, but B absent in GltKonobaeier. The cnyyncs
of glycolysis cither do not exist or only partially
exist in acciic acid bacterid.
Oxidation by the hexose monophosphate path-
way begins with the phosphorylation of sugar, fol-
lowed by two successive oxidation reactions. The
second is accompanied by a decarboxylation. The
xylulose 5-P cnlcrs a group of transkcioli/ation
and uunsaldoli/alion reactions (l-igurc 7.2). The
overall reaction is the degradation of a glucose
molecule into six molecules of CO> In parallel. 12
Fi£ 7.1 Degradation of phkinc by acetic .u Hi Rule ru (home mono phosphate palhuay)
Handbook of linology: The Microbiology of Wine anil Vindications
c«x'n/>iic molecules air reduced. TV: transfer of
elections aid protons by the cytochrnmK chains in
iu in reoxidizes Ike coenzymes The transfer gener-
ates In ice molecules of ATP per pair of H r and c~.
36 ATPs for Ibc oxidation of a molecule of gbneose
inn CO?. Tin metabolic pathway is tcgulaicd by
Ihc pH of Ihc environment and the glucose con-
centration. Ii Is significantly inhibited by a low pH
( <3S) and a glucose concentration above 2 g/1 Ii
these conditions, gluconic acid accumulates in ihc
medium.
7.4.2 Metabolism oflCthanoI
Among the transformations carried onl by acclic
acid baclcria. cnotogists arc most interested in ihc
transformation of clhanol. Ii is the source of an
increase In volatile acidity In many cases In fact.
Ihc oxidation of cthaaol leads to the formation
of acetic acid. The transformation tikes place in
two steps; the intermediary product is cthanal
(acculdchydc):
CH3-CH2OH CHbCHO CHiCOOH
cthanol cthanal acetic acid
(7-1)
Acettibticter arc also capable ofoxidizing acetic
acid, bm this reaction Is inhibited by clhanol. It
Ibcrcforc docs not exist in etiological conditions.
Acetic acid slows Ihc second slcp. when II accu-
■!■ tiles in the nKdium. in which case the cthanal
concentration of the wine may increase Accord-
ing to Asai (1968). this second slcp is a dB-
mutitmn of cthanal into clhanol and acctK acid.
In acrobiosis. up lo 75'< of the clhanal leads in
the formation of acetic acid. In intense aeration
conditions, the oxidation and the dismutation con-
vert all of the clhanol into acclic acid. When the
medium grows poorer in oxygen, clhanal accumu-
lates in the medium. Furthermore, a pH-dcpcndcnl
metabolic icgulalloa preferentially dircch Ihc path-
way towards oxidation rather than towards disuiu-
tiltoa in an actdK environment.
The enzymes involved arc. successively, alco-
hol dehydrogenase tADHl and acclaldcbydc dehy-
drogenase tALDH). These two enzymes wcic
pcoven to excu in Acetobucter and Gluconohur-
ler. Two kinds have been distinguished: an NADP
cocnzymc-dcpcndcnl ADH and ALDH and a
soluble coenzyme- independent ADH and ALDH.
The liist arc soluble and cytoplasmic: the sec-
ond aic linked lo Ihc plasmic membrane, lor
Ihc tiller. Ihc electrons generated in Ibc oxida-
tion reaction aie conveyed 10 oxygen by an elec-
tion transport sysKm iutcgmlcd in the membrane.
These membrane enzymes are incapable of reduc-
ing the NADP coenzyme (or NAD) but in rinv
they reduce electron acceptors such as ferrocya-
nnrc and methylene bine. They arc probably the
most involved in Ihc oxidation of clhanol in wine
since they function at low pHs. Convciscly. cyu-
plasmic enzymes, which function at the interior of
Ibc cell, have an elevated optimum pH of approx-
imately 8.0.
7.4.3 Metabolism of Lactic Acid
and Glycerol
hi vim), all of Ihc species of Ihc genus Aceltiboeter
oxidize d- and L-laclic acid. Certain strains com-
pletely oxidize it into CO> and I I.O. but moM slop
at Ihc acclic acid stage. The two isomers aic trans-
formed, bit the activity is exerted more effectively
on the d isomer Pyruvate R Ihc tlrsi intermedi-
ary. Il Is first decarboxyfcilcd into clhanal. which
Is oxidized into acetic acid by the ALDH.
Two types of enzymes have been identified, one
in the membrane and the other In Ihc cytoplasm
(Asai. 1968). The i> and i.- lactate oxydases ate
mcmbranal enzymes which do not require acofac-
kx toil function with the cytochrome chain. The
membranes also contiin Ihc pyruvate decarboxy-
lase, catalyzing the transformation into cthanal In
Ihc cytoplasm. i> and 1.- lactate dehydrogenases
ensure Ihc oxidation of laclatc into pyruvate The
pyruvate decarboxylase ensures cthanal pmduc-
ton Finally, the NADP-dcpcndcnl ALDH leads
to the formation of acetic acid. This metabolism
docs not seem to be particularly active in wine; il
has never been proven that il is the source of wine
spoilage
The oxidation of glycerol leads to Ihc formation
ofdihydioxyacctonc(DHA:CHi0H-CO-CH?0H).
Acetic acid baclcria. except A ptoteitritinus. pro-
ducc ibis compound This reaction requires an
Acetic Acid Bacfcra
],V.<
intense oxygenation or ihc environment and is
inbibikd by cthanol. h is ■■likely ibal il occifs
la wIk. bm the conditions arc mote favorable on
spoiled grapes In fact, acetic acid bacteria ate
present on the grape alongside BiHrylis cinerea.
glycerol being one of Ik principal metabolites
7.4.4 Formation of Acctoin
Acctoin is the compound at the intermediary
oxidation level in the gtoap of three accioiiK
molccnlcs ptcsent in wine: diacctyl. accuin and
butaucdiol. It Is formed fmm pyrevatc. itself a
metabolic intcniKdiaiy product having different
origins in microorganisms.
Acclic acid bacteria piodncc acctoin fmm lactic
acid that is oxidized beforehand. For most strains,
this pathway is not very significant, but some form
np to 74'i of the theoretical maximum quantity.
Two synthesis pathways arc believed u exist
tAsai. 19681
1. Pyrnvatc is dccarboxylatcd in the presence of
thiamine pyrophosphate (TPP) and krads to
the formation of cthanal-TPP. The traction of
cfhanal and cthanal-TPP forms acctoin:
2CHb-CO-COOH + 2TPP
2CHjCHO-TPP+ 2COi (7.2)
CH,CHO-TPP . CH3-CHO + TPP
0.3)
CH,CHO-TPP + CH 3 -CHO
CHjCHOH-CO-CHj + TPP (7.4)
ace loin
2. The other synthesis pathway bypasses the
intermediary step of forming a-acctolactak
(idntical to the lactic acid bacteria pathway),
by the reaction of cthanal-TPP and pyruvate:
ch 3 cho-tpp + ch,-co-cooh
CHi-CCMCHi)COH-COOH (7.51
o - ace lolac late
CH3-CCMCH1 )COH-COOH
*C02+CH,CHOH-CO-CH, (7.6)
ace Din
G. oxyrians can also oxidize balancdiol into ace-
loin (Swings. 1992). The presence of accloin in
wine is less problematic than thai of diacctyl.
which has a much more pronounced aroma. More-
over, it has never brcn proven that acclic acid bac-
teria are capable of oxidizing acctoin into diacctyl
7.5 ACETIC ACID BACTERIA
DEVELOPMENT
IN GRAPE MUST
Acetic acid backria arc present on a ripe grape
The populations vary greatly according to grape
health. On healthy grapes, the population level is
1 1 ■■-.-. .iiuiiii.! I" 1 "I''-iul. .("..: liis.Lhvsu-iKiK'h
made vp of GhieonobtKieroxydtua. Rotten grapes,
however, are wry contaminated: populations can
reach upwards of 10' to 10* UFC/ml and arc
mixed, comprising varying proportions of Glu-
conobaeler and Arelobucter (Lafon-Lafou trade
and Joycnx. 198 1).
This bacterial microflora modifies mast com-
position by metabolizing sugats and sometimes
organic acids Atetofoicier partially degrades cit-
ric and 111.1].. acids (Joycux et til . 1984). How-
ever. Ihc most significant activity of these backria.
especially those in the Glucanometvbaeter oxy-
iliins species, is producing substances that combine
strongly with SO? (Scctk*s8.4J; 8.4.6). They
transform glucose into gluconic acid and its lactone
derivatives. •• and A gluconolactone. may combine
np to 135 mg/l SO?, in a must containing 24g/l
glnconk acid with a free SO. content of 50 mg/l
tBarbc el of., 2000). These bacteria aLso oxidi*
glyccml 10 form dinydroxyaccunc. which com-
bines with SO; in an unstable manner This com-
pound is mctaholi/cd byyeastdaring fermentation
and is no longer a factor in forming combinations
The most abundant SO, combination due toC/tr-
contrtxtcler rcsalts in 5-oxofrncKsc. which Is not
metabolized by yeast, so it remains unchanged in
Ihc wine. It is formed by oxidation of any fructose
in the medium, as is the case in grape must. In a
bolryti/cd mnsl where the fungus has developed
to ils most advanced stage, this compound alone
1VO
Handbook of Enology: The Microbiology of Wk and Vinifications
account) lot (if* of all combinations. In wines
made ln'in this lypc of must. 5-OXOfruc lose a hi
t>- and <f-gluconolactoncsarc involved in St/I of
Dm SO? combinations, while clnanal and kchxicids
(orated by yeast account for most of ihc tenialndet
(Barberry*.. 2000).
In addition to acetic acid bacteria. yeast, con-
taminate £ rapes. Although alcohol piuluclion Is
limited, these strains do produce small quan-
tities or ethanol directly on extremely rotten
grapes or immediately following crushing and
pressing This alcohol is immediately oxidized
by acclK acNI bacteria. Some musts can there-
fore have a relatively high volatile acidity before
fcrmenuttoB.
Furthermore, acetic acid bacteria produce yeast-
inhibiting substances Lafon-Lafonrcade aid
Joycux( 1981) demonstrated this tact for GhieoiHi-
bacier. Cultivated during 3. 7 and 14 days in
a mist then inoculated by S cerevisiae. this
bacterium supped alcoholic fermentation. There
remained 15 g. 9g and 18 g of non-fermented
sugar per lilcron average, respectively, as opposed
u 05 g/l in the control Gilliland and Laccy ( 1964)
identified the same inhibiiive cITccI of Aeelabac-
ter uward strains from seven yeast genera pos-
sibly present in must, including Saccbtwmxces.
As a general rale, however, this effect is very
limited In ract. acetic acid bacteria activity in
grape must is obligatorily short-lived: it slops at
almost the same lime that alcoholic fermentation
begins.
In summary, the principal inconvenience of
grape contamination by acetic acid bacteria is the
production of volatile acidity and ke Ionic suh-
stinccs. This con liiii inatiou results from sigars
liberated through Insures on the grape caused
by fungi during their proliferation on the berry.
Part of the sugar is fermented into ethanol.
which is oxidized into acclK acid: the rest
is oxidized directly. In all cases, the wine-
maker's -I'- is complicated: the volatile acid-
ity can be high before fermentation, and musts
altered in this manner strongly bind with sulfur
dioxide.
7.6 EVOLUTION OF ACETIC
ACID BACTERIA DURING
WINEMAKING AND WINE
AGING, AND THE IMPACT
ON WINE QUALITY
The principal physiological characteristic of acctK
acid bacteria is their need for oxygen to multiply.
In wine. Aceltibtiaer itceii and A puxteuriama
draw their energy from the oxidation of ethanol.
Acetic acid concentrations indicate their activ-
ity Finished wines contain around 0J-0.5 g of
volatile acidity (HjSOi) per liter, resulting from
yeast and lactic acid bacteria metabolisms Above
this concentration, acetic acid accumulation most
often conies from acctK acid bacteria: this prob-
lem is called acetic spoilage. This contamination
must be avoided not only because of ie> negative
effect on wine quality but also because of the legal
limits on the concentration of volatile acidity per-
mitted in wine AcclK spoilage Is accompanied by
an increase in ethyl acetate The perception thresh-
old of this ester is aronnd 160- 180 mg/l Yeasts
also form it in concentrations ot up to 5(1 mg/l. An
excessive temperature during wine storage accel-
erates this spoilage.
Acetic acid bacteria multiply easily in acro-
biosis. i.e. in grape mnsl or wine at the sur-
face in contact with air. but Ibis is not the case
during fermentation As soon as alcoholic fer-
mentation begins, the environment grows poor
in oxygen and the oxidation-reduction potential
talK
Lafon-Lafonrcade and Joyenx (1981) observed
the evolution of bacteria during the production
of two kinds of wine. In a white grape must
parasitized by It. cineivu. the initial population
of 2 x 10* UFC/ml fell to 8 x 10* UFC/ml. five
days after harvest. At this stage. 90 g of sugar
per liter was fermented. The population was less
than 10" UFC/ml on the 12th day. after the
fermentation of 170 g of sugar per liter. Similarly
in the red grape must, the initial population
of 2 x 10 4 UFC/ml progressively diminished to
Acciic Acid Bacteria
20 UFC/ml by ihc time Ike wIbc was run off. The
acetic acid bacteria arc therefore km involved in
alcoholic fermentation The sane Is troc diinip
malolaclic fermentation. Yd in all cases, ihcy
never totally disappear
During banc) or lank ;k my . Ihc wine should
be protected from air to avoid both chemical
and biological oxidalion. In addition to oxidative
ycasis. acetic acid baclcria slill viable ahcr bolb
fermentations an: capable of multiplying in the
presence of air. To avoid this problem, the
con tuners (tanks or barrels) should be filled as
coniplclcly as possible. Topping off should be
practiced with a wiic of excellent microbiological
quality to avoid ion lain 1 nation An inert gas may
also be nscd lo rcplace the atmosphere present at
the up of the uinks.
Aging ab*t entails racking for ctarifying and
aerating the wine— causing limited oxidations that
arc indispensable lo wine evolution. In the absence
of air between rackings. acetic acid baclcria remain
present in the entire wine mass at concentrations
of I0 2 n 10* UFC/ml Dnring traditional barrel
maturation, the dissolving of oxygen is more
significant than in tanks, due *> diffusion across
Ihc wood and the bung (when bung is on lop).
This slight oxygen dissolution suffices to ensure an
oxidalion -reduction level compatible with bacteria
survival
At the time of racking, the conditions arc
radically modified The transfer from one lank or
barrel lo another is accompanied by the dissolution
of 5-6 mg of oxygen per liter in the wine,
unless very careful precautions arc laken. The gas
dissolves more quickly when air contact is favored
and the temperature is lowered This oxygen
Is at lirsl rapidly and then more progressively
consumed by the oxidi/ablc substances in wine.
The oxidation -reduction potential follows the
same evolution. Tabic 7.3 Illustrates the evolution
of the dissolved oxygen conccnlrarion and Ihc
acetic acid bacteria population — the growth of
which is very active Just after racking. Afterwards,
the bacteria slowly lose their viability until the
next racking, several months later The same
Tabic T.J KvoUni
acid bacteria conceit
barrel to amine.
i of diuobeiL
mucin v. be. ni
oxygen 4-1 acciic
Stipe Dm
sited m
(mgfl)
yB"
Acetic acid bacteria
(I'FC/ml)
Before racking
During racking
AVer J day*
Alee 20 day,
Aie.00 da>s
02
OS
0J>
0J
1.0 x ID'
1.2 x 10*
2.0 x 10'
10
phenomenon occurs al each racking during the
IS months of aging
Acetic acid B always synthesized during each
growth phase of Ihc bacteria. In a scries of
observations, its concentration increased from on-
to 0.04 g/1 following rackings. These values vary
greatly. They arc linked to the baclcria population
level and multiplication rale. For example. 0.02 g
of acetic acid per lilcr was formed when a
population was doubled from 35 x I0 4 UFC/ml lo
7.0 x 10* UFC/ml. TV acetic acid concentration
increased by 008 gfl when Ihc initial population of
50 Uf<7ml multiplied to 1.5 x I0 1 UFC/ml. These
observations prove that acetic baclcria play a key
role In the increase in volatile acidity dnring aging
(Milleltfof., 1995).
The principal factois affecting acciic acid bac-
lcria development (as with lactic bacteria) arc
Ihc alcoholic content, the pH. Ihc SO, cotccnira-
Ihmi. the temperature, and the oxidation-reduction
potential The more the pH and Icmpcralnrc arc
Increased. Ihc more easily Ihc bacteria survive
Their m amplication Is quicker in the case of
aciuuOo.
There is no effective method for eliminating
acetic acid bacteria Currcnt observations shmv that
even when protected by 25-30 mg of free SOj
per liter, wines always conserve a viable baclcria
population— up to I0 1 to 10* UFC/ml during bar-
rel aging Only a relatively low temperature of
around 15 C can eventually Until this problem
To avoid spoilage related u acetic acid bac-
lcria. Ihc wlncmaker should first of all concen-
trate on winery hygiene in order to eliminate
192
Handbook of Etiology: The Microbiology °f Wiie anil Vnifialiois
potential con taml nation sources Furthermore, all
the other parameters (alcohol. pH. etc.) being
equal, the influence of storage conditions on the
oxidation - reduction potential is a deciding lac-
ur In laig c-capucity cmks. the Increase in volatile
acidity is lower than in battels. Similarly, even
when the population is around Kl' UFC/ML at the
tintcof holding, it decreases slowly but inexorably
daring bottle aging as the redox potential becomes
wry rcslriciiw.
REFERENCES
AniT. (1908) I* Attic Acid ftnmi. Cl-aatinaion
mil biodifiiiiad iicihiiiei. 1'nivcnay of Tokyo Picm.
Tokyo.
Baibc J.C.. dc icvcl G.. Jojeu* A.. LonvMal-KincI A.
and Hen rand A (2000) / Apic. Food Onvu.. 48.
1113.
Dc Ley J. Gilln, M. and Swings J. (1984) In Jhr#V.t
Mnnui ofSMvMtc Bttfcriotogy. cd> NR. Krieg
and J.<;. Hot. pp. 207-208. U ii|.i m , and Wilkin*.
BnkinoK.
Gillifand RP. and Lacey J.P. (1904) Naure. 202.
727-728.
Joycux A . I afon-l albuivadc Sand Rtocreau-Gayon P.
( 1984) Si. Aliments. 4. 247-255.
[jioii-ljfi.iiv.ulc S. nnd Joycut A. (1981) Rait 0FV
008.803-829.
Millet V..Vivu N.andUnvaud-PunelA.(l995>./.Sri-
Te.ii. Tmmellerie. I. 123.
Rbcicau-Gavtin I. Pcynaixl V... Rfecicau-Gattin P. and
Sudaud P. ( 1975) In TrtM tTQ-jwtogr: Sciences n
Tnfaiiauei rfu Ho. \ft>l. 2. Dunod. Pnriv
Skvcn M.. Ludv-tp W. and TcubciM. (1992) 5m.
Appl. Microbial.. 15. 380-392.
Skvcn M.. Gabcnuucl C. Bocich C. Luda >g W. and
TcubcrM. (I99S) FKMS Microbiol. Lett. 120.
123-126.
Sv.Iin» J. (1992) In Vie Proaryata ed% A. Balou*.
!! i ! i..i|;. , V I ■■ - . .1. . .i . W.llnnlci nnd K II
Schleilcrpp. 2208-2280. Springer Vertyt.Ncv. York.
Yaaada Y.. Kaiwra K. Kawasaki H.. Widyauui Y..
Siiono S.. ScklT.. Yamada Y.. Uchiaiun T. and
Komagula K. (20021 hi. / Syti. Eiol. Microbial..
52.813.
The Use of Sulfur Dioxide in Must
and Wine Treatment
HI Introduction
8 2 Physiological effects
8 3 Chemistry of sulfur dknklc
8.4 Molecules binding sulfur dioxide
8 5 Practical consequences: the stale of sulfur dioxide i:
8 6 Antimicrobial properties of sulfir dtoiktc
8 7 The folc of sulfur dioxide in wincntaking
88 The use of sulfur dioxide In the winery
Z'r,
KB
8.1 INTRODUCTION
The general ist of snlfur dioxide (SO;) appears
to dale back to tie cud of the ISlh century Its
many properties make it an indispensable aid in
wincaiaking. Perhaps some wines could he made
in total or ■car-wed absence of SO. but it would
certainly be prcsumptuovs to cfciim that all of the
wines produced in the varkws wineries throvgnout
the world could he made in this manner. It must
also be taken into account that yeasts produce
small quantities of SO; during fermentation In
general, the amount formed is rarely ntorc than
10 mg.'l. bit in certain cases it can exceed 30 mg/1.
Consequently, the total absence ofsilfnr dkixKIc
In wine R rare, even in the absence of sulfiiing.
Ik principal properties ate as follows:
I. Antiseptic: it inhibits the development of
microorganisms. It has a greater activity on
bacteria than on yeasts. At low concentra-
tions, the inhibition is transitory. High con-
centrations destroy a percenttge of the micro-
bial population The effectiveness of a given
n»- .,-. ...■..■A.. ■ •
r r*, ,.i (» ,„
Handbook of Etiology: The Microbiology of Wine anil Vinifications
concentration is increased by lowering ihe
initial population, by filtration for example.
During storage. SO; binders Ihe development
of all types of microorganisms (yeasts. tu.Ui
bacteria, and. toalcsscrcxicni. acetic bacteria),
preventing yeast ha/c formation . secondary fer-
mentation of sweet white wines (Section 8.6.2).
Brelianmiyret contamination and the subse-
quent formation of ethyl- phenols (Volume 2.
Section 8.4.4). the development of mycodcr-
mic yeast I flor) (Volume 2. Section 8 J.4). aid
various types of bacteria spoilage (Volume 2.
Sections 8 J. 1 and 8.3.3).
2 Antioxidant: in the presence of ealalyecrs. it
binds with dissolved oxygen according to the
following reaction:
S0 2 +$Q 2
- S' >,
;S I i
This reaction is slow. It protect* wines from
chemical oxidations, but it has no effect on
enzymatic oxidations, which arc very quick.
SOj protect* wine from an excessively intense
oxidation of iis phenolic compounds and ccrEiin
clemcnb of it. aroma. It prevents madciri ration.
It also contributes to the establishment of a
sufficiently low oxidation- reduction potential.
i.i'.' i/iiil wine aroma and UsK development
during storage and aging.
3 Anboxklasic: it insCinlancously inhibiLs the
functioning of oxidation cn/ynics (tyrosinase,
laccase) and can ensure their destruction over
time Before fermentation. SO; piotccls musts
fiom oxidation by this mechanism It afc>o helps
to avoid oxidasK cassc in white and red wines
made from rotkrn grapes.
4. Binding ethanal and other similar products,
it protects wine aromas and makes Ihe flat
character disappear.
Adding SO; u wine raises a number of issues.
Excessive doses musl be avoided, above all for
health reasons, but also because of their impact
on aroma High doses neutralize aroma, while
even larger amounts produce charac tensile aroma
defects, i.e. a smell of wet wool lhat rapidly
becomes suffocating and irritating, ugclncr with
a burning sensation on the aflertasle. However, an
insufficient concentration docs not ensure Ihe total
stability of Ihe wine. Excessive oxidation or micro-
bial development can compiomisc )b> presentation
and quality.
Il is not easy K> calculate the precise quantities
required, because of the complex chemical equilib-
rium of this molecule in wine. Il exists in different
forms that possess different properties in media of
different composition.
The concentration of sulfur dioxide in wine is
habitually expressed in mg SO; per liter (or ppmi
although this substance exist, in multiple forms in
wine (Section 83).
The wonLs sulfur dioxide, sulfur anhydride or
sulfurous gas can all be used equally, or even sul-
furous acid, though Ihe corresponding molecule
cannot be isolated. The expression sulfur', how-
ever, is fundamentally incorrect . Additions made
to wine arc always expressed in the anhydrous
form, in mg/I or in g/hl. regardless of the form
effectively employed —sulfur dioxide gas or liquid
solution, potassium bisulfite ( KHSO, I or potassium
mctabtsulftlc (K.S.O.i The effect of the addition
lowinc is the same, regard less of the form used The
equilibrium established between the various forms
is identical It depends on the pH and Ihe presence
of molecules lhat bind with the sulfur dioxide.
Substantial progress in the undcrsGinding of
the chemistry of sulfur dioxide and lis properties
have penniik'il the winentakcr to reason in use
in wine As a result, the concentrations of SO;
employed in wine have considerably decreased.
Simultaneously, this technological progress has
led to a decrease in authorised concentrations.
In 1907. Erench legiskition set the legal Until
in all wines al 330 mg/1 increased, in 1926. to
450 mg/1. Today. Erench wines anr subject lo
EU legislation (Table 8 11. which has gradually
reduced the permitted level to 160 mg/1 for most
red wines and 210 mg/1 for the majority of while
wines Higher doses may only be used in wines
with very high sugar content They arc generally
premium wines produced in small volumes and
consumed in moderate quantities.
In practice, the concentration used isevci lower.
Eor while Erench wines (excluding special wines)
The Use of Sulfur DKixklc in Musi and Wine Trcutttciit
Table Kl. Minimum «il«ir diovidc concern ml ion* depending on «inc ij
lb*. (vikicicipitucd la mgil)
A. EU iraubtniB no: 1 4.93/1999 ••,! 1622/2000. modified in 1655/2001
<Sg/l = o->5g/1
Red « urn IOO(t-IO)*
Whne and n»C = Ine* 210 (r40)*
Red >«»<(-- jmys I2S
White and n»e iftu ric pan ISO
Dcucn win ISO
Km rfpp./vi (TAV > IS'i vol.; augur >4S p/ll
Wttc AOC wine*
Boidcmn wipcricur. Gave* de Vhyn.Ctta dc Bonlcaui
Suiu-Mutairc. frcmicKi Cole* dc Bordeaux. Siimc-Foy
BordcMn.C<McbOx Benierac Mirvk ou sonde h dc*omi*aik>n
Coin dc Sjimijni,. lima Mantra vcl. Cote* dc Momravclet
Ro*ctlc. ' a illi.
\V hie DO >i»a
Allcb. U Mnncha. Savnna. Pencdc*. K»b. Rcutfa. Ij^mh.
ci Valencia
\li> .dkje. 1 renin, -paib" ->endcmmiaiaidiva
Vqprd Mom-sIo di Pamelkria ntfumk and Mowala di Pamcllcm
Unfed Kingdom V<ip-d deserted » follow*:
boiiytit. noble hir.cn. noble bic harvested
German wine*
SeSksc
Amine
and Mime Rum.innn white wine*
Beeicivt
iu*k*c.Aushiuch.Au*b(u
en-wein. Tro,.cnbccrcn.iu*l
Eton
cin
While AOC winci
Inutc
rncs.BaiNac.Cadilbc.Ce
10*-.. Iixipbe
Saint
c-Cmlx-du- Mont. Gove*
tupciKUn. Mantn/ilbc.
Jurancoa. Pacne-cnc du Vic Bilh. Anjou-Cotcnu. dc b Loi
Bonn
env , Quart* dc Chuum
r.Coteauxde 1 Aubancc.
( oicaui du Ijiyon ml 1 . 1 du noi
n dc In commune d rig me.
Cbtn
ail ill Ijvoii Miivl du noi
n dc Chnuma. CoIcmii dc
\-..H
eel Abaecgrandciuuii'
.idcbmc.nn-vcad.i.K*
budii
c* ou "*clcci»ndcgraii
a nobles"
Swcci '
line* from Grccc (sugar
= or >4Sg/l)
Samo*.
Same
Rhode*, hum*. Rki hrnn
inn. Nemc*. Daphinc*
a. Ccp halonic. linn, 'di
Certain
Cin.iiiiins.hiewj.eMk
.-1.-..1
>4g/l
Red « ine*
Whne and n*c wines
Ccui.»«i«hic»ii
l'Jf,
Handbook of linology: The Microbiology °f Wi»e anil Vindications
Ibc average concentration is 1(6 mg/1: for icd
wines ii is 75 mg 1 The Office International dc la
Vignc el dn Vin iOIV.i recommends slightly higher
values than those advocated by ihc EV in lis mem-
ber cm n tries. In ccnain countries, ihc regulation
of sulfur dioxide dictates a common limit for all
wines. For example, this value is 350 mg/1 in ihc
USA. in Canada, in Japan and in Australia
Due Id ihe fluctuating equilibrium between free
and boand forms of SO., ii general, the leg-
islalion of different countries exclusively refers
lo Ihc total Miliar dioxide concentration. Ccrtiin
countries, however, have icgnlations for the free
fraction.
Today, especially for health reasons. Ihe possi-
bility of further reducing the authorized concen-
trations in different kinds of wines Is sought after.
Such an approach consists of optimizing the con-
ditions and perfecting the methods of using this
product. This sapposcs more in-depth knowledge
of the chemical properties of Ihc sulfar dioxide
molccalc and its enological role. Substitute pred-
icts can also be considered Due to the varims
effects of snlfur dioxide in wine. Ihc existence
of another substance performing the same roles
without the disad vantages seems very unlikely,
bat. the existence of adjuvants, complementing the
effect of SO? in some of its properties, is perfectly
conceivable. Enological research has always been
preoccupied by the quest for such a product or
substitution process (Chapter 9).
In conclusion, sulfur dioxide permits the storage
of many types of wine known, today that would nol
exist without its preelection. In particular, it permits
extended barrel maturation and bottle aging. In
view of is involvement in a wide variety of
chemkal reactions, it Is not easy to dcKrrmine the
optimum dose to obtain all the benefits of SO;
without any of ic anfortanatc side-effects. The
adjustment shmkl be made within pins or minas
10 mg/1
8.2 PHYSIOLOGICAL EFFECTS
The addition of sulfur dioxide to wine raises
health-related objections. These should be taken
inu account, although this product boasts a long
history of use Its asc has always been regulaKd
and enological techniques have always sought
methods of lowering its concentrations Since the
beginning of the century, the possible toxicity
of sulfar dioxide has been Ihc sabjeel of much
rescareh (Vaqucr. 1988).
Acute toxicity has been studied in animals. The
ataorptioa of a single dose of saltitcs is slightly
toxic. Depending on the animal species, the LD V
(lethal dose foi .«' ' I of individuals) is between 0.7
and 25 g of SO. per kilogram of body weight.
Sodium sulfite would therefore have an acute
toxicity similar to inoffensive products such as
sodium bicarbonate or potassium chloride.
Chronic toxicity has also been studied in ani-
mals (Til c/ ill.. 1972). During several generations,
a diet containing 15 g of SOj/kg was regularly
ateorbed Three kinds of complications resulted: a
thiamine dclicicncy linked to ic. destruction by sul-
fur dioxide, a histopathological modification of the
stomach, and slowed growth. This study permitted
Ihc establishment of a maximum nontoxic concen-
tration for rats at 72 ntg/kg of body weight. This
value led the World Health Organization Id set the
RDA (recommended daily allowance! al 7 mgof
SOi/kg of body weight.
Concerning its toxicity in humans, studies carried
ont indKafc the appearance of intoxication symp-
toms such as nansea. vomiting and gastric irritation
at significantly high absorbed concentrations (4g
of sodium sulfite in a single concentration) No
secondary effects were observed with a cotccntra-
tion of 4tt) mg of sulfur dioxide during 25 days.
In humans, is possible uxKily has often been
attributed lo the well- known dcslruc lion of thiamine
or vitamin Bl by sulfiks. but the corresponding
reaction has been observed lo be very limited at a
pH of around 2. which corresponds to stomach pH.
In 1973. allergic reactions to saltitcs were pro-
ven lo cxBt. They occar at very low ingested
concentrations (around I mg) and primarily con-
cern asthmatics (4-IOf of Ibc human popula-
tion). Asthmatics are therefore urged lo absttin
from drinking wine. Although SO? sensitivity has
not been clearly demonstrated for non-asthmatics,
these allergic reactions led Ihc US FDA (Food aid
Drag Administration) to require the mention of the
The Use of Sulfur Dioxide in Mum .mil Wine Treatment
presence of sulfites on wine Libels in the United
Stiles when Ine coaccnUuiioi exceeds 10 mg/1.
Considering an RDA of 0.7 mg/kg/tfay. ine
acceptable coaccnUuiioi for an indi mla.il is
between 42 and 56 mg per day. depending on body
weight (60 and HO kg. respective))'). Thccousump-
Ikii of half a bottle of wine per day (375 ml) can
supply a quantity of SO. higher than the RDA
If the fcttal SO> concentration is at Ihc ■minium
limit authorized by Ihc EU ( 160 ug/1 for red wines
and 2 10 mg/l for while wines). Ihc quantity of SO;
furnished by half a bottle is 60 mg for reds and
79 nig for whites. The average SO, concentrations
observed in Fiance are much lower: 75 mg/1 for
led wincsand 105 mg/l lor while wines. Therefore,
■he daily consumption of half a botllc furnishes 28
and 39 mg of SO>. respectively.
In any case. Ihc figures clearly indicate that,
with respect to World Health Organization nonius,
wines can supply a non- negligible quantity of
SOj. (t is therefore understandable thai national
and international health authorities recommend
additional decreases in the accepted legal limits
Experts from the OIV estimate that the concen-
trations recommended by the EC can be decreased
by It) mg/l. at least for Ihc most conventional
wines In this perfectly justified quest for lower-
ing SOi concentrations, specialty wines such as
bnliytifcd wines must be taken inu account Due
to their particular chemical composition, they pos-
sess a significant combining power with sulfur
dioxide. Consequently, their slat* li ration supposes
extensive saltiling. The EU legislation authorizing
400 ntg/l is perfectly reasonable, but this concen-
tration is not always suflicicnt. In particular, it
docs not guarantee the stability of some batches of
bolryti/cd wines and will not prevent them from
secondary fcrmcntilioa.
8.3 CHEMISTRY OF SULFUR
DIOXIDE
&3.I Free Sulfur Dioxide
During the solubilization of SO;, equilibria are
established:
HSO,
i SOi J
r ri-
ff. J)
The HjSOj acid molecule would not exist in a
solution. It nevertheless possesses two acid func-
tions whose pKs are IS I and 6.91. respectively
at 20'C. The neutralisation of an acid begins at
approximately pH = pK — 2. The absence of neu-
tral snllifcs(SO ( J " | at the pH of wine can therefore
be deduced But Ihc first function Is partially ncu-
Iroli/cd acconling to the pH. Knowing the propor-
tion of free acid (active SO;) and bWilfitc i II SO, " )
is important, since the essential cnoligical paipcr-
ties are attributed u the tiist. The calculation is
made by applying Ihc mass action law:
IHTIIHSO. |
= Ki
(8.4)
The water concentration can be treated
constat! or very near to I :
Log-
ISO. |
= pH-pK,
(8.6)
Table 82 indicates Ihc results for Ihc pH range
corresponding lo various kinds of wine. The pro-
portion of molecular SO?, approximately corre-
sponding to active SO;, varies from 1 to 10. This
explains the need for more substantial saltiling
when the must or wine pH Is high.
Tabic Rl Molecular \O r 1*1 hiwlntc
acconlimi ti> fll mi 20 C > in aujucou* m>U
X«U(JC\
pll
Mokcubr SO.
Biwilfilc til SO. i
("1
0.00
94.94
3.10
428
93.12
3 JO
.i.'H
MD9
3 JO
1.13
96117
3 JO
231
9749
3 JO
200
9800
3JJ0
100
9840
3.70
\ 2-
98.73
320
10]
9899
3J0
> 1 ,S 1
99.19
4A0
004
H u
I landbook or Etiology: The Microbiology of Wine anil Vindications
Tab k- 8.3. Sulfur
( Uucplio-Tom uv
dioxide pKl
*. 1995)
value JinirJiiw to ikoholK'
Mtciqah .
■■die
""""""
■kohol
Tcmpcnturc ( Cl
e "•!•) 19
22
25 28 31
H
37
40
2.14)
225
2JI
1.98 206
2JJB 2.10
2.18 226
2J7
2JB
224 2.31 2J0 2J7 230
2.34 244 230 237 260
2J5 254 201 207 2.70
233 204 2.72 278 280
The pK value is also influenced by Icmpcralnrc
and alcoholic strength (Table 8 1 1, and equally by
tatK lone — ihc concentration in sails. Usscglio-
Tomassct (1995) calculated ihc effect of these
factors on the proportion of sulfur dioxide in the
fom of active SO- (Tabic 8.4)
The bisulfite ion (HSOil represents the corre-
sponding fraction of the acid neutralized by bases,
this almost entirely in the form of ionized salts.
Active SO; tot sulfuious acid in lie free acid stile)
represents free sulfur dioxide as defined in etiol-
ogy. The difference between the chemical notion
of a free acid and a salificd acid should be taken
into account
As a result, the antiseptic properties of a given
concentration of free SO; lowaids yeasts or bacte-
ria vary in function of pH. even if the USD, form
Is altribalcd with a ccrctin activity. In the same
manner. Ihc disagreeable last and odor of sulfur
dioxide, for the same value of free SO., increase
the more acidic the wine The disagreeable odor of
SO; is sometimes less the result of an exaggerated
SO; addition than Ihc nature of the wine— inferior
quality, an absence of character and aroma, and
wry high acidity
TuhlcK4. 1 .".., Ml.'. Of .Cll.f iimI... ].-.: SO, ill
(rl I 3D M-coiding to alcoholic Mniuth and tcmpccuurc
(UttcglM-Toaaucl. 1995)
alcohol
Temp,
:imuk TO
■:\ vol.)
19
28
38
488
m
7 JO
I5J0
2733
20
10.95
8,3.2 Bound Sulfur Dioxide
B (sulfites possess the property of binding mole-
cules which contiin carbonyl groups according to
the following reversible reaction:
■ R— CH— SOiV
R-C-R" + HSOj"
II
O
' 'II
These additional forms represent bound sulfur
dioxide, or bound SO? as it is defined in i n- .-
ogy. The sum of free SO; plus bound SO;
is equal lo total SO;. With respect (u free
SO;, bound SO; has much less significant (even
insignificant), antiseptic and antioxidant properties
(Section 8.6).
In Ihc reactions forming these combinations.
Ihc equilibrium point is given by the formula in
Eqn (8.9). for Ihc reaction in Eqn (8.7). This for-
mula presents the molar concentration relationship
between the diffctcnl molecules:
|R-CHO||HSO,-|
IS CHOII-SO,!
= K
<M)
K is a constant characteristic of each substance,
with aldchydic or ketonic functions, able lo
bind SO;.
This tclalionship can be written as follows:
IR-CHOH-SO,-
[HSO,
(8.10)
The Lfcc of Sulfur Dioxide In Musi awl Wine Treatment
For example, a concentration of 20 mg of free
SO2 per lilcr represent! 25 mg of HSO»" per liter
(moleculur weights 64 aid 81. respectively) The
molar concentration is ihcicfoic:
The it* la l ion ship
10--JH
324
Eqn (8.10) becomes:
[CI _ |R~CHO~SOri
|A1 ~ IR-CHOI
i 24*
(8-11)
(8.12)
ll expresses ihe proportion of carbonyl gtonp
molecules bound to SO; (C) and ii their free
form (A).
First case; K has a low valnc equal to or krss
than 0.003 x lo ' \\. m equilibrium:
1" ■
3.24 x 0.003 *
-T-
. = 100
(8.13)
In this case. there cxnfe 100 nines more of
lac bonnd form lhaa (he free form. The binding
molecule is considered to be almosl cniircly in the
combined form. Free SO. can only exist when
all of tie molecules in question arc completely
bound. Furthermore, in is combination is stable and
definitive, (he depletion of fire SO; by oxidation
docs not cause an appreciable displacement of the
equilibrium.
Second case: K has a
>r greater than 30 x 10"
elevated value equal l
|AI 3.24x30x10" 100
In this case, their exists 100 limes more of the
lite form than the combined form The binding
molecule Is considered to be slightly combined
and the corresponding combination is not very
stable When free SO. is depleted by oxidation,
the dissociation of this combination, necessary for
reestablishing Ihe equilibria, regenerates free SO;.
Of course. |C| pins IAI represents the total
molar concentration of the combining molecule
as given by analysis, expressed in millimolc per
liter. It is therefore possible lo cstibltsh overall
reaction values of bound SO; for different free
SO; values In fact, by determining the quantity
of each combining molccnlc. Ihe amonnl of bonnd
SO? can be calculated using the valnc of K and
the concentration of free SO; (sec Figure 8 3).
The sum of the individual combinations must
correspond with the total bonnd SO. determined
by analysis (Section 8.4.3).
Flgnre 8.1 gives SO; combination enrves for
different values of A' and for a combining
molecular conceit nation of 10* m. The maximum
bound SO> concentration is also It) ' vi.64mg/1.
Fig 8.1. Sulfur dioxide cumbittiion time* in accc
oftartxinyleduilwuntc = 10 ' M (Blouin. I96S|
Handbook of Etiology: The Microbiology of Wine and Vhiitialiwa
Fifi 82 Tbcdiflcic«*iMCT,ufuiliur<ln\)ilc la wine (RfccmtKIuyoii et ti.. 1977)
oiMM*M» ♦ ♦ ♦ ♦
ool I m\l .in liiihlionof Ikcu
Id conclusion. foe different fonts of Miliar di-
oxide existing in wine air summarised in ihc
Figure 8 2. Active SO, is located to Inc left its
separation (a) wild HSOj " vanes according (o the
pH To the right, sulfirous aide by die acid rep-
rcscnis ihc SO, fracliot combined with cihanal.
Since K is low. this combination is very sta-
ble and depends on the cihanal concentration.
The (c) separation line is definitive. On Ihc other
hand. Ihc lb) scparaiion between silfnr dioxide
and sulfnr dioxMlc combined with other sub-
stinccs varies, moving in one direction or ihc
other accoiding k> icuipcrulnre and the free SO;
concentration.
8.4 MOLECULES BINDINC.
SULFUR DIOXIDE
8.4.1 1' 111 ana I
The reaction:
CHi-CHO+HSO," =CH 3 -CHOH-SCV
|8.15)
gcnciully represents Ihc most significant portion
of bound so, ii wine. The value of K is
extremely low (0.0024 x 10') and corresponds
to a combination rat of greater than ffk The
cthanal concentrations between 30 and 130 mg/l
The Use of Sulfur Dioxide in Musi and Wine Treatment
:ni
correspond to possible bound SO? values between
44 and 190 mg/l.
In wine no longer containing free SO., a weak
dissociation of sulfurous aldchydic acid liberates
a trace of clhanal. This ethanal is said to be
responsible for the Hut ckaraclcr in wine, but
lie picscice of free ethanal is considcicd to be
impossible in wine containing fire SOi.
The combination is rapid. At pH 3 3. 98*i of it
is combined in 90 minutes and the combination is
total in 5 bonis. Within normal limits, the combi-
nation is independent of temperature. The amount
of free SO; liberated by raising the temperature
is very small. Concentrations in bolryli/ed musts
are of the order of 10 mg/l. up to a maximim of
20 nig/I. These concentrations may explain a mean
combination of under 10 mg/l SO;
Alcoholic fermentation is the principal source of
ethanal in wine. It isan intermediary product in the
formation of cthanol from sugars Ik accumulation
is linked to the intensity of the glyccropyruvic
fcnticitilion. It principally depends on the level
of aeration, but the highest values are obtained
when yeast activity occurs in lie presence of free
SO, The formation of sulfnrous aldchydic acid
is a means of pioteclion for the yeasts against
this antiseptic Consequently, the level of grape
snltiting controls the ethanal and ethanal bound to
SOi concentration.
Considering these phenomena, the addition of
SO. to a fermenting mast should be avoided It
would immediately be combined without being
effective. When the grapes arc botiyti/cd. the vari-
ation in Uiccihanalconicntof different wincswhen
50 mg/l of SO; Is added to the must accounts
for a combining power approximately 40 mg/l
higher than that of non-sulliied control wines.
When stopping the fermentation of a sweet wine,
a sufficient concentration should be added which
stops all yeast activity. This concentration can be
decreased by initially reducing the yeast popu-
lation, using ccntrifngation or cold stabilization
I-4"C). for example. The highest ethanal con-
centrations are encountered when successive fer-
mentations occur. The necessary multiple sulfitings
progressively increase bound SO? concentrations.
The chemical oxidation of cthanol. by oxida-
tion-reduction in the presence ol a catalyzer, may
also increase the clhanal concentration during stor-
age — for example, during rackiugs The combin-
ing power of the wine therefore also it
8.4.2 Retook Acids
Pyruvic acid and 2-oxoglntarK acid (formerly a-
keuglucuic acid) arc generally present in wine
tTable 83). They arc secondary products of alco-
holic fermentation. Considering their low K value,
they can play an important tolc in the SO?
combination rale For example, a wine contain-
ing 200 mg of pyruvic acid and 100 mg of 2-
oxoglucuic acid per liter has 93 mg of SO; per
liter hound to these acids for 20 mg of free SO?
Those two substances may combine with very
different anoints of SO?. In wines made from
bouyti/cd grapes, for a free SO? content of
50 mg/l. 2-oxoglutirie acid Is likely to combine
with an average of 43 mg/l and pyruvic ackl with
58 ntg/UBarbe. 2000). The average pcrccntagcsof
pyruvic and 2-oxoglntaiic acids in the SO? combi-
nation balance arc 20.7'i and 16 Ti . respectively
It is therefore interesting to understand the for-
mation and accumulation conditions of these acids
during alcoholic fermentation. They are formed at
the beginning of the fermentative process. After
initially increasing, their concentration decreases
lowaids the end of fermentation This explains the
higher concentration of these molecules in sweet
Tabic B.5. The kclua
ic ackk of wiac <Uucgli>-Tum*MCi
:. IOT5)
Ante
Fbmuhi
It
Avcaajc
Pyiuvic Mid
2-OiupluarN- acid
CHi-CO-COOH
COOH -CO-C H »-C Hi-COOH
0.3 x 10"' U
0J x 10"' M
10 -SOD au/1
2-350 an/1
Handbook of linology: The Microbiology of Wine anil Viuitications
Tuhb &<i. Action of Ikbmitc »n [clonic ackb and Hcc Milfur do
ofiotal SO, perlMf(Ru>e.eau-<iiyi>n«irf. 1977)
.ideco*
vnti
««*iif'li.
calculated fai 250 m?
Origin Cotf ml
-Thou
„.-,-
•h wMmM pymvfc 2-0*ogu*:aric Free SO,
acid Kid far tola! **) m^l
Pyruvic
■cid
frOxoglluk
acid
Free SO,
foti«al2S0 mgJI
wines wilb respect to dry wines. Blcvatcd temper-
.units and pits, along will aerations, favor ihc
synthesis and accumulation of kclonic acids. Ii
numerous lc nucn unions. Ihiaminc (al a couccntra-
lion of 05 mg/1) has been shown h> diminish ihc
concentration of these acids and consequently suV
lm dioxide combinations Thccffcclof thiamine Is
not surprising II is an essential clement of ihc car-
boxylase which assircs decarboxylation of pyruvic
acid ink) cihanal Thct is an csscniEU slop of alco-
holic fc mien til ion. The accnmulaiion of ke ionic
acids appears k> result Iron a thiamine detkiency.
The figures in Table 8.6 show ihc effcel of thi-
amine on Ihc accumulation of kctoatc acids and ihc
corresponding combining power In Ihc first three
wines made from slightly rotten grapes. Ihc sulfur
dioxide equilibrium is not modified alter Ihc addi-
tion of ihiaminc. In ihc whereases, the presence of
ih Limine decreases Ihc kclonic acid conccnliution
and oflcn improves the snlfur dioxide equilibrium.
To be effective, thiamine needs lo be added to
clarified and sulfilcd must siffkicnily early. Ii has
•o action on ihc accumulation of cihanal. In certain
cases, nscfnl secondary dice is arc observed:
activation of ihc fcnncntiiioa and diminution of
volatile acidity. On average, in eight cases out of
10. Ihiaminc increases Ihc free SO? concentration
in sweet wines by 20 mg. for ihc sane bound SO;
con cen (ration.
8.4.3 Sugars and Sugar Derivatives
Considering Ihc existence of aklchydic and kctonie
functions in different sugar molecules, they can
be expected lo have a combining power with
snlfur dioxide. Fructose and saccharose, however,
practically do nol react.
Arabinosc binds SO; at a rate of approximately
8 mg of SO; per gram of arabinosc for 50 nig
of free SO, per liter. Since ihc concentration of
arabinosc in wine is low (less than I g/1). Ibis
combination is nol generally taken into account.
Glucose has a much lower combining power. One
grani combines J mg of SO? for 50 nig of free
SO; per liter. Due u> Ihc high concentration of
glncose in musts and sweclwincs.ihrs combination
should be taken into accoanl and it Is inclnded in
the IntcrprcciiKan of the decrease in free SOi after
salliting ihc grapes or ihc must.
Burroughs and Sparks (1964 and 1973) iden-
titied the following substances: kcto- 5- fructose
(5-oxofructose). xylosonc. kcto-2-g Income (2-oxo-
glnconK) and dikcu-25-gluconK (25-dioxoglu-
conic) acids (Table 8.7). line In their concentra-
tions In some wines (capable of attaining several
dozen milligrams per liter), and Ihcir K values,
some of them can play a significant role in bind-
ing with sulfur dioxide. These substinccs exist
naturally In healthy, ripe grapes and they are
also formed in large quantities by BiHryris cinerea
and acetic acid bacteria (Acerobacler and Pteii-
(AmKinof). Their development frequently accom-
panies various forms of tot.
According to more recent findings (Barbc el «/..
2002). among all Ihc previously- mentioned com-
pounds. 2-oxo and 25-dixogluconic acids always
present In a ratio of 25/1. do not have a significant
affinity for SO ? . In contrast, al ihc pH of botrytized
The Use of Sulfur Dioxide in Mnsl and Wine Trcatttcnt 203
Tabic K7. Snllui>.l»)xak hiii(Iiiuuv-»<kriv.-U l >'»<lia%c<l«ii Ilurmt^kniHl Spuli 1901 40.1 1973)
Dm
Kiickl
J
ujaiovkkuk
OflXHild*
Gafactiwak-
ackl
GbHumnlc
■cid
Kct»
2-ghKM
■cid
« Dikcic
-2J-pkno»i
Kid
Kcro-5-f incline
XyUnoK
cn/m cit/m CMpti
04 x 10"' U 04 x 10"' U OJx 10"' II 0.15 x 10"' I
Kifi &'- romuibn of J"- amld-|thx.n
mnsis and wines, gluconic acid (20 g/1) is In equi-
librium with two lactones, y and d-glKonobctonc
(Figure 8.4). representing aboni KM- of Ihc con-
centration of ihc acid. The affinity corresponds to
■hat of a monocarbonyl compound with a bisullilc
couibiualion dissociation constant K =4.22 111M.
Thus, the lactones of gluconic acid arc likely Id
combine win up to 1.15 mg/1 SO; for a free SO2
conienlof 50 mg/1.
The 5-oxofraclcw coiicnl is also frequently
of Ihc order of 100 mg/l In wines made from
hoiryti/cd grapes (Barbe. 2000). Concentialions
increase win ihc combining power (Figure 8.5)
According to Barbe (2000). 5-«>xofructosc may
account for the combination of 4~78'i of the
snlfur dioxide. Concentrations of this compound
arc not altered bv alcoholic fermentation or
any other aspect of ycasl metabolism. Excessive
conccnlraiions can. therefore, only be avoided by
monitoring grape quality. In the special case of
must made from grapes aliened by rot in Ihc
mature stage, ii contributes, on average, over 60*
lo Ihc combination balance This compound is
produced from free hue by acetic bacteria in Ihc
genus Gtuconobacier (Section '5).
8.4.4 Diearbonvl Croup Molecules
Ingiupesaffecled by rot. Guillou-LargctcauU996)
identified molecules with two carbonyl groups
(Tabic 8.8). They arc probably formed during
Ihc development of Bolnlis ciaerea and olhcr
microorganisms involved in various types of rot
In view of ihc facl thai concentrations do not
Handbook of Hnology: The Microbiology of Wine anil Vinific.uions
tip, «S- ("fcamw, iaibc hu
,«inUniJto(hcco«hInInppoiie<ortlx mm (Baibc M .rf.. MOD)
Tabic R8. Some dkaiboit)! primp mol«iiki involved in uilfar doxidc «imh)aul»nb
(hviinnypnipunciUil n .1 rmsoaci Ibrat of itdutli>ac)<Cu)lkiu-l.j<£Clcau. I9IXi)
Name C'IkbkjI loirnub llcalrh; grapw Bum'"™"" nope*
exceed 3 mg/l. Ibe contribariau of gryaxal lo the
SO; combination balance Is praclically negligible.
Mclhylglyoxa) makes a more siguilkanl conlribu-
lion aid may be responsible Tot combining over
30 mg/l SO. for a Tree SO. cotlent of SO mg/l
< Barbe. 2000). Glyoxal anil, especially, mcihylgly-
c during alcoholic rer-
an? iwly responsible Tor insignificant b
combined SO; in wltc.
8.4.5 Other Combinations
OlncrsubsEinccs likely to tix small
In r dioxide kavc been itlcatilicd gl
tsitfsil-
.galac-
:nlaiion. so these two a-dicarboiyl compounds tnronic acid and xylosoie (Table 8.7). glyoxylic
The Lte of Sulfur Dioxide in Musi anil Wine Treatment
:i."
acid. "ul'n.L". acid, glyceric aldehyde, ace-
tone, diaccfyl. 5-inydimy methyl tfuri'iiral. etc.
Thclc individual contribution Is insignificant In
the eusc of dihydroxyacelonc. 100 mg/l accounts
fur the combination of approximately 16 ntg/1 for
50 mg/l free SO;, although this value may be as
nigh as 72 mg/l in certain types of mast (Barbc
el of., 2001c). White glyccraldchydc has a greater
affinity for SO; ( K =04 mM. Blouin. 1995). it is
only present in liny amounts, so it makes a negli-
gible c ■ ■ i.i !■ ■ ii. ■ n K> the SO; combination balance
SO. can aba bind with phenolic compounds.
In the case of pminlhocyanic tannins, a solution
of I g/1 binds with 20 mg/l of SO; per liter The
combinations an: significant with anthocyanins.
These reactions aic directly visible by the dccol-
oration produced. The combination is rcvcisiblc:
the color rcappcats when the frcc snlfur dioxide
disappears. This reaction is related n> temperature
tSection 83.2) and acidity (Section 85.1). which
affect the quantity of free SO;. The SO; involved
in these combinations is probably limited by iodine
along with the free SO;. In fact, due to their
low stability, they arc progressively dissociated to
reestablish the equilibrium as the free SO; isoxi-
ili.vil by iodine.
8.4.6 The Swlfut Dbxklc Combination
Balance in Wines Made from
Botrylired (Jrapes
Bnrronghsand Sparks (1973) calculated the Si i.
combination balance for two wines on the basis
of the concentrations of the various constituents
involved, determined by chemical assay and
expressed in millimolcs per liter (Section 8 J 2)
The combined SO? calculated by this method was
in good agreement with the combined SOi assay
results, so it would appear that the SO? combina-
tions were fully known in that case.
Blouin (1965) had previously demonstrated the
particular importance of ketonic acids in this rypc
of combination In spile of all these findings.
Ihc snlfur dioxide combination balance cannot
be considered complete and satisfactory Progress
has been made in establishing the combination
balance for wines made from bolryti/cd grapes
by finding out about other compounds, such
as dihydroxyaic tone, which is in balance with
glycciuldchydcs (Blouin. l995;Guillou-Largclcau.
19961. and work on neutral caibonyl compounds
in wines (Guillou-Laigctcau. 1996). Finally, more
recent research by Barbc and colleagues (2000:
2001a: b; and c; 2002) has improved control
of sulfur dioxide concentrations by adding to
knowledge of the origins of these compounds
In wines made from bolrytixd gtupes with
high or low combination capacities, almost all of
these combinations arc acconntcd for by the con-
centrations of 5 -<>xo fructose, dihydroxyacetonc.
■- and <t-gluconolaclonc. cthanal. pyruvic and
2-oxoglutarK acid, glyoxal. mclnylglyoxal. and
glucose (Table 8 9). In contrast, in must made
from the same type ofgrapes. Ihc high combining
power is precisely accounted for by the quantities
of SO; combined by 5-oxofmctosc. dihydroxyace-
tone. and gluconic acid lactones (Table 8.10).
Carefully-controlled fermentation of bolryti/cd
musts minimizes ihc accumulation of yeast meta-
bolic products combining SO;, although much
big her concentrations of these compounds arc impli-
cated in stopping fermentation than those present in
dry wines. Varions technological parameters dur-
ing fermentation make it possible u reduce the
quantities of sulfur dioxide, by affecting only those
combining compounds produced by fermentation
yeasts Wincswithalowersulfurdioxidc combining
power may be obtained by not sulfi ting must, adding
05 mg/l of thiamine lo must, choosing a yeast strain
known to prodncc little cthanal or 2-oxoacids. and
delaying nialagc until the yeast metabolism has been
completely shut down (e.g. by filtering or chilling
Ihc wine) (Baibe el of.. 2001c).
These compounds arc prodaccd due *> the
presence of microorganisms in botrylizcd grapes
Although yeas*, represent a preponderant part
of the microorganisms present. acclK bacteria,
especially those in the Glucanobacler genus, arc
responsible for producing large amounts of these
compounds, which act as intermediaries in their
metabolism of the two main sugats in holiyti/cd
grapes (Barbc el id.. 2001a).
I I and book of Etiology: The Microbiology of Wine anil VnifkatioaB
TjhleR'i. C<.«brai«p powca uf cuapnmb In J wime. SO, coMbimblc by all ibe
coapouad* uiajrcd or only ufihcm icikm.il. pyi\k acid. 2-oxogkiark acid. — .mil
*9luc«iwUci<ine. and 5-oxofiudo*e) la 9 wines (Bite. 2000)
umbinahk SOj
SI), ,-omhioihlc by Ik
com poind* accounting for
Tabic 8 1(1. A'
renffe
tpuaiitic
i (mn/l) of
uiltut dktxidc ..!
mbincd bv 1 he compound), umki
audi in diffcrcnt mu
iu<B*ihe «-i..
j. ■!
c °" p "" rf
Mum
> .1/
isbi
= 24) i >h to»
nlinr power
Muds (a =7) win high
combining P""«'
CLSOi
= 11
1 ma/lloial SO,
C1J0 = 49S m$)\ loul SO,
5-OVOUUCIOSC
24
258
V- 30.1 A-gUEE
Mbcii
i:
SS
dihydioxvaccii
7
Ml
gklCOSC
48
45
metbylnb/oul
1?
9
ftmal
2
2
cl banal
10
14
2-oioghJaric i
icid
1-
IS
pyaivic acid
5
9
..■.IlL-l
12
11
The SO; combination balance varies cotsidcr-
ably between different musts (metabolism of ihe
acetic bacteria) aid wines (fermentation paianic-
tiM. Furthermore, the total content of these com-
biiant compounds in wine may result fiom boU
soniccs. as shown in Tabic X I i
Finall>'. Boirytis cinerea indirectly plays two
major roles in the accumulation of substances
that combine with SO,. Fiistly. it cusses in-depin
mod ilk at ions in Ihe giapc skins. whKb become
permeable, thus facilitating access to Ihe varions
substrains for acetic bacteria. Secondly, noble rol
causes glycerol to accumulate in the grapes and
is thus indirectly responsible for dihydiuxyacclonc
pnduclioa.
Table 8 12 recapitulates all the substances that
combine SO? idcnlilicd in musts and wines made
from botry tired grapes
8.5 PRACTICAL CONSEQUENCES:
THE STATE OF SULFUR
DIOXIDE IN WINES
8.5.1 Equilibrium Reactions
In a su I filed wine, an equilibrium exists between
Ihe free sulfnr dioxide and Ihe bound sulfur
dioxide— more precisely, the boand salfur dioxide
with a high dissociation constant K. Sulfurdioxidc
The Lfcc of Sulfur Dioxide in Mum and Wine Tn-aintcnt
■■ .1, M.-.l.H 11 i
eA:TLS0 =310 raa.'Il.
r AiTLSO =340 mg/lli
I SO, (it. CLSO = 360 np/1 uml SO,)
I SO, (ic. CLSO = 290 ng/1 lot *l SO,)
S-oxnfniclDSC
i'- and A-ghxonDhctouc
iricMk(»1y«nldchydc-t DMA)
ahanal
pyuvk acid
2-oxoghua lie acid
u-i!«,j*onvU (mcihylphuvul + pi
Btacnc
(Hit I
Tabic 8. 12, CoiKCrtniioiu luui.1 and K 4 nictitated furlhe uuin mokmb idea inc. 1 in h«liyti*ed
•noU and aino. (Bumwpln and Spirit. 1961. 1973: Bloutn. 1965. 1995: auilluu-Laigcteau 1990:
Iia«V. 2000)
ci ha nil
::. ICMI
nnuvE acid
20-330
2-<)\uj)kilirit ac'il
50-330
glyoul
02-23
■elh) bdyo.al
0.7-0
galaclwonicacid
100-700
phKumnic acid
incci-60
S-tixotuiclinc
1 occv 2500
dihy dim vacci n nc
incci-20
p ly cc ra klc hy dc
ll3i.CS- 1"
phiconk- acid
l'"l 2-1
2-axogaiconic acid
Irae«-I20G
5-axopkiconk ackl
incu-500
\— and J-phjconuluciunc
tfi and J'i ol
ihc pbiei
BjhjMMC
;IOO p/l
<> hi mianam oi u»- imifinii u
bound to clhanal docs not participate in ihis
equilibrium, since ils combination has a very tow
K value and (has is very* stable
Any addition of sulfur dioxide to a wine rcsulLs
in ihc combination of a pan of this snlfur dioxide.
Conversely, the depletion of free sulfur dioxide
by oxidation results in a decrease of ihc boand
fraction lo such a desire lhai ihc loss of free
snlfur dioxKlc Is less than (he amount oxidized.
This liberation mechanism is advantageous, since it
automatically prolongs Ihc effectiveness of a given
concentration of snlfur dioxide
When the free salfnr dioxide concentration of
a wine decirascs to a very low level, it rarely
falls com pic tcly to rcio. unless ycasR arc involved
or other factois modify wine composition. The
decombination of boand SO? progressively rc-
places Ihc missing five sulfur dioxide.
As a result of these equilibria, the total sulfur
dioxide con ccnirationsol different wines cannot be
Humlhrok of Enology: The Microbiology of Wine anil Vmiikaliuaa
n odhchiihlint) of sulfur dioiHk il
n CL20. CLSD tkl CI. 100
compared if ihey <lo 10I have ihc same ficc sul-
fur dioxide concentration. For example, ifaswccl
wine has a (oral SO; conccnlmlk* close 10 ihe
legal linii. ihc consequence is not at all the same if
il only contains 10 nit: office SO? pcrlilcrtinsnf-
licicm fi i ensuring its stability I or 50 nig (largely
sufficient).
To remedy this difficulty, fllouin (1965) recom-
mended ihc use of Ihc expressions CI. Ill aid
CL50' (Figure 8.6) which icpicscnt. respectively,
the quantities of bound SO? necessity to have
20 or 50 mg of free SO? per lilcr. Known sul-
fur dioxide additions arc used to obtain these
numbers experimentally i Kiel holer and Wnnlig.
I960). These considerations arc mosl important in
the caw of sweet wines (Saulcrncs. Monba/illac.
Cotcaux dc Layon. and Tokay), which require rela-
tively high free SO? concentrations to ensure their
stability In practice, the combining power (TL50>.
of the amount of tola! SO? necessary in a must or
wine to obtain 50 mg/l of free SO?, R calculated
by drawing a graph of total SO? agalnsl free SO?
(Barbc. 2000).
8.5.2 Influence of Tempera lure
The detain in aim of the free sulfur dioxide
concentration in samples of a botryli?cd sweel
while wine with a siting binding power varies
according to temperature, although the total SO?
concentration remains constant (Table 8.1.1). The
results for determining free SO? concentrations arc
Ihcrcforc variable. Depending on the conditions.
Ihe results obtained can differ by as mnch as
20 mg/|.
The suragc Icmpcralurc of ihc wine mast also
be laken into account in the evaluation of Ihe
effectiveness of sulfiting. at least in the case of
sweet wines Finally. Ihc influence of icmpcra-
lurc becomes particularly important when beat-
ing wine. The SO> concentration can doable, or
even more. This liberation of sulfur dioxide sin-
gularly reinforces the effectiveness of healing.
Tank 8. 13 Utluence of temperature
■Jiltui dioxide ( ■j,*'lt in J h-nnuJ ■
74g/l:cthanal70 nur/l|
Total ttlhtirdntidc
Free Milbir dioxide
Bound uiiair dnxidc ( SO,C )
SOrfT (to ct ha lull
SO,C (loothcriubMjiKoi
The Use of Sulfur Dioxide in Mum and Wine Treatment
:i->
Al the time of bottling, wines can lv sterilized
at rcttlivcly low temperatures (between 45 and
50 C. fot example), due in pail K> this phc-
8.5.3 Kmpirical I.a
of Cum bin a I km
For a lonj: linic. cnology has Hied *> dclcrmiuc
applicable combination rules, bolh for saluting a
new wine immediately following fermentation and
for adjusting Ihe five SO? during storage.
The most satisfactory solution consist! of adding
increasing concentrations of SO? lo various sam-
ples of lie same wine to produce a curve as
in Figure 8.6. ThR operation is king and difH-
cult: consequently, il is not always feasible. Lab-
oratory tests are. however. recoaiiKnilcd before
Ihe first sulliiing of unknown wines immedi-
ately following fermentation Due lo the diver-
sity of harvest, a sondard SO. conccnlration can
lead lo an insufficient free SO? concentration for
ensuring seibility. or. on the contrary, an exces-
sively high concentration thai would be difficult lo
lower.
The combination curve O-tgarcS.6) clearly
reveals lhat the bound SO? increases with the free
SO2. Yet the increase is slower and slower as the
free sulfur dioxide concentration increases
To increase the free SO; concentration of a
wine already containing some, the combination
of Ihe added concentration mnsl be taken inlo
acconiL The lower the free SO; coKcnlratiou.
Ihe more the added concentration combines. Asa
general rule in standard wines already con tuning
free SO?, rwo-lniids of the supplementary con-
centration remains in a free stile and one-third
combines. As a result. 3 g/hl arc necessary lo
increase the free SO? concentration by 20 mg/1.
l-vcniual abnormal cases must also be anticipated,
corresponding with a much higher combination
■ale.
In practice, a few days after the addition of
SO? to wine, the free SO? concentration should
be verified lo ensure thai il corresponds with the
desired concentration and thai the siabili'aiion
conditions arc obtained
&6 ANTIMICROBIAL PROPERTIES
OF SULFUR DIOXIDE
&6.1 Properties of the Different Forms
The cnological properties of sulfur dioxide were
summarized al Ihe beginning of this chaptri Sec-
tion 8.1 1. It b essentially a multifacclcd antlscptK
and a powerful reducing agcnl lhat piolccb against
oxidation. 1c antifungal and antibacterial activities
will be covered in Sections 80.: and 86.3: the
autioxidi/ing and anlioxidasic properties will be
covered in Section 8.7.2. The various forms of
sulfur dioxide do nol share these properties lo the
same exicnt (TableS. 14).
lis varioas properties can make sulfur dioxide
seem indispensable in wincmaking The goal
of ciology is not to eliminate Ihis subsctnee
completely btt rather lo establish responsible
Tnile R 14. W)nc comci
Gjyon«(rf.. 19T7)
'"»■ pwpeiti
» of the diffcicut Ibra*
of lulfar dknide 1 Rkcrcau-
Pnifcay
SO, II SO.
R-SO,"
I'liDOhHllI
It.iilciKiilil
A mo. id*-
-t-
AiUktxUuait
tiiMJton um
Reducibn-
Ncutialiiat
rlbcukin:
axkbikm plica Ul
Mnofcihaml
t
+
GuMttorv 1
ok of SO,
biinp odor.
iiltoiku. taMcku
Handboak of Enology: The Microbiology of Wine anil Vinificalions
concentration limits. ThR supposes a sufficient
knowledge of its properties and conditions of use.
8.6.2 Antifungal Activities
The antiseptic action of SO; with respect to yeasls
can appear in different ways. On one hand, it can
be nsed to stop Ihc fermentation of sweet wines
(mnlagc) (Section 1425b) It effectively destroys
the existing population (fungicidal action) On the
other hand, it protect, these same sweet wines
from possible refcmicntalions— evaluated by the
growth of a small rcsidnal population It effec-
tively inhibib cellular multiplication (fungistatic
activity). Moderate sulfiting is also known to
inhibit yeast growth temporarily without their KUl
destruction The .subsequent disappearance of free
SO? permits the revival of yeast activity In prac-
tice, in the winery, new yeast activity may also
come from new contaminations resulting from con-
tret with non-sterile equipment and containers
For these different reasons, the results concent-
ing the action of SO- on wine ycasLs cited in
various research work and obtained in different
conditions ate not always easily computed. More-
over, the data on this subject seems incomplete.
Bound sulfur dioxide docs not have an anti-
septic action on yeast*. Ycasrs make use of the
formation of this combination to inactivate Si v
HSOj~ also possesses a low hut undetermined
anlLseptic activity Table 8.15 indicates the con-
centrations of free SO-, liiratablc by iodine, that
must be added to wines (according to their pHs) to
have an antiseptic activity equal to 2 mg of active
molecular SO? per liter. The antiseptic activity of
the bisulfite form IIS' 'i is mote or less signif-
icant, depending on the various hypotheses being
considered According to experience obtained <>•
wine subility. HSOi seems to be 20 times less
active than SO; . notably in wines containing
reducing sugars
Sulfur dioxide is fungistrlic at high pHs and
at tow concentrations, and it is a fungicide at
tow pHs and high concentrations The HSO, ■
form is exclusively fungtsttlK. Each yeast strain
Table H. 15. Ftec lultur dtDiidc co*cc«im»m. nccn-
2 ■£ of active Biok.ul.ii.Sti, per INcilRtKicau-OiYoii
a <i.. 1977)
WUK |ill
HypotbcbU: HSO,
probably has a specific sensitivity u the differ-
ent forms of sulfur dioxide. Romano and Suzzi
1 1992) considered possible mechanisms that conld
explain these differences According to these same
authors (Suf/i and Romano. 1982). sulfiting mnsl
before fcrmenttlton increases yeast resistance to
SO?. Yeasts fiom a non-sulfilcd mast, isolated
after fermentation, are ntore sensitive to SO? than
those coning from the same mnsl which is snlfitcd
before fermentation.
Concerning the immi&e of sweet wines (fungi-
cidal activity). Ihc fcrmentition seems u stop
abruptly after the addition of 100 mg of SO? per
liter. The conccniraiion of sugar remains coastal t.
although carbon dioxide continues to be released
for about an hoar. During this time, the yeasts do
not seem to be affcclcd by Ihc sullitiug — they arc
still capable of multiplying (Table 8 16). whatever
the concentration used. It is necessary to wait al
least 5 hours, and mote often 24 bouts, to observe
a decrease in cell viability
To ensure a complete cessation of fermenta-
tion. Sudraud and Chanel (1985) estimated thai
1 50 mg of molecular SO; per lilcr must be added
to wine. According to the same authors, alter
Ihc elimination of yeasts by different treatments.
1 20 mg of molecular SO? per lilcr seems suffic rent
for ensuring the proper storage of wines contain-
ing residual sugars (fungistatic activity). Lower
concentrations conkl be recommended for wines
The Use of Sulfur Dioxide in Mum and Wine Treatment
:m
Tabic R in ^ulfti)n|j h> )ah*t* yraati la * *«cci wine
01 Ihccnduf fcimcnUlkin I'.ikuiu aimK-f ■■!"' isl>!c
celb.. up.il>k of piakKif cubaics Id Petri diiho..
per nl; (nisi popubtunSK - lOVnlKRIbciriU-Qiyoa
el «V. 1977)
SO, en ikxoir.il kin
Time
I bur
5 noun
24 bun
100
Six ID*
8x 1(1*
10'
ISO
58 x ID*
Jx 1(1*
ii.i
SSx ID*
10*
D
stored .o law temperatures having ;i low yeast
population.
Romano and Sn/ri (1992) summarized the eur-
icnt understanding of Ihe action ol the: sulfur diox-
ide molecule on yeast. Molecular SO; penetrans
Ihc cell by either active transport or simple dif-
fusion. Considering the intracellular pH. it must
exist in the cell in the form of H SO , " . Once inside
Ihc cell, it reacts with numcrons constituents such
as coenzymes INAD. FAD. FMN). cofactois and
vitamins (thiamine) It would also haw an effect
on numerous enzymatic systems and on nncIcK
acids Finally, a significant decrease in ATP is also
attributed to it.
8.6.3 Antibacterial Activities
The activity of free SO; on lactK ackl bacteria Is
well known. It Is even more Influenced by pH than
the activity with icspcci to yeasts. Yet Ihc fraction
combined with cthanal or pyruvic acid is also
now known to possess an antibacterial activity.
The combined SO; molecule has a direct action
on bacteria. The mechanism is not explained by
Ihc decomposition of the combination by bacteria,
resulting In the liberation office SOi.
The sulfnr dioxide combined with cthanal (or
pyruvic acid) seems to possess an antibacterial
activity 5-10 times weaker than free SOi. ycl it
can be 5-10 tines more abnndant.
A large number of bacteria ate eliminated by
5 nig of free SO? per liter. The same concentration
In the combined form towers the population by
50%. Oenocoevia ifai is less resistant to sulfur
dioxide than UxtobacilhB and PedHKOCCia.
Significant technical applications for controlling
malolaclic fermentation and storing wines haw
rcsnllcd from these observations. Sulfillng Ihc
grapes docs not only act rapidly on bacteria in the
pic- fermentation period: it acts by leaving a certain
concentration of combined sulfnr dioxide which
effectively protects and re tint, bacterial growth
until completion of alcoholic fermentation In this
manner, the medium that still contains sugar is
protected from an untimely bacterial development
which could lead to ihc production of vnLuilc
acidity (Section 38.1).
When malolaclic fermentation is not sought
tin dry while wines, for example), it shoukl be
noted that wine stability is not due solely to
Ihc bactcricRtal action of free SO; but rather to
Ihc concentration of combined so. that Ihc wine
conserves after fermentation, its action Is king-
lasting during storage. In certain types of wine
with kx> low a pH. combined SO; concentrations
of 80- 120 mg/l can make malolactic fermentation
Impossible.
Sulfur dioxide is also active on acetic acid
bacteria but additional studies on this subject
arc needed. These bacteria resist relatively high
concentrations In Ihc winery, acetic acid baclcria
arc most effectively prevented by avoiding contact
with oxygen in the air and controlling temperature
i lhe\
cry.
8.7 THE ROLE OF SULFUR
DIOXIDE IN WINEMAKING
8.7.1 Advantages and Disadvantages
Although the use of sulfur dioxide in the storage
of wine seems t< be fairly ancicnl. its nsc in
wincmaking Ls more recent. II was recommended
at the beginning of Ihc 20th century— essentially
for avoiding oxidasic cassc. The very appreciable
Improvement In wine quality by snlfiling rottn
grapes was an csscntEU factor in the gain in
2i:
Handbook of linoUigy: The Microbiology of Wiic anil Vinificalions
popular) ty of this process. [Is antiseptic ptopenics
and Ik role in the prevention of bacterial spoilage
were discovered htlcr
Nevertheless, ihc g cncrali/alioi of salfiling in
wincmaking. «c at leas) the establishment of a pre-
cise and homogeneous doctrine from ok viticol-
tural region lo another, look a long linvc lo conic
about. Besides its many advantages, sulliliug also
presented sonic disadvantages; therefore, a sufli-
cieitly precise understanding of the properties of
salfur dioxide bad to be obtained bcfoie defin-
ing the proper conditions of iLs use. These con-
ditions pciimi the wine maker to ptofil fully from
its advantages white avoiding ifi disadvantages.
When used in excessively high concentrations,
this prod net has a disagreeable odor and a had taste
which it imparts K> the wine; the taste of hydrogci
salfidc and mercaptans in young wines can also
appear when they arc sKired too long ot their Ices.
The most serious danger of improper sulliting is
the slowing or definitive inhibition of the malo-
laciic fermentation of ted wines. Incidentally, for
a long lime sillited grapes were obscivcd lo
produce red wines wilh higher acidilies. Bcfoie
the understanding of malolaclic fermentttioa. Ihis
observation was aliribuled » an acidifying efTecl
of salfur dioxide or an acidity fixation.
8.7.2 Protection Against Oxidation
The chemical consumption of oxygen by SOi is
slow. It corresponds to the following reaction:
SOj+ $0 2 SO, (8.16)
In a synthetic medium. SO; has been shown
lo Dike several days lo consume 8 0-8.6 mg of
oxygen per liter (this amount corresponds with
the saturation of this medium) Such oxidation
requires the presence of catalysis, notably iron
and copper ions Vet musts ate very oxidirablc
and should therefore be rapidly aid effectively
protected against oxidation Silliting accomplishes
this Sulfur dioxide, however, cannot acl by its
anti-oxygen effect, thai Is to say by combining
wilh oxygen which is no longer available for (be
oxidation of other must constituents.
[) n heme l and Ribrjrcau-Cayon ( 1974) continued
this hypothesis. The experiment consisted of satu-
rating a white grape must wilh oxygen and mca-
saring the oxygen depletion rale clcclromclrically
(Figure 8.7). In ihe absence of snlfiting. the deple-
tion of ihis oxygen is vciy rapid and Is com-
plete within a few mimics (4 lo 2D on average)
This phenomenon demonstrates the extremely high
oxidabllily of grape must If al a given montcnl
Fig 87. Oxygen conn
of <B) Slopping potato
DUlubcr-cl j»IR*»b«k;ivoii. 1974). I A) Adrian
cccMaiy for oxygen comimpiion lo uop)
The Use of Sulfur Dioxide In Musi and Wine Treatment
Tabic 8 17. Kmei
uiUtlingiSudmHl.
lion of color of led »ik
1963)
nude " D "
baliyl
isd papa, by
Level of h.r. cm
Com foi.0 n
■ oftlewiK
, ..hi;
ilncd
uilbino
Total f hciBlkr
compound* lindcx)
Color
i«e»n>-
OxkhukaHC
potential
Wak>u IO,
-t- ID* SO/hl
32
41
45
033
033
t-t
the must is snlfitcd. oxygen isio longer consumed
and IS concentration remains constat aflcragrvcn
mi ic I, which varies depending on the conditions
hoi is always fairly short. As an initial approxima-
liai. the value ; varies bclwcen I anil 6 minutes
when su lilting varies between I'" and 10 nig/l.
The valnc* is much greater for nwsl obtained from
loilcn grapes
In summary, although the anti-oxygen effect of
sulfur dioxide is involved in wine storage, its rote
is insignificant during wincmaking In this case.
SO. protects against oxidations by destroying oxi-
dases (lactase) or. al least blocking iheir activity.
If desiTKlion Is not lolal. The cn/ymalK oxida-
tion phenomena are inhibited in this manner until
the start of fermentation. From this poinl. the
reductive character of the fermentation continues
to ensure the protection Yet oxidative phenomena
can resume at the end of fcrmentilloa insofar as
active oxidases lemain after the deplelion of free
SOi. The oxidasic casse test. or. even bctlcr. the
determination of lactase activity, permit, the eval-
uation of Ihc risk and the necessary precautions to
be taken.
In must, enzymatic oxidations arc more signif-
icant than chemical oxidations because they arc
mote rapid. In wine, however, chemical oxida-
tions pfcty an unquestionable rote, since oxidative
enzymes no longer exist In this case. SO? reacts
with oxygen in protect the wine
Rot is responsible for Ihc most serious oxidative
phenomena. In ract.ffrtnv«c7HP/wisccrclcsalac-
case moic active and stable than the tyrosinase of
grapes. It Is responsible for the oxidaslc casse in
icd wines derived from rotten grapes An appropri-
ate su I tiling can piotccl against this phem
to some extent The figures in Table 8.17 show
thai intense snltiting of rolten grapes (since they
ton hi be used in Ihc past) increases the total phe-
nolic compound concentration and the color inten-
sity while decreasing the risk of oxidaslc casse
Progress in pnylosanitiry vincyaid protection has
made such situations extremely rare.
Prom the start of fungal development, the oxi-
dase sccietion by Bonyiis cinerea inside the berry
can be considerable whereas the external signs arc
barely visible. This situation can be observed in
the case of red grapes. The first brown blcmcthcs
arc more difficult lo observe on icd grapes than
on white grapes. During cold weather. Ihc external
vcgclation of Biitryris cinerea is less developed
These factors must be taken into accounl when
choosing the corresponding sulllting conccnuauon
8.7.3 Inhibition, A clival km
and Selection of Ycasls
Snlfur dioxide Isa general antiseptic with a mulll-
facclcd activity on different wine microorganisms
Its mode of action has been described In previous
sections.
With respect lo ycasls. sulllting is used first
and foremost to ensure a delay in the initiation
of fermentation, allowing a limited cooling of the
grapes The fermentation is also spread out over a
longer period in this manner, avoiding excessive
Icmpeialnies More and more often, natural tink
cooling Is complemented by controlled refrigera-
tion systems
In the case of while wincmaking. Ihc delay in
Ihc start of fermentation permits Ihc settling and
racking of suspended particles In musl
:.4
i landbook of Etiology: The Microbiology of Wine anil Vinific.ii«>ns
Sullitiag also makes u.sc of Ihc stimulating effcel
of sulfur dioxide when used ia km concentrations.
Consequently. Ihc fermentation speed accelerates,
as showi by Ihc curves in Figure 8.8. Alter an
Initial slowing of ihc fcrmcntitioa al ihc stm.
Ihc List grams of sugar arc depleted more rapidly.
Finally, ihc fermentation iseoniplclcd more rapidly
in ihc llghily sullitcd musi.
During Ihc running off of a tink of red wine
thai still cob tains sugar, a light sulDUng (2-i g/hl)
docs not block Ike completion of the fermentation:
oa ihc contrary it R known to facilitate it moic
often then nol.
This long-proven effect of sultiling has been
continued time and time again. It has been inter-
preted as the destruction of fungicidal substances
by sulfur dioxide These substances arc toxic for
the ycasl and coald conic from the grape. Biiry-
lis cinerea or even the fermentation itself An
increase in Ihc mast prolcasic activity has also
been considered. ThR activity would put assim-
ilable amino acids al the disposal of Ihc ycasl
(ScclKii 9.6.1). Sultiting probably acts by main-
tuning dissolved oxygen in the must. Nol being
lied up in oxidation phenomena, it is available lor
ycasl growth (Section 8.7.2).
Sultiling has also been considered to affect
yeast selection ApKulatcd yeast. (KUteckem and
Honseiuiiipimi). developing before the others,
produce lower qaality wines with lower alcohol
strength These yeasts arc more sensitive lo
salfur dioxide. Therefore, a moderated sultiling
Mocks their development. This result has been
confirmed by numerous experiments (Romano and
Sum. 19921. bat the research of Heard and
Fleet ( 1988) cast doubts oa this general! cilion. In
spile of salfiting. these strains attained an initial
population of Hf* io 10 cells/nil in a few days
before disappearing. Moreover, the advisability of
eliminating apKulatcdycas&and the interest of the
successive participation of different yeast species
for the production of qaalily wines arc still being
considered.
The problem of sleriti/ing musts by the kxal
destrection of indigenoas yeasts through massive
salfiting. or other processes sach as beat treat-
ments, followed by an inoculation using selected
vcasts will be covered elsewhere.
Fifi 81* Effect nl modecue
(B)5ulfiicd«uM(Sg/nl>
lifting! S Id g/hl) tin ilcohnlu: fcimcnuiinn lioaioof gape
The Lsc of Sulfur Dioxide in Mum and Wine Treatment
8.7.4 Selection between Yeasts
and Bacteria
Sulfur dioxide aco more on w]k baefcria than on
yeasts Lovvci concentrations arc con nqicilly suf-
ficient Tot hindering their growth or suppressing
■heir activity. Nosystcmatic studies have been ear-
ned out on this subject but this Tact is well known
and is often demonstrated in practice, l*or example,
in the case of a red wine still containing sugar (a
site for simultaneous alcoholic and tactic fermenta-
tion), a moderated sb Ih imy (3-5 g/bl)can initially
block the two fermentations Afterwards, a pure
alcoholic rcfcmicntalion can take place without the
absolute necessity of an yeast inoculation.
One of the principal rotes of suUiliig in wine-
making is lo obtain musts much less sasccplibtc
to bacterial development, white undergoing a nor-
mal alcoholic fcnucnctlion. This protection is most
necessary in the case of musts that arc rich in sugar,
low in acidity and high in temperature The risks
of stack fennen tit ions arc highest in these cases.
In summary, sulfur dioxide delays, without
blocking, yeast multiplication and alcoholic fer-
mentation The txtclcria. supplied by grapes at the
same time as the ycasfe. arc killed or at least suf-
ficiently paralyzed to protect the medium from
their development while the ycasK transform the
totality of the sugar into alcohol. The serious
danger of bacterial spoilage in the presence of
sugar is an important factor in wine microbiology
(Section 3.8.2).
In white wincmaking and for wines in which
malolaclic fermentation is not sought, silfiling can
be adopted to inhibit bacteria completely. Inciden-
tally, the light so I filing of white mush undergoing
nialolaclic fcmienttlion can be iusaflicicat id pro-
tect effectively against oxidation
In red wincmaking today, malolaclic fermen-
tation has become common practice. Generally
speaking, the snlliting of red grape musK favors
wine quality. However, snlliting must not com-
promise malolaclic fcrmenuiKw due to its con-
ditions of use and the concentrations employed.
To ensure the successful completion of alcoholic
fermentation the amount of the sulfamus solution
added to grape must should be regulated accord-
ing lo the pH. temperature, sanitary conditions and
Tabic K 18. IdBuckcc of muu uiliisiiu <in (be lime nee
(wjii KVfidWtl in da. 1 1 for maktUclic IcnucMJliu
iaiiaiio* in wine »fiei manlng oil ( Rfccfcmi-QiYu
mi.. 1977)
SulHtinp
Wine No. 1
wi
ii. '.,■.
2
Control
40
30
~2S g/hl
45
*
+5 g/hl
70
i.;
-io uyi
ion
Ii. i
other factors Baclerial development mast initiate
rapidly alter the depletion of sugar for exclusive
malic acid degradation The exact SO; concentra-
tion is difficult to determine, and it varies depend-
ing on the region. For red wincmaking in the Bor-
deaux region. 7-10 g/hl seems to be an effective
range: below this, the nialolaclic fermentttion is
not compromised, above this, it can be consider-
ably delayed (Table 8.18).
8.7.5 Dissolving Power and General
Effects on Taste
In red winemalang. saltiling favors the dissolution
of minerals, organic acids and especially pheno-
lic compounds (anlhocyanins and tannins) which
constitute the colored substances of red wines. The
dissolvent acliviry Is due to the destruction of grape
skin cells, which yield their soluble constituents
more easily in this manner In tact, the dissolvent
effect of saltur dioxide seems to have been exag-
gerated in the case of healthy grapes. The better
color of wines derived from sullilcd mast is prob-
ably due to a better protection against the oxidasK
cassc in slightly rotten grapes.
The effectiveness of sulfide maceration for
extracting grape pigments Is indispatablc. and this
process Is used for the industrial preparation of
commercial colorants. Yet when rigorous experi-
ment! arc carried out on healthy red grapes, using
classic winemalang techniques, no significant color
improvement (anthocyanin and tannin conccntra-
IIoh and color intensity value i is observed in the
presence of a normal snlliting. Since only the free
SO; n active, aid since this form rapidly dis-
appears in crushed grapes, this effect of snlliting
appears to be exerted for only a brief moment At
21'«
Handbook of linology: The Microbiology °f Wiie anil Vinifications
(he end or fermentation, the effects of maceration
linic. Icmpcralurc and pumping-over arc moic
significant.
Nevertheless, ihc dissolvent effect of sulfiting.
with rcspcel lo phenolic compounds, is obvious in
ihc case of limited maceration. This operation is
not recommended for crushed white grapes before
■last extraction by pressing The sulfiting of grapes
also has an impact on the color of rose wines.
Suirilitg also has certain effects on wine qual-
ity which still rcniaii poorly defined. The general
properties of sulfir dioxide may possibly have
indirect conseqiences i protection against oxida-
tions aid the binding of ctbanal). In Ibis way.
salfiling oflcn improves the tastof wine — notably
in the case of rollcn grapes or mediocre varieties.
Il also protect certain aromas of new nines.
Moreover, grape sn lilting docs not have an obvi-
ous impact on the subsequent development of the
bonquct of mature wines.
Certain conditions, such as fermentations in
strict anaerobiosis and especially prolonged aging
on yeast lees, can lead k> the formation of hydrogen
sulfide and ncrcaptans from Ihc added SO?. The
odors of these compounds are disagreeable and can
persist ii wine.
8.8 THE USE OF SULFUR DIOXIDE
IN THE WINERY
8.8.1 Wine-making Concentrations
Considering the rapNliry of oxidative phenomena,
grape and must su lliting isonly effective if Ihc sulfur
dioxide is iniimak'ly and rapidly incorporated ink)
the total volume before Ihc start of fermentation. If
a fraction of the grape must fenucnts before being
snlliled. it is definitively shicklcd i n mi the action of
the SO> . because it immedialcly combines with the
clbanal pmduccd by the fermenting ycasls
In fact, a homogeneous distribution before the
stirt of fermentilkw is not suflicicnl Considering
the rapidity of the oxygen consiimplioa by grape
mnst. each fraction of the grape harvest or the must
should receive the necessary quantity of sulfur
dioxide in Ihc minutes that follow the crashing of
the grape or Ihc pressing of the harvest. This n the
only truly effective method of protecting againsl
oxidations. Il can be more effective to add 5 g of
sulfur dioxide per hectoliter correctly to the harvest
than i:> add 10 g/bl added in poor conditions. A
poor snlliting technique is certainly one of the
reasons in the past that led to the use of excessive
concentrations.
Based on these principles, the only rational snl-
liting method for wincmaking consists of regularly
incorporating a sulfnious solution into the while
grape mast as it B being extracted, or for red grapes
as soon as they arc crushed. A few successive addi-
tions of SO. into Ihc lank as it is being tilled arc
not truly effective, even after a homogeni onion al
Ihc end of filling During homogcni/alion. pari of
Ihc added sulfur dioxide is already in the combined
form and thus inactive
Il is Ihcrcforc also accessary lo use a suflicicnlly
diluted sulfur dioxide solution, capable of being
correctly incorporated and blended inlo Ihc must.
The direct usage of mctabisulfilc powder or
sulfurousgas in Ihc tink should be avoided. When
a tin k of red grapes is snlliled byafcwadditionsof
aconccnlraled product during rilling. Ihc complete
decoloration of certain fractions of the pomace
is sometimes observed during the running off. In
these cases, the sulfur dioxide was not properly
blended, bul was instead fixated on ccitain parts
of the grapes, leaving the other parti unprotected.
When choosing the SO> concentration k> add lo
ihc grapes or the must, grape maturity, sanitary
stale, acidity (pH). temperature and eventual con-
tamination risks must all betaken into account. The
choice can sometimes be difficult. Table X. 19 gives
a few values for vineyards in temperate climalcs.
The generalization of tank cooling systems and
increased hygiene in the wineries, combined with
a belter understanding of the properties of sulfur
dioxide, permit the lowering of the concentrations
used in wincmaking Today. saniEuy practices in
Ihc vincyanl avoid grape rot. which once justified
Ihc intense sulfilings indicated in Table 8.19.
During the harvest, progressive increases in
Ihc snlliting concentrations can compensate the
increasingly significant inoculation (notably bac-
terial) resulting from the dc\ck>pmcnt of microor-
ganisms on the equipment— the inner surface of
The Lsc of Sulfur DWxidc in Musi and Wine Trcutttciil
Tabic ft l«>. Sulivn .(>.... k- ,1...-. for win
SullU I .ln.«lc.k>M
Ik-.illli- i-L-ix-.%. ;i'ii,ipc cn.lunl.. hipli niiliC. 5 --'111 ill' "in
Hcuhhy ;'■:■■■. h«b ■-.,i-..iii . . low ..i.lc. 5-8 g/hlol-lnc
Rotten grape* 8- ID g/bl of »lr
-ikhy ficifcv average imiiuniY. huih ackli
r.idhy f niftt. high b.iIuiiIy. low niiild,
5 p/hl of muu
6-8 g/hlolmi
8- 10 p/hl of a
Ihc buks and the walls of ihc winery. Problems of
difficult final stipes of fermentation and microbial
deviations arc frequently observed in Ihc lasibinks
tilled. Sutlkicil must suHiiing should avoid these
cou tun (nations.
In while wine making, excessive concentrations
(>1S g/nl) followed by a significant sultiling at
the end of fermentation <>4g/hl) can be a sou tee
of reduction odors and shoukl be avoided. Picss
wines, however, should be more intensely mI-
litcd — especially in the case of continuous presses
which cannot be disinfected regularly.
Concerning the Minting technique for red wine-
making, the solution shoukl be added aficrgrapc
crushing lo facilibilc the blending and Id avoid
evaporation losses and atticks on metallic equip-
ment. Taking into account the transfer of the
crushed grapes by a pump with a constant delivery.
Ihc sulfnrous solution shoukl be injected ink) ihc
tube ini mediately alter Ihc pump oulkl The sal-
liting is suitably distributed and homogenized in
this manner. Of course, the injection pump for the
sulfurons solution musl be properly adjusted and
perfectly synchronized with the grape-pump.
The add) ton of the sulfnrous sol n lion after
each grape load, by rcgilatty spraying Ihc surface,
can only be practiced in small tanks and musl
also be sufficient in number. Even if il is nol
completely effective, a honvogeni ration pumping-
over is necessary after filling.
In Ihc case of white wincmaking.sulliling musl
tike place after must separation Snlliling of ihc
crushed grapes is not recommended since il entails
Ihc risk of increasing the maceration r
and a fraction of the SO; is fixated on the solid
parts of the grape
Considering the oxidation speed of while grape
musl. sultiling (which ensures the appropriate pro-
leclioni should be carried onl as quickly as pos-
sible. Musl extraction equipment (the press cage,
mechanical drainer and continuous press) docs nol
supply a cons tin I delivery. Consequently. SO? can-
not be injcclcd with a pump adapted directly lo
these outlets. In order to suUilc in this manner,
the must has to pass via a small tmk throigb a
constat I delivery pump The corresponding manip-
ulation of the must, in particular the pumping, docs
nol protect Ihc mast from a slight oxidation before
sn I tiling.
The Minting of while grape mnsls can also he
calculated front the volume of the juice Iruy at the
outlet of the press During filling, a homogeneous
distribution should te ensured
The necessary volume of a sulfnrous solution
for sultiling an entire tank during its filling at
the chosen concentration should be prepared in
advance. If Ihc system Is correctly adjusted, the
entire volume of the sulfnrous solution should have
been injcclcd in » the tmk by Ihc lime ihc link
is fill.
8.8.2 Storage and Bottling
Concentrations
During storage, sulfiling is. first of all. thought lo
protect wine from oxidation. As an approximation,
oxidative risks are present during prolonged stor-
age below 5-10 mg/l for red wines. 20 mg/l for
Handbook of linology: The Microbiology of Wine anil Vindications
TnifcH.2u. Recum-coded free Milfur dioxide concern alio nv <-u'li In nine*
Dux type
Red wine*
Dry wane wines
Sweet white win
Bottling)
I'Apcdina dine*
20-30
10-30
23-35
30 -40
20-30
35 -45
40-80
30-50
80- 100'
111 ctfmllMt;
white wines made from healthy grapes and 30 inj;. 1 "!
for while wines made from more or less roitci
grapes.
Al Ike microbiological level, su lilting dry wines
must avoid yeast and bacterial development during
suragc. In diy while wines and red wines hav-
ing undergone malolactK fermentation, the con-
centrations ased for protection against oxidations
arc generally saflkicut u avoid microbial devel-
opments. In red wines that have not undergone
malolactK fermentation, the habilaal free and tola)
SO; coKcitrations can be insufficient to shield
the wine completely from a malolactK fermenta-
tion— al least a partial one— during storage.
Of course, the snlliting riles do not apply to
certain kinds of wines (red or white, dry or sweet)
with qualities derived from a certiin oxidaikin slate
or conciining cthanal
Sulrlling ab>o binders Ihc icfcrnicntalion of
sweet wines, generally provoked by SOj-icstseml
yeast strains The n- fermentation risks arc indepen-
dent of svgar concentrations, but arc influenced by
alcohol strength. In satisfactory storage conditions.
SO mg office SO< per liter is required loensure the
storage of a sweet wine with a relatively low alco-
holic strength ( I \'i I and 30 mg/l for wines with a
high akohol content ( 134).
In practice, carefully adjusted sufficient concen-
trations must be used to avoid accidental risks. The
identic matron of a sweet wine can sun in the
Ices of a tank containing a sufficiently high ycasl
population to ensure the combination of the SO..
Simultaneously, al least for a certain amount of
time, all of the liquid remains limpid without a
re fermentation, wiih 60 mg of free SO; per liter.
If the fcrmcutilivc process begins from the Ices,
the re fermen ration seems possible in spite of ihc
high concentration of free sulfur dioxide.
The sice of the yeast population should always
be tiken into account to evaluate the effectiveness
of a snlliting All operations (lining and filtration)
that eliminate a fraction of the yeast- permit the
lowering of the free SO; concentration necessary
for conserving sweet wines.
The possibility of lowering free SO? concentra-
tions for stabilizing sweet wines results from steps
taken in storing wine. Clean (if not sterile) condi-
tions have diminished contaminating populations.
These criteria for cleanliness should be applied
noi only to Ihc product but also to the building,
the containers and the material— all contamination
sources. Microbiological controls that indicak the
number of viable yeast cells ate useful tools for
adjusting sulfiling
Table 820 iudkaks free sulfur dioxide con-
centrations thai can be recommended in different
situations
8.8.3 Diminution of Sulfur Dioxide
by Oxidation during Storage
The free sulfur dioxide concentration docs nol
icii.ii n conscinl in wines stored in bariclsor tanks.
There is a continuous loss month after month.
Over Ihc years, its concentration decreases even
in bottled whte.
The decrease in barrels or tanks results from
an oxidation catalyzed by iron and copper ions.
Although il is very volatile, a negligible quantity
of free sulfur dioxide evaporates during storage
in woodci banels. Nor Is it combined. A fairly
common error Is to consider that any decrease in
free sulfur dioxide is the result of a combination
with wine constituent. In reality, after the four
or five days following the addition of SO;, the
wine constituent no longer bind. An equilibrium
is attained and decreases occurring afterwards ate
The Lsc of Sulfur Dioxide in Mum .mil Wine Treatment
due Hi oxidation. For a new combination to occur.
Ike tliaiiii.il composition of the wiic must be
modified. For example, new binding molecules
must be formed, such as clkanal. doting a limited
yeast development or by the oxidation of etnanol
when a poorly clarified wine in racked.
The oxidation affecting sulfurous acid forms
sulfuric acid. At the pH of wine, it is almost
entiicly in the form of sulfate In boii\ii/cd and
non-bouytircd sweet wines with elevated free
SO? concentrations, a considerable amount of
snlfaK can be formed (05 g/I). Less is formed in
dry while and red wines, especially Ibose stored
in Conks. In the case of barrel-aged wines, the
formation of sullale by the oxidation of free SOi
accumukties with the amount resultiig from the
combustion of sulfur in the empty barrels This
formation lowers the pH and haishcns the wine.
This phenomenon contributes to the decrease in
quality of wines stored in barreLsforai
long time
When sulliling is effected without a ■
ment beforehand, the wine can be excessively sih
lised and its tasie affected. In general, the charac-
teristic odor appears al or above 2 mg of active
molccnlar SO? per liter. Table 8. 15 indicates the
corresponding free SO; conccn nations To lower
the conccn (ration of free SO; of a wine, the most
effective solution, when possible, is to use this
wine to increase an insufficient concentration of
free SO? of a similar wine
If such an operation is not possible. Inc most
generally recommended method is to aerate the
wine. The effectiveness of this method is based
on the slow oxidation of sulfur dioxide During
the days that follow, the higher the temperature.
Ike more rapidly the concentration decreases
Aeration has a limited effectiveness, and 16 mg
of oxygen per liter is required io oxidize 64 mgof
total sulfur dioxide per liter. This approximately
corresponds « a decrease of 42 mg of fiec SO;
per liter, taking inlo account the dissociation of
combinations
The use of hydrogen peroxide is a radical
means of eliminating an excess of fiee SO;. This
method is loo severe and is therefore prohibited: it
compromises wine quality for a long time.
8.R4 The Forms of Sulfur
Dioxide Used
This antiseptic has the advantage of being available
in varKws forms capable of responding to diffcient
situations gaseous stale (resulting from the com-
bustion of sulfur). IN|uc fled gas. liquid solution and
crystallized solid.
Snlfnrons gas SO; liquefies al a temperature of
— 15 'C al normal atmospheric pressure or undcra
pressure of 3 Kirs al normal ambient temperature
It is a colorless liquid with a density of 1 .396
at I5'C. Pkiccd in 10-50 kg metallic bolllcs. this
form Is used for large-quaility additions that can be
measured by weighing the bottle, which Is placed
directly on a scale. A siil)kk>seur' is used to treat
smaller volumes of wine. The graduated container
can be precisely lilted from the metallic bottle
by regulating a pair of small faucets — permitting
the addition of precisely measured quantities of
Inc gas.
Liquefied sulfur dioxide Is still delivered in vials
comaining 25. 50 or 75 g of sulfur dioxide for
example adapted for sulfiiing wine in barrets with
capacities of several hundred liters. A special tool
perforates these small metal cup- stoppered bottles
when they aic inside Inc bartcl to be treated.
For SO? additions n small volumes of wine,
or to have a bcllcr incorporation. 5-8* solu-
tions prepared in water or must (to avoid dilution)
Irom liquclicd sulfurous gas are used. The quantity
needed is weighed. The concentration of the solu-
tion is regularly verified by measuring its density
(Table 8.2 1 1 or by chemical analysis. It lends lo
decrease in contact with air.
Handling these solutions is disagreeable, since
Ihcy give off a strong SO; odor. Prepared on
the premises, they aie well adapted k> large
wine making facilities such as bulk wineries
Concentrated 10* solutions, or 18-20* potas-
sium bisulfite solutions, are also used They arc
mote easily handled than the preceding since they
arc less odorous. Being more concentrated, how-
ever, they arc less easily incorporated into wine
and must Legislation limits their use to a sin-
gle addition of 10 g of SO> per hectoliter. They
acidify less than the preceding since the acidity in
these solutions is partially ncnlrali/cd. Potassium
Handbook of linoUigy: The Microbiology of Wiie anil VinUuralions
TubbH.21. Dciuuv <ai IS-C) of Hil&it db»kle *oIu-
ih>iu pre piled by ihc diuok**>a of witiur dkixkk fiai
in wMct
SuUurdbikk Dcniv Sulfur dioilik Dciuiv
tC'HKi ml) (p/100 nl)
2.0 1.0103 05 1.0352
25 1.0135 Til 1.0377
3.0 1.0108 75 1.0401
35 1.0194 SB 1.0*20
*.0 1.0221 85 1.0*50
*J 1.02*8 90 1.0*7*
5.0 1.0275 95 1.0*97
S5 1.0301 Ht" 1.0520
0.0 1.0328
mclabciuUilc(K?SiO>) solutions al llf.i diluted In
watr can also be used. These solution* contun
approximately 50 got sulfur dioxide per liter (5'* i
and arc salable for limited-volume wincmaking.
The mctasultitc powder should be diluted in water
bcfoic use. When added directly, it is difficult to
blend iuio the must
8.8.5 Sulfiting Wines by Sulfuring
Barrels
Sulfuring barrels, or small wooden tanks or con-
tuners, consist, of burning a certain quantity of
sulfur in these containers. II is probably the old-
est form of using sulfur dioxide in cnology. Ii is
used for adjusting the free SO; conccnlralion of
wines at the momcil of racking and also for avoid-
ing microbial couttmination when storing empty
containers It has a double slcriliying effect. II is
exerted at once on the wine and the internal surface
of the contiincr. This practice is part of normal
winery operations and could not be replaced by the
simple addition of sulfurons solution lo Ihc wine.
Dnc to the unptcasanl odor imparled to the wine
cellar by burning sulfur, its usage can be piohib-
itcd by the safely legislation of ccrciin countries.
Instead of coming from lac combustion of sulfur,
sulfurous gas can also be delivered as a bottle of
compressed gas.
In any case, sulfur combustion is only applicable
u wooden containers. In fact, sulfurous gas.
coming from the combustion of sulfur, attacks the
internal surface ol cement tanks and the coating of
metallic tanks II also accelerates Ihc deterioration
of sEiinless steel.
The sulfur is generally supplied in the form of
a wick or ring. It may be coaled on a cclluktsK
weave or mixed with a mineral fuse (aluminum
or calcium silicate). The units most often used are
25.5 and 10 g of sulfur Chatonnct el<il. (19931
demonstrated a certain heterogeneity in the quan-
tity of SO? produced by the combustion of the
same weight wick or ring according lo their prepa-
ration conditions or storage (fixation of humidity).
From Ihc equation:
S + O, SO;
32 + 32 = 64
(8.171
the burning sulfur combines with its weight in
oxygen to give double the weight of SO;. In
reality. 10 g of sulfur burned in a 225 I barrel
produces only about 13-14 got SO,— a3f» loss.
One part of the difference is accounted for by
the portion of the sulfur that falls u the bottom
of the hairc! without burning, and the other part
by the production of sulfuric acid— a strong acid
without antiseptic activity. The sulDting loss and
the acidification of wine (by repealed sulfuringsi
arc explained in this manner
The combustion of sulfur docs not exert its effect
by eliminating all of the oxygen from the barrel.
The maximum quantity of sulfur that can burn
in a 225 I barrel is 20 g. for the maximum pro-
duction of 30 g of sulfurous gas. At this stage,
the combustion stops because the sulfurous gas
has the property of hindering ic own combus-
tion ll has been determined thai approximately
325 liters of oxygen are present in the barrel al
Ihc moment when the combustion slops, compared
with 45 liters beforehand
These observations lead to ihc conclusion thai
Ihc combustion of sulfur R limited. When a 40 g
sulfur wick is burned, not all of the sulfur is
consumed, even if the wick is burnt in a cinder.
About half of it falls to Ibe bottom of the barrel
without burning.
The production of SO? by the combustion of
sulfur in a barrel is therefore irregular It is espe-
cially hindered in humid barrels; for instance 10 g
The Lte of Sulfur Dioxide in Must and Wine Treatment
sulfur bnrncd In dry barrel give 12 g S 1 >? and only
5g in humid IxirrclfRibcrcan-GayontVoV . 1977).
In addition, ihc dissolution of Ibc SO; formed is
generally irregular during ihe filling of ihc barrel
Depending on Ihc tilling speed and conditions (by
Ihc lop or bollom. for example), a more or less
significant pan of ihc sulfnrous gas is driven out
of the harrcl. Moreover, the distribution of the sal-
liting in the wine mass is not homogeneous The
liisl wine that flows into the barrcl receives morc
SO. ihan the last. In one example, the free SO,
increased by 45 mg/l at Ihc bottom of the barrcl.
by 16 mg/1 in Ihc mkklk and noi at all in Ihc
upper portion. Consequently, the wine should be
homogenized after racking — by tolling Ihc barrcl.
for example This snlfiting ntefhod should only be
used for wines stored in small-capacity contain-
crs — say up to 6 hi.
As Chatonnct el ill. 1 1993) stated, the couibuv
llan of 5 g of sulfur in a 225-liter vvooden barrcl
increases the SO, in wine from 10 to 20 ntg/1.
Sulfur wicks are less efficient ( 10 mg/l) but more
conswent than rings 1 10-20 mg/l). The latter are
more sensitive to their external environment, i.e.
moisture
The combustion of sulfur for Ihc storage of
empty barrels will be covered in \blumc 2.
Section 13.6.2.
REFERENCES
Bmbc It'. (2000) Let combiniisms ila t&oxyile de
uxiiro sou lei moult n les inu i.utti de ruiiiti
boiriaiiex Role des biieieries i/ceiiouex Dtese tie
Oociortt. liuieruie Victor Segitoi Bordeaux It
BaibcJr.. de Revel G. and Ben and A. (2002) J.
A/gric. Food Cltem.. SO (22). 0408.
Bmbc Jr.. dc Revel G.. Joycut A.. Louvaod-Funcl A.
undlknand A.(2000U A/fie. Food Cbem.U& (8).
3113.
Bmbc Jr.. dc Revel G.. JoveuiA.. BcnaailA. and
Lorn jial-liiat I A i5>Hj|J fypi Microbiol. .90 . 34.
Bmbc Jr.. dc Revel G . Joycuv A.. Unvaud-Funel A.
and Be •rand A. (2001b) l" r funic. Rente F.
iFtEaatogie. 189. 26.
Bmbc Jr.. dr RcvcIG.. Peiclk. M< . Lonvaud-
Uincl A and Bctlawl A. (2001c) f f ilk . Rewe
F. d'{FJiolo#e. 190. 10.
EI liium I 1 19051 Cowrihulkin a I elude des coabr
n»ae dc I Anhydride uimukux d» k* aalls el
lei vitiv These Docicui-Ingenkui. UninanH dc Bor-
deaux.
EIli i« m I (1995) iA Jouniee Teititiioue du CIVB.
Rnnkauv. Fancc.
Bumupfe. l.T. and S^rt. A.K. (1904) J. Sci. Food
Api.S. 170
Bumupb. l.f. and Spuria A.H. (1973) /. Sri. Food
Agfi. 24. 187. 199 and 207.
Chakmnei P.. BohIidb JJ>". and Dubounlicu D. (1993)
J. hi. Sri. Vpte Vht. 27 (4). 277.
Dubeinct M id Ki>tn.iu-G»vo n P. (1974) H/ii 13.
233.
Guilku-I.aipeicuii I. (1990) Bude tie substance* dc
Libit post, aokcublic «>tnb)*am k diov.de dc
miuIit dans les vim hbm- Ksus dc venduit|xs
bi*i>lnccs. Mnccncvkkncccl imponanccdu nikdc
I hydro Vi f lofuacdul 1 bese DoelOCU dc I U'nivcoic
dc BonJeauv II (oftkm OEnulogk-Aafclobgk).
Htjnlll jal Ike? Gil . ( 1988) A*«r. ( VZ l«w fod. J. .
3.57.
Kklbolcr E. and WuoUu. G. ( 1900) Wmberg V. Keller .
7.313.
Rfccmu-Gayon J.. Pcynaud E.. Rfccmu-Gayou P. and
Sudnud P. ( 1977) Sciences ei Tedmiaaes da Ma.
\o\.\: Claificition el Stabiliuiioa. Mcteriels ei
bisiilttion. JXinod. Pari*.
Roaano P. and Suzzi G. (1992) In Wne Microbiolo*-
•nd Bioicchiiolo&y (cil. G.K. Flea). Harwood Aca-
demk PuhlBhee.. Chui. Swijvriand.
Sudcuid I'.i 1903) Bude cvpcrimcaakdc la viuiEcatM
en i'nii.'c. Tbcse dc DueieurlniKnkur. Fwukc des
Seknc»dc Bonlcaui.
SudoxidP. **d Cluiuvel S. ( I98SI Com. lip- Ma.
19(1). 31.
Sii.-.-i G. and Roaann P. ( 1982) Villi nTAofia, 24. 138.
TU H.P..Femn I'J.andDegniui A.P.(l972)frf. Costal.
7b wrW.. 10.291.
I ■«( l ?IWi- lom.s.ti 1.. 1 1995) Cnimk ocnnlopknic. Tee
and Doe Lntwkr, Pan,.
Vm»ic( JA1. ( 19881 Tlieie .le Itonorit eit Plutoim-ie.
t'miciMiC dc MoiHpcllici. Fcinec.
Products and Methods
Complementing the Effect
of Sulfur Dioxide
9 1 Introduction
92 Sorbic acid
9 3 Octanoic and dccanoK acids (salmutcd short-chain Liny ;
9.4 Dimclhyldicarbotatc (DMDC)
95 LysoAHic
96 Destruction of ycasfc by beat (pastcuriaition)
9 7 Ascorbic acid
9S The use of inert gases
9.1 INTRODUCTION
Considering ibe legitimate desiie to tower sal-
fur dioxide concentrations, il is normal to search
for adjuvants thai complement let action by rcin-
forcing ibe effectively of one of its properties.
This chapter covets such chemical produce* and
physical processes thai have been or arc likely
to be authorized by ihe legislation of different
coi ■tries. Others arc likely to be proposed in com-
ing years.
Sorbic acid, which can be used to increase
the antimicrobial properties of snlfur dioxide,
is now well known and authorized in many
(onirics. The possibility of using octanoic and
decanoic acids will also be covcrcd. though they
arc not cuncilly authorized. They do not seem
to pose any hygiene problems, and they exist
224
Handbook or Etiology: The Microbiology of Wine anil Vindications
naturally in wine, this treatment only reinforces the
existing concentrations Lysozymc from cm; white
has similar properties. This enzyme Is capable
of destroying certain haetciia. especially ktctic
bacteria in wiac. Ik capacities have bcci knowi
for a long i : 1 1 1 l bul Ik practical application in the
winery has been developed in icccnt ycars.
Nnmerous antibiotics and antiseptics known to
acton wine ycasfchaw not yet been authorised, for
reasons of hygiene One loose most recently pro-
posed. 5-nitrofurytacryllc acid, is a powerful fun-
gicide bit is highly carcinogenic and induces clhyl
Intoxication. Another, pimaricinc. is ailodcgrad-
able aid has a fungistatic effect, without knowi
secondary effects, il is already authorized )■ the
food Industry A proposal requesting the official
approval of this predict has beei filed ii Prance
In the 1970s, the isc of ethyl pyrocarbonale
( Baycovin) in wine was pcmiillcd in sonic coun-
tries. Tils fuagtcidc is wry effective for cold-
sicrilmng wine at the time of bottling It disap-
pears rapidly, breaking down, mainly. Into clhyl
alcohol and CO,, bit also releasing tliy quantities
of ethyl carbonate that were, nevertheless, signif-
icant enough that its use was soon giwn up alto-
gether. The Bayer pharmaceutical company later
marketed a dimcthyldicarbonak: prodKt. Vclcorin.
thai was jist as effective without aiy of the health
risks.
Among the physical processes capable of com-
plementing the antimicrobial properties of sulfur
dioxide, the destruction of genus by heat (pas-
teurization) can be used Recently, the possibility
of destroying germs by high pressure has been
demonstrated This method scents to be effective
and affects qiality less than heal treatment The
practical conditions of i& use remain to be defined
and the appropriate equipment to be designed. Of
course, all operations that work lowaids eliminat-
ing microorganisms, even partially (racking, ecu-
trifugatioi. filtration and pastcu nation), facilitate
mKrobEd stabilization and permit the wlncmaker
u lower the SO> concentration used
Ascorbic acid is the most used adjuvant, con-
tributing K> the antioxidant properties of sulfur
dioxide. The storage of wine with an Inert gas is
another eff cell w means of avoiding oxidations.
9.2 SORBIC ACID
9.2.1 Physical and Chemical Properties
The formula for sorbic acid contains two double
bonds:
CH,-CH=CH-CH=CH-COOH
(9-D
Foir isomers exist bit only the trans-Irons Ri-
mer Is ised. Due to its effectiveness and lack of
toxicity, its use is authorized in many c onirics.
In panic ■ ku in the BC. at a maximum concentra-
tion of 200 mg/l It remains prohibited in a few
countries (eg. Austria and Switzerland).
Il exists In the form of a while crystallized pow-
der. In a water- based solium. It can be entrained
by steam, for this reason. It is found in wine distil-
late aid fabely increases volatile acidity. It has a
slightly acidic flavor Its dissociation constant cor-
responds loa pK of 4.76. In other words, in wine.
It Is essentially In the form of a free acid.
Sorbic acid is not very soluble in water ( 1 .6 g/l
at 20'C:5 g/l at 51) O bul it Is soluble in ethanol
1 1 12 g/l at 20'C). lis sodium and potassium sills
arc very water soluble In this form, concentrated
soIiUobs arc prepared for treating wiic. Potas-
slim sorbatc contains 75% sorbic acid A solution
at 200 g of sorbic acid per liter is prepared by
dissolving 270 g of potassium sorbatc per liter in
water One liierof this solution can treat 10 hi of
wine at 20 g of sorbic acid per hectoliter. Sorbic
acid can also be dissolved in alkaline solutions, and
200 g will dissolve in a liter of cold water contain-
ing 100 g of KOH. These concentrated solutions
mist be prepared Immediately before use They
become yellow with lime.
Certain precautions must be taken when incor-
porating the concentrated solution lnk> the wine
being treated Die to the pH of wine and the pK
of sorbic acid, the latter will be liberated from its
salt as soon as the concentrated solution ct intro-
duced in Ihc wine However, this acid is not wry
soluble: If its concentration at a giwn moment is
mo high, it will precipitate The concentrated solu-
tion must therefore be added slowly white being
constantly mixed. The use of a dosing pump is
recom mended
Products and Methods Complementing the Effect of Sulfur Dioxide
9.2.2 Antimicrobial Properties
The fungicidal activity of soibK acid hits been
tested on yeasts in different circumstances (Rib-
crcan-Cayontffl*.. 1977).
The lung ic idal concci (ration . slopping a fermen-
tation (mulagc). is relatively high (5 g/1). From
05 g/1 on. the fcrmcntilKw P> observed lo stow
and slop before its successful completion These
elevated concentrations do not pcraiit the use of
soibic acid in sweet wlic vindication.
The fungistatic concentration, which hinders fcr-
mentation In grape must, varies according to must
composition (in paiticuktr pH). the six of the
inoculum ami the nature of the strain. The con-
centration limits cited in the litcratuic arc between
ICO and 1000 mg/l. with an average value of
30O-5OD mg/l.
The inhibiting concentration for yeasts in wines
con tuning sugar Is lower and depends on the al-
cohol strength and the pH. Concerning the alcohol
conk'ni. numerous tests arc citd in the litcratuic.
taking into account the si/c of the inoculum and
the nature of the yeast strains (Table 9.1).
The pH also has a strong influence on the fun-
gicidal activity of sorbic acid, which increases
as the pH decreases. In laboratory experiments
i SplilMocsscr el ill.. 1975: Devc/c aid Ribc'reau-
Gayon. 1977. 1978). a concentration of 150 mg/l
of sorbic acid was needed to hinder the iclcnncn-
talion of a sweet wine at pH 3.1 . All other factors
being equal. 300 mg/l was accessary to obtain the
same results at pH 35. At a pH >3.5. Ike maxi-
mum concentration authorized. (200 mg/l) may be
Tabic*). I. Sortie acid ik*c% (mg/l) ncccuaiy (<■>
%iv eel- wine caincmriita (bhoalaiv lew. with .S'iiiv
/*.><*.«.vi(Kl>cieiiu-Givoo« of.. 1977)
Alcoholic laoiublDn popublion
insnflicicni lo ensnte Ike proper stabilization of a
The effect of the pH on the stale of sorbic acid
has been analyzed The non-dissocialcd free acid
molecule is known to possess the antiseptic char-
acter. Between pH 3.0 and 38. the proportion of
soibic acid (pK =4.76) in iK non-dLssocEilcd free
acid stile passes from 98 lo f XH . Thts difference
is »» small to explain the much more significant
impacl of the pH. Cell pcrmcabilily and penetra-
tion phenomena of sorbK acid rcgnlalcd by the pH
of Ihe ■it'll i mn may also be involved.
In addition lo its action on classic fermentative
yeasts, sorbic acid acls againslftorycasis develop-
ing on the surface of wine iCniHliiki) In capped
bodies of red wine containing Hf.i vol alcohol
wiln a rclalivcly significant head space and stored
upright. 150 mg of sorbic acid per liter ensures
Ibeir storage for three weeks. Higher concentra-
tions may be needed for lower alcohol strengths,
longer storage periods, higher temperatures or
wines containing tcsklnal yeast.
The antibaclcrial properties of sorbic acid ate
less significant. It exert* practically no activity
against acclic acid and be tic acid bacteria Conccn-
Irationsof 05-1 g/l wonkl be necessary lo have a
significant effect.
Sorbic acid therefore exerts a selective effect on
wine mK morgan crtis and opposes yeast develop-
ment wiihonl blocking bacterial growth. It has the
opposite effect to thai of salfur dioxide (which
favors yeasts at the expense of bacterial Con-
sequently, sorbic acid mast never be used akanc
but always associated with an anllbaclcriul prod-
uct (sulfur dioxide). In wines exposed to air. the
amount of volatile acidity formed by acetic acid
bacteria can be greater in Ihe presence of sorbic
acid, due k> the absence of an antagonism wiln
ycasK.
In conclusion, sorbic acid only present* a suf-
ficient effectiveness in praclKc when associated
wiln a certain concentration of cthanol and free
snlfur dioxide. Soibic acid is an effective adjuvant
lo sulfur dioxide, since il reinforces its action, but
it is not a replacement Most pioblcnis cncounleitd
in employing sorbic acid come from its incorrect
I landbook or Enology: The Microbiology of Wine anil Vinificaiions
use. A lack of effectiveness and Ihc appearance of
strange tosics arc generally reported
9.13 Stability and Gust alive Impact
Fresh solutions of sorbic ackl have no odor aid
Ihts pnxluci docs nol Influence wine aromas. Up
to a concentration of 200 mg/l. sorbK acid docs
nol modify gustatory characters of correctly sH>rcd
wines, either immediately after its addition orafler
several years of bottle-aging l-or certain wines, its
impact on taste is perceptible above this value.
b»t it is distinct only for conccn nations above
400-500 »g/l. It docs not increase the appar-
ent acidity of wine or thin il but docs accen-
tuate impressions of astringency. bitterness and
harshness, which arc perceived in particular in the
aftertaste.
In the 1960s, as early as Ihc litsi treatments,
strange odors and lasts were sometimes noted—
especially in red wines treated with sorbic acid.
For ihts reason. itsauthorifation was reconsidered.
However, when it is correctly used, experience
has shown that in normal storage conditions the
evolution and development of bottled wine is nol
affcclcd. These observations do. however, lead k>
Ihc problem of the stability of ibis product in wine.
The sorbic acid nvotccakr. like unsaturated laity
ackl molecules, possesses two don He bonds. As
explained in organic cbcmLsliy. these bonds can
be oxkliird by air lo form molecules with aktchy-
dK fane tions. This reaction explains the unpleas-
ant tastes impaired h> tatty substances by oxida-
tion. Concentrated aqueous sotitions of sorhatc
effectively become yellow and take on a pun-
gent odor. This observation Is proof of a ccrtiin
chemical Instlbility of sorbic acid Yet dilated solu-
tions are noted to be significantly more stable:
in wine, in particular. Ihc same quantity initially
added Is found after three years of bottle-aging.
Consequently, the chemical Inseibility cannot be
presented as an explanation of the organolcptical
dev unions attributed to sorbic acid.
The appearance of a disagreeable, infcnsc and
peistNtcni odor, similar to Ihc odor of geraniums,
in wines treated by sorbic acid was quickly deter-
mined *> be related to bacterial development. In
fact, this olfaclivc deviation appears at the same
lime as an increase in volatile acidity or sim-
ply malolactic fcrmentatun II can also occur in
pooriy stabilized bottled wines. LaclK ackl bacte-
ria arc responsible for this spoilage, and numer-
ous isolated wine strains are capable of ntclabo-
li/ing sorbic acid. The molecule responsible for
the gcramnm-like cxlor Is a derivative of the
corresponding alcohol of sorbic acid (Volume 2.
Section 8.7 11 This strong- smelling molecule has
a perception threshold of less than I n.g/1.
Il is almost impossible to remove the geranium
odor from a wine. Since il is still perceptible after
significant, dilution, blending is not recommended.
The most drastic deodorizing treatments fail (fixa-
tion an active charcoal, extraction by oil. etc.). The
odor passes during distillation and Is concentrated
in spirits — only a severe oxidation with potassium
permanganate eliminates il. Fortunately, the neces-
sary conditions to avoid this scnoas spoilage (the
rational use of SO.i arc now well known and this
problem has practically disappeared
9.2.4 Use of Sorbic Acid
Sorbic acid must be used exclusively for the con-
servation of wines containing reducing sugars. It
serves no purpose in dry wines. In the case of rcd
wines in particular, the development of tactic acid
bacteria in the presence of sorbic acid can result
in the appearance of an extremely scnoas olfac-
livc ftiw.
Sorbic acid is not a wincmaking tool. Il docs
nolaffccllhc rules of aattagf for sweet wines It is
incapable of supping fermentations that arc under-
way. Sorbic acid is exclusively used for Ihc conser-
vation of svvcel wines to avoid ihcirrcfcrmcntaiion
Il can be added lo wine alter the elimination of
yeasts by racking, ccntrifugation or nitration
The conccn nation used is generally 20 g.'hl II
can bcdccrcascd in wines with little residual yeast,
high alcohol content and/or a low pH. Dae k> the
low solubility of this acid in walcr. a concentrated
solution of Ihc much more soluble potassium
sorbalc Is prepared immediately before treatment.
The solution must be introduced slowly ink) wine
and mixed quickly to avoid ihc iusolubilinilion of
Ihc acid
Produce, and Methods Complementing ihc Effect ol Sulfur Dioxide
22~
In wines ircalcd by sortie acid, lie free SOi
concentration nasi be maintained between 30 and
•40 mg/l Id protect against oxidations. baclcrEi
and Ihc gustatory neutralization of aldchydK
substances. This SO? concentration alone would be
insafltcical lo protect wine against n-tc mentations
9.3 OCTANOIC AND DECANOIC
ACIDS (SATURATED
SHORT-CHAIN FATTY ACIDS)
Ccrciln long-chain fatly acids (Ci* and Ci») acti-
vate it men unions Convctsely. other shorter-chain
tally acids, in particular C& aid Cm acids, pos-
sess a significant fungicidal action (Gcncix el id .
1983) They are formed by yeasts during alcoholic
fcnucitition and can contribute to difficult final
fcmicitalion stages. This property. combined with
their complete innocuousness. led to them being
proposed as an adjuvant to sulfur dioxKIc to ensure
swccl-winc stability (Lame el of., 1986). Their use
asa wine stabilizer shonld he approved by official
icgnlation.
Different options are possible Their uul con-
ccntralioi added to wine shoakl not exceed
10 nig/I: tor example. 3 mg of octinoic acid plus
6 nig of decanoic acid per liter. Octinoic and
decanoic acids arc prepared for use by beiig sol-
nbilizcd in ethanol at Hfi volume. The concen-
trations arc calculated so thai the additm of the
solution docs not exceed II. Il ml/1).
Al Ihc indicated conccnttulion. these acids
possess a fungicidal effect complcmcnling sulfur
dioxide For the nailage of sweet wines. 150 mg
of SO? + 9 mg of Kitty acids per liter has the
same cffcciivcncss as 250 mg of SO? per liter
(Table 9.2). The SO? saving is significant. The
tally acids should be added 24 hoars before
sulfiting. In these conditions, the SO? addition acts
while yeasts ate pirdominaally (if not completely)
inactivated Also, a fraction of the lathy acids
is eliminated by fixation oa the yeast cells
After this kind of licatmcal. sweet wines can be
conserved with a tree SO? concentration of around
40 mg/l.
It should be noted that fatly acNls arc more
effective than sorbic acid, which docs not permit a
decrease of the SO; conccnlralNin used fatmntiige
Due to the aromatic intensity of these acids and
I heir esters, the organoleptic effect of sach an addi-
tion had to be determined. An increase of the lour
principal wine fatly acids and their cthylic esters
has been observed (Tabic 9.3). hul ihc increase did
nol icprescnl Ihc lolalily of the fatly acids thai were
added. A laigc portion of them wcte fixed to the
yeast cells during nuilti&e and consequently elim-
inated during clarification. This treatment leads to
an increase of a few milligrams (per liter) of the
constituent! naturally existing in wine The varia-
tions observed aic well within the limits caused by
Ihc action of certain classic wincmakiug methods
and operations acrotnosis. Ihc addition of ammo-
nium sale., temperature variations, etc
Numerous sensory analysis tcse were carried
out This addition Is not diluted to be completely
without effects bnl. provided that Ihc addition
docs not exceed 9-IOntg/l. Ihc effects on the
1 .id I.-".:. >■■>-.-.•■■ 1 -.
atocianoK aaddccai
n <■".. I9KA)
■ of miIIui dlnvkt I Lam
1 Vuhk \csl.
Ociaook: acid (3 ati/li 24 baflertieaimci
Decawik acid <0 mg/l) (cchVml)
Handbook of Enology: The Microbiology of Wine anil Vinifie.uions
Ibble "J. Effect ol Mcnllalbn a>*d**>iual the rmtlal
akxibulK tcmcvjlkiD<SO, 200 mg/l. SO, 100 -pi -
fall) ntiiK 9 mji.1i o» m iK cnnptnkbn (tally .nKK aid
rhcitahylcMco,.mvi/l)<Iaiuc<T.V. 1986)
Wioc
Tun
1 (in-, Kkb
Tciu
Icihvlcuci-.
numbci
n wibc*
in nine'
S> i.
SO, 1- buy
wuk 4
so;
SO, +■ fatly
Mid).*
n'l
2d
J.I
020
050
■°2
4.7
02
OJft
OSO
■"3
40
80
0.34
095
■"4
3.3
4.9
0JB
OX
■°S
30
4.7
ii
Hi
I4J
081
IX
■°7
48
02
020
0J4
■"8
2A
32
D.17-
OJO
Aveagc
4.9S
586
DJ3
0.70
organoleptic a I character are judged to be .slight aid
on the whole positive.
9.4 DIMETHYLDICARBONATE
(DM DC)
The use of dicthyklicarbonalc (DEDCl. or cUyl
pymcarbonaK (Baycovin). was authorized in the
United Stiles anil Germany Toe a few years in
the caily 1970s. This fungicide is very effective
i>n yeast,, after acting it is hydrolyzcd in a few
bonis after application, at a rale thai varies with
temperature, according to Ihc following reaction:
CjHj-O-C-O-C-O-CjH,* HjO
II II
O O
- ICHi-CHjOH+iCOi (9.21
This product, at doses of a few hundred mil-
ligrams per litr. was very c flee live for steriliz-
ing wine during bottling iRibcrcan-Gayon el ill .
1977). It was less useful during wine storage as il
disappeared rapidly and ceased u provide protec-
tion from repeat contamination.
Il rapidly became apparent thai the reaction of
ethyl pyrocaiboaate ii wine was more complex
■ban indicated by Ihc reaction shown above. Ethyl
alcohol and carbon dioxide were certainly the main
degradation by-producls. but small quantifies of
ethyl carbonate were also formed, and its fruil
aroma was perceptible above a certain threshold.
Mosl importantly, ethyl pyrocarbonatc is a highly
reactive molecule and combines with certain sub-
stances in wine (otganic acids, polyphenols, and
nitrogen- based compounds) to produce urethanes.
e.g. ethyl carbamate, which is loxic and carcino-
genic. Quantities never exceeded 2-4 mg/l. signif-
icantly below the official threshold of 30 mg/l in
Canada. However. Ibis risk wussaflicicnl for the
pioduct to be completely abandoned.
Bayer, the company that prodnced Baycovin.
replaced it with dimcthyMKarbonalc (DMDC. or
Vclcorin). considered to haw Ihc same sterilising
piopcrties in wine without any of Ik problems
tBcnrand. 1999). DMDC breaks down la form
methyl carbamate, which is considered to have
practically bo toxK effects. Ougn elitl. (1983)
demonstrated thai 100 mg/l DMDC sterilized wine
completely at pH below 3.8 in the absence of SO),
even if the initial yeast population wasgrcater than
10 r cells/ml
On the basis of these findings. DMDC was ini-
tially authorized in the United States, then in other
countries. LikcclhylpyiDcarbonatc. DMDC is most
effective al the time of holding, although it has also
been suggested for use in snipping the fermentation
of sweet (botrytized) wines (Bcnrand and Guillou.
1999). this reducing the amount of SO; required.
In any case, a certain quantity of free SO? is always
necessary to protect the wine from oxidation
In the European Union, this pioduct is currently
authorized for use in unfermentcd beverages at
doses bckiw 250 mg/l. In view of its properties,
especially the possibility of reducing the use of
SO,. DMDC R currently being tested with a view
to rcgBtralion in the OIV Infcrnatiotal Code of
Wine making Practices.
DMDC has proved effective not only on fer-
ine ntition yeasts, but also on those respoisi-
hlc for contamination (Brcttauomyccs. Volume 2.
Products and Methods Complementing the Effect of Sulfur Dioxide
Section 8.45). as well as. to a lesser extent, bac-
tcnKOugh rial.. 1988).
DMDC decomposes Id form mainly methanol
and carbon dioxide.
CHi-Q-C-Q-C-O-CH.-t- HjO
2CH.-OH -2CO>
Methanol is the n
A mi]' Hani ln
■ this i.
as it is in: hly toxK. TheoietKally. the breakdown of
200 mg/1 of DMDC produces 96 mg/1 of niclhanol.
There is no regulated limit for this compound
(excepted in Ike LIS ) whkh is present in all wines
(Volume 2. Section 2 2.1). The OIV has set two
limits. .100 mg/1 for red wines and 150 mg/1 for
whites. In the United Suites, the maximum pcrmlttd
methanol content Is 1000 mg/1 Consccjncntly. the
use of DMDC may be considered not to have any
scoots impact on the methanol content of wine.
The breakdown of DMDC abo prodnccs non-
toxic methyl carbamate, as well as several methyl,
ethyl, and mclhyl-ctky) carbonates. The killer arc
odoriferous motccitcs bni arc present in insuffi-
cient quantities to modify wine aroma.
Finally, il shoikl be Liken into account that
handling this undiluted product involves some risk
as it Is daugcroas if inhaled or allowed to come
into conlacl with lac skin. It ts advisable to use
proper equipment to ensure safe handling.
ipcrlk?
9.5 LYSOZYME
9.5.1 Nature and Pr
of Lysozymc
Rittfrcaa-Gayon end. (1975 and 1977) wcic ap-
parently the first to describe Ike winemaking
propenics of lysozymc. Tic authors described a
tour of the Mcdoc wineries in the 195% with
Alexander Fleming, winner of the 1945 Nobel
Prior for Medicine for his discovery of penicillin.
During a lining operation using egg white, he won-
dered whether lyso/ymc. which he had discovered
in egg whit a lew years earlier, could ptiy a role
in the microbiological stabilization of wine.
Lysozymc is an enzyme capable of dcsbDyiig
Gram- positive bacteria (Section 43.2). such as
lactic bacleria in wine. This natural, crystallized
substance is capable, at low doses, of causing
lysis of Ike bacteria. I c dissolving their cell walls
l:gg white contains approximately 9 g/l lysozymc
and the standard method of lining may introduce
5*8 g/l into Ike wine. Ribcrcan-Gayon (1975 and
1977) observed that this agent had no effect on
acetic bacteria in wine, which is nol surprising, as
Ifccy arc Gram- negative . However, (key confirmed
that the ux of crystallized sample of lysozymc
very well parihed at doses above 4 mg/1 achieved
its maximum effect within 24 hours and was
capable of destroying almost all Ike tactic bacleria
in a wine ITablc 9.4).
However, it was not possible to detect any effect
of ly.No/yinc during fining with egg white, even at
very high doses. There was no difference compared
to lining witk gelatin or bentonite The lysozymc
in egg while is probably not released into the wine
during fining and is precipitated, together with the
albumin, by contact witk tannins.
At that lime, the use of lysozymc for the
mtcrobtotogtcal stabilization of luetic bacteria was
not envisaged, piobubly due to the facl thai the
crystallized pnxluct was not wklcly available and
Ike cost of treatment seemed unacceptable.
H>bk ".4. Effctt of purified ]■■..■
IRfeercau-Cayon el <i.. 1977)
c (Number of lit jap butlcru per i
IXiWtof
I mg/1)
Red win
c 18000 0D0
Red vrii
k 520000
Whic nine 20000
A tic f 1 i
Alter 24 h
A act 4 h
Alter 24 h
Alter 24 ■
4
2000000
2300
490000
12000
30
8
141" 110"
2250
200000
S'ln:
10
12
19700
1 750
4200
•4tii:
!■■:
Id
13 BOB
:t.:.i
4700
::>■:
10
230
Handbook or Etiology: The Microbiology of Wine and Vwifiiations
The use of lyso/yme in the dairy aid cheese
industries has gradually become widespread and il
has been demonstrated (o have no »xK effects 01
■■mans. The mulling increase in availability has
ted u new interest in ibe ase of this product in
wincmaking since 1990.
Further research has established lhal increasing
ihc dose of lysozymc accelerates lysis of ihc baclc-
ria bit has link impact on the number of icsisCinl
cells The bacteria aic not all desimyed. irrespec-
tive of Ihc dose, so il is impossible 10 achieve
pcrfecl stlbilizalion of the wine (Gcrbaax ■■/ of.,
1997: Gerlaid el ill.. 1999). Crystallized lysozymc
is paid need by Fordas (Lugano. Switzerland) and
marketed, al least for wincmaking purposes, by
Marti u-VEilatlc (Epcrnay. France!, under Ihc ' Bac-
lolysc' biund. Lysozymc is a prolcin consisting
of 125 amino acids (molecular weight 14.6 Kdal
thai ace almost immediately, bal. at Ihc conditions
prevailing in wine (pH). il is rapidly piecipilatcd
oul or dcactivalcd (eg. following bcnlonilc ticat-
nKnl). In contrast to SO,, the activity of lysozymc
incicascswith pH.
9,5.2 Applications of Lysozymc
in Wincmaking
Several situations in which lyso/yme has useful
i-iiei is dnring fermentation and wine storage have
been described in the literature (Gcrbanx el al..
1999: Gcrland el al.. 1999). even if stabilization
in terms of laclK bacteria is not perfect.
I) Inhibiting makilactic fermentation i
* ll.lv
In view of the fact that Ihc high dose of SOi
required in any case k> prevent oxidation also has
.iuiiniiiRihi.il effect, picventing malolaclK fer-
mentation in while wines is not usually a problem,
except in wines with a high bacicria conicnl and
high pH. sach as press wines. However, the use
of lysozymc makes il possible to achieve the same
protection with lower doses of SO; (4-5 g/hl>. It
is necessary loadd lysozymc cilher once (500 mg/l
in the musl). or twice (250 mg/l in the must and
the same in Ihc new wine), as one 250 ntg/1 dose
is not sufficiently effective.
Adding lysozymc docs not affect fermenta-
tion kinetics. The wines have Ihc same analytical
piramctcis and arc unchanged on lasting, provided
they are adequately protected from oxidation.
but only 30 mg/l of total SO> are required, for
example, instead of 50.
As lyso/yme is deactivated by bentonite. this
treatment should be delayed For Ihc same reason,
wines treated with lyso/yme react to the heal
tcsl, indicating prolcin instability. However, protein
cassc develops al high tempcralurcs(50'C). which
do nol normally occur during wine shipment and
storage.
2) Delaying Ihc development of luetic bacteria
and malolaclic fcmicntalion in red wines
One case where Ibis applies is the fcrmcnla-
boi of whole grape bunches, i.e. carbonic mac-
eration and Bcaujolact wincmaking methods. It is
nol unusual for malolaclic fermentation to start
in Ibis type of medium favorable to the devel-
opment of lactic bacteria before Ihc end of the
alcoholic fcrmcntition. Lyso/yme ( 10 g/hl) added
Id the crushed gtapes ts al least as effective as
saluting (5-7 g/hl) and minimi/cs the risk of an
unwanted increase in volatile acidity 1 .
Another situation c that of wines vatted forking
periods, when bacterial development increases the
risk of malolaclic fermentation on Ihc skins. This
should always be avoided in view of Ihc risk
lhal there may be trace amounts of residual sugar
present, especially if the grapes were not com-
pletely crushed prior to fcmicnEition.
Lysozymc s effectiveness al this stage b less
clear, in view of its gradual elimination by pre-
cipitation with the phenolic compounds. Adding
lysozymc earlier in fermentation is immediately
effective, bul there R always a risk of reconum-
inalion. eg dnring pumping-ovcr. while delaying
is addition raises ihc risk of premature malolaclic
fermentation.
3) Use in cases of difficult alcoholic icrmcnia*
Bn
If conditions are unfavorable to ycasls. a de-
crease in ihcir activity causes fcrmcntibon kiskiw
down. This may. in turn, promote the ptulifcrubon
of lac Ik; bacteria, which are likely to slop alcohol k
Products and Methods Complementing the Effect of Sulfur Dioxide
231
fermentation completely (Section 12.7.4). making
it extremely dillkiilt to restart (Scctiou 3.8 I). It
is well known that kictcrial growth in a sugar-
containing mediam provides the nuist favorable
circumstances for lactic spoilage ki occur, together
with an incicase in volatile acidity.
Sulfiling R the mosl common technique (Sec-
tion 8.7.4) for controlling unwanted bacterial
development A 200-300 nig/I dose of lysozymc
provides a nsefnl complementary treatment, espe-
cially in white wines, where lysonmc is more
stable than in red wines. I.yso/ynic iol only pre-
vent lactic spoilage, but is also effective just after
spoilage starts the addition of 23 g/h) reduces the
bacterial population wry rapidly from several mil-
lion bacteria k» fewer than 100.
4> Microbiological stabilization after malolaclic
fcrmcitation
Microbiological stabilization of wines is nec-
essary to avoid the many problems that may be
caused by microorganisms. Lysozymc docs not
piotcct sweet wines fiom fermenting again or elim-
inate contaminant ycaso tBrcltanomyccs). nor is
it effective against acetic bacteria, which cause
volatile acidity in aerated wines.
Lysozymc is. however, effective in eliminating
lactic bacteria. A dose of 200 mg/l has a similar
effect to salfiling at 50 mg/l As this level of so-
ts generally required in any case to benefit from
the other properties of this product (preventing
oxidation, as well as eliminating yeast and acclK
bacteria), it seems unnecessary *> add lysozymc
as well.
It has been observed that delaying sulliting in
icd wines by adding lysozymc resulted in a more
intense color, which also remained moic stabte
iCcrfcwd «7 «/.. 1999).
9.6 DESTRUCTION OF YEASTS
BY HEAT (PASTEURIZATION)
9.6.1 Introduction
The destruction of microbial germs by heat has
been known for a long time, but in cnology
there have not been as many applications of
pasteurization as in other food industries The rea-
sons arc easy to understand.
Bottled wine conserves relatively well due to
it alcohol content and acidity, provided that it is
conditioned with a sufficient SO ; (and cvcnlaally
sorbK acid) concentration after the satisfactory
elimination of microbial germs. Hot* bottling can
contribute to wine sCibili miiou but. anlikc beer,
the paste uri ration of wine was never widespread
Hot bottling ensures the stability of bottled red
wines with respect to bacterial development, and
sweet white wines with respect u rcfcrmcnutioiis
It is generally nscd with wines of average quality
that have microbial stabilization problems. Heating
to 45 or 48 'C sterilizes the wine and the bottle
The presence of free SOj avoids an excessive
oxidation Of course, a space appears below the
cork after cooling I Volume 2. Sec mm 12.2.4).
Sweet wines arc difficult to store during their
maturation phase between the completion of fer-
mentation and bottling, l-or this more or less long
period, the wine is stored in bilk, in tanks or in bar-
rels; it normally acquiics its stability and limpid-
ity and improves qualitatively, but re fermentation
risks ccrctinly exist. The destruction of germs by
heal should be able h> contribute to the required
stabilization, while at the same time limiting the
snlfur dioxide concentration. The heat conditions
necessary for wine stabilization arc easy lo sat-
isfy withont compromising quality. However, the
eqaipmcnl available in a winery makes it difficult
to ensure satisfactory sterile conditions for wine
handling and storage
Despite these difficulties, the Ihermoresistincc
of wine yeasts is well understood and the practical
conditions for microbial stabilization of bulk
sweet wine by heal treatment arc also clearly
defined ( Spliltstocsscr end.. 1975: Dcvcze and
Ribircan-Gayon. 1977 and 1978). Therefore, the
constraints and advantages of these techniques
with respect to the desired decrease in sulfir
dioxide concentrations can be correcllv evaluated.
9.6.2 Theoretical Data i
Resistance of Win
i the Heat
Yeasts
The Ihermorcsislancc of microorganisms can be
characterized by two criteria: the decimal redaction
Handbook of Etiology: The Microbiology of Wiic anil Vniftatkws
lime il) i which represents the duration nl healing,
al u i" >nst:m i temperature, irquiitd 10 reduce ihc
population to oic- tenth of it. initial valve; and ihc
Kmpcrature variation (Z) whkh permits ihc nuf-
liplicalion or division of D by 10 The value of D
depends on ihc mkrootg um, ihc culture condi-
tions aid ihc beating environment. Ihc valve of z
depends almas! exclusively on Ihc mkroorganism.
The following four ksicd vtast strains me
classed according to an increasing resislanec k>
Kmpcrature in Icons of ; aid D al -HI'C:
Sacehanmnrades z=323*C D* = I0.8mii
***"*Jf*
Saeehtimmyres z =33t°C 0*=8.45mii
Ixmnats
Zygtisaceharanmrs ; =426X D n =46.1 mil
Ixiillii
Stwehammwes c=434'C D„ =65.7 mil
Studies of ihc inductee of different factors oi
the ihc ni loirs is tunc of a Sacch baytuaa strain
have demonstrated Ihc following.
• YcBEts ate half as resistant in a wine at ii'i as
ii a wine at ll'i.
• YcasLsate about three times more rcsistail in a
wine containing 100 g of sugar per liter than ii
the corresponding dry wine.
• A wine al pH 3.8 must be heated for a period
three times greater than the same wine at pH
3X)
• Yeast resistance is 10 times greater diring the
linal phase of fermentation than during the tag
phase of the cclLs multiplication
ThcclTcctivcncssofalicat ireatmcuiiscxprcsscd
in pasteurization units These units represent the
diralioi of a treatment at 60 : C having the same
effectiveness on the given mkruorgailsii as the
irealn>cnt bciig considered. Thcgraph in Figure 9.1
indicates, for wine mlcroorganisnis (.: =45*C).
the mmbcr of pustcuri/aiion units conespoidiig
withagiven llitc/tcmpcratire combination. Heating
for 3 mm at 55 5 'C and for 1.4 mm at 57 'C arc
equivalent and correspond with 0.3 paslcuriAitiiw
nils.
Kift ft I. ft
It ill -. rirn.v . .. . i -L..I ■ ' (II 1 .mi,
did Kil-. .v,.ii-: ,i- ,-n I"-".
Mipplicd by > i<>o.i2ia
iml icmfcrjiun ( lie > c.i
The number of pastcuri&ilion nut. required
to obtain a ccrtaii level of dcstruciioi can be
calculated from >east Ihcmioreslstance criteria ii a
given medium Laboratory siadics anticipated thai
0.05 paste uriAilioi nic. would be sufficient for
the dcstruciioi of yeasts in a dry wine at \2'i
volcthanol. In practice. 03 pastcurifaiiou units
are required to sterilize such a wiic. The uneven
heal supplied by industrial heating equipment and
Ihc existence of particularly resistant yeast strains
at the final stages of fermentation can cxptiin
this difference Table 95 shows the necessary
conditions for Ihc dcstruciioi of germs according
Id the constitution of the w inc.-
In addition, a iKxlcnilc sulfiting is required lo
protect sweet wiics from oxidation This sulfiliig
iicreascs yeast sensitivity to beat. The effective-
ness of a heat trcatmcilcai therefore be improved
by injecting sulfur dioxide al Ihc ink! of the tem-
perature exchanger, al elevated temperatures, the
proportion of free SOi i:
Products and Methods Coin plcnvcn ling the Effect of Sulfur Dioxide
233
Table 9.5. Number ol fiMcunnilun uni). (PU> a
Oxyoa. 1978)
Alcuholic
CoKCMMba i* supiMalj
uicntsb (*i vol.)
a SO 100
ID8PU 1.74 PL' 2S3 Pli
0.73 PU 1.19 PL' 1.93 PU
05(1 PU 081 PL" IJI PU
0.34 PU OJS PL' 0.90 PU
0-23 PU 0.38 PL' Ofil PU
These results show the relative rase of dcslroy-
ing yc:is(s by heal They pcnuil the establishment
of operating conditions adapted to each particular
rase. In practice, the principal difficulty arises alter
the treatment that is. avoiding subsequent contam-
inations.
9.6.3 Practical Applications
In practice, stability is satisfactory if the viable
yeasl population Is less than I cell/ml. It might
be preferable to set a lower limit (for example
less than I cell per 100 ml) but in this case the
sample would have to be tillered for foe germ
count This operation can be very difficult, if not
impossible— for example, with new sweet botry-
lizcd wines The number of pasteurization uniLs
required to sterilize a wine in lerms of its constitu-
Ikii is given in Table 95 . Tic heating time directly
depends on the industrial pastciri/aiion flow rale
From the graph in l-igurc 9.1. the required temper-
ature can be predicted.
After paslciri/alion. the wine nasi be stored in
sterile conditions. The tanks and all of the material
in contact with the wine must therefore be steril-
ized— preferably with steam, and if necessary with
a chemical disinfectant. In the first case, sterile
air must be introduced into the tanks during cool-
ing In the second case, they must be energetically
rinsed with sterilized water.
The results obtained from applying these tech-
niques to a large volume of wine (several thou-
sand hectoliters) led to the proposal of a sweet-
wine elaboration method comprising several skrps
ilX'Vc/cand Ribcrcau-Gayon. 1978):
1 . A well-adapted heal sterilization at the time of
mutiige. when the sugar-alcohol equilibrium is
a l mined This heat treatment is preceded by
the addition of a sufficient amount of SO?
to obtain a free SOj come n nation of around
30 mg/l. The sterilized wine is then placed in a
sterilized tank.
2. Clarification and stabili/alion of the wiic dur-
ing the winter and spring following the harvest
The risks of yeasl multiplication are limited
during this period. After these treatments, the
wine is sterilized again and placed in a sterile
tank. During these operations, small concentra-
tions of SO? arc added to maintain the required
free SO; concent rat ion for avoiding oxidation
phenomena.
3. Regular monitoring of the microbiological stale
of the wine to verify sterility. If the viable
yeasl population increases exaggeratedly and
attains 1000 cells/ml. at additional sterilization
Is effected This increase of the yeasl population
(Table 9.6) can be explained by the presence
of an excessive residual population in the wine
( > I cell/ml) preventing a sufficient sterility . or
by subsequent contaminations
4. Sterile bottling, cither by miration or by pas-
teurization, using established techniques.
If appropriate equipment (which remains rela-
tively expensive) is used, bottled sweet wines can
be obtained with the same free SO. concentra-
tions as those used for dry while wines. Conse-
quent)*, the total SO; concentration is significantly
lowered. Sweet wines stored with only 30 mg of
Tabic 9.0. \liin*ii>l<vii.iUi»Blml.ov(ilmic.ol VCMI
populal*>in in two *wccl-»inc lunla. M*bihrcd by
put curl nit kin (Dock and Rfccirau-Gayon. 1978)
Viable ycuu/ml Dun
Date
Viable ycuH/al
Mi r. 1 1
<l
Apr. 18
<l
Am. 23
70
Id. ':
no
Jul. 20
aw
234
Handbook of linokigy: The Microbiology of Wine anil VinUkalkws
free SO; per lifcr contain oa average 60 nig less
uttil SO? per lilcr ihiia those stored with 50 ntg
of free SO? per lilcr. The killer concentration is
indispensable lor avoiding the rc fermentation of
non- pasteurized wines.
9.7 ASCORBIC ACID
9.7.1 Properties and Mode of Action
Ascorbic acid, or vitamin C. exists in fruits aid
in small quantities li grapes (about 50 mg/l of
Juice I bit it rapidly disappears during fcrmcuuikia
and initial aerations Wines generally do not
contain any.
Ascorbic acid Is essentially used in cnorogy
(Ribcrcan-Gayonr'/crf.. 1977)asaredncing agent.
Ewarl el erf. (1987) proposed replacing ascorbic
acid with its isomer. crythorbK acid. The latter
docs not have vitamin properties but possesses the
same oxidation -reduction properties Its industrEil
production costi arc less.
Ascorbic acid was authorized in France as an
antioxidant for fruit juices, beers, carbonated bev-
erages and wines, in 1962. Its use docs not raise
any health- related objections It Is now used in
most viiKuliHr.il countries at a maximum concen-
tration of 150 mg/1. always in assocErlion with
snlfnr dioxide The recommended concentrations
arc between 50 and 100 mg/l: higher addition can
affect wine taste. As it Is completely water soluble
(130 g/i). Ik preparation docs not pose a problem.
The solution shoald be prepared at the time of ils
•se. Homogcni onion should be complete in avoid-
ing all oxygenation, for example by mixing it with
snlfnr dioxide.
The oxidation mechanism of ascorbic acid has
incited much research (Makaga and Maujcan.
1994). It functions like an oxidation --reduction
system. Ik oxidized form Is dchydroascorbk acid
(Figiic 9.2):
ascorbic acid ■
dchydroascorbk acid
This react in is theoretically reversible but. due
lo us instability, dchydnuscorbic ackl disappears.
+ 2H r + 2e" (9.4)
V-
Cllptl citpii
Fig, 9.1 Oxidation of awoiblc acid lodehydw
The two electrons that appear in the course c
the reaction reduce certain wine constituent, i
particular the ferric ion:
2tV*
l-2e
-2IV"
<9.5)
The effectiveness of ascorbic acid in the preven-
tion of iron cassc wbkh is exclusively caused by
Fe ,r ions is explained by the above reactions
In the presence of oxygen, the oxidation of
ascorbic acid leads to the formation of hydrogen
peroxide — a powerful oxidant that can profoundly
alter wine composition The presence of a snfl>
cical amount of free sulfur dioxide protects wine
from the action of this molecule It Is preferentially
oxidized by hydrogen peroxide (Figure 93):
Si ■ +
- H.SO,
(9.6)
The oxidation reaction of ascorbic ackl Is
catalyzed by iron and copper. But. contrary lo the
direct oxidation of SO. by notccnlar oxygen, the
icaclkii Is rapid. It constitutes a simple means
of almost instantaneously eliminating dissolved
oxygen and preventing the corresponding flaws.
Of course, lo remain effective, the amount of
dissolved oxygen must nor be too considerable.
V-.
Fig, 9.X OxklMtoa
hyit*>gcn fcmxalc
Products and Methods ComplcuKniing the Effect of Sulfur Dioxide
235
Sulfur dioxide and ascorbic acid therefore have
different antioxidant pmpcrlies. The first hah a
delayed, but stable, effect which continues over
time even in the presence ofasubscqucaloxygcna-
lioa It cannot prevent iron casse. which rapidly
appears arte ran aeration The second has an imme-
diate effect: il can instanEtncoasly compensate the
damage of an abrupt and Intense aeration (iron
casse). but it acn only as long as the wine is not
in permanent contact with air.
Due to the high oxidation sensitivity of ascorbic
acid, lis cffeclivencss Is only guaranteed when
its contact with air is limited. In other words, it
protect well against small, brief aerations bat not
against intense or continued oxidation. Its rote is
limited to protecting wine from light aerations,
following bottling, for example. It is not effective
for prolonged storage in ranks or barrels
The danger of the oxidation of ascorbic acid,
especially in the presence ofa large amount of oxy-
gen, should also be considered In these conditions,
hydrogen peroxide and sometimes other peroxides
arc formed. Coupled with the presence of catalyz-
ers, they can cause a thoroagb oxidation of certain
wine const Hue ii is. which in the absence of ascorbic
acid would not be directly oxidi/ablc by molecular
oxygen. The inverse of the desired result can unfor-
tunately be obciined in this manner This explains
some of the problems that can be encountered
when ascorbic acid is used incorrectly For this
reason, ascorbic acid should only be used in wines
con tuning a sufficient concentration of free sulfur
dioxide, available for the elimination of the hydro-
gen peroxide formed in the course of oxidations
use is particularly justified for the protection of
mechanically harvested grapes, since il does not
act on the maceration, as does sulfur dioxide
(Section 1323). This use P not permitted by EU
regulations, which only authorize the addition of
ascorbic acid in wines
An effective protection can be obtained in red
wines sensitive lo oxidask casse as well as while
musts during wincmaking. At present, however.
the use of ascorbic acid is not widespread in wine-
making and not authorized in France probably
because the required concentrations are too high
to protect musts against oxidations and because
sulfur dioxide Is more effective.
9.7.3 Protection Against Iron Casse
The aeration of wine oxidi/es iron The amount
of ferric iron formed (several milligrams per liter)
can be sulIKicnt to induce iron casse. Protection
against iron casse can Iv ensured if the wine
receives SO-lCOmg of ascorbic acid per liter
beforehand (Table 9.7).
Simultaneously, when a wine containing ascor-
bic acKI is aerated, the oxidation- reduction poten-
tial slightly increases and then rapidly stabilizes,
whereas il continues to increase in Ihc control
wine (Figure 9.4). Reciprocally, if ascorbic acid
is added to an acralcd wine possessing ferric iron.
Ihc iron is reduced in the ensuing hours and the
Table ".7. Protect i-ii Iimiti ii-.- ... ■.■.'.!.- by the .1.1:11 1...-
of UCOlbic ami bcfUR jcral»>« 1 Kihf rt.iLi-tiiviio d ,i..
1977)
9.7.2 Protection Against Enzymatic
Oxidations
The addition of ascorbic acid will limit (if not
eliminate) must oxidations catalyzed by tyrosinase
and lac case. Moreover, il is a suhsiralc of lactase
Il docs nolact by inhibiting the enzymes, as docs
sulfur dioxide, but rather by monopolizing the
oxygen, due lo its fast reaction speed Ascorbic
acid is used in this manner in certain countries lo
complement Ihc protection given by sulfur dioxide
againsl the oxidation of white grape must, lis
While wiac (total Ye 18 rau.lt
Control
*25 rap awoibie acid/1
-50 mp ucoihfc ackl/1
-KM mp ascoibic acid/I
Red wine (total Fe 15 mp/li
Control
*25 rap awoibic ackl/1
-50 rap aworhk *ckl/l
-HKi ap ucoibk ueidfl
Cloudy
Llmpkl
Llmpkl
Llmpkl
Sl^'hlv cloudy
Llmpkl
Llmpkl
Llmpkl
Handbook of Isitology: The Microbiology of Wine anil Vlnifii.itions
Fifi 0.4. Evolotkinofoxkbibo-ieduttkinfoicalHlof
Rdiccil -inc. acralcd ,iHci ike ukUkin of 100 mg
of awoihk atrid »cr Iter. co»fao:d ».b toMrol »inc
(Rbereuu-Gayon el ti„ 1977). (A) Red wine coiriml.
lB)Rcd»ine - awottSit acid. fC) While -i* raBml.
(D) Ahfc aloe + ascorbic acid
oxidation - reduction potential rapidly decreases.
The beginnings of an iron cassc can be reversed
and the corresponding hare cliniinalcd in ih is man-
ner (Tabic 98)
Ascorbic acid cffcciivcly prolix is against in*
cassc. which can occur aflcr opcralions that place
wine in contact with air. snch as pumping-over.
transfers, filtering and especially bottling li ihc
same conditions, sulfur dioxide acts wo slowly h>
Hock tic oxidation of iron Bat. if the wine must
be aerated again after a treatment following a first
aeration. Ihc ascorbic acid no longer protects the
wine. When a wine that has received 100 mg of
ascorbic acid per liter a month earlier is acralcd.
the cvolation of iron III rs idcitKal to that in Ihc
con t ml These results lead to the supposition that
the added ascorbic acid has disappeared.
9.7.4 Organoleptic Protection
of Acralcd Wines
In certain cases, ascocbic acid improves the taste of
bottled wines. Wines generally last worse when
they contain dissolved oxygen and have an ele-
vated oxidation -reduction potential. Ascorbic acid
permits a better conservation of wine freshness and
frii tin ess— especially in certain types of dry or
sparkling white wines. It also decreases the crit-
ical phase that follows bottling, known as bottle
sickness'. The effect is not us considerable or spec-
tacular for all wines but wine quality is never
lowered by its nsc.
The lisle improvement due to ascorbic acid
depends on several factors. The first is Ihc type
of wine. Ascorbic acid is of littk merest in the
case of wines made from certain varieties or very
evolved wines— for example, barrel-aged wines:
oxidised while wines, bolrytinrd sweet wine, and
line red wines. On the contrary, it improves the
stability of fresh and fruity wines (generally yoang
wines), having conserved their varietal aromas
Another important factor is the concentration of
free sulfur dioxide. Itshonkl be situated in an inter-
mediate range between 20 and 30 ntg/1. to ensure
a refinement of the wine, which in tarn presents
a fresher aroma with a floral note For higher
or lower free S< i. concentrations. Ihc qualitative
improvement of the wine is less obvious.
Tabic VK KcitKMoB of inm «-.i
uccubn cawing the ha* (Rfrcn
uc by addikin of aworfik at
au-Giyon <* i**.. 1977)
Hi. 4S h alter the
Fr III imp.'])
t-lmiUllty
At lime of 48 h alter
addaba addibn
48 haltc.
addition
Con ml
+50 mpaMroihkackW.
Rrd Htw lloui Fc lb mg/ll
Con ml
+51) maaM-mbkackLO.
9 7
9 2
5 4
S 1
Very cloudy
Limpid
Cloudy
Limpid
Products and Methods Complementing Ihc Effect ol Sulfur Dioxide
:;-
Sutisfintory rcsulLs were obtained for sparkling
wines produced by the champagne method, by the
transfer method, or )■ pressure Links. The neces-
sary amounts of sulfur dioxide and ascorbK acid
arc added to the dosage to ensure concentrations
of 20-30 mg/l and 3O-50 mg/l. respectively. The
coupled addition of the two substances ensures
an optima) aronia and improves the finesse and
longevity of the wine.
In sparkling wines, ascorbic acid act> not only
by in reducing properties but also by iK capacity
as an oxidation - reduction buffer. Their potential
remains stable at 240 niV for several years In
the absence of ascorbic acid, it varies between
200 and 265 mV. according in the effectiveness
of corking. This phenomenon clearly affects the
oigunolc plica! characters of wine (Malaga and
Man jean. 1994).
The use of ascortHc acid insignificantly modifies
Ihc use of salfnr dioxide, it permits a slight
lowering of in concentration. Yet il possesses other
advantages.
9.8 THE USE OF INERT GASES
9.8.1 Wine Storage using I ncrl Cases
Even before the nsc of antioxidants (sulfnr dm-
idc and ascorbic acid), ihc first rccommcndalion
for proKciing wines against the adverse effect
of chemical or mKtobtotogkal oxidations was to
limit their contact with air. Wines were stored in
completely filled containers, sometimes equipped
with a system pcrmitling difcitition compcnsalion.
This rccon intend at ion cannot always be follavved.
if the availability of tanks of a satnfaclory size is
limited or wine is regularly taken fiom the same
Link for several days Tanks equipped with a slid-
ing cover which always remains in contict with
Ihc sarfacc of the wine were introduced, but the
joints between the cover and the inner surface of
Ihc tink are rarely satisfactory and their effective-
ness Is questionable
Satisfactory results arc obtained by storing wine
in a partially filled tank with an inert gas. in the
n>Lil absence of oxygen. Wine storage asing inert
gas also permits the carbon dioxide concentration
(lowering or increasing) b> be adjnslcd. Although
not directly related to piotcction from oxidation.
Ibis subject will nevertheless be covered in this
section.
The following gases are authorized for storage:
nilrogen. carbon dioxide and argon Aigon Is rarely
nscd: il Is more expensive than Ihc others and
its solubility is limited in wine (4 l/hl). Carbon
dioxide e very soluble in wine (107.2 l/hl) and
therefore cannot be used alone in partially filled
containers The carbon dioxide concentration of Ihc
wine woukl significantly increase by Ihc dissolu-
tion of the gas. Il is sometimes used in a mixture
with nitrogen (for example. 15'.* CO, + $5'i N.)
lo avoid Ihc degassing of certain wines that must
maintain a moderate CO; concentration Nilrogen
is Ihc most commonly used gas R* quality nitro-
gen is used which contains a lilllc oxygen as an
impurity bni has no impact on wine. It Is less
soluble in wine than oxygen (IX l/hl compared
with 3.6 l/hl j bat contrary u oxygen, which reacts
with wine constituents by oxidizing them, nilrogen
accumulates wilhoul reacting. The wine sponta-
neously becomes saturated in nilrogen during han-
dling in eon lac I with air. Storage in the presence of
Ibis gas therefore cannot increase its concentration
Several principles of inert gas systems for tmks
exist bui Ihc system adapted lo the winery must
be well designed. In particular, the instillations
must be perfectly airtight. Maintaining a slight
overpressure is recommended in older lo monitor
for possible leaks. This method is essentially
applicable to perfectly hermetic tanks.
The gases are stored in compressed gas hol-
lies. At Ihc ouilcl from Ihc bottles, the gas gen-
erally undcigocs a double expansion. Initially, it is
reduced to a pressure of 2 to 8 bars and circukilcs
in copper piping up fc> the storage tank A second
expansion reduces Ihc pressure ta 15-20 mbaror
100-200 mlxir. This second solution permits easy
identification of piping and tink leaks. Bach tink
has a separate line and a manometer enabling ver-
ification of the pressure and thus the airtightness
Finally, a pressure release vulvc avoids the unfor-
tunate consequences of operation errors
Handbook of Enotogy: The Microbiology °f Wine and VinificaUons
Tabic 1.9.
Ev»hf»i
nif the ciiKi
ndbx
idc
CDMCMWiD
a in wan Moied in a
..I.VHII
dioiide oi
ntn>n»i
1-o^bC.C
. jironliDjf to
-:i k.
icl
ufcouuinc.
ll.on>aud-J-unel. 19701
1 ill lend
CO, at a
,... r ^
*
S", »imo!
*ucic
(»)
ni.
ConCCMRItkin
i
bi
cue (fi )
CO, concern twbn
Dccra*
m
.Incim^l)
ianiaclmg/1)
98
3(W
7
2B1
15
82
589
100
234
17*
30
1132
297
1-1
49*
IS
1708
499
SI
SIB
Instill: i lions have been specially designed (or
wine storage using inert gas. Metal tanks are
connected together by gas Dies, but the tanks can
be isolated and iidivklnally maintained al a slight
overpressure 1 100-200 mbarl This overpressure
attests Id the hemcticiry of the Ginks. It is verified
by the manometer reading.
Al the beginning of the opcraton. the tank is
completely tilled with wine and the hermetic talk
vent is secured. A hccmlilcrof wine is then drained
from the faucet at the bottom of the tank Simul-
taneously, nitrogen gas Is spatged in the upper
portion of the tank— replacing the drained wine
and creating a nitrogen atmosphere buffer Next,
the internal pressure R adjusted The talk R then
ready for storage To remove wine, the nitrogen
bottle should first be opened, then the gas valve for
the tank and finally* the wine tank valve. Perfectly
clear and stable wines are conserved and pro-
tected from oxidations and evaporation for several
■Km Us iu these wine tanks. Niliogcn consumption
•s extremely limited. The evolution of the taste of
these wines is identical to that of wines stored in
completely filled tanks.
When there is insufficient wine to fill the lank
completely, thus expelling all of the air contained,
another solution consists of completely filling
the lank with water. It is then emptied under a
nitrogen counter- pressure before introducing the
wine. In many cases, after partially lilling the lank
with wine, residual air is expelled by spacing
nitrogen in the tank hcadspucc. If these operations
are carried oul in non-airtight tanks, the inert
gas must be constantly renewed — resulting in a
considerable consumption of gas. These conditions
are not recommended and can lead to a false sense
of security for wine storage. Al the end of a few
weeks, the wine is oxidized and has lost its CO?:
its odor and taste become insipid.
In any case, this storage system docs not
release the wincmaker from using sulfur dioxide
or even permit conccn (rations to be lowered.
This antiseptic remains indispensable for lighting
against yeasts and laclK acid bacteria. It must be
used al the same conccn (rations as in full tanks.
Storage under inert gas can cause either an
increase or a decrease in the amount of car-
bon dioxide naturally existing in wine The data
in Table 9.9 show the impact of the wine \of-
umcz^ascous atmosphere ratio. The variations are
slight, especially with nitrogen, if the lank is
practically full. Storage under a carbon dioxide
atmosphere easily leads lo an excessive increase
of its concentration, with corresponding changes
in the organoleptic characters of the wine. Con-
versely, stoiage under nitrogen causes a consKlcr-
able decrease in the CO? concentration for half-
lllk-.l I.1I1-.S
The sludy by Lonvand-Funcl and Ribcrcau-
Cayon 1 1977) gives the factors pcrmilling the esti-
mation of the CO</N; mixture which must be used
at a given temperature so that a wine conserves its
Initial dissolved CO? conccnlrauou during storage,
whatever the tank fill level. Inert gases can also
be used for ensuring that wine transfers are pro-
tected from oxygen, by injecting nitrogen in the
lines while pumping wines, for example.
9.&2 Adjustm-; thc Carbon Dioxide
Concentration
Wine tasters are very sensitive to taste modifi-
cations caused by the presence of this gas. even
Products and Methods Complementing the Effect of Sulfur Dioxide
below the organoleptic perception threshold For
example, in a red Bordeaux wine, more than 50*
of the tasters correctly pnl in order three samples of
a wine conttining 620. 365 and 20 nig of carbon
dioxide per liter (RibCrcau-Cayon and Louvaud-
Fuel. 1976). The characteristic pricking sensation
of CO; was only perceptible in the first sample.
The third simple appeared more insipid than the
second, which was judged the best. Yet nothing led
the tasters *< believe that the difference was related
in the CO, concentration.
Dry while wines tolerate higher carbon dioxide
conccn Ira lions. Around 90* of the osiers correctly
pnl in order three samples of the same wine
con tuning, respectively. 250. 730 and 1 100 mg of
COi per liter. The second sample was preferred
overall: the carbon dioxide increased the aroma
and the freshness of this wine. Ycl the carbon
dioxide concentrations should mi be exaggerated
dm centra (Km of 1001) mg/l air not as appreciated
in dry white wines as one might think
Due to ifc organoleptic impact, the carbon di-
oxide concentration should tc correctly adjusted.
Fix each type of wine, there is a corresponding
optimal concentration. Red wines tolerate lew CO;
laround 200 mg/l) than dry while wines (aronnd
500-700 mg/l). TV more tannic and adapted for
aging the wines arc. ihe less they tolerate CO?.
It can be ascful Hi eliminate excess carbon
dioxide rapRlly. by agitation, in young red wines
intended for early holding. Racking in the presence
of air can dec rcasc the CO, concentration by 10*.
bnt this is not always sufficient
The injection of line nitrogen bubbles in wine
cntrainsa certain proportion of devolved gaslcar-
hon dioxide or oxygen) in a wine. The wine deliv-
ery rale, with the device in Figure 95. can vary
from 30 to 120 hl/h Temperature plays an impor-
■ant role in the effectiveness of this treatment.
Below 15 'C. the degassing yield is insufficient.
The temperature of the wine should preferably be
at IS C. The wine, emulsified with very line nitro-
gen bubbles, should then be exposed to air by
flowing in a thin film through a shallow lank with
a large sarfacc area so thai the nitrogen is easily
released and en trams the dissolved carbon dioxide.
;=n_
ViH 9.5. (h\ iajcciof. <i)IIuwng very line buhhlct J
vine iinul.ilin^ Ihitiinrh f ipimf (Kihtit.iu-ikivoii et li
I97J)
In tests, simple racking permitted the elimination
of 26* of Ihe CO> Treating with half a volume
of N, for a volume of wine eliminated 43** of the
CO,. Four times more nitrogen 12 volumes) only
eliminated 54*. It is therefore more reasonable t»
carry ont two consecutive treatments with lower
volumes of nitrogen.
In certain cases, the carbon dioxide concen-
tration must be decreased: in others, it mnsi be
increased It can be increased by sparging with
carbon dioxide gas: the gas can be injected in the
winery piping. The same result can be obtained by
placing wine in a partially lilted tank, its hcadspacc
tilled wilh mixture of a N, and CO;. Lonvand-
Fnncl 11976) has given Ihe mixture required for
obtaining a certain CO; concentration according
to the respective wine and gas volumes
These operations ate normally carried out at
atmospheric pressure. If they were to take place at
higher pressures, a gasification would be effected
The operations would no longer be considered as
ordinary wine treatments, since gasified wines arc
subject to special legislation.
The dissolution of carbon dioxide in wine dors
not differ much from that in water. It depends on
the temperature and ranges between 2.43 g/lalS'C
and 1.73 g/lal 18'C These values correspond with
i amount of CO, that can be devolved
The sparging of wine by carbon dioxide has been
suggested. This method can be useful for avoiding
oxygen dissolution during transfers and u ensure
a protection against oxidations The wine must be
degassed before holding.
Humllkxkk of Etiology: The Microbiology of Wme anil VnincatioBS
REFERENCES
Bcnmad A.(I999) Wfi.,249. 25.
Bcitnad A. and Cullbu I. ( 1999) Bull. OIV. 72. 84.
Dcvcie M. and Rfee'n:au-Gi>uo P. 1 1977) Cum. Mgie
Mi, II (2). 131.
Doric M and Rfce'ieau-Gijoo P. ( 1978) Cum. Mgie
Mi, 12 (2), 91.
i:«ait A.J.W.. Sitcn. J.H. and Bncu C.J. (1987) Avtfr.
NZ \lbie b*l. J. 1,59.
Gcncix C. Lafoa-LafouKadc a.andRbcicau-Gayoa P.
1 1983) CH Ac.*!. Sri.. SAfe ///. 290. 943.
Geibaui V.. M.i-.i, .-.-., mi B., Coireieau P.. Barriirc C.
CuinierC.Bcigcr J.L. and Vilfa A.(I999)fl««. OIV.
72(819). 319.
Gcihaui V.. Villa A.. MomMyC. and BcrnaodA.
( 19971 /Inn J. F.iwl. Vtric . 48 ( I ), 49.
GcdandC-.Ceihaui V.aad Villa A. <I999> /trim- <fcv
Om-l-g/iet. 931 . 44.
I .uiR- F.. Murakami Y . Boidmn IH. and K>hi L( 1980)
Cora.. Ifjnr Uii. 20 (2). 87.
lnnvaud-Rmcl A. (1970) RccbcKko, hii k gae c*i-
Kn.|ii; du via. 1 IKw dc Doctnral (I no logic. Ini-
vcolf dc Boidcaui II.
l«nvaud-Fuocl A. and KaScrcau-Oivoa P.I 1977) Conn.
\tpie \m. 11(2), 105.
Malaga E.andMaujcao A. (1994) ft*//. 0/V. 153.703.
CXigaC.S. Ku.it.ee ki:. VUnH.IL, llonkii II aad
Huang MXT. (1988) Am. J. Eaol. Miic. 39, 279.
Rfecrcau-Gayon J..Peynaud E., Rlncrcau-Gayon P.aad
Sudau.1 P.(1975)SVi«*i-irf Tecimiaae* du XWi. Vol.
II: CiriKieiet da lias. Miimiaioti du niiiin. tfmifi
et birieriet. Duwd.Pam.
Rbcitau-Gayon J IV. ii.idI i: .. Rfccrcau-i ;..mii P.aad
SudcuidP. (1977) Sri™™ ei Technique* da Mn.
Vol. IV: Clirifieitiai el S-Jtirintion. MiierielM el
bwtiUtkuti Duaod.Pam.
Rbircau-Gayon P. and Unvaud-Puacl A. (I97A) CR
Acini. Auric. . 02 (7 ). 49 1 .
>i!.hi,..-...L-ii' r. 1..-11I. : .: . WiikiaMin.il ,,ui su=.-i
iK.fl975)Appl. Microbiol.. Sep.. 309.
10
The Grape and its Maturation
10.1 Intixxlnclion
102 Description and composition of inc mature g rape
10J Changes in the grape timing maturation
10.4 Definition of maturity— notion of vintage
103 Impacl of various other faciois on maturation
10.6 Botrytis cincrca
10.7 CoKlnsion
259
276
10.1 INTRODUCTION
The giupc constitutes the raw Bi.tu.-ri.il for produc-
ing wiits Ik maturity level is ihe Him factor,
and certainly ok of the most deciding ones.
ii determining wine quality It is the itsull of
all of the complex physiological and biochem-
ical phciomena whose proper development and
intensity air intricately related to environmental
conditions (vine varieties. soib>. climate) (Pcynaud
and Ribcrcau-Gayon. 1971: Ribficau-Gayon «•/«/.
1975: Champagnol. 1934. Huglin. 1986: Kancl-
Irs and Roubclakis-Angcfcikis. 1993. Flaazy, 2000:
Roabelalas-Angelakis. 2001).
Compared with other fraio. the study of the
grape presents many problems. Berry growth and
development arc the result of a long and complex
leprodnction cycle. Tic ovary, and then the seeds,
attract the hormones necessary for their develop-
ment from inc leaves., wheic they an- mainly syn-
thesized. The triggering of the maturation process
docs not correspond with a true climacteric cri-
sis It ts linked to the drop in growth hormone
levels and the appearance of a stress hormone,
abscisic acid. In consequence, the behavior of the
eniite plant strongly influences the development
of these processes. Certain studies can be carried
out on fruiting miciocntlings or polled vines under
:-:
Handbook of Etiology: The Microbiology of Wine anil Vinificaiions
controlled conditions, bni ihc preponderant iiflu-
enceof cnvin>nmcnta) parameters on vine behavior
requires that a large number of experiments be car-
ried out in Ihc vineyard. The study of maturation
therefore comes up against diflicullies dK to ihc
cxlremc variability of berry composition, al any
given time and for ihc same variety.
In spile of these diflicullies. Ihc observations
made each year by researchers al ihc Faculty
of Biology at Bordeaux and by other learns in
different wine- producing regions have permitted
• fit] km and compare the chemical composition
modifications of Ihc grape during maturation.
• compare ihc maturation kinetics over the years,
in terms of meteorological conditions.
• compare Ihc evolution of different vineyards, in
tents of kxal eivironmcital conditions:
• forecast maturity dales and thus establish the
harvest dates
These piclimitary observations directed subse-
quent research towards a more thorough study of
maturation mechanisms. This chapter will cover
ihc biochemical phenomena characterizing grape
maturation and Ihc process of the development of
rol ll will also focus oa ihc influence of c
mental factors on maturation
10.2 DESCRIPTION AND
COMPOSITION OF THE
MATURE GRAPE
10.2.1 The Bern
The grape is a berry, classed in a group of several
seeded fleshy fruits The berries air organized into
a cluster Bach berry is attached to the rachis by
a small pedicel con tuning ihc vessels, which sup-
ply ihc berry with water and nutritive substances
(Figure 10. la). Cluster structure depends on ihc
length of ihc pedicels: if they arc long and ihin.
the grapes arc spread out (Figure ID lb), if ihcy
arc short. Ihc bunches are compact and ihc grapes
ihc gape vine: (a)oapc beny 41
.n of i! rape chmci
arc packed together Varieties used for wincmak-
lug often belong to the Latter category. Cluster
compactness Bone of Ihc factors affecting rot sen-
si tivily.
Genetic factors and environmental conditions
thai characterize berry formation greatly influence
its development and its composition al maturity.
10.2.2 Berry Formation
Bruit development is closely related (o ihc modali-
ties of ovule fertilization. Eknvcring corresponds
The Grape and Its Maturation
to ibe opening of Ihc corolfci and ihc ejection
of ihc calypHa (antacsis). The pollen liberated in
this manner can reach (he ovary and Higher its
growth fnnuastm or berry selling! The liberation
of pollen is facilitated by warn), dry weather. In a
cool and humid climate, flowering can be spread
onl over 10- 15 days and sometimes more.
Pollination is normally followed by fertilization,
pcrmiiting Ihc development of a berry possess-
ing one to four normal seeds. Poor fertilization
can lead to the formation of rudimentary seeds
Islcnospermocarpic sccdlcssncss). The absence of
fertilization piuluces seedless berries (partneno-
carpic scedlcssncssl. Sccdlcssncss can be a varietal
genetic character, sow hi after for the production
of tihlc giupcs (Thompson seedless) or for the
preparation of raisins tConatac) Non- pollinated,
unfertilized ovaries arc deficient In growth regula-
tors (polyamlncs) and fomi tiny berries that remain
green (Colin el til.. 20021.
In general, not all of the flowcis borne by the
cluster arc fertilized and become berries. The berry
setting tatio decreases as the number of Dowers
formed on the grape cluster increases. The causes
of this phenomenon have been known for a long
time Asa gene nil rite, a plant can onl)* supply ItX)
to 200 berries per bunch with sugar, depending on
Ihc variety.
After berry set. a variable proportion of appar-
ently fertilized young berries no longer grow and
fall from the plant. This abscission is caised by
the hydrolysis of pectins of the middle lamella
of the cell walls forming the separation layer
at the base of the pedicel. The phenomenon,
called shatter Utntlitre in French), ts often diffi-
cult to distinguish from berry setting in the case
of cold weather and overcast skies, which cause
an abnormally long flowering— sometimes up lo
i weeks (Figure 1021 Shatter depends in partic-
ular on sagar avaifcibility and the effect* of cli-
matic parameters on ib> avaifcibility (photosynthe-
sis, sagar migration in Ihc plant). Climatic shatter
constitutes the principal cause of yield variability
In northern vineyards. In warm climate /ones, a
water deficiency can bring about Ihc same result
A vanclal- spec Hie sensitivity* also exists Shatter
can be complete with Crenachc. Mcrlol. Muscat
E''iii 10.1 Ftaucrand fail cvnlubn during bloom and
berry acuta? (Reus, and rroraiaux. 1902)
Ottoncl or Cbardonnay. Other varieties, such as
Carignan. Chcnin. Sanvignon Bfctnc. Folic Blan-
che. Pinot Blanc. Riesling and Cabernet Sanvi-
gnon. are much less affected.
Millfrimibnige is related to poor flowering con-
ditions, involving a defective pollination with dead
pollen that does not lead to fertilization
10.2.3 The Developmental Stages
of the Crape
In Ihc course of its development, from ovary loupe
fruit, the grape follows an evolution common to
all berries It is generally divided into three phases
(Figure 103). taking into consideration parameters
such as berry diameter, weight and volume:
I. An initial rapid growth or herbaceous growth
phase Listing 45 to 65 days: depending on
vine variety and environmental conditions. The
Intensity of cellular multiplication depends on
the existence of seeds. Growth hormone con-
centrations (cylokrnins and gibbcrellins) cor-
respond directly with the number of seeds
The application of gibbcrelllc acid on seedless
grapes has become a common vilicultural prac-
tice (llo el til.. I%9). Cellular growth begins
about 2 weeks after fertilization and continues
Handbook of Enology: The Microbiology of Wine anil Vinifieatmns
Tank- Id I. RcbiMiuhlf bduccn number of iecdi »*l
btrr, ii/c ul ■Murky: Mciln variety unpci sampled in
1982 icij Sa)ai-EmUb*v)K> B nl(FniiKC)
Fift 10- J- K-'.. Ii,iiti.ii1i: "ji.\-. of I.-.: ,-litc berry;
B. bloom BS. berry *el; V. %rriiani; II. harvcit
umil ihc cad of ibe lirst phase li the course of
this Him period, chlorophyll Is the predominant
pigment The berries have an intense ntclabolic
activity, characterized by an elevated respira-
tory intensity and a rapid accimilalion of acids.
2. A slowed growth phase during which rtrtuum
occurs. Wiiiiunt Is characterized by Ihc appear-
ance of color ii colored varieties aid a translu-
cent skin in while varieties. Il is an abrupt phe-
nomenon al the berry level but takes place over
several days when different berries of Ihc same
bunch are considered. In a vineyard parcel, ibis
phase lasts 8 in 15 day or longer if flowering is
•i'iv slow. It corresponds with Inc depiction of
growth substance synthesis and an increase in
the concentration of abscisic acid.
3. A second growth phase corresponding to matu-
ration. Ccllilar growth resumes and is accom-
panied by diverse physiological modifications.
The respiratory intensity decreases, whereas
certain enzymatic activities sharply increase.
This Una! period lasts 35 to 55 days, dur-
ing which Ihc grape accumulates free sugars,
cations s»ch as potassium, amino acids and phe-
nolic compounds, while concentrations of malic
acid and ammonium decrease. Grape si/c al
malnrity depends largely on these accumula-
tion processes bul also on the number of cells
per berry. There is a very close telalioBship
between the dimensions of a ripe grape and the
number of seeds il contains (Tabic 10 I )
Number
Berry
Juice tokiac
Supir
DfKXk
weight
perheny
(uatcra ration
1P>
(■■>
iBfl)
0-1
1. 10
0.75
235
2
155
11)1
233
3
1.94
i i:
221
10.14 Grape Morphology
Each grape comprises a group of tissues (the
pericarp) surrounding Ihc seeds. The pericarp is
divided into Ihc exocarp < ihc skin), the meso-
carp (Ihe pulp) and the endocarp (the tissue thai
lines the seed receptacles containing the seeds bul
is not distinguishable from Ihc rest of Ihc pulp)
(Figure 10.1a). The fruit is nourished by a branch-
ing vascular network of Ihe rachis. which traverses
the pedicels. This vascular bundle then branches
oui in Ihc pnlp This network can be observed due
to Ihc transparency of certain white varieties al
maturity.
The skin of the grape forms a heterogeneous
region constituted by the cuticle, the epidermis
and the hypodcrmis (Figure 10.4). The cuticle is a
continuous layer whose thickness varies depending
on the variety: 15-4 mi forccreiin \itis vimjera
varieties and up to 10 in for certain American
vines. It hcginMo develop. I weeks before an thesis.
In the course of berry maturation and develop-
ment, it becomes increasingly disorganized and its
thickness diminishes. The cuticle is generally cov-
ered by cpicuticukir wax (bloom) in the form of
stacked platelets, visible by electron microscopy.
Wax thickness is relatively constant throughout
the course of berry development (about 100 p.g of
wax/cm 2 of surface).
The epidermis is constituted of one or two lay-
ers of tangcntially elongated cells whose thickness
varies depending on Ihc grape variety. The hypo-
dcrmis comprises two distinguishable regions: an
outer region with rectangular cells and an inner
region with polygonal cells.
The pulp is composed of large polygonal cells
with very thin, distended cell walls There are
The Crape and us Maturation
24S
I'ifi Hy.1. I'llt.-irnt -|-!|K- lu-ll. ituUCl ir lllirn.M;.
25-30 cell layers, organized into three distinct
legions.
Each normally consul! td seed comprises a
cuticle, an epidermis and In ice envelopes covering
lac albumen and ihe cnibi>u
Giapc berry consistency depends on skin and
pulp cell wall thickness Generally, bible grape
varieties produce plump, thin-skinned grapes (the
pulp having thick cell walls), whereas winenuiking
varieties have lough skins and juicy pulp (pulp
with thin cell watts).
On the grape surface, there aic between 25 and
40 slomata per berry, depending on Ihe variety
After Yt'rtiiitm. these stomati no longer function
and they necrotize Rapid fruit enlargement creates
tension, resulting in lie development or pcristotv
atic m iui> leisures.
10.2.5 Grape Cluster Composition
at Maturity
The stalk rachis rcprescnLs around J-7% of the
wctghl of a ripe grape clislcr. lis chemical com-
position rs similar to Ihe composition of leaves.
Il contains little sigar (less than 10 g/kg) and
an average acid concentration (180-200 mEq/kg).
These acids aie in Inc form of salts, die k> the
large q nan lily of cations present
Sulks arc rich in phenolic compounds. They can
contiin up to lift of lie total phenolic compound
coKcilration of lie grape cluster, even tbotgb
ihcy repicsent a lower proportion of He lotil
weight These phenolic compounds arc more or
less polymerized aid have a very aslringcni taste
The stalk attains irs dctinilive si/c around the
iriK of vfrtiison. Although il loses most of irs
chlorophyll, it remains green during maturation. It
is often completely lignilicd well after maturity.
The seeds represent 0-tV* of Ihe weight of the
berry. They conuin carbohydralcs (35'* on aver-
age), mitogen compounds (around (ft-) and min-
erals (4'i| An oil can be extracted from the seeds
1 15-20* of the lotal weight) whkh is essentially
oleic and linokic acid The seeds aic an important
source of phenolic compounds diring red wine-
making Depending on the varieties, they contain
between 20 and 55'* of the total polyphenol of
Ihe berry.
The seeds attain their definitive sis before
rtfmisim. Al this time, they have reached phys-
iological maturity During maturation, the tmnin
concentration of the seeds decreases whereas their
degree of polymcrizaliot increases. Conversely.
Ihe nitrogen compounds are partially hydiolyzcd
The seed can yield up to one-fifth of its nitrogen
lothc pulp, while still remaining richer in nitrogen
lhan Hi! ' 'in i" solid parfe of the grape cluster.
Depending on ihe giape variety, the skin repre-
sent from 8 to over 2iYf of berry weight. Being
a heterogeneous tissue, it importance depends
gtcatly on the extraction method nsccl. Scpatal-
ing the skins by pressing the grapes icsulls in the
extraction of the pulp and seeds. This method cor-
responds best to cuncnl cnological practices. The
sugar concentration of skin cells is very low For
the same weight, the skin is as rich ii acids as the
pulp bnt citric acKI is predominant Malic acid, in
significant quantities in Ihe skins of giccn giapes.
is .n : lively metabolized in Ihe ionise of maturation
The majority of tartaric acid is estcrilicd by pheno-
lic acids iiafcie. coumaric). A significant qnantity
of cations cause the salillcalion of these acids The
contents of the skin cells always have a higher pH
lhan Ihe pulp.
:_,,
Handbook of Etiology: The Microbiology of Wine anil Vindications
The siin is especially charactcri/cd by signif-
icant quantities of secondary products of major
etiological importune (phenolic compounds aid
aromatic substances). II accnnmlatcs these sub-
sCinccs during maturation.
Tbe following phenolic compounds arc present
in Inc grape skin at maturity: benzoic and ciinamic
ackt. flavonok and tinnins. They arc dislributed
in the cells of ihc epidermis and Ihc liist sub-
epidermal layers in both while and red grapes. In
addition, ihc red grape skin contains anthocyanins.
essentially located in Inc bypodcrmal cell layers.
Exceptionally, in certain years, the celts adjacent
to the pulp can be colored. The pulp itself is col-
ored in the case of TcnturKr varieties and sonic
American vines or direct producer hybrids. Antho-
cyanin composition varies from cultivar to cultivar.
depending on the anthocyanidin substitution and
■ctciDsidic nature of the cultivar (sec Volume 2.
Section 62.3).
The ripe grape skin also contiins considerable
anion ii is of aroma lii suhsGinccs and annua pre-
cursors In ccrtiin muscat varieties. Ihc skin can
contain more than half of the free tctpcnols of the
berry (Bayonuvc. 1993) Other chemical families
of aromatic substinccs may also be contained in
the skin. Finally, tbe skin is covered by cpKnlicu-
fctr wax. essentially conslituKd of okauolic acid.
All of this information is very important from a
technological paint of view. All methods increas-
ing Ihc solid- 1*| s id con tic t for color extraction
or aroma dissolution shouM be favored during
wincmaking.
The palp represents the most considerable fmc-
tioH of the beny in weight (from 75 Id 85'iJ.
Tbe vacuolar contents of the cell contiin the grape
uiusi — the solid pans (cytoplasm, pccloccllulosic
cell walls) constituting less than \'t of this tis-
sue. Tbe must is a cloudy liquid, generally slightly
colored, having an elevated density due to the
many chemical substances that it contains. Sugars
ate Ihc primary constituents— essentially glucose
and fructose. ErucMsc is always predominant (the
glucose/fructose ratio is around 09) Saccharose,
which is Ihc migratory form ol sugar in the plant,
exists in only trace amounts in the grape Other
sugars have been identified in the grape: arabinosc.
xylose, rhamnosc. maltose, raffinosc. etc. (see v. -i-
n me 2. Section 3.3.1). The reducing sugar concen-
tration in normal ripe grapes varies from 150 to
240 g/l.
Most of the acids of the metabolism arc found
in trace amounts in ripe grape pulp (pyruvic.
D-kcloglutaric. fnmarK. galacinronK. shikimic.
etc) Must acidity, an important clement of
cnotagKal dati. is essentially constituted by three
acids tartaric, malic and citric acid (Volume 2.
Section 12.2). It can vaty from 3 to 10 g/l in
sulfuric acid or from 45 to 15 g/l in tartaric acid,
depending on Ihc cultivar. the climate and grape
maturity. Phosphoric acid is the preponderant
inorganic anion.
The pulp is particularly rich in cations. Potas-
sium, the principal element. Is much more abun-
dant than calcium, magnesium and sodium The
other cations are present in much knvcr concentra-
tions, with iron representing 5tfi of Ihc remaining
cations Concentrations of metallic trace cICBtcntS
such as lead arc infinitesimal, except in the case
of accidental pollution In spile of this concentra-
tion in cations, part of the acids remains unsalilicd.
Must pH currently varies between 28 and 35
The pulp contains only 20-254 of the total
nitrogen content of the berry The must conttins
40-220 dim of nitrogen in its aininoni-ic.il or
organic form. The ammonium cation is the most
easily assimilable nitrogen source for yeasts and it
is often present in sufficient quantities (Volume 2.
Section 5.22) The amino acid fraction varies from
2 to 13 mvi in leucine equivalents (2-8 g/l). Most
amino acids arc found in grape must al variable
concentrations, and a feu ot ihcmt proline. arginine.
threonine and glutamic acid) represent nearly 90C*
of the total concentration. The relationship between
the must amino acid concentration and its organic
acid come ni ration has been known fora long lime.
The most acidic grapes ate always the richest in
amino ac ids Soluble proteins of Ihc iiiusl represent
15-I00mg/I.
Al maturity, the grape is characterised by a law
concentration in pcclK substances with respect to
other fruits Pectins represent from 0.02 to "«■•
of fresh grape weight. Differences from cultivar to
cultivar and from year to year can be significant.
The Grape and )K Maturation
:-r
Only Ibc Tree pcciic fraction, associated to diverse
soluble oscs. is likely b> be found in must This
fraction also contains small amounts of insoluble
proteins
The skin is considered to be the principal source
of aromatic suhscinccs. bnt the pnlp docs contain
significant concentrations of these compounds. In
certain muscat varieties, the must can conciln up to
two-thirds of the Icrpcnol hetcrosides The pulp is
characterized in particular by the accumulation of
a diverse variety of alcohols, aldehydes and esters
which participate in grape aromas
There is considerable heterogeneity between dif-
fcrcnl grapes on the same grape cluster Similarly.
the diverse constituents of must arc not evenly
distributed in the pulp. Asa primary technological
consequence, the chemical constitution of the juice
evolves in the course of grape pressing in while
wine making. The peripheral and central nines
l near the seeds) arc always richer in sugar than the
Intermediary /one of the pulp (Figure 105). MolK
and tartaric acid concentrations Increase towards
the Interior of the berry Potassium is distributed
differently within the grape and often causes the
salilication of the acids, with the precipitation of
potassium bitartrate.in the course ol pressing This
heterogeneity seems to apply lo all must con-
stituents Finally, the half of the grape opposite
E-ifi 10.5. Breakdown <■( principal .ohm it
cxprcuctl in m.- perg in»k uciybll (a) n
Handbook of linology: The Microbiology of Wine anil Vraiikaliuna
(he pedicel is generally richer ■■ sagars and poorer
in .kills ihan ihe proximal half.
10.3 CHANGES IN THE GRAPE
DURING MATURATION
10.3.1 General Characteristics
of Maturation
As early ;is 1897. daring his studies on grape n-v
pi ration. Gcrbcr discovered a icspiraury substrate
change in berry pulp al ve'niisim These obser-
vations were lalcr continued by Ihe use of l4 C-
marked molccalcslRibcrran-Gayon. 1959: I960).
Al present, ihe dominant role of niolK acid in the
metabolism of ripening frail is Tally established
(Rnffucr. 1982a).
Most of the primary mcubolK pathways have
been clncidalcd through progress in Ihe cxlrac-
lion and slndy of rniRnns cn/ymalK activities.
High-performance analytical mclhods. capable of
determining nanograms <if volatile sn tetanies. ;irc
currently being developed and shot hi be able to
provide much supplemental Information on the
secondary metabolism of grapes in coming years.
The biochemical processes of maturation have
traditionally been summarized by the transforma-
tion of a haul, acidic green grape ink) a sofl. col-
ored fruit rich In sugar and aromas. As already
indKakrd. these transformation can only occur
when Ihe grape is attached lo Ihe rest of Ihe plant.
In Ibis case, the increase in the concentration of a
substance in Ihe berry can be die lo importation of
this substance, on- location synthesis or water loss
in the vegetal tissue Conversely, ils diminution
can rcsalt from exporEilion. degradation or water
gain in Ihe tissue.
During maturation, the grape accumulates a sig-
nificant quantity of solutes, principally sugars.
In spile of berry enlargement (cellular enlarge-
ment). Ihe percentage of solid maKrial increa-
ses — indicating that the solutes air Imported In
greater quantities than water The amoani of walcr
thai accumulates each day in the grape rs the
snm of the phloem (elaborated) and xylcm sap
flux minus the water loss due lo transpiration
(Figure 10.6) At the start of maturation, the berries
FtA I "- G «*r beny
simaltancoasly Import walcr with the sugars, but
the amount of water transpired rapidly dimin-
ishes as the stonuta degenerate, then, transpiration
uniquely occais actoss the cuticnlar wax Sugar
accumulation then occais against the diffusion gra-
dient, of Kb up u considerable concentrations cor-
icsponding to a sabstantial osmotic pressure In
addition. Ihe xylem solute supply strongly dimin-
ishes after Tpw/irw. This phenomenon, due lo
a partial vascular blockage (or embolism), has
an impact on the accumulation of certain sub-
stances, especially minerals Peripheral vessels
(Figure 10. Lit then become responsible for most
of ihe food supply to the grape.
The grape is more Ihan an accumulation organ:
It maintains an intense activity (respiration and
biochemical transformations) during maturation.
Veniison also corresponds to the synthesis of new
eii/jme activities and the release of inhibition
of other ones. These variations in gene expres-
sion cause profound changes in grape inctiMism
(Robinson and Davlcs. 2000).
The Grape and Its Maturation
10.3.2 Sugar Accumulation
The iii'si spectacular tiologica pber
maturation is ccnainly ihc rapid accumulation of
sugars in ihc grape from vemistm onwards From
the start, the inflorescences, due K> their growth
hormone concentrations, have a strong demand lor
Ihc products of photosynthesis However, during
Ihc entire herbaceous growth phase. Ihc sugar
concentration of green grapes does not exceed
10-20 g/kg in fiesh weight (around the same as
leaves). The sugars imported daily arc mcEibolized
at a high intensity for fruit development but in
particular for seed growth and maturation. The
nutritive substance demand uwards the grape is
even more considerable in the days that precede
249
The depletion of growth hormones, notably aux-
ins. and the increase in abscisic acid concentrations
correspond with Ihc lifting of Ihc inhibition of the
principal enzymatic activities involved in Ihc accu-
mulation of sugars in pulp cell vacuoles. Saccha-
rose phosphate synthetase, saccharose synthetase
and hexokinase are no longer blocked (Ruffncr
flat., 1995). This accumulation occursagainsl the
diffusion gradient The transport requires energy
to counter Ihc growing osmotic pressure as the
sugar concentration increases. Up to 30 burs of
pressure can be attuned uwards the end of matu-
ration An cn/ymatic complex associated with the
lonoplast of the pericarp cells ensures this trans-
port (Figure 10.7). The sugars, synthesized in the
leaves, migrate exclusively in the form of saccha-
rose through the phloem to Ihc grapes (Lavcc and
;::-'-
Fuji 10.7. Biochcm
return* a of ui£4r fcnciotfio
250
Handbook of linology: The Microbiology of Wine anil Vinifications
Nir. 1986). A litst invcttasc. linked lo the plas-
■iK membrane, hydrolyacs (he imported saccha-
rose iiio glucose and fructose The free sugars air
Ihcn phosphory kited by ihc cytoplasmic hexoki-
■asc. After the formation of UDP-glncosc. Ihc sug-
ars combine again lo form saccharose phosphate
with Ihc help ol a saccharose phosphate synthetase.
The energy accumulated in this molecule and liber-
ated by a saccharose phosphate phosphatisc linked
u Ihc touoptasl permits the accumulation 01 sugars
In Ihc vacnolc. These enzymes maintain a sufli-
ileat activity during Ihc entire maturation paxes*
u accumulate a maximum of 23 niniol of sugars
per hour per berry. However, lie direct transfer
of cytoplasmic bexoscs via lonoplasiK transporters
cannol be excluded (Robinson and Davics. 2(«')>.
Profound changes in Ihc mctibolic parkways
also occur al tvriastw. lacilititing the storage of
imported sugars. The study of respiration evolution
during grape development provides information on
these changes The respiratory intensity increases
in proportion to cellular multiplication during
Ihc first growth phase It then remains relatively
sable until maturity (Figure 108). It docs not
increase during maturation, as in many other
fruits. The most active respiratory sites simply
change location. He tore nVn/irw. the palp and in
particular ihc seeds are primarily responsible for
respiration, but during maturation ihc respiratory
activity is highest in the skins. TV respiratory
quotient (ratio between the carbon dioxide released
and Ihc oxygen consumed) changes at vfriiisoii.
indicating a change in the respiratory substrate .
During the entire herbaceous giowlh phase, the
respiratory quotient remains near I .
In reality, the respiratory quotient of the pericarp
of green grapes is slightly higher than I. whereas
it is near 0.7 for the seeds. Seeds arc rich in
•v^
l'i(i 10.8. Kvoknba of rtipirariua duriap. gape deve
upmenr Olairu mi. 19711 -. tcbplmory qinrlci
t . mpinirurY i Measly
falty acids, which are mostly likely their respiration
substrate. In Ihc pericarp, on Ihc other hand, this
quotient results from Ihc combustion of sugars,
primarily, but also organic acids (Tabic 10.2).
After veroisim. Ihc rcspiraloiy quotient increases,
teaching 15 towards the end of maturation. On the
whole, it can logically be considered that Ihc grape
essentially uses organic acids as its respiratory
substrate during maturation
Supplementary information on metabolic path-
way modifica lions is provided by observing the
evolution of the glicose/fnclosc ratio during
grape development. Saccharose is Ihc principal
transport form of photosynthesis products: this
ratio should be near I in Ihc grape where these
products accumulate Yet in the green grape al
the stui of development, glucose predominates
and represents np lo S5'4 of grape reducing
Tabic Ifl.2. Ru
mpktc
C*lli,O*-t0O,
:. :ll„l ■ ■ SO,
C«H|O,-i-45 0,
C,H»0, -t 25 O,
C,iH*0*-f2eO*
-OCOj-t-O 11,0
- 4 COj + 3 HfO
-OCOi-f 1 HjO
- 4 COj + 3 HiO
- 18 CO, - 18 H,0
The Grape and in Maturation
251
sugars This ratio, near S. decreases to 2 al
reritisim and ihcn to I at the beginning of
maturation It Ihcn remains relatively constant until
mam my (between I.D aid 0.9). Since glucose
is more likely to enter into cellular respiration
than fructose. Ihc latter piefcrcu ttdly cniers into
cellular synthesis reactions. Tils phenomenon
explains ihc elevated glucose/fructose ratio during
the herbaceous giou th phase of Ihc grape and lis
decrease afler w'rttistm. related lo a slowing of
biosynlhctic activity
10.5.3 Evolution of Organic Acids
From the stirl lo Ihc end of its development, the
grape contains mosl of the acids involved in the
glycolytic and shikimic acid pathways as well as
in the Kicbsandglyoxylic acid cycles. This aitcsls
lo Ihc fanctioning of these different pathways.
However, their concentrations ait generally very
knv. TartarK and malic acid represent on average
W* of the sunt of the acids. These two acids
arc synlhcsi/cd in the leaves and grapes, with a
majority produced in the grapes prior to veraison.
There Is no formal proof of Ihc transport of these
diac ids from the leaves to the grapes (R ■finer.
1982 b)
In spile of their chemical similarily. these
two ackls have very different metabolic path-
ways. Their evolution Is not identical during
grape development and main ration. The malic
ackl/tartaric acid proportion varies considerably
according to the grape cullivar and the maturation
conditions.
The grape Is the oily cultivated fruit of Euro-
pean origin lb.it accumulates significant quanti-
ties of tartaric acid. Specifically, the /.-(+! tar-
taric acid stereoisomer accumulates in the grape,
attaining 150 nm in Ihc must al veraison and
from 25 to 75 mm in the must at maturity (3.8-
llJg/1).
This acid is a secondary product of the mcia-
bolisni of sugars. In fact, there is a significant
lag line before obtaining radioactive tartaric acid
from Ihc incorporation of l4 COi in Ihc leaves. Thus
phenomenon only occurs in the presence of light.
Ascorbic acid is considered to be Ihc main inter-
mediate in the biogenesis of tartaric acid and small
quantities are still present in ripe grapes. Even
■hough the ascorbic- tartaric acid transformation
is well understood at present, the origin of ascor-
bic acid is not known with certainty — in spite of
all the research carried out over the List .10 ycais
Two btosyilhctic pathways of ascorbic acid appear
to exist: one is dependent on plant growth, the
other Is not. The kinetics of Cirlaric acid during
grape development and maturation are consistent
with this dual pathway hypothesis. The hcrbuccous
growth phase is characterized by a rapid accumu-
lation of urtaric acid, related lo intense cellular
multiplication. During maturation. Ihc tartaric acid
concentration remains relatively constant in spile
of the increase in berry volume. A small amount
of this acid is therefore synlhcsi/cd during this
period. Convciscly. there is no formal proof of ils
calabolism during maturation. The small variation
in levels seems rather to be related lo ihc plant s
water supply.
Malic acid Is a very active intermediary product
ofgrapc metabolism. The vine contains the /.-<—)
malic isomer The viae assimilates carbon dioxide
in thcairbyaC, mcchanismtRnlTncref «(.. 19B3).
In this manner, during the dark phase of photosyn-
thesis, the leaves and young green grapes fix CO?
on ribulosc 15-diphosphatc to produce phospho-
glycctic acid, which condenses to form hexoscs
and may also become dehydrated into phosphocnol
pyrnvic acid. CO;, catalyzed by PEP carboxylase,
is fixed on this acid to form oxaloacetic acid, which
is. in turn, reduced into malic acid.
The significant malic acid accumulation during
Ihc hcrbuccous growth phase of the grape <up
lo 15 mg/g fresh weight-about 95 nmol/bcrry) is
due in pari to this mechanism, but a non-ncgligibfc
proportion results from its direct synthesis by the
carboxylalion of pyruvic acid. This reaction is
catalyzed by the malic enzyme, whose activity is
very high before veraison.
In any case. Ihc imported sugais arc the pre-
cursor of the malic acid found in grapes. The
malic ackl is produced by cither catabolic path-
ways (glycolysis, pentose phosphate pathway) or
by p -carboxylalion
Grape maturation is marked by an increase in
Ihc respiratory quotient, which suggests Ihc use of
252
Handbook of linology: The Microbiology of Wine and VnifkalkMS
ihnacid for energy production )■ the grape (Harris
el al.. 1971) li fact, during maturation. DialK acid
takes on Ihc role of an energy vcckirlFlgarc 10.9).
During ihc herbaceous growth phase, (he sugars
coming from phoktsyn thesis aic iransforaicd into
malic acid, which accumulalcs in ihc pericarp cell
vacuoles (ihc grape being incapable of stocking
significant amount of starch, as many olhcr fruits
do). Al ven ii son. due to ihc severe inhibition of
the glycoly lie pathway, malic acid importilwn
froii Ihc vacuole permits encigy production to be
maintained. Tic aclivalion of a specific permease
ensures ihis liunspon. The tie now synthesis
of diffcicni malalc dehydrogenase isoenzymes
support* i his hypothesis
In order to maintain a normal cytoplasmic pH
value when energy needs drop (al night, or al a
low Icnipcralaic). the excess imported malic acid
Fit; Hi" Ri>k al milk acid la ihc piodiclkio n
grape . HulliKf. 1982b i MIKI . mulaic dc hydm^aj
PEPCK. phnpbncn>lpym>ilc cjiboivkiuac
cflcaiy (ATP) ind Ihc fomulbn of .iilkicnt uiburaid in the
: ME. milk enzyme; PEPC. phaspbocnulpyaiMic ij*o.vl»«:
The Grape and us Maturation
253
Is eliminated and transformed into g Incuse by glu-
o "i-i genesis PEP carboxy kinase dccarboxylalcs
pan i>f lac oxaloacetic acid formed Glucose is
then formed by Ibc inverse glycolytic pathway.
This gluconcogcncsis is particularly elevated dur-
ing Yemistm. bui Ihc amouni or nudK acid trans-
foniKd into glucose decs not exceed 5'* of the
Mucked ".'.-.ili- acid, it less than 10 g/1 glucose.
The presence of abscisic acid in Ibc grope incirascs
Ibc enzymatic activity ofgluconcogcncsis (glucose
6- phosphatase, line Rise 1 .6-diphosphalc. maloK
dehydrogenase) (Palcjwalaeidf. 1985).
Malic acid can also be dceartioxylatcd by Ibc
malic cn/ync. lis affinity constant*, which arc
different after reriihmi. invert the activity (Ruffncr
ei <il.. 1984). The pyrovic acid formed also contri-
butes Ri energy production.
During the herbaceous growth pbase. tartaric
and malic acid essentially ptty an kuk role
Cation importation and prolon consumption during
metabolic icactiots impose organic acid produc-
tioi from sugars This KuK regulation seems b>
occur indifTcrcnlly with the help of cither malic
or tartaric acid. Consequently. Ihc sum of these
two acids is rcbiivcly cousttnl at rertiii&n from
one year to another for a given cullfvar. In spile
of Ibeir close chemical similarity, these two acids
behave very differently in the course of matura-
tion In spile of their chemical similarity, these
two acids behave very differently during ripening:
the tartaric acid content of grapes varies very lit-
tle white that of malic acid follows Ihc decrease
in lotd acidity. At ■laiuriiy. the sum of these
two acids is highly variable, depending on vintage
conditions.
10.3.4 Accumulation of Minerals
Poussium is one of Ihc rare minerals transkicaKd
by Ihc '.'M. i in sap In Ihc phloem, it permit Ihc
translocation of sugars derived from photosynthe-
sis Consequently, during maturation, ihc potas-
sium concentration in Ibc grape increases with
icspecl to sugar accumulation kinetics (Schallcr
el of., 1992).
The xylcm sap translocates most other cations
in relationship m Ibc amount of water transpired
by the grape. Yei transpiration inlcnsily strongly
diminishes alter rfrinum because of grape skin
modifications and sttmata degeneration. Mosl
often, calcium accumnlation ceases al Ihc start of
maturation because of ibc above grape modifica-
tions (Doncchc and Chardonncl. 1992). This phe-
nomenon is Klcnlica) for magnesium, builo a lesser
degree. Consequently. Ihc calcium and magnesium
concentrations per liter of juice dccieasc most of
the time during maturation
Being a natrophic pfcml. Ihc vine accumulates
little sodium. This permits a ccrctin level of icsis-
lancc in sally soils. The concentration of metal
trace elements (Zn. Cu. Mn. eic I is likely lo
decrease during maturation. The inorganic anion
concentration (sulfates, phosphates, chlorides. etc.)
continues Ri increase with the cation concentration .
but ihc incorporation of phosphates, as with mag-
nesium, has often been observed to slow during
rtfmisiHi (Schallcr and Lohncrlz. 1992).
The distribution of minerals in the grape berry
is not insignificant and it has an impact on the
composition of musl at maturity. Potassium is
essentially kxaKd in Ihc pulp cell vacuoles, bul
Ihc skin cells also sometimes conttin significant
amounts.
In theory, the sum ol the at ids and cations deter-
mines must pH. However, in hoi years, it depends
mainly on the tartaric acid and potassium con-
centrations, according R> the following relationship
(Champagnol. 1986):
pH=f.
tartaric acid]
1 10. 1 1
10,3,5 Evolution or Nitrogen
Com pounds
The grape nitrogen supply depends on both the
phloem and xylcm saps In these two cases, ni (rates
are rarely involved. They ore oily present in small
quantities because of their reduction in the rood
and leaves
Nilrogcn transport lo ihc grape essentially occurs
in the form of ammonium cations or amino acids
Clulaminc represents about 5i¥i of Ihc oiganic
nilrogcn imported.
254
Handbook of linology: The Microbiology of Wiac anil Vindications
There ate mo intense nitrogen incorporation
phases during grape dcvclopaKnt: Ihc first follow-
ing berry set. aid the second .Stirling al venuum
and llnishing ai mid- niata ration. Towards (be cad
of uiatarily. lie toul niliogcn concentration may
increase again. As a result, al harvest, half of
Ihc niliogcn in Inc vegetative pun of Ihc pfcinl is
slocked in Ihc grapes < Roubclakis- Aigelakis aid
Klicwcr. 1992). In unripe fmil. Inc ammonlam
calion represents more than half of Ike util nilio-
gcn. l-roni wrinum onwards. Inc aninionium con-
ccnlralion decreases whereas Ihc organic fraclion
increases. The free amino acids iKirasc by a fac-
tor of 2 lo 5 during maturation, altaining 2 -8 g/1
in leucine equivalent. Al maturity, the amino acid
fraction represents 50- l Xff of ihc lotil nitrogen in
grape Juice.
The iKorporalioa of lac aninionium cation oa
a-kctoglutarK acid appears to be Ihc principal
nitrogen assimilation pathway by Ihc grape. It is
catalyzed by glatimlnc synthetase (GS) aad glnla-
nalc dehydrogenase (GDH) enzymes. Other amino
acids arc synthesized by Ihc transfer of nitrogen
incorporated oa glulaailc acid.
Research carried out by numerous authors show
thai even though the annuo acid composition
vanes greatly, depending on conditions, a small
nambcr of amino acids predominate: alanine. "-
aminoburyric acid, arginlnc. glutamic acid, proline
and threonine
Al maturity, arginiac R often the predominant
amino acid and can represent froai 6 in 44*4 of
Ihc total niirogca of grape jnlce. In fact, this amino
acid plays a very important role in grape berry
nitrogen metabolism (Figure 10.10). A close rela-
tionship exists between arginiac and diverse amino
acids (ornithine, aspartic and glatimic acid, pro-
line). As a result, the proline coaccnlratWn can
increase during maturation by a factor of 25-30
through the transformation of argininc. Moreover,
aspartic acid constitutes an oxaloacetic acid reserve
which, depending oa the demand, can be trans-
foratcd into malK acid or into sugars during
maturation
Maturation is also accompanied by an uciive
prolcosyn thesis. The soluble protein conccnlralion
reaches its maximum before coaiplclc maturity and
i,nni. ' B <|"ilrcwlir. «|niu:
Kift 10. IO :■...■ k .■ I Jipiniot ,ii the ■Impel m.i.il-'l.'
ufpmpcifRuibchkU-Anttcbl.)*.. 1991)
then diminishes towards Ihc cad of maturation.
The concentration of grape Juice protein can thus
vary from 1.5 to 100 mg/l. The concentration of
high mokxuLir weight insoluble proteins, often
attached k> the cell wall, is high from the start
of development and continues to increase during
maturation.
The juice from mature grapes contains barely
2('f< of Ihc total berry nitrogen. The remainder
is rctiincd in Ihc skins and seeds, even though
Ihc latter arc likely to liberate soluble forms of
nitrogen t ammonium calKins and amino acids) in
Ihc palp towards Ihc end of maturation.
10.5.6 Changes in the Cell Wall
The softening of the grape daring maturation is
the result of sigailicant changes in parietal con-
stituent composition— notably al the cellular level
of the palp Cellular multiplication and enlarge-
ment during grape development and maturation
arc not accompanied by a proportional increase in
the parietal polyostdcs (Chardonnct end.. 1994).
Depending on Ihc varieties, cither cell wall deteri-
oration or a relatively constant purKlal polyosidc
concentration results, until the approach of matu-
rity The pulp leviurc differences between varieties
arc explained ia this manner
Al Ihc beginning of grape development, the cell
walls arc primarily composed of ccllatosc. The
«Wi.7.k>;i period is characieri/rd by considerable
pectin synthesis to such an extent that it becomes
the majority polyosidc in some varieties (Silacci
The Grape and Ik Maturation
:ii
ami Morrison. 1991)) Like a cement, pectins
ensure cellulose titer cohesioa Tbcy arc formed
by the polymerization of galacturouK ackl and
diverse neuiral -oscs (rkaninosc. gaLicur*.- and
arabinosc). A high percentage of the acid functions
of gatacturoaK acid mills arc mclbykilcd.
Maturation is accompanied by a solubilization
of these pectins under (he Influence of several
factors, First, pectin mclhyl esterases (PMB) liber-
ate the acid functions of galacluroaic acid, result-
ing in lac augmentation of the grape methanol
concentration. Cell wall hydration, characterized
by swelling, is Inns facilitated by increasing the
K'yCa 2 ' rabo (Possncr and Klicwcr. 1985). As
a result, the pectins are less chelated by cal-
cium: the free acid fanctions of the galaclmuaic
icstdaes arc the site of attack by other enzymatic
ac II vi tics— poly galacturonascs and pectin- lyases.
Al though pectin methyl cslcrases arc prcscnl in
majority in the grape skin, all of these enzymes arc
ills.- active in the pulp. This explains the diminu-
tion of total pec (ie subsCiaccs during grape mat-
uration. This phenomenon is accompanied by an
increase of the soluble peclic fraction which is
later found in mast The pulp cells arc solubiliord
first At the end of ripening, variable proportionsof
pcclinolytic enzymes are located in the grape skins.
At maturity . the grape is characterized by a tow
pectin conccnlralioa with respect to other fruit..
10,3.7 Production of Phenolic
Compounds
One of the most remarkable characteristics of mat-
uration is the rapid accumulation of phenolic pig-
ments, which give the red grape its etiological
importance These phenolic pigments arc second-
ary products of sugar diabolism Their biosyn-
thetic pathways arc present and partially active
right at the start of grape development
Phenolic compounds denied from a simple unit
k> a single benzene riag arc cicatcd from the
condcnsalKia of crylhrosc 4-phospbalc. an inter-
mediary ppxluci of the pcnlosc phosphalc cycle,
with phosphocnol- pyruvic acid. This biosynthctK
pathway, known as the shikimic acid pathway
(Figure |0. 1 1), leads Id the production of benzoic
c acid. as well as aromatic ammo acids
tPHE. TYR). The condcnsaiwa of three acetyl
coenzyme A molecules, derived from Krcbs cycle
reactions, also leads to the formation of a benzene
ring. The condensation of this second ring with a
cinnamic acid molecule produces a molecule group
knowa as the flavoaoids These molecules possess
two ten /en c rings foiacd by a C« carbon chain,
most often in an oxygenated helcrocyclic form
Various uaasformalions tnydroxylatiou. mcthoxy-
laiion. cslc rit leal ion and glucosiditication) explain
the presence of many substances from this family
in the grape (sec Volume 2. Section 6.2).
In tncse metabolic pathways, phcaylalaainc
amnion toryasc (PALI is lac en /vine, which, by
eliminating the Nil, radical, diverts phenylala-
nine from protein synthesis (primary metabolism I
lowaids the production of Inms -cinnamic acid and
otner phenolic compounds. PAL is located in g rape
epidermal eclbt as well as in the seeds, k- maxi-
mum aclivit)* in the seeds occurs during the herba-
ceous growth phase, its activity then decreases
after w'roisim to become very low during matu-
ration PAL activity contained in the grape skin
is very high at the start of dcvclopatciit. then
decreases np to rfrttistni. In colored grapes. PAL
aclivit)* in the skins increases again at the start of
r&misim. There is close relationship between its
activitj* and the color intensity of the grape (Hraz-
dina el id.. 1984). Chakone synthetase is the first
specific enzyme of the fktvonoid syntheses path-
way (condensation of the two rings): its activity
strongly increases at the beginning of renown and
then rapidly decreases
The biosynlhctic pathways an active as early
us the start of grape development Consequently,
the ioliI phenolic compound conccnlralioa con-
tinues to increase during this period. The rapid
increase in tannin concentration at the beginning
of development, however, is followed by a slower
accumulation during maturation The biosynthesis
may therefore be less active than the increase in
ben)' volume
The i". -. ;■ .in ii.liiui tannins. derived from ftavanol
polymerization, attain a maximum concentration
in the seeds before rtntiwn. ThB then strongly
decreases to a lower and relatively stable value
Handbook of linology: The Microbiology of Wine anil Vindications
Fifi I'lii I!...
s fnihuiiys tif p he ■■> IK
wbci the seeds ;iic mature. AI teraium. the skin
tannin concentration is already high— sometimes
corresponding u over half of Ihc concentration al
maturity IF^nrc 10.12).
In while grapes, (he concentrations of phenolic
acids cstcriflcd by tartaric acid. flavan-3-obi aid
ollgoitcric procyanidins are high at the beginning
of development They then diminish to minimal
concentrations al malnriiy
In colored varieties, the anlhocyanins begin to
accumulate in Inc skins about two weeks before
the color is visible. The concentration increases
dnring maturation . but. as with tannins, it attains a
maximum and generally diminishes at the time of
maturity.
This appearance of anlhocyanins Is linked to
sagar accumulation in lie grape but no dirccl
relationship has yet been established. Diverse
parameters, sach as sunlight, increase tie antho-
cyanin accumulalioa speed without alfecting the
skin sugar concentration (Wicks and Klicwcr.
1 983).
10.3.8 Evolution of AroMatk
Substances
Several hundred different chemical substances par-
ticipate in grape aroma. In this complex mix-
lire, bydrocurbides. alcohols, cslers. aldehydes
and other carbon- based compounds can be distin-
guished tSchrcicr <■»«/.. 1976).
Nearly all of the compoands identified at present
arc found in numerous varieties that do not possess
a particularly specific varietal aroma I -or example.
a trace of terpenk alcohols Is found in ncutral-
lasling varieties, ycl let concentration can alttin
The Gr.ipc ;md Its Maturation
Fig 10.12. Evolution ...t phci
aMhm-vaniiu: * . ricni unnini: |
o-fou-U (Dam. I<*9I> (r
iifiniicd la -,- , V dry ■■■■> - c -rt ■
3 mg/l in certain aninialK varieties (Gcwnr/Ka-
miner. Miscall.
For certain varieties, however, ihe characteris-
tic an * ■i.i is Ihe result or a limiKd mini her of
specific compounds in low concentrations (from
nanograms ki micrograms) The following com-
pounds and ihcir varicGd origins fall into ihis
category: clhylic and mcthylic esters of anlbronilic
acid in varieties issued from \$iii ItihnuciK aid in
particular Ihe Concord grape iSleri etal., 1967):
2-mcibo\y-3-isobutyl pyra/inc in the Cabernet
Sauvignon (Bayonove elal.. 1976): and +mcr-
capto-4- methyl pcitan-2-onc pa-sent in ihe Chcnin
varicly(Dn Plessis and Aignsiyn. 1981 land idcn-
lificd in Sauvignon Blanc (Darnel. 1993).
The grape aromatic potential is divided ink):
• free and volatile odorous substances:
• non-volatile and non-odorous precursors (glyco-
sides, phenolic acids and fatty acids):
• odorous or Boa-odomus volatile compounds
which by Iheir instability arc transformed inlo
olhcr odorous compounds (tcrpcnols. tcrpcik
diols. Cn norisoprcnokls. etc.).
Tcrpcnic compounds have been studied in par-
licular. Their biosynthclk pathway is schematised
in Figure 10 13. The first sKp produces mcval-
onic acid from glucose by the acetyl coenzyme
A pathway. This principal pathway is generally
recognized although another exists by the interme-
diary of amino acids snch as leucine or valine. The
second step prodKcs isopcutcnyl pyrophosphate
i IP!') from mevalonic acid. All of the terpenoids
arc built from thrsC> isoprcnic base unit. With the
help of the isopciiicnyl pyiophosphatc isomerosc.
IPP is isomcri/cd ink) dimethylallyl pyrophosphate
( DM APP). These two Isoprcnic uniti play an active
role in terpenoid synthesis. One IPP unil con-
denses with a DMAPP molecule wilh the help
of a prenyl transferase (hcad-uil condensation of
Ihe two molecules) to produce a Cio molecule,
gc ran y I pyrophosphate (GPP), which constitutes an
important junction in terpenoid synthesis. Prom
Ibis compound, the synthetic pathways can form
Handbook of Etiology: The Microbiology of Wiie anil Vindications
Fifi 10. IX Tetfc
either acyclic or cyclK monolcrpciotds or more
i omk-n.sc J terpens
The grape con rains maiy icipcnic- bused cont-
ponnds (sec \bluurc 2. Section 7.2). Tick
moaolcrpcnoids exist in a Tree stale aid in a
bound form of ;i bctctosrdrc nature. The bound
and free tcrpcnol concentration increases duriig
berry dcvelopnrcnl I ignrc 10.14) The Icrpcnic
■etc resides are abundant very early, when ihe
berry R still green 1250-500 u-g/kg in fresh
weight), whereas the free Kipcnols exist )■
only small quantities (30-90 |tgJkg in fresh
weight). Some are lot present at this stage [a-
tcrpincol aid citioucllol) but begin to appear
)■ significant amount from wriusmi onwards
tlinalol. for example) The bound fractions
outnumber the free fractions during tic entire
n til u ration phase and cvci increase beyond
maturity, whereas the increase in tic free fraction
slows and its concentration can cvci decrease
The conccnlrattoa of sonic tcrpcnoLs. snch as free
linalol and a-tcrpincol. diminish in this way duriig
The iir.i|v and in Maturation
Kift 10.14. Ave age cvnkiliin of lerpcnc i to Ink dui-
m^.' NUncul unpc malueubn I Hay.) bom. 1993): « , free
tcifcnc iikuhob.: ■ . bouaal terfene ukuhob.
ovcrripcnlng. This evolution seems *< indhatc thai
the Mocking of Ictpenol occurs for ihe until pail
ii a bound limn All of the Ktpcnols behave in
Ihis way will ihe exception of linutol. whose ficc
fraction sometimes remains stealer lhan the boand
fraction throughout main ration
Other aroma precursor compounds— carote-
i" 'its — arc well known loday (sec also Volume 2.
Section 73.1). These substances share the suite
origin as Ktpcnols but have a higher molecular
weight The caroKnoid concentration In the grape
bcrrj' varies fioni 15 lo nearly 2500 |ig/kg in fresh
weigh i (Ra/nnglcs. 1985). The most important,
in decreasing order, arc lutein, /J-carofcnc. ncox-
anthyn and luKln-5.6-cpoxidc. These molecules,
generally enclosed in cellular oiganiics. arc csscn-
Itully located in the solid pan* of the berry: the
skin is two to three limes richer in caroKnoids
lhan Ihe pulp. Tic carotcnoids are found in dif-
ferent proportions in the different pins of the
berry, depending on their structure During mat-
uration, a decrease in Ike carotcnokf concentra-
tion and an increase in certain carotcnoid-dc rived
molecules such as norisoprcuoids arc observed
(Figure 10.15) The metabolic pathways in the
grape leading to the production of odorons sub-
stances such as nocisoprcnoids from camicnoids
arc not yet known, but carolcnotds arc known to
be sensitive to biochemical oxidation — resulting
in the production of ionone-typc molecules Some
nortsoprcnotds arc also found as glycosylated pre-
cursors (Volume 2. Section 7.32).
Information on other aromatic substances, speci-
fic to varietal aromas, is at present very limited.
According to Harriet (1993). the 4-mcrcaptr>4-
mcthyl pcntan-2-onc seems to evolve similarly
to free KipcnoLs. with a slight decrease in its
concentration towards the end of maturation
Conversely, the unripe grape contains a high
concentration of mcthoxypyra/incs (a few do/en
nanograms per liter) in certain varieties, such as
Cabernet Sauvignon (sec Volume 2. Section 7.4)
The concentration of these compounds drops
signilkrantly in the course of maturation. The
highest concentrations arc found in the coldest
maturation conditions (Laccy el ill.. 1991). They
develop in a very similar way lo malic acid
(Roujou dc Boubcc. 2000).
10.4 DEFINITION OF
MATURITY— NOTION
OF VINTAGE
10.4.1 State of Maturity
The various biochemical processes just described
arc not necessarily simultaneous phenomena with
identical kinetics. Environmental conditions can
modify eertun transformation speeds, sometimes
>,,,;,
Handbook of linology: The Microbiology of Win: anil Vinifications
vi ihc poinl of upsclliig ihc older or physiolog-
ical changes in (he ripening (ran DilTcring from
iVr<nK<n. which is a fully ik lined physiological aid
biochemical incidcil (Abhal end.. 1992). grape
maturity don mi constitute a piril.se physiolog-
ical stage. Yd diffcicni degrees of maturity can be
distinguished. B iologisis consider thai the different
parti of ihc berry reach maturity successively. The
seeds me the lirsl to altaii physiological maturity
(foe ability lo germinate) during the period pre-
cediag vt'inison Over scver.il weeks. Ihc pulp aid
the skin continue lo evolve through a maturation
pcocess simiikir lo senescence (alteration of Ike cell
wall, accumulation of sccoadary nictabolilcsl
In ciotogy. pulp maturity conrsponds to ai
optimal sugar/acid ratio, skin maturity is Ihc stage
al whkh the phenolic compounds aid aromalic
substances altiii a maximum concentration. These
two kinds of maturity can be distinguished, bit the
dissociation of Ihc cell wall fioni the skin iniisi be
.sufficiently advanced to permit easy extraction of
these essenllal constituents.
Consequently, the definition of maturity varies,
depending 01 foe objective For example. Ihc pro-
duction of dry while wines requires grapes whose
aromatic sibstinces ait al a maximal concentration
and whose acidity Is still sufficient. It certain sit-
uations, an early harvest can be interesting Con-
vciscly. when the elaboration of a quality red wine
is desitcd. grape development mist be lei) to con-
tinue to obtain the most easily ex tradable phenolic
compounds
In general, grape maturation results from several
biochemical transformations thai arc not necessar-
ily related lo each olhcr. To simplify matters, the
increase ii sugar concentration and the decrease
in acidity ate monitored. The accimlalion aid
refinement of while grape aromas and phenolic
compounds in red grapes should also be taken iito
account. The essential pfopcrty of a quality winc-
pcoducing area Pi lo permit a favorable maturation.
This corresponds with a harmonious evolution of
the various transformations lo reach the optimum
point simultaneously al the lime of the harvest
In too cold of a climate, the maturation cannot be
satisfactory, but in very warm climate Ihc increase
in sugar concentration can impose a premature
harvest even though the other grape constituents
are- nol al full maturity. Of coarse, environ menial
conditions (soil, climate) arc involved in Ibese
phei
10.4.2 Sampling and Study
of Ma (ura (ion
Monitoring maturation poses problems relating lo
Ihc large variability of berry composition. When
precise data an: sought in order 10 compare the
diverse constituents of grapes from ok viicyanl lo
another, from one week to another or even one year
to another, grape sampling methods are of prime
importance Nothing is more heterogeneous than
grapes from Ihc same vincyanl al a given moment,
even if the same variety is considered
On a grape cluster, the grapes arc formed,
change cok>r and ripen one after another over a
period of up Id 2 weeks, or mote in certain dif-
ficult conditions Oi the same vine, the diffcicni
grape clusters arc never at Ihc same maturity level.
The clusters closest to Ihc trunk contain more
sugar than those at the extremity of the branches.
The ripest grapes aic in general the furthest from
Ihc ground, as the sap is preferentially conveyed
towards the highest aid into the longest blanches.
These differences are even greater when vari-
ous vincsmcks are considc ted — some viics always
develop more quickly than others II is therefore
risky lo determine the harvest dale from a single
vine sampled at random.
Due to Ibis great heterogeneity, a proper moni-
toring of the maturation of the same parcel requires
regular sampling ofasnflicicnl quantity of grapes:
15-2 kg. or about 1000- 20fX) grapes. A larger
number of samples are required lo ensure thai the
icsiIk are representative of the plot (Bmuin aid
Guimberlean. 2000). The most common method
consists of gathering, wilb sbcais. three or four
grape cluster fragments from 100 viics. Grape
clusters under the leaves as well as those directly
exposed lo sunlight should be gathered, taking
them alternately f mm each side of Ihc row al dif-
ferent beigho on the vine. When sampling varieties
with compact clusters. Ibis method docs nol gen-
erally tike into account the berries located at the
The Grape and Its Maturation
261
interior of ihc cluster These hemes air often less
ripe than the others la this case, whole grape clas-
Ic is should be sampled. K> obtain a precise idea of
Ihc maturation level of the parcel.
In Ihc laboraury. ihc berries are separated,
counted and weighed The juice is extracted with
Ihc help of a snail manual press or a centrifugal
I'm! Juice separator. The juice volume is measured
and the result, are expressed per liter of must
The juice sugar and acKI concentrations arc ihcn
determined.
The study of red grape phciolK compounds
requires Ihc manual separation of Ihc skins from
Ihc seeds of about 200 berries taken at random in
Ihc sampling Once separated, the skins and the
seeds arc dried aid lyophili/cd to facilitate the
extraction and the dcKinti nation of their phenolic
con lent.
Aromatic substance monitoring, noubly of
while grapes, requites the maceration of the solid
grape i'iii.s with Ihc must beforehand. After a light
crushing of the grapes, this maccialion is usually
earned ont for 16 or 24 hours at a low temper-
ature under a carbon dioxide atmosphere. These
techniques require adapted equipment and cannot
yet be routinely monitored.
10.4.3 Evaluation of the State of
Maturity — Maturation Index
Grape monitoring during maturation helps vinc-
yaid manage isto set the nan est date and maxim i/c
Ihc eflicicncy of their harvest teams according
to the ripeness of diffcicnt cnllivars and divene
parcels.
Dclcrmining the grape sugar concentration is
essential. It is most often effected by an indiiccl
physical measure snch as nydromctty or refractom-
ctry. If Ihc Icmpcralnic rsioiat 20 C. a correction
is theoretically necessary, but has little effect on
Ihc sugar concentration. The results arc expressed
in varioas units, depending on Ihc instruments
used This does not facilitate the interpretation
of data originating from different wine- producing
countries (Bloain. 1992: Boulton rtof., 1995).
These assorted mcasurcmcni scales arc com-
pared in Tabic 10.3. The degree Occhslc corre-
sponds to the third decimal of the relative apparent
density (D) The relative apparent density per-
mits the evaluation of Ihc sugar concentration. The
degree Baamc is approximately converted to rel-
ative apparent density by the following formula:
'Baumc = 14432(1 - I/O). The degree Baamc
of a mast corresponds fairly well with the percent-
age alcohol, al least for values between 10 and
12. The degree 8rix (or degree Balling) gives the
weight of must sugar*, in g rams, per 100 g of mast.
In reality, it is a percentage of the dry matter in
must, measured by refiactomcliy or dcnsimclry
This measure is only valid from a certiin matu-
rity level onwards (15' Brix). Before this matu-
rity level, orcanic acids, amino acids and certain
precarsots of parietal polyosidcs can have similar
refraction indexes k> sugar and interfere with the
measurement.
In the same way. the relationship between must
density and alcohol conkrnl R always approxi-
mate, since sugar Is not Ihc only chemical must
constituent that atlcets density. This measurement
is more accurate in while wincmaking with non-
mucilaginous musts having few suspended parti-
cles The values obtained for mast from roiKrn
grapes arc inaccurately high. Moreover. Ihc estima-
tion of potential alcohol should take inio account
Ihc sa gar/alcohol transformation ratio. The figures
in Tabic 10.3 nsc the relationship of 1683 g/1 of
sugar prr liter for \'i alcohol— the official value
retained by Ihc EEC.
Empirical observation of the inverse variation
of sugars and acidity during maturation led to the
development of a sugar/acidity ratio, called the
maturation index This index is very simple but
it should be used with precaution, since there is
no direct biochemical relationship between sugar
accumulation and acMlity loss. More specifically, a
given gain in sugar docs nol always correspond
with ihc same drop in acidity. Thct ratio is
nol suitable for comparing different varieties,
since varieties exist that are rich both in sugar
and in acids. In France, this ratio b. calculated
from the must sugar concentration (g/1) and the
titration acidity expressed in grains of sulfuric or
tartaric acid equivalents per liter Other modes of
expression are used in other countries according
to the measurement unit used lo express the
Ibbfc 10.3. ("on.
Handbook of Etiology: The Microbiology o( Wine anil Vinific.uions
Relative
*PpaiCM
:wiy <3TC)
5u,«.
fotcniul ak-ohol
Igrt)
110 83 gat
MlfrjB'J for l*r
alcohol)
82.3
925
4."»
S3
I03A
62
: 14 1
OS
l'.VI
7-4
136 Jl
8.1
147*
S7
158.1
94
109.3
10.1
180.5
ID.7
191.9
114
203.3
a i
214*
128
2204
13.5
238.2
u :
249.7
US
I B37I
i ..ii:
1.0454
MUM
1.0538
1.0581)
1.0623
1.0000
1.0710
1.07S4
1.0798
1.0842
1.0880
1.0932
1.0978
1.1029
1.1075
1.1124
1.1170
1. 1219
1.1208
1.1310
1.1305
1. 14 10
1.1405
37.1
412
454
49.5
53.8
580
02.3
660
710
754
792
117.0
121.9
1208
I3in
136.5
I41/i
140
150
I0O
170
180
190
232
244
25.5
200
27.7
322
334
34.5
3208
1" 1
332.9
IIS
345.7
20S
(57.7
2IJ
sagar concentration Ii Germany, for example,
the Rilio obtained by dividing the 'OcchsuJ of
■in! by ihc acidity, expressed in tartaric acid, is
currently used
Attempts have been made )■ the pasl lo describe
(be st iic or malnrily. taking into acconnl the
respective variations of malic and tartaric acid
or the accumulation of cations, but none of the
indices developed have signilicautly improved the
evaluation of the maturity level II scenis sensible
lo take into acconnl the iidividnal variations of
each berry constituent separately.
More recently, researchers have focused oi the
evolution of phenolic compounds dnring matu-
ration, bui the technique of scparaling the skins
from the seeds is awkvvanl and exacting lim-
iting its practical applicability. There R now a
rapid whotc-bcrry grinding technique The grape
grinding is followed by a differential phenolic
contpoind extraction, in cither a pH 3.2 buffer
(compounds easily ex tractable) or a pH I buffer
(total potential in phenolic compounds). The den-
sity of the solutions obtained Is then mcasaicd at
280 mi Information on the lotal phenolic com-
pound concentration and Iheir cxlractabiliiy is thus
obtained
Unfortunately, no simple methods cnncnlly
exist that permit an aromatic substance malntation
index. Tasting the gtape remains, in this respect.
Ihc only avaifciblc criterion lor judgments bal this
docs not estimate the subsequent revelation of
other aromas
Micro-imagery by nuclear magnetic resonance
has recently been shown to give detailed informa-
tion on the chemical composition and degradation
level of grape cell walk (Pope et id . 1993) but
this technique will remain reserved for scientific
experimentation for a long-time.
The Grape .mil us Maturation
263
Fourier UuBstom inflated spectrometry, which
has recently been developed. should make il
possible Io assess grape quali(>' mote accurately
(Dubcrnct eliil . 2000). This method is easy b>
iiiptcmcii and tlocs only require prior filtration of
Ike samples. Il provides a satisfactory evaluation
of ihc potential alcohol, lolal acidity. pH. and
nitrogen conicnl. as well as Ibe color index Tor
Mack grapes, in a single operation. In addition u>
Ihls general analysis of Ihc grapes, it is possible to
detect the presence of ml (gluconic acid, kiccasc
activity, etc.) or fcmcnlaliou activity (lactic acid,
pytvvic acid. etc.). This new technique, however,
only gives reliable results after a long, laborious
calibration process using samples analyzed by
standanl methods.
10.4.4 I IT., i of Light on the
Biochemical Maturation Process
Three facto is have major roles in maturation dyna-
mics: light, heat and water availability. In general,
they affect vine growth and metabolic activity:
their action is well known Yet these also act
diicctly on grapes, and their elicits oa metabolic
pathways translate ink) changes in grape chemical
composition.
In esciblished grape-growing aiks. the avail-
ability or natural light docs not. in general. Until
photosynthctic activity and thus the overall func-
tioning of the pLmt In facl. photosynthesis is opti-
mum at a sun radiance (expressed in cinsleins.
E) of abont 700 E/m 1 *. Below 30 hfvrts. leaf
energy consumption is greater than net photosyn-
thctic piodKlion (Sman. 1973). In the absence of
clouds, sun radiance Is greater than 2500 Ei/nr.v
On cloudy days. Ihc radiance varies from 300 to
1000 fi/m'/v A icdnclion In photosynthctic activ-
ity can thnsocenr. resulting in a nutrient deficiency
in the grape However, in practice, certain vine trcl-
Itsing methods still cause radiant energy loss For
this reason, wine-growers shonld ensure that the
spacing between vine tows is in proportion to the
height of the foliage (06-08) and should avoid
leaf crowding by thinning unwanted shoob in the
center or Ihc canopy.
Light has a direct effect on (Viral induction
Grapccultlvur fertility dcpcndsgrcally on bnd light
exposure during this induction period
The c fleet, of sunlight on grape composition are
even more numerous and complex. In addition io
furnishing the enctgy for photosynthesis and still t-
lating certain lighl-dcpcndcnt mctibolic processes.
its radiant effect heats not only surfaces but also
the airsnrrounding vegetal tissue. Grape clusters
grown with little light exposure (shade grapes)
always contain less sugar and have a lower pH
and a higher total acidity and malic acid con-
centration than grape clusicis directly exposed to
sunlight Light Is also essential for phenolic com-
pound accumulation, and phenylalanine ammoai-
alyasc (Section 10.3.7) is a pholoinductive enzy-
matic system In normal conditions, this pho-
loaclivation docs not seem to be a factor that
limits coloration or phenolic compound concen-
trations in most varieties. Crippcn and Morrison
il'J86) showed that (he phenolic composition of
shaded and light-exposed grape clusters remained
Ihc same in Cabernet Sanvtgnon. Only certain sen-
sitive red varieties I AhmcnrBou Ahmcnr. Cardinal
or Emperor) may exhibit color deficiencies when
their grape clusters are not exposed to light In cer-
tain northern vincyanls. wines nude in climatically
unfavorable ycais arc always poorly colored.
The amount of light reaching Ihc grapes also has
an impact on the composition and aromatic quali-
ties of Ihc grapes. Exposure to the sun accentuates
the decrease in mcthoxypyra/inc content during
the ripening of Cabernet Sauvignon grapes. Con-
versely, partial shade preserves the floral a
in Muscat grapes.
10.4.5 Influence of Temperature
on the Biochemical Processes
of Maturation
Tempera m a- is one of the most important param-
eter of grape maturation and one of the essential
factor that trigger, it Temperature affects pnc*>
synlhclic activity, metabolism and migration inten-
sity in the vine Io action is not limited to the
period of grape development It. influence on bnd
burst and flowering dates also has important indi-
rect consequences on grape quality. It r. easy Io
264
Handbook of Enology: The Microbiology of Wine anil Vindications
understand thai the later Hie tripe develops, the
greater ine risk ihal ihc accompanying maturation
conditions will be on favorable
Crape growth aid development arc directly
affcclcd by temperature. High temperatures me
unfavorable to cellular multiplication. Daring the
herbaceous growth phase. Ihe optimum tempera-
ture is between 20 and 25 C During maturation,
lemperature affects migration intensity and thus,
indirectly, cell growth. Vine temperature require-
ments during this period arc around 2(1 C (Calo
el ill. 1992) Too high of a Icnipcralnrc. even for
a shon time, can irreversibly alter sagar accumu-
lation. Scpulvcda and Klicwcr (1936) found thai
Kmpcratures of -IOC during Ihc day and 20 : C
al night favored sugar accumulation in other parts
of the vine to the dclrinicnl of Ihc grapes, which
received only a small percentage (about 25'i).
with respect to the coMol (25 'C day/15 'C night).
As vines have difficulty growing and produc-
ing grapes below 10'C, lempcralnrcs above this
threshold arc known as "active temperatures" A
strong correlation exists between the nam of the
active temperatures during grape development and
the grape sugar concentration in a given location
This measurement permits Ihc evaluation of the
climatic potential ofa given location to ensure sati-
able grape maturation. Various bioclimatic indexes
have been developed lo evaluate this potential.
Crowing degree-days (Winkler. 1962) arc the
sam of the average dally temperatures above l<> C
from April 1st lo October 30th. a 7-itonlh period.
This sam Is often calculated using monthly aver-
ages. Initially established for classifying Califor-
nia into different viiiculiur.il zones, this index has
become widely used in other coanlries. The cli-
matic dati lor the month of October arc not useful:
In warm /ones, the grape has already been har-
vested: In cool roacs. Ihc average temperature In
October is often below lot. Furthermore, this
index docs not bike the duration of light exposure
Into account
ThcBranasHcliolhcmiic Product (8 ranaser rrf ..
1946) corresponds lo the formula X x H x 10".
where X is Ihc sum of the average active
temperatures above in c for the entire year, and //
a- presents the sum of the length of Ihc days for the
corresponding period. Vine-growing is practically
impossible when ihc product is below 2.6. This
index gives the most precise results for vineyards
established in cool temperate climates where the
end of Ihc period containing active temperatures
more or less corresponds with harvcsl time. In
extreme cases of warm climates, this period covers
Ihc entire year.
In order lo obttin a better correlation between
bioclimatic data and final grape sugar conccntra-
toons. Huglin (19781 proposed a hcltothcrniic index
(HI). This index tikes into account the maximum
daily temperatures over a 6-month period from
April Isi to September 30th In this relationship
S-p. »
HI = £ (|(ADT-IO)+(.y/jr-10)lx K)/2.
Vpfl t
<I0.2)
where ADT represents (he average daily tempcra-
larc. MDT (he maximum daily Icmpcratarc. and K
is Ihc day-length cocffkicn( — varying from 1.02
to 1 .06 between latitudes of 40 to 50 degrees. An
HI of around 14(1) is (he lowest limit for vine-
growing. This index has permitted (he specification
of Ihc needs of different varieties for attaining a
given sugar concentration.
The comparison of these dlffcrcm Indexes
(Table 10.4) shows (he difficulty of evaluating (he
viiicnllaral potential of an area based solely on
a icnipcralnrc criterion, even when corrected for
light cxposnic time. These indices arc. however,
useful in choosing early- or latc-ripcniig grape
varieties to plant in a new vineyard
In most of ihc European vlticnltural /ones.
cultivars arc chosen that reach maturity just before
Ihc average monthly lemperature drops below
10'C. In warmer climates, this drop occurs later.
Consequently, the maturation lakes place during
a warmer period. Viticnllural /ones can thus be
classified into two categories: Alpha and Beta,
depending on whether Ihc average Icnipcralnrc
during grape ntalnrity of a given variety is below
or above I5 C (Jackson. 1987).
Temperature also strongly influences many b*>
chcmKal mechanisms involved in grape matura-
tion For example, malic acKI degradation is con-
siderably accelerated during hoi weather: malic
The Grape and ]& Maturation
Tub* 10.4. Comparison of dilfc«e
method, of tvakiii in
Vnk-uiur.il mm
Sua hi ' i*anc-.lj. i
Hcliotbxnk produii
HclinhcrmK index
(A'iokkr. 1902)
iBmnai. 19461
(Himlin. 1978)
Zone 1 aln^ihan
U90"C
Gcncnhcim
99S"C
2A
Geneva
1030"C
25
Dijun
1133%
1710
I20S'C
—
_
Bordcaui
I3VC
*0
zioo
Zone 2 = I3WC t
» I6T0"C
Orfnu
1433'C
—
1850
!•!■:. :
::•<:
N.pa
lMXTC
_
2130
Bukni
I040^C
—
—
Zone 3 = I070"C t.
a IWC
Mo v pel Ik i
I785'C
524
2250
Miba
I839*C
—
—
Zone 4 = 1950"C 1
a 2220'C
VenkeA'ctoM
I9OT 1
—
2250
HnbB
2022'C
2.7 * 78*
2000
The Capc/SlelleoboKh
2066-C
2350
Zanc5 = BuieinB
«2220^C
SpU
22T2"C
_
—
Palermo
227H"C
—
(Ban) 2410
I'ltUI
2000*C
—
3170
A«»
28S9"C
2000
c activity (Section 10.2.3) steadily increases
between I0T and 46'C. Temperature does not
illicitly inllicncc tartaric acid concentrations. Ele-
vated respiratory quotients, witnessed at tempera-
tires greater than 35 C. were initially intcipfcicd
as the respiratory oxidation or tartaric acid, but
at such a tcnipcralnre this activity corresponds
more K> the initiation or fermentative phenomena
in crape pulp— essentially acting on malic acid
tRomicu ei<il.. 1989)
Tcmpcratutc also has an influence on the com-
position of grape phenolic compounds. Intensely
colored wines are known to he difficult lo oMain
in extreme tcnipcralnre conditions (too low or
high) though the phenomenon involved can at
lirst appear paradoxical. High temperatures stim-
ulate metabolic reactions, whereas low icmpc-
ra tires curb migration. In cither case, however, this
corresponds with poor grape sugar alimentation
and thus increased competition between primary
metabolism (growth) and secondary metabolism
(accumulation). The concentration of phenolic
compounds is also affected by thennopcriod
(Klicwcr and Tones. 1972). Raising the night-
time temperature from 15 to 30 C white main-
taining a daily Kmpcrature of 25'C results in a
decrease in grape coloration The anthocyaninsaic
therefore not a blocked metabolic product bit.
on the contrary, arc reversible. Thus, temperature
and sun exposure determine phenolic compound
.Ul --'.'. i.ltl.'P
Temperature also exerts a considerable effect
on aromatic substances The aromatic potential of
certain white cultivars (Gcwirt/iramincr. Riesling.
Sauvignon) arc known *< be fully expressed only
in cool climates, where the ai.iiiir.uion period is
slow and long. By comparing a cool vilicultural
A>ne with a wanner /one in Soith Australia.
Ewart( 1987) showed Inat the utal volatile lerpcnc
quantity increased more slowly in the cool nine
266
Handbook of Enology: The Microbiology of Wive anil Vindications
bni was higher al maturity In a cool climalc. aid
especially with shaded grapes, mcthoxypyra/inc
concentrations can attain unfavorable organoleptic
thresholds (Laccy el al. 1991). Conversely, warm
climates can lead ft» high concentrations of ccrtiin
phenolic compounds in white ciltivais snch as
Riesling These compounds confer an excessively
astringent chaructcr to the wine and lead to the
development of a dicsel-likc odor during aging
(HcrnckandNagcl. 1985).
Despite the lack of specific experiments, exces-
sive Kmpcratures arc known not to be the most
favorable conditions for aroma quality.
10.4.6 Impact of the Vine's Water
Supply on Grape Ripening
la) The Effect of Water Availability on the
Biochemical Processes- Imohcd In Grape
Ripening
Unlike mosl plans, particularly annual crops, vines
are generally grown under less than optimum con-
ditions. Various lypcsof envinwmenta) constraints
arc considered to icdncc vine vigor and yields,
while maximizing the wincniaking potcntEU of the
grapes. Antong these constraint!, a limited water
supply plays a major role in vine behavior and
grape composition A moderately restricted water
sapply. known as water delicti", generally has a
be tic I kia! clfccl on wine quality The expression
water stress" should only be used in situations
where an excessive lack of water has a negative
impact on grape qaalily or threatens to kill the
Most high-quality wines arc produced in areas
where annual precipitation does not exceed
700-800 mm. Evidence indicates that high rainfall
and excessive irrigation arc detrimental u grape
qualify.
Before veriiisim (color change), wafer R mainly
Iran si erred to the grapes via the xylcu aid
there arc close hydraulic relationships between the
grapes and the rest of the vine. Any change in
the vine's water supply affects sap circulation and.
consequently, grape development. The resulting
irreversible reduction in grape size is positive from
a qualitative standpoint bat also rcdnccs yields.
In some countries, the climalc may necessitate
controlled irrigation of the vines *< conrpcnsalc
water losses via Iranspirattan Alter veraison,
the deterioration of xylcm circulation leads lo a
concomiiant increase in Hows via the phloem. Al
that stage, the phloem provides the main wafer
sapply to the grapes. As phloem sap circulation
is not directly related lo the vines water supply,
grape growth becomes much less dependent on Ibis
fiic lor. A minimum water supply is still necessary,
however, for the biochemical ripening processes lo
proceed normally.
Matthews and Anderson (1989). Dulcau el ill.
1198 1), and Van Lccnwcn and Segnin (1996)
showed that water stress caused an increase in (he
phenolic content of grape juice and skins, with a
higher concentration of proline and a lower malic
acid content. Inadequate water supply also leads
r» higher concentrations of terpenic compounds
tMacCarlhy and Coombc. 1984) Conversely, an
abundant water supply leads lo an increase in
grape volume, with a concomitant decrease in
phenolic content Although the acid concentration
is often higher, the jnice still has a higher pH
t Smart and Coombc. 1983). This is due lo an
increase in imports of tannic acid and minerals,
especially poctssinm The aromatic componndsarc
also modified. c.g excess water gives Scmillon
grapes a strong herbaceous aroma (Urcta aid
Yavar. 1982).
While walcr deficit docs not prevent grapes from
ripening satisfactorily in terms of their sugar aid
acid content, excessive water delays the ripcniig
process and alters the chemical composition of
Ihc grapes to a considerable extent. In vineyards
where irrigation is used, it shoald be reduced lo
a minimum after nrvvtfww to maintain a moderate
water deficit.
Finally, heavy rain when the grapes arc close
lo ripening is likely lo cause them to burst due to
a sudden absorption of water directly through the
skins This phenomenon is less marked at lower
temperatures and depends on rcspiratury intensity.
(b) Monitoring Vine Wafer Levels
Studying the vines response K> different lev-
els of water supply requires reliable, easily used
The Grape .mil us Maturation
2(>~
itdicalofs of water availability in ihc *.»l or ihc
wilier stilus <>f ihc vim.
The tlrsi sludies of vine reactions 10 watcrsupply
ii ihc kite 1960s were bused oi wafer balances,
carried out using a neutron moisture IcstcrlScg ulu.
1970). A piubc cmilUBfj fasl neutrons Is inserted in
an access lube ih.ii stays permanently in Ihc soil.
The neutrons arc slowed down to a stale of thermal
agiGiuou when they meet hydrogen aunts The vasl
majority of hydrogen atoms In soil arc In water
molecules: the number of thermal nculronscountrd
per unil time is thus proportional *> the dampness
of the soil (humidity by volume) The vines
wilier consumption between two nKasurcnKnLs
Is calculated by subtracting Ihc second reading
from the first and correcting, if necessary, for any
precipitation during the interval. Neutron moisture
tester studies were used h> obtain a detailed view of
the water supply In gravel solbt in the Haui-Mcdoc
tSeguin. 1975) clay soils in Pomcrol.andastcriaKd
limestone In Sainl-Bmilion (Duieau etiil.. 1981).
Although this was a highly innovative technique at
the lime, it bad several disadvantages. The water
balance calculated using this method docs not
take Inn account any horizontal Inflows of water
through Ihc soil or runoff, which may be significant
on slope vineyards Alter a period of lime, roots
develop around the access tube and distort the
results (Van Lccuwcn eiiil.. 2001a). Finally, the
vine root systems are often very deep and vineyard
geology (gravel, rocky soil, etc.) may make il
particularly difficult to install the access lube.
Even if neutron moisture testers arc used In some
New World countries to con irol vineyard irrigation,
the complexity of ihR technique prevent it from
being used more widely. Using Time Domain
Rcllcclomctry (TOR) m csttblish the vineyard
water balance Is subject to the same difficulties.
Producing a theoretical water balance by mod-
eling is another approach to determine the vines
water supply The aim is to simulate the water
reserves remaining In the soil during the sum-
mer on the basis of data on Ihc water avail-
able al the start of the season, plus any prccip-
itiiion. minus losses vu cvapoltanspiralioa. The
most advanced model was developed by Rlon and
Lcbon (2000) In ihrs formula, prccipitilton could
be determined accurately and cvapotranspiralion
estimated correctly. The main difficulty with this
approach is estimating the wakr reserves al the
beginning of the season, which is particularly com-
plex due to Ihc specific conditions In which vines
are grown (deep root systems, rocky soil. etc.).
In view of ihc difficulty In assessing Ihc vines
water balance on the basis of measurements in
the soil or modeling, it seemed more practical to
measure water levels in the plants themselves. A
water dentil causes several measurable alterations
In Ihc vines physiological functions: variations
In xylcm sap pressure, ckislng of the stoma,
slowdown in the photosynthesis process, etc. When
a plant is used as an indicator of its own water
stilus, we refer to physiological indicators"
Among these indicators, leaf water potential is
undoubtedly the most widely used because it
Is reliable and easy to implement Water potential
Is measured by placing a freshly picked vine
sample (usually a leaf) In a pressure chamber,
connected to a bottle of pressurized nitrogen. Only
the leaf stalk remains outside the chamber, via a
small hole. Pressure in the chamber is gradually
increased and the pressure required to produce a
sap meniscus on the cnl end of the stein is noted
This pressure corresponds to the inverse of the
water potential: the higher the pressure required to
produce Ihc meniscus on the leaf stem, the more
negative ihc water potential and Ihc greater the
walcr deficit to which Ihc vine has been subjected
There are three applications for water potential
measurement, using a pressure chamber leaf
potential, basic leaf potential, and stem potential
iChonc <•(<«'.. 2000).
1. Leaf potential is measured on a leaf that has
been lefl uncovered on a sunny day. This value
only represent ihc walcr potential of a single
leaf. Even If this potential depends on the water
supply to the viae, the considerable variability
from one leaf U> another tin ihc same vine
(eg. dnc to different sun exposure) leads » a
large standard deviation oa this measurement,
making the value less significant as an indicator
2. Basic leaf potential is measured la the same way
as leaf potential, except thai Ihc leaf is picked
Handbook of linokigy: The Microbiology of Wine anil Vindications
jusi before sunrise The stoma close in ihe
dark aid ihe water polcniial In Ihe vine conies
back Ink* balance with thai in ihe soil matrix.
Basil leaf potential reflects water availability
in Ihe most humid layer of soil in contact
with the toot system, providing, ihcrclbfc. a
more stable valnc that Is easier n> iiicrprct than
leaf potential measured during Ibc day. It is.
however, more ditricilt Id apply, as it requites
spec! lie conditions.
3 Stem potcniial Is mcasircd daring the day. on
a leaf thai has been covered by an opaqne.
airtight bag for at leasl one hour before the
measurement is made. The leaf stoma close in
the dark and the leaf potential balances with
thai or the xylcm in the stem This measurement
gives a close approximation of the water sipply
of the whole plant during the day. Provided
certain conditions arc observed ( measuring time
and weather conditions), stem potential is the
most accurate or the three pressure chamber
applications (ChOK el (rf., 2001aa>).
Carbon 13 isotope discrimination is another physi-
ological indicator or water balance. This isotope
represents approximately )'i of the carbon in
atmospheric CO? and the lighter isotope. ,2 C. is
preferentially involved in photosynthesis Water
delicti causes the stoma to close for pan or the
day. which slows down CO; exchanges between
the leaves and the atmosphere and reduces iso-
nr* disc rim inalton Under these conditions, the
"Cf'C ratio(knowias AC13) becomes ctoscr to
the ratio in atmospheric CO;. Manning AC 13 in
the sugars in must made from ripe grapes i analyzed
by a speciali/cd cnology laboratory) piovidcs an
IndKaur or the global water dclicit to which the
vines have been sabjcclcd during ripening. ACI3
is expressed in '(• in relation to a standard Values
range from -21 to -269.. where -21% indi-
cates a considerable water delicti and — 26Sr. the
absence of water dclicit. The advantage of this
indicator Is that li does not require any field opera-
tions other than taking a sample of ripe grapes (Van
Lecuwcn el ill . 2001b; Gandillcrc el ill. 2002).
There is a good correlation bclwccn the AC13
value measured in must male fiom ripe grapes
and Ihe stem potrnllal.
tc) Impact of Water Balance on Vine G rowth
and the Composition of Ripe Grapes
A water dclicit during the growing season causes
profound changes in the physiological functions
of the vine, ll may progress at varying rates, as
shown by the changes in stem potential measured
in the same plot or Saint-Emilion vines in 2000
and 2002 (Figure 10.15). When there ■ a water
deficit, the stoma remains closed for pan of the
day. increasingly restricting photosynthesis as the
deficit becomes more severe. A redaction in water
sipply tends to stop vine shoot and grape growth,
affecting the grapes especially before r^ria'sim
(Becker and Zimmcmiann. 19841 When the soil
dries out aroand the roots. Ihe tips produce abscisK
acid, a hormone that promotes grape ripening.
Restricting the water sipply to the vine has both
negative (restricting phousynlhcsis) and positive
(abscisic acid production, less competition for
carbon compounds from the shoot tips, and smaller
frail) effects on grape ripening. If the water dclicit
Is moderate, the positive effects are more marked
than Ihe negative factors: the grapes contain higher
concentrations of reducing sugars, anthocyanins.
and tannins, while the malic acid content is lower
(Van Lecuwcn and Scguin. 1994). For example.
Sainl-Emilion wines from the 2000 vintage, when
there was an early drop in slcm potential, are better
than those from the 2002 vintage tFigire 10.16).
In cases of severe water stress, photosynthesis
Is too severely restricted and ripening may stop
completely.
In viticulture, it is essential to know to what
extent a water deficit has a positive effect on
quality and locate the threshold of harmful water
stress. The answer to this question depends on
the type of production, the types of suhstinccs
considered, and vine yields.
Mosl studies concerning the link bclwccn the
vines' water balance and grape composition have
dcallwith red wine grapes. It Is generally accepted
that ihe red wine grapes can benefit from more
severe water dclieits than white grapes. Oi an
estate producing both rypcsofwinc.it Is. therefore.
The Grape and in Maturation
Fig 10.10. Companion of vaiaibiu. ja Mem pmcoiiul la * Slim- Km Mb a viaeyanl i* 2000 tad 2002 (gravelly v
ami McrVx grapci) Ihc more negative the vakiei.thc more severe the unci defied
I'ifi 10.17. Coreebtkm bci.
ten 1 1- (Mcmiiy
<l redudmr >ugiin
1 of water deficit Iwnied by ibe %i
n [- " ■ t 11 1 ■' hen ihc : -ii ["■■
logical id plant Ihc icd varieties on soils wiih less
plentilil w-alcr reserves.
Among ihc substances thai promote red wine
quality, sugar accumulation reaches maximum lev-
els when ihc waicr balance is modcmlcly restric-
tive. Giupc sugar con tcnlR lower both when there
Is an unlimited waicrsupplyand in cases of severe
walcr stress (Figure 10.17). The anlhocyauin con-
range of waicr deficits, reaching a maximum when
Ihc walcr stress is grealcst (Figure 10 181. The
qualily of a red wine depends more on Ik phe-
nolic content lhan on the sugar content of the ripe
grapes, so red wine grapes may have the potential
lo make excellent wine, even II severe walcrstrcss
has penalized the sugar level of Ihc must
The issneof Ike effect of walcrdclicils on qual-
ify cannot be scltkd without dc^cussing yields. The
same waicr deficit may have a positive effect on
quality in a vineyard with yields of 30 hi 1 lev Lire
and lead « blocked ripening with disastrous results
at 60 hl/hcctare
id ( Impact of Walcr Deficit on Karl) Ripening
The date when grapes ripen depends both on the
phcnological cycle, which may be assessed by the
dale of mid- vfraistm. and the rale al which they
Handbook of Enofcgy: The Microbiology of Wine anil Vlnifkations
is (<■ Mini |-uil Dl < MPai
Fit; 1(1.18. Conelaln
i*d 1 he anthocytaia c
r ikiii a (aucucil by sic« jmcxal when ihc gra
mature, calci tiled according to Duk'au (1990).
The carlincsrt or lateness of Ihc pbcnologlcal cycle
depends mainly on Ihc soil tcnipctutnic. which is
tcblcd to its moisture content (Mortal. 1989). The
ripening Rile is largely determined by the vine's
water balance I Van Lccuwcn aid Scgnin. 1994). A
water dclicit promotes rapid ripening by keeping
the grapes small (this making them easier to till
with sugar) and reduces the competition between
grape* and shoots for lie carbohydrate supply.
Figure 10.19 shows an example of the impact
of water availability on the ripening rate and
early/late maturity in thice plots with very different
soils (Van lccuwcn and Rabasscau. unpublished
icsultM To cliniinaK the impact of tempcratnte
on Ihc ripening rale. dates arc indicated on Ihc
abscissa by Ihc sum of active lcmpcra(arcs starting
on August I '-each day Is represented by 'he
average temperature minus ten degrees. The vines
on gravel soils and plauosol were subject to water
deficit and the sugar-acid ratio evolved rapidly
towards ripeness. The water snpply on the luvisol
was not restrictive and the pulp ripened slowly.
Although the niid-T^riffiitff dales wcie very close
Fig IU.I0. Ripening g
m ol ,M d c It D^aTBUI ■'> Uiirtl^i in .««« I":
liili. hiq*t-Mitnlli> 1". mailje <<-ni|xi-.iluii'
i SaiM-Emilna (MctkM Noir. 300 1 )
The Grape .mil us Maturation
on all ibc plots, the difference )■ ripening dan?
was as much as 70" days in the sum of at live
icmpc rail res. or nearly 7 days after 4 weeks.
The '.im iii.ii' ni> of daiip soiLs are cool and
provide a non-reslriciivc water supply. Grapes
ripen lak' on these soils as the phonological cycle
is delayed and ripening is slow. By the same
reasoning, most dry soils are conducive to early
ripening. Then- are a number of highly reputed
estates in Bordeaux, especially in Pomcrol. bul
also a few localized cases in Sainl-Emilion and
the Haui-Mcdoc. plan led on soils with a high
clay content They arc unusaal as they have high
water contents (and arc thus cool) bul still cause
an early water deficit in the vines. This type
of clay (smectite) is unusual in that, although it
contains large amounts of wakr. it is unavailable
for use by the vines These soils are conducive to
early ripening and although, for historical reasons.
Mcrlol has been planted on them in Pomcrol.
Cabernet Sauvignon ripens perfectly on the same
type of soil in the Haut-Nttdoc. This example
snows that water del kits play an essential role in
the early ripening of grapes and have a greater
impact than soil temperature. Tnc choice of a
giupc variety to snit a particular type of soil
sin 'Li lil depend mainly on its conducivencss u>
early ripening and. thus, on its water balance (Van
Lccuwcn. 20011.
(c) Wafer Balance and Vintage Variation
The water stalasof a given vintage can be assessed
by calculating the water balance Table 103 shows
the water status of several vintages in Bordeaux,
calculated using the method developed by Riou
and Lcbon (2000) To eliminate Ibc effect of
Ike soil, wc introduced a value. 0. for the water
reserves at the beginning of the season, which
explains the negative values of the waler balance.
These values indicate a theoretical water deficit,
corresponding to the difference between precipita-
tion and real cvapolranspiration ( in Ihc absence of
slomalal regulation) All the lesser quality vintages
without exception had only a slightly negative
water balance al Inc end of September (correspond-
ing approximately to the harvest period). Seasons
in which the vines were subjected to a significant
Table I a 5. Concbiion between the Ibenietkal water
hikincc from April I in September 3a a*l ihc •fiiliiv
of ihc vi« J( tc The more -ailed the negative water
hikincc. Ihc drier ibc via ape (b.i«.l on uapuhlnbcd
work by van Lccuwcn and Jacck)
Vi-ape
TnaMdkml
Viuupc
voter balance
on September 3d
(Maris cm of 201
199D
-300
19
:
2'- 1
i"
I960
-271
IS
1998
2. v.
18
1995
:n
17
1902
2*i
17
1904
22"
17
1997
211
IS
1968
211
17
1970
-210
IX
1901
-207
20
1991
-200
13
1969
-204
I"
1965
-198
IS
[<>77
-87
II
1993
-80
14
1954
-80
9
1971
-40
17
1950
-39
9
1908
-33
1958
II
12
1909
14
12
1973
12
12
1905
II
3
l'«:
S
3
1992
-4
12
1900
-1
12
walcrdcfkil were all great vintages, liven if ripen-
ing may be halkd on some plots (especially those
with young vines, i.e. shallow root syslcmsl in a
very dry summer, which has a detrimental cffccl on
Inc wine, it is interesting to note thai, since 1950.
there has been no overall quality loss due to water
stress in Bordeaux, al Icasl in red-wine producing
vineyards
(0 Wais or Modifying Water Supply
in a Vineyard
The ideal waler stilus for producing grapes to
make high-quality wine consists of a moderate
waler deficit, starling early in the season (before
27:
Handbook of linokigy: The Microbiology of Wine and Vinifkalions
reniium). The grapes will show less wincmaking
polcmial if Ihc vines arc not subject lowalcrdclicil
al all. as well as in cases of severe water stress
Loss of quality Is much more commonly due
to a plentiful water supply thai dnc lo excessive
water stress, even if il is generally unnoticed.
When summer rains and water reserves In the
soil arc such thai the vines do not regularly
sifter a moderate water deficit, leaf surface must
be increased to promote cvapolranspi ration and
vines should be planted on roofetocks that do not
tike advantage of the plentiful water supply (eg.
Riparia Coirc de Monlpcllicr) Quality can also
be in. i\. mi ■i.'.l lv. selecting an appropriate grape
variety <carly red and white grape varieties. Van
Lccuwcn. 200l|.
In situalMms where excessive wairr stress causes
a drop in quality In ccrtiin vintages (very dty
climate and lack of waicr rescivcs in the soil),
it is possible to minimise the negative impact oa
tbc vines by adapting the vine training system and
vegetative growth (Chonc el al., 2001b). The best
way to protect vines from the negative effects
of water stress is by restricting yields. When
yields aie low. a relatively small leaf surface
docs not penalize Ihc leaf/fnlt ratio The most
widespread form of adapuikin to dry conditions
Is the use of a drought- resistant rootsmck (e.g.
110 Richtcrl Itshoikl also be noted that reducing
the vines nitrogen supply reduces their water
requirement, by reducing vigor and restricting the
leaf surface.
Under extreme conditioas. vine-growers may
accd to irrigate, if permitted by local law. It Is
considered difficult to grow vines producing viable
yields if annual rainfall Is under 400 mm. This
value may vary, however, depending on the dis-
tribution of rainfall throughout Ihc year and the
soil's capacity to retain walcr. In very dry cli-
mates, rational irrigation may be a quality factor,
while poorly controlled irrigation may also lead
to a reduction in wincmaking potential Irrigation
should be gradually reduced, so as lo produce
a moderate water deficit in the vineyard before
Ye'rtrisvn. while avoiding severe watrsUcss Mon-
itoring the vines' water status by testing stem
potential Is essential lo ensure that irrigation is
perfectly controlled (Chonc el of.. 2001b). Other
promising monitoring methods aie currently in the
experimental stage
For many years, the concept of growing vines
under restrictive conditions was purely European,
mainly in AOC (controlled appellation of origin)
vineyards. It Is interesting *< observe that this
idea is being introduced in some New World
v incyards The Australians have successfully tested
two irrigation systems that deliberately restrict
the vines' water supply In Regulated Deficit
Irrigation' (RDI). a water deficit rs deliberately
caused after flowering by stopping irrigation for
a period of time (Dry eld. 2001). This is
particularly aimed at reducing grape size Partial
Root zone Drying IPRD) involves irrigating both
sides of each row separately, alternating at two-
week intervals Thus, part of the root system
is always in soil that R drying out. This has
been observed lo have very clear impact on the
grapes' potential to produce high-quality wine,
probably partly due to synthesis of larger amounts
of absctsK acid than in vines not subjected lo any
water deficit (Stoll el al., 2001 ).
10.4.7 Meteorological Conditions of
the Year — the Idea of Vintage
The three principal climatic parameters (light, heat
and humidity) vary considerably from year to year.
Their respective influence on maturation processes
is consequently of varying importtnee and leads
fe) a given grape composition at maturity The
etiological notion of vintage can thus be examined.
Variations in niclcorokigical conditions do not
have the same influence in all climaks. The princi-
pal European viticultur.il regions have been classi-
fied into different o>ncs(Figuie 1020). Examining
only the sugar concentration in ihc northern conti-
nental /oactAlsacian. Champagne and Burgundian
vineyards in France, and Swiss and German vine-
yards for the most part), the length of sun exposure
scents to be the principal limiting factor during
giapc development t.Caki .■( al., 1992).
This factor R also important during matura-
tion in the North Alfctnlic /one (Loire and south-
western Fiance vineyards), but Is less iniporcint
The Grape .mil us Maturation
273
Fig lalO. Bliopeai
)■ ihc southern zone (Mediterranean vincyunts
ii Spall. France and Icily). In ibc taller /one.
Ihc hydric facur interferes with Ibc relative
consistency of temperature and sun exposure.
High temperatures In this case do noi posi-
tively alien sugar accumulation, if a considerable
hydrK stress exists. Ii ihc opposite case, incy
can limit this accumulation by favoring vegeta-
tive vine growth when ihc walcr supply is nol
United.
In the Rtofa vincyanls of northern Spain, ibc
respective iniporlancc (varying from year to year)
of ihc opposing influences of ihc Atlantic and
Mediterranean climate determines wine qualify.
Thus Ihc climatc'qnaliiy relationship can only
be represented approximately The sum of ihc
lenipe rati res. rainfall or length of lighl exposure
docs nol have the most influence on grape quality,
ralhcr. il is their distribution in Ihc course of ihc
vine growth cycle.
wkipKaliitn
nl fnf* <kvch>pm
tnleaui (lunc I (vintage* ilnsilicd )■ onlci
I ..1,1 11
).(MciUand
<B>
(C)
1997
1990
1989
:i.'i
21 May
27 May
29 May
31 May
I nine
1970
4 tunc
I"..-;
4 tunc
1994
4 kinc
1993
4 nine
1994
4 tunc
1983
3 lunc
1993
hinc
1992
hinc
XIII
-.' hint
2002
1 hinc
1981
12 June
1988
i: hint
1983
It tunc
1973
14 lunc
1983
13 hinc
1991
13 tunc
1974
13 hinc
1987
13 tunc
1984
III lunc
l"S'i
20 lunc
1979
21 hinc
1980
2.3 tunc
1978
20 tunc
1977
2? hi IK
31 July
4 Aupuu
7 Aupuu
7 Aupuu
i<l . i.'i iu
Itl Aupuu
9 Aupuu
9 Aupuu
14 Aupuu
12 Aupuu
12 Aupuu
20 Aupuu
17 Aupuu
19 Aupuu
20 Aupuu
Id Aupuu
20 Aupuu
19 Aupuu
Id Aupuu
20 Aupuu
19 Aupuu
25 Aupuu
.1 September
2 Scptc-bcr
2 Icplc-btr
15 September
24 September
10 September
20 September
24 September
IS September
25 September
19 September
23 September
28 September
23 September
20 September
28 September
30 September
I October
5 October
October
28 September
1 October
I October
4 October
October
8 October
October
3 October
8 October
13 October
12 October
12 October
274
Handbook or finology: The Microbiology of Wine anil Vindications
In northern vineyards. clinialK conditions favor-
ing a forward growth cycle permit grape matu-
ration during a warmer and sannicr period. Ihas
bcncliting grape quality Recent yean have permit-
ted the verification of this simple observation in the
Bordeaux region (France).
Among the mosl forward years forgrapc devel-
opment. 1982. 1989. 1990. and 2000 produced
wines of outstanding qnalily (Tables 10.6 aid
10.7). The climatic conditions of these years arc
patlicularly favorable, with warm and sunny days
and very little rainfall. At Ike harvest. Carbcr-
nct Sauvignon grapes had high sugar and low
malic acid concentrations (Table 10.8). A high
cation concentration, as shown by an elevated ask
alkalinity, indicated a suitable circulation of water
in the plant and led to relatively high pHs The long
length of maturation in 1990 (55 days on average)
resulted in one of the lowest nialK acid coKCilra-
Iiobs in recent years.
Conversely, during Lite years such as 1980 and
in particular 1977. grape development and matu-
ration occurred in unfavorable climatic conditions.
The grapes obtained in the same parcels studied
were poor in sugar and rich in acids — especially
malic acid. TV* importance of an early growing
season for grape quality has also been demon-
strated by wine-growing rcglons with similar cli-
matic conditions, like the Loire Valley in France
and New Zealand
But in a temperate climate, like thai of Bord-
eaux, the moment at which (he best or worst
Tufck? 107
((Wen.
. Companion between
indue* cbutilicil in on
■eccm vinaiK tpiali, and .linuiic condubi
Icrof fe(uanljK»)
b> from April ■<> Sept'
ahci In Bordeaux
V.ouge
Sim of avenge
Icapeoiuru
Durutimof Number of cxccitb
*un expouitc warm dqm
(h) l>30"C
nalk Kjinlall
l-m)
Wine quilily
1997
.1491
1210
24
500
Oood
1990
3472
1490
38
319
Except bnal
NK'I
3403
1403
35
304
Except bnal
1999
2000
1970
1998
.U-:S
3447
3384
3373
1420
1454
1430
1220
17
25
:-
25
523
477
278
537
Vctv good
Except bnal
Very good
Very good
1994
I99S
3341
1143
1149
2D
30
020
303
Very good
Except bnal
199(1
3207
1207
24
531
Except bnal
I9B2
1331
1202
18
289
Except bnal
1993
3231
||[M,
17
498
Good
1992
3325
1219
•2
557
Medbcie
2001
3357
1503
32
438
—
2002
3309
1414
is
405
—
1981
.!*•.!
11-1
17
289
Very good
1988
3288
1249
15
302
Very good
I98J
3354
1182
24
437
Very good
I97S
3250
1250
M
302
Verv good
1985
1185
1320
10
III
Except bnal
1991
3419
1370
28
319
Good
1974
3129
1279
17
301
Good
1987
1300
l?;:i<
28
308
Good
I9K4
3111
i 1 8
15
423
Good
I9SC
3129
li.i
:i
438
Very good
1979
2938
us;
3
300
Good
I9W1
3057
1020
9
343
I bod
ITS
3029
1153
12
320
Good
1977
3044
1135
2
407
Fairly p>od
The Grape .mil lis Maturation
Tabic 10.8
Weigh of
100 heme*
P H
Atkilmiiy
of**b
tmEq/ll
3.38
SO
3.33
48
357
52
3,03
SO
TatUrk
1997
1990
I9B9
1999
:■■
1970
1998
1991
I .;.,-,
1990
1982
1993
1992
:::n:
1981
1988
1983
I97S
I98S
1991
1974
1987
1984
I960
1979
I960
1978
1 1,77
3.10
3.20
3.S9
3J9
3.30
3.3S
3.23
climatic conditions occur has a greater influence
on grape qualify than Ilic absolute Knipcr.nu re and
tic total rainfall during the entire growth cycle
I Figure 10.211 .Jnis grape quality depends on a
favomblc clinutlK period towards the end of matu-
ration The 1978 vintage at Bordeaux is a paradox-
Kal example of one of lie latest yean (Table 10.6):
unfavorable climatic conditions at Ike beginning
of the i! muii cycle n* tinted ftowcriug and grape
development, bai from iwYj/iononwaids. although
Ihc temperature was slighUy lower than the sea-
sonal average, a lack of rainfall and consider-
ablc sin exposure permitted the grapes to ripen
correctly and attain suitable sugar concentrations
(Table 10.8) Among the tale vinttges of the last
30 years. 1978 is Ihc only year when Ihc grapes
reached a satisfactory maturity.
The quality acquired at Inc beginning of devel-
opment can be compromised by severe bad
weather during maturation. In 1992. grape devel-
opment was initially precocious thanks to high
Icmpcraluics and mouciulc rainfall in the months
before flowering. After a July will normal cli-
matic conditions. August was very hot but suf-
fered from an extremely high rainfall (about three
limes the normal rainfall for Augnsl ai Boidcaux)
Handbook of linology: The Microbiology of Wine anil Vindications
Tabic 10.9. I'hcnolH- compmiiiua i>f Cabefoa Sauvt-
gaua gape* .11 amuny for Ihicc diiitnrn i mjpc%'
iBonkjui FaiKcXAugimla. IWCb)
ViAgG
Anihocyjnla* Tannla*
(p/loabcrric*)
Ska
Seed*
1985
1983
1984
lift 0.35
132 0.25
129 0.33
0.39
:: -J
052
V linn jrt
cbucd hv ilccraulB inki <i
-IK+MI.V
Climatic conditions evidently have an influence
on all grape consliticnis— in particular, secondary
metabolites mbIi as phenol* compounds aid aro-
matic substances. Slndics on these substances arc
incomplete and have often been carried out will)
very different KchiKJics— especially extraction.
Tabic 10.9 gives an example of the phenolic
composition of Cabernet Sauvigion grapes at the
lime of harvest for the 1983. 1984 and 1985
vintages. The skin anthocyauin conlcnt is higher
in qualify vintages Tkis relationship is not valid
for tannins
Fig 10.21. Monthly icmperaiurc and rain nil differ-
no wild nspcti (u 30-yeur i.cnjv foi (be period
fmm March to October. 1978 and 1992. nl Bontaui
(Fnitec): :.tcmpcnriurcri)iic<catc (C):# niinfilldlf-
accompanied by a lack of sunlight Despite
more element climatic conditions in September,
sagar coaccnlraliois icinained low (Table 10.8).
In (act. sugar accimilation is generally rapid
in the weeks following veraison. aid migration
becomes stower aftcrwaids. Sugar concentraliois
rarely increase rapidly during the days before the
harvest.
Yet. the harvest should occur )■ favorable
climatic conditions. In 1993. this period was
characterized in Bordeaux by heavy rails (more
than ISO mm in September) The quality of the
vinttgc dropped substantially during the last days
of maturation. Similarly. 1976. a forward vintage
benefiting from a warm, dry summer, did not attain
the exceptional quality hoped for. because of rails
at the end of maturation
10.5 IMPACT OF VARIOUS OTHER
FACTORS ON MATURATION
The variability of the matiratKiu process, in terms
of vintage climatic conditions, is also icgulatcd. if
not controlled, by other parameters.
Some of these parameters arc fixed aid exert a
constant aid permanent action the nature of the
soil, the variety and possibly the rootstock as well
asplantdcnsity and trcllising methods. All of these
factors arc established during the creation of the
vineyard Vine age. to a certain extent, can also be
placed ii this first category.
Other parameters can be continuoasly changed.
Their modification most often corresponds with
a desire to adapt plant reactions to vintage cli-
matic conditions vilicultural practices such as
pruning, cluster thinning, hedging, leaf thinning.
phytoNinitary treatments, etc Fertilizing is also
often placed in this second category. These prac-
tices can modify the nalirc of a soil for a long
lime.
The Gr.ipc and It. Maturation
Finally, vintage climatic conditions can produce
accidcntil factors— bolh meteorological (frost,
bale) and sanilaiy (cryplogamK diseases!
10,5.1 Variety and Rootstoek
Rooistocks aic used in vine-growing when the
chemical composition of ihc soil or the presence
of pesfc (such as phylloxera) prevent ihc varicly
fiiiai developing on its own tools. The rooKtock
develops a different tool system than ihe graft and
this results in changes in ihc water and mineral
supply. Grapes of a given variety, grown on the
same soil, are known lo have a different ionic
composition accoiding to Ihcir roocdock. but these
diffcrcnecsarc not sufficiently important to cause a
significant variation in must acidity iCarbonncau.
1985)
They are. however, capable of influencing graft
photosynthctK activity. The vigor may cither in-
crease or decrease, depending on Ine type of soil.
In the richest soils, rooistocks sach as NOR.
140 Ru. I 103 P. S04 and 41 B confer an execs-
sivc vigor to the graft. In is heightened vegetative
growth slows and limits Ihc maturation process
(Pougcland Dclas. 1989). In contrast. Ine Riparia
Gloire and 161-49 C rootslocks create a relatively
short vegcutive cycle, favoring maturation and
respecting Ine general specificity of the variety.
Grape composition at maturity differs when a
variety has developed on its own rood as opposed
to on a roonuock. These differences essentially
affect maturity: 'hey concern the concentrations of
sngais. acids and phenolic compounds. Vet if the
rootslock isjudkiously chosen, ihc differences that
result from divers rooistocks for the same cullivais
are always slight (Gmlktux. 198 1 1. Only the
nitrogen concentration appears to vary significantly
iRoubclakis-Angclakisand KIKwcr. 1992).
Choosing the variety to suit Ihc climat is a
deciding factor for obtaining a good maturation
and quality wines In general, early ripening vari-
eties are cultivated in cold climates iChassclas.
Gcwurrtramincr. Pinot) and relatively laic- ripening
varieties in v»arm /ones ( Amnion. Carignan. Grct-
acbe). In both cases, matnnty should occur just
before the average monthly temperature drops
2"
bckiw l()'C. The maturation process should not
lake place too rapidly or abruptly in excessively
favorable conditions.
Quality cullivars such as Cabernet Sauvignon
and Pinot Noir lose much of their aromatic sub-
stance and phenolic compound finesse in warm
climates. Figure 10.22 indicates the phcnological
behavior diversity of these two varieties in Ihe dif-
ferent viticullural regions of the world. In a warm
climate, characterised by average monthly tem-
perature always above 10'C (for example. Penh.
Australia), the duration of development is partic-
ularly short — notably at maturation Conversely,
the cycle grows longer in cool and humid temper-
ate climates (Preach viucyardsandinChrisichurch.
New Zealand).
Choosing a variety for a given jna depends
greatly on its ability to reach a sagar concentra-
tion of 180-2(10 g/l during maturation, but ripe
grape quality is also affected by other chemical
constituents
The tartaric/malic acid ratio varies consider-
ably from one varicly lo another. Ai maturity, the
grapes of most varieties contain more tartaric than
malic acid. Some varieties, however, always have
a higher concentration of malic acid than of tar-
taric acid: Chcnin. Pinot and Carignan. In a warm
climak:. varieties having a high Girtaric/malic acid
ratio arc preferably chosen
Pcynaud aid Maorie ( 1953) had already noticed
that variability in organic acid concentration causes
a very variable nitrogen concentration from one
variety to another liven more than Ihc total nitro-
gen concentration, amino ackl composition varies
greatly — to such an extent that it is used by certain
authors as a means of varietal discrimination.
Anj in me and proline concentrations can vary by a
factor of 10 to 15. depending on the variety — for
example, from 300 to 4600 nig/I for proline. The
proline/arginine ratio is relatively constant from
one vintage to another in the same grape variety.
The different varieties also seem lo have a large
diversity in phenolic composition A study carried
out on the principal French red grape varieties
cultivated in the Mediterranean or Atlantic climate
snowed that concentration variations accoiding lo
Hamllxxik of linology: The Microbiology of Wiic anil VhUiailiuM
<<» MttOI (O
Fift I".:: Phcnolojikfllbctavbrof Cabernet Sauvipnon j«I Pit** Ndi
|t>| I..-.--L .» A US Ail,!' ■luiU.l.ii.h.Nc.-. Zcabud:(d) Hi.nfcjui.H-jn
and Lombani ( l"'.H BB. budbunl; B. bhxim: V. imiinn; H . banal; ::
cycle of Cabernet Sauv^nan;| | tcgcuint cycle nl Piw Nalt
lag 10 climate: (a) Pcah. Auunila:
caunc. France, (a )-<i> alter be b-m
mfccuuK ain't: f^^~ i vegetal ive
inc climate arc significantly lew. (fc.ui according lo
the variety (Bhoi and Ribcrcan-Gayon. 1978).
Similarly, fluctuations in grape phenolic con-
ical from one village lo another and for a givei
variety arc less than Ihc variations between vari-
eties. The gcuotypk effect of Ike variety is this
pic pontic rait on grape phenolic compound rich-
ness. Aithocyanidic and pmcyanidK piofiles vary
greatly with respect lo Ihc variety aid can there-
fore be iscd in varietal discrimination (Cak> el ill..
1994).
The variability of grape aromatic con tcit is even
gicatcr. Some varieties ixkscss characteristic aro-
mas. Al present, not all of the molecules respotsl-
blc for these aromas have been identified. In cer-
tain varieties, such as Ihc Concord, descendant of
The Grape and Its Maturation
279
native American vines ( VHis tabruuu. Sin's mtun-
ihftilia). the grapes always exhibit a foxy odor due
hi methyl ;ui ill ramble (Bailey. I9H8).
Similarly, disease resistant hybrids such as Cas-
tor and Pollux, obtained by crossing Ifrti viiafera
and native Autcrican vines, ate often charac icri/cd
by a strawberry odor resulting ln>m the presence of
a few nanograms of 25-dimc!hyl-4-hydiaxy-2.3-
dihydro-3-fntanonc (fnraneol) (Rapp. 1993).
The Cabernet family of varieties possesses notes
of vegetal aromas die *> the presence of pyia/jnc
derivatives (sec also Volume 2. Section 7.4) (Bay-
oioverrnr.. 1976).
Filially, within Ihc muscal gioup. Ihc free and
glycosidic Icrpcnol profile and concentrations vary
greatly with respcel lo the variety Iscc aLso \o(-
nmc 2. Section 72) (Bayonovc. 1993) Sulfurous
compoinds and volatile phenols responsible for
diverse aromas (medication, blackcurrant, etc.)
most likely vary in the same way (Rapp. 1993).
Varieties differ considerably with respect lo
each other, and choosing a variety suitable for
local environmental conditions is a deciding fac-
tor In wine quality When such choices are well
established by viticultural tradition and scien-
tific observation, quality improvement depends on
clonal selection within the variety Clonal selec-
tion has been successfully developed for Caber-
net Sanvignon. Pinot Noir. Chardonnay. Riesling
and Gcwir/lramincr(Schacffcr. 1985). It focuses
on limiting varietal shatter sensitivity, increas-
ing membrane selective permeability (higher sugar
concentrations) and modifying berry volume and
grape cluster morphology.
Although many empirical observations note the
influence of vine age on wine and grape quality,
little scientific work has been devoted lo (his sub-
ject. According to Dring 1 1994). (he young vine
develops a root system adapted to i& environment
during Ik first yeais. At the end of it* fourth year,
a functional equilibrium between the roots and the
metabolic activity of the aerial pans of the vine is
established. A uiycorrhizum most often facilitates
Ihc mineral nutrition of the young vine (Possing-
bam and Grogl Obbink. 197 1 }
If the vine-grower Iben succeeds in respect-
ing the vinc-soil-cliuiatc equilibrium and in
regulating Ihc harvest volume, notably by pruning,
the plant can develop snflicicnl reserves in the old
wood to ensure the prerequisites for proper matu-
ration each year. Old vines are this less sensitive
lo yearly climatic variations and most often pro-
duce grapes rich in sugar and secondary products
favoiublc lo wine quality
10,5.2 Soil Constitution
and Fertilization
The influence of soil on grape composition and
wine quality ■* dcliniKly the most difficult lo
describe. Tic soil, by its physical structure and
chemical composition, directiy affects root system
development and consequently the vine water and
mineral supplies. It exerts an equally important
effect on the microclimate. Soil color and its
stone content profoundly modify the minimum
and maximum temperatures as well as Ihc light
intensity in ihc lower atmosphere surrounding
Ihc grape clusters Whether wilh schist plates,
limestone pebbles or siliceous gravel, vine-growers
have long made the most of this second sun lo
improve grape maturation
As mentioned previously (sec Section 10.46).
a regular water supply is needed for grape
development and maturation.
This water from the soil transports the minerals
that are necessary for growth in the plant. The
Ionic concentration of this solution is re-bled lo
the nature of the soil and Ihc fertilizers added,
bul a large amount of the available minerals is
the result of biological activity In the soil. A
potential disequilibrium can seriously affect vine
growth . The best- known example is lie increase in
the exchangeable phytotoxic copper concentration
In old. traditional vineyards that have received
many sulfur and copper-based treatments H> ensure
Ihc sanitary protection of the vine. Under the
Influence of bacteria in Ihc soil, the sulfur is
oxidized inlo sulfaKS which accumulate In the
soil The resulting soil acidification causes copper
solubilization i Donee he. 1976).
Many synthetic pesticide residues can similarly
disrupt certain soil reactions, notably the biological
mineralization of nitrogen, but there has been no
:>n
Handbook of Isiiology: The Microbiology *** Wi»e anil Viniftcaudns
research en ihc consequences of these phcr
on grape maturation and composition.
Many studies have recused on Ihc influence
of different levels of nitrogen and potassiam
fertilization. Tbe removal of ihcsc minerals by
the harvest is relatively low. compared with other
crops Since the roots exploit a large volume of
soil, vine mineral needs arc relatively low
For example, annual nitrogen fcnilicitionshoald
•ol exceed 30 kg/ha. which Is largely sufficient for
meeting the plant, needs Above this valnc. nitro-
gen exerts a considerable effect on vine vigor, aid
excessive vegetative growth blocks the maturation
ptoccss. In this case, the grape crop is abundant,
bnl sugar and phenolic compoand concentrations
arc low and the grapes arc rich in acids and nitro-
gen compounds Excessive addition of nitrogen
also increases the concentrations of ethyl carba-
mate prcciisoraid hE&imiac. which arc likely to
tower the hygienic quality of wine (Ough el irf .
1989). The elicit of nitrogen on vigor cai be lim-
ited by walcrsupply deficiencies in warm climalcs.
Tcmpoiury or permanent cover crop between vine
rows may lead to a deficit in the vines' nitrogen
supply die to competition, but also as a result of
mineral nitrogen fixation 'ordcnitrilicalioul Niln>
gen deliciency in Ihc grapes may lead to fermenta-
tion problems in the mast and may also, above all.
have a detrimental effect on their synthesis of phe-
nolic compounds and a large number of aromatic
substances.
The problem of potassium is more complex.
This catkin predominantly participates in must and
wine pH and acidity. Facing Ihc fairly general
increase of wine pH during recent ycais. much
research lends to show that Ihc soil is responsi-
ble for this high potassium supply, due h> exces-
sive soil richness or fertilization However, a direct
refctlKmship between excess potassium fertilization
and decreased grape acidity has not been demon-
strated definitively in all cases
Potassium actively participates in grape sagar
accumulation In years with lavoniblc climatic con-
ditions, the ripe grape imports large amounts of
potassium Due to Ihc high malic ackl degradation
characterizing such a maturation, must acidity is
principally the result of the tartaric acid c
■ration. Insolubilizalion of tartaric acid sail in tbe
course of wincmaking greatly lowers the acidity
Some viucyaids arc established on sally soils
High sodium chloride concentrations increase the
osmotic potential of Ihc soil solution. As a resell,
the plant must strongly increase its respiratory
intensity to ensure Ihc necessary energy for its
mineral nutrition. In hydroponics, this levers vine
vigor and result! in a more forward maturity. The
sagar concentration increases but not the amount
of phenolic compounds. In vineyards, these effects
arc modified by asing specific roobdocks Isall-
crcck. dodridgc and 1613) On salty soils, potas-
sium, magnesium and oiganK acid concentrations
decrease whereas calcium and chloride concentra-
tions increase.
In conclusion. Ihc primordial influence of soil
has been recognized for a long time in Ihc form of
viiKullaral lemiirs. The soil must create favorable
conditions for grape development and maturation
(mineral and water supply and microclimate). The
Icmpcrature above Ihc soil and i& waurr content
also have an impact on Ihc carliness or lateness of
Ihc vines' growing season (Barbcau rial.. 1998:
Tcsic r! itl.. 20111 J. These rvvo parameters give an
initial indication of the quality of a temrir Bui a
quality lermir mast also limit the consequences of
weather variations from one year to another. Soil
study is difficult since all of the factors likely u>
influence the biochemical processes of maturation
should be taken ink) account.
Our understanding of the role of soil in the
intrinsic quality of wine still rests essentially on
empirical data. Each grape variety docs, however,
excel in particular soil types. Thus Cabernet
Sauvignon predominates in the Medoc appellation
(France) where this variety ripens on sandy.
gravely hilltops and produces rich and complex
wines, bul tradition shows thai the best results on
clay-rich panels in the Mcdoc fill land and daks
are obtained with Mcriot.
10.5.3 Management of Vine Growth
Grape maturation Is also influenced by other per-
manent factors, established during the creation of
Ihc vineyard. Planting density, row spacing and
The Grape and its Maturation
canopy placement (existence and piisJiioniM^ of
wire Utilising) condition plunl physiology through
toot development with rcspccl to the soil and uscot
sunlit: hi hy the leaver These factors diicclly affect
vine vigor Their action on (he grape on only
be indirect, notably acting on Inc niictocliniaic
surrounding (he grape clusters (temperature und
sun exposure)
Rigorous experiment in Ibis domain arc diflkull
to carry oat The existing criicria for establishing
a vineyard arc primarily empirical bnl vine vigor
has been shown to increase when plant density
decreases, with the risk or a retarded maturation
In northern and temperate regions, tradition
(vcrilied by research) recommends rctalivcly high
planting densities of around 10(H) vines/ha In a
drier. Mediterranean climate, optimum quality is
often obtained with a density of between 3000
and 5000 vincs/ha. In spile of the water deficit,
the high density restricts potassium imports and
mainlainsa good acidity level in wines made from
these grapes
In conditions favoring vegetative growth (irriga-
tion in warm and sunny climates), excessive leaf
crowding should be limited by low plant densities
(IOOO-2000 vincs/ha). and adapted training and
pruning methods. Canopy management is of major
importance in this case (Carbonncau. 1982).
As well as the climate, soil fertility imposes
ib own rules According to Pclit-Lafiltc (1868).
the pooler the soil, inc higher Inc plant density*.
A very compact mot system thus permits the
maximum cxploittlioa of Inc soil potential The
same reasoning shoukl be used in dry soils.
10.5.4 Vineyard Praclfccs
for Vigor Conlrol
Vine management and growing arc characterized
by severe measures limiting vegetative develop-
ment and the amount of fruit A ccrtun canopy
surface is required for grape alimcnttlion and a
relationship exists between this surface and grape
quality.
The development of the canopy surface to fruit
weight ratio can be used to evaluate grape quality.
In an example with Tokay. Klicvver and Weaver
I't 10.23. KlpnuitHi ol'tolil M>k*lc uilHh < Brixiflf
Tokiv berry juice at Ion cm (September 21) on leal
area rerun* crop "eight (em'/p). (Klicwnand Weaver.
19711
(1971) showed that grape sugar concentration
diminished sharply when Ibis ratio was lower
than 10 enr/g (Figure 1023). Proline and phenolic
compound concentrations were similarly affected
Di Stcftino el ol. ( 1983) obtained identical results
for IcrpenK compound concentrations in white
muscat In general, the necessary canopy surface
for the maturation of I g of fruit varies fiom 7
to 15 cnr*A; for most \Vtis viniferti varieties, but
increasing Ibis ratio above these values has Utile
cffcctoigrapc composition, as shown by the sugar
concentration curve in Figure 1023.
Winter pruning is the first operation carried out
in the vineyard. It consists of controlling vine
production by leaving only a certain number of
buds capable of producing inflorescences. Vines
respond very differently to pruning. Some varieties
have such fertile buds at the base of the shoots
that pinning isoficn ineffective for yield control
Bud fertility varies greatly at the same position
on the shoot, depending on the variety. Colli var
productivity can also vary with respect *> the
climate. Many factors must thus be considered. Vet
most specialists agree that kiw yields arc needed
to obtain proper grape maturation
Increases in yield have long been known
to affect grape sugar concentration negatively
(Table 10.10) This problem is especially trou-
bling in vineyards that use varieties close to
I landbook or Etiology: The Microbiology of Wine anil Vindications
1'iiliU.- M I". Rcbibftthlphcio
Gape <> ciphr
Su^re
«*iBmW
fcrwiM
Pta
VIK
Per tier of mini
tbeir cultivation limits In very warm regions,
the harvest can be delayed to attenuate this phe-
nomenon In northern vineyards, the high variabil-
iiy of annul cliuialk conditions determines the
ratio of grape yield to sugar concentration. Expe-
rience shows that great vintages, obtained in par-
ticularly favorable i lunatic conditions. Ulost often
correspond with abnndant grape crops.
In a warm and snnny climate, inctcasing canopy
sarfacc and improving snn exposure combined
with controlled irrigation will often increase yields
without lowering grape quality (Bravdo el id.
1985). but several consecutive abundant crops can
lead to depletion of vine reserves.
After berry set. excess grapes can be removed by
thinning Manual thinning is expensive. Thinning
iPfA of the crop before veraison results in a
X5H increase in the sugar concentration and a 5'i
drop in acidity. Chemical berry thinning currently
re ■tuns experimental and is an extremely delicate
operation, undertaken at berry set.
Other icchniqucs ate available u the vine-
grower to modify the physiological behavior of the
vine. Depending on soil fertility and climatic con-
ditions, trimming or hedging can slow vine vigor
and limit leaf crowding. A single topping (remov-
ing recent shoot growth), at the end of flowering,
diminishes the risk of shatter by limiting sngar
competition between young grape clusters and api-
cal shoots.
According to Koblct (1975). considering that
a shoot bears about 200 g of grapes. 10-14
leaves per shoot arc necessary U> ensure their
maturation, assuming a canopy surface to grape
weight ratio of 8-10 em'/g Trimming, which
leaves a maximum of 14-16 leaves per shoot.
improves the maturation process and increases
sugar and secondary metabolite concentrations.
Trimming loo severely produces opposite resale.
Trimming also presents the advantage of lowering
the evaporation surface, thus limiting hydric stress
risks in certain environmental conditions (tack of
rainfall, soil water deficiencies).
Older, less active leaves can also be removed
from the base of the shoot. Correct leaf thin-
ning essentially exposes grape clusters to sun-
light, improving grape maturation and limiting
the risk of rot Leaf thinning around grape clus-
ters thus reduces malic acid concentrations and
the pyrarinc character while augmenting grape
anthocyanin concentrations for Cabernet Sauvi-
gnon. The vegetal aromatic character of Sauvlgnon
Blanc can also be lowered in this manner (Arnold
and Bledsoe. 1990).
Leaf thinning seems to he very effective, espe-
cially when practiced just after wnnstm. The result
of this operation depends greatly on climate, vari-
ety and canopy placement A partial leaf thinning
before berry selling lowers the future crop vol-
ume. due to flower- fall. But this delicate technique
can lower fertility during the following vegetative
cycle (Candojfi-Yasconcclos and Koblct. 1990).
Finally, chemical substances are now available
to the vine-grower Ki stow vine vigor and accel-
erate the maturation process. These are usually
growth hormone biosynthesis innibiursOtcynokls.
1988)
10.5.5 Effects of Disease
and Adverse Weather
I .at frosts and hailstorms occurring in the spring
often produce the same effects as shoot removal
by lopping, but talent bud development is dts-
organized, resulting in bnshy vegetation. Flow-
ering is considerably extended and grape clus-
ter maturation is uneven. The latest grape clus-
ters have difficulty in reaching maturity. Dam-
age caused by summertime bail alters grape clus-
ter alimentation: affectd grapes wither or arc
attacked by parasilcs. and a rapid harvest may be
necessary
The Grape .mil us Maturation
Diverse causes can mull in more or fcss
severe vine defoliation. Maturation is diflicull due
to insufficient grape alimcnttlion. A tile downy
miklcw attack can cause total leaf loss in ccttain
vcty sensitive varieties, such as Grcnacbc. Simi-
larly, leaves infected with powdcr>* mildew always
lower grape quality Parasite dcvelopuicni leads
m significantly reduced crop yields, and vcty laic
attacks hinder grape maturation.
A potissinm deficiency can cause leaf-scorch
llavcsccnce and premature leaf-drop — and in con-
sequence a decrease in grape sugar and phenolic
coin poind concentrations l( Lick leaf, encouraged
by overproduction and soil dryness, often accom-
panies potassiim dcliciencies.
Bnnch stem necrosis also lowers crop qual-
ny and can cause crop loss. It is often linked
to excessive yields and magnesium deficien-
cies Ccmin varieties arc particularly sensitive:
Gcw tint ram incr. Sanvignon Btanc. Ugni Blanc
and. notably. Cabernet Sanvignon. This necrosis
icsalts in a decrease in sugar, antnocyanin. Tally
acid and amino acid concentrations whereas the
grapes remain rich in organic acids (Urcta el ill .
1981)
10.6 BOTRYTIS CINEREA
10.6.1 Gray Rot and Noble Rot
In addition lo the diseases already mentioned
Idowny and powdery mildew), one of the principal
causes of crop quality degradation is grape rot
dnc to Ihc development of various microorganisms
(bacteria. yeast, or other faigi).
The principal microorganism responsible is usu-
ally Botrytis cinerea which is a ubK|iitous fun-
gus except perhaps in dcscrl macs' (Gatcl. 1977).
Endowed wilh a great polyphasy, (his saprophyte
can exist on senescent or dead tissue such as vine
wood It is also capable of waiting for favor-
able conditions in diverse resistant forms tsclcrotEi
or conidia. with a high dissemination capacity)
The presence of water on the surface of vege-
tal tissue and an optimal temperature of IS C arc
ideal conditions for the germination of the resis-
tant forms and mycelial growth. Conidial germi-
nation is possible at temperatures between 10"C
283
and 25 'C In these conditions. Ihc contamination
area of this fungus is very large and covets a great
number of the world s vilicullnral regions Grape
gray rot thus remains one of the major concerns of
vine- growers.
In a few areas of the worki. particular conditions
permit Biitryrit cinerea K> develop on mature
grapes This process results in an overripening that
increases the sugar concentration while improving
qualify. The parasitised grape dehydrates and the
sugars .ire more concentrated than the acids. Most
importantly, the grape acquires the characteristic
aromas thai permit the production of renowned
sweet white wines such as Sanlernes-Barsac.
Colcanx du Layoi (Fiance). Tokay (Hungary) and
Trockenbccicn anslesc (Germany and Austria)
Noble rot requires specific environmental con-
ditions. The many studies undertaken have not
yet been able to define these conditions, pre-
cisely bul. in general. B. cinerea development in
the form of noble rol is thought to be favored
by alternating dry and humid periods Night-
time humidity, dew and frequent morning fogs
in the valleys of certain rivers stimulate fungal
development, whcicas warm and snnny windy
afternoons facilitate water cvaporaton — limiting
fungal growth.
Many factors participate in this phenomenon:
• The soil, by its nature and possibly let drainage,
should permit the rapid elimination of rain
water.
• The canopy placement and surface should per-
mit a maximum number of grape c listers to be
aerated and exposed to sunlight.
• The grape cluster structure shoukl be fairly
dispersed
The nature of the variety also greatly affccR
grape sensitivity lo B cinerea. bit no direct
relationship seems to exist between variety type
and noble rot quality Differences essentially
originate from Ihc level of maturation precocity
Puchcu-PlauK and Lcclair (1990) showed the
importance of the nature of the clone on noble rot
quality.
I l-imllx»'k of linology: The Microbiology of Wine anil Vindications
10.6.2 (irapc Sensitivity to Bolryth
Vine Inflorescences can suffer from roi alticks If
ibc climatic conditions air favorable to It cinetea
development. Peduncular lot causes flowcts to
fall aid consequently a sharp drop in future
cn>p volume. During the entire herbaceous growth
period, the grape Is rcsisEinl to thts parasite
Cray ml rarely occurs between frill set aid
reruiwn. In 198.1 and 1987. nonhent European
vineyards suffered early attacks, sometimes affect-
ing np to M'.' of the berries but the teasois for
the loss of rcsisttnic in green grapes air still not
known.
In certain cases, of compact grape cluster
varieties with elevated grape setting rates, a few
berries can become detached from their pedicel and
remain imprisoned Inside the grape cluster. These
damaged grapes constitute a direct penetration path
for the fungus.cireumvcnting the natural resistance
of the grape. More often, the contamination Is the
consequence of another phenomenon, snch as hail
or other parasites. After veroison. the grape rapidly
becomes more or less sensitive to B cinerea.
These behavioral differences of the grape are
die to multiple causes that will now be examined
briefly without an in-depth study of the pathology
of the grapc-# cinerea relationship.
In the first place, the green grape skin, covered
by a thick cuticle, constitutes an effective barrier
against parasites. Since Bonnet's (1903) initial
research, a icstsuncc scale of the principal Yiits
species has been established bused on the cuticle
thickness of their respective berries American
varieties whose cuticle thickness varies from 4 |tm
tMlit nuJfiWHl to 10 urn (W/« ciiiacea) have
better protected berries than European species
( Wis vimferti). whose cuticle thickness is from 1 5
to 3S |iin This observation led to the production
of V. vim/em and American species hybrids thai
arc effectively more resistant u gray rot. bnt these
hybrids do not usually prodncc quality grapes on
the bcsl lerrtws.
The same relationship berween cuticle thickness
and fi. cinerea resistance was encountered in
V. viw/era varieties but on a smaller scale The
sensitive varieties all have a cuticle thickness of
less than 2 |im ( Karadimtcbcva. 1682).
During maturation, the cutin and wax qnantily
per surface unit increases. This accumulation is
more intense when the grapes are exposed to sun-
light in an environment relatively low in humidity.
Contact between berries isalwayscharactcrivd by
a lower cuticle thickness
Although the fungas possesses a culinofytic
activity, it is very low. In fact, direct penetration of
the grape cuticle by B. cinerea enzymatic digestion
has not been proven Only a developed mycelium
produces sufficient amount! of cutinasc to attack
a neighboring berry cuticle (often less thick if
the grape cluster is very compact). In the surface
of the cuticle there are perforations that are a
potential point of entry for mycelial filaments
tBIaich etal.. 1984). Resistant hybrids have fewer
perforations than sensitive varieties. The number of
these cuticle perforations increases in the conisc of
maturation.
Under the cuticle, the exocarp also participates
in the resistance to B cinerea. According to
KaradimKhcva (1982). the external layer of the
hypodcmiis in certain varieties resistant to gray rot
comprises more than seven rows of thin. cmngakrd
cells, with a utU thickness exceeding I") pm. In
sensitive varieties, it contains only four to six cell
rows, with a total thickness of 50-60 *n.
The extent of the thickening of the epidermal
cell walls, occniring In the course of maturation,
varies depending on the variety. The most sen-
sitive varieties have the thickest cell walls This
phenomenon r. caused by the partial hydrolysis of
pec tic compounds by endogenous grape enzymes
(Section 10.25). The increase in soluHe pectins
varies greatly, depending on the variety In conse-
quence, grape skins exhibit varying degrees ofsen-
silivity to enzymatic digestion by the cxocellular
enzymes of B. cinerea (Chatdonnct and Donecbc.
1995).
In addition to this mechanical resistance, the
grape skin contains preformed fangal development
inhibitors. All epidermal cells possess tannin vac-
uoles. These phenolic compounds cxctt a weak
fungistatic effect on the pathogen
The Grape and us Maturation
285
As in many Hulls, an inhibitor of the cndopoly-
galacturonasc of I-', cinenta is also contained in
the crape skin cell walls. ThR glncopfotcK sub-
stance is liberated during cell wall degradation by
lie pathogen The concentration and pcisislcacc of
th is inhibitor vary according to the sttgc of devel-
opment and the variety considcied
The gnren grape skin is abio capable of syn-
thesizing phytoalciins in response lo an infec-
tion (Langcakc and Pryce. 1977). These stilbenic
derivatives (Uuns-rcsvcralrol and is glycoside,
traks-piccid. diner i-vinifcrin. o-vinifcrin trinKr.
0-vin)fcr)n Iciranien have fungicidal patpctlics
(Figure 10.24).
Rcsvcratrol is obtained by the condensation
of /i-couniaroyl CoA with three makiiyl CoA
units in the presence of the slilbcne synthase
iFignie 10.25) Vinifcrinc formation R then cn-
suicd by grape peroxidases.
At the time of a purasile infection, the normal
Havonoid mclabalLsni (Section I0J 6) rs divcrtd
towards stilbenic derivative production by the
action of the slilbcne synlhase. Remarkably, after
nVmirw. this capaciry R vety rapidly lost even in
pathogen- tolerant Amerkai vines (Figure 1026).
Thus during maturation. Ibc grape loses most of
it. physical and chemical defenses. The parasite
sensitivity differences observed for diverse vari-
eties and clones essentially result from differences
in thcirgrapc development time Microperforations
of Ihe cuticle and sUuialK Insures I Section 10.2.4)
also constitute a passageway for the efflux of
grape cxndatc. which Is indispensable for coni-
dial germination and the proliferation of W. ciaerea
(Donccbc. 1986) Chemical modifications of the
grape during maturation— notably an incieasc in
Ibc sugar, amino acid and soluble pectin concentra-
tions — furnish the fungns with essential nutrients
for mycelial growth
10.6.3 Noble Rof Infection Process
Sonic observations have led to the conclusion that
Boiryth ciiteren is sometimes present inside grapes
as soon as they scl in (PctcI and Pont. 1186)
When It comes out of the latency phase. It develops
II& 10.24. PrliKifiluibcnlcdcii'
t. identified in the v
c (Langtake und Piycc. 1977)
Handbook of Fnology: The Microbiology of Wmc anil Vindications
l-'iji Hi. In. Evulubn of lotcvml pnxaiciiia in m-
fomc In Inlcclkin h. ,i pirasic imiriag hem' develop-
■cb liciixkt ct .**.. 1991)
picfcfciliully towards ihc sk.m . doc to Ihc presence
i>f antagonist cnzymcslchitinusc antl B. thieonmei
Id ihc ifcsb cells. An exogenous nuuicnl sipply
(ac ill tiles ihc elongation or ihc germ native lube.
When Ike mycelial hypba reaches a mKroflssuic. II
pent* Irak's Ihc grape. Tnns II ciaerea development
occurs mainly in ihc grape s superficial cell walls.
More precisely. Ihc mycelial lllamciits ait locaKd
In Ihc micldk' lamclLi of Ihc pcctoccllulo&ic cell
walK. Tbc laitcr an- degraded by Ihc enzymes
of Ihc fungus (pcclitolylK. ccllutasK complex,
protease aid phospkolipusc en /vines i
When ihc mycelium has lotally overrun ihc
pcclocclliknK cell walls of Ihc cpidcrm;il and
immcdiaic snb-cpldcmial cells, while grapes lake
on a chocolate- brown color thai is characlcrislK
of the ptnirri piein stage. The mycelium ihcn
produces filaments ihai cmcigc from the skin
sirfacc by piercing Ihc citKIc or by taking
advantage of the diverse fissures nscd for Ihc inilEil
penetration The lilamcnl extremities diffcrcntialc
by producing conidiophorcs. whose conidia later
detach and coniaminaK nearby berries
The cell walls of vegetal iLssnc arc so greatly
ntml i lied lhal I hey can no longer ensure their
functions In particular, berry cell hydration is
no longer regulated It can vary with respect lo
climallccondilionsand.il Ideal conditions, should
lead to a characteristic desiccation accompaiicd by
the cytoplasmic death of ihc epidermal cells The
sigar concentration of these cells is considerable
Due lo ihc high osmotic pressure, ihc fnngus
can no longer sihsist and stops developing. This
shriveled aaifu stage, known j&pmrriroti. is used
for elaborating sweet white wines (Table 10.11).
The infection process, from healthy grape lo
ptnirri rAii grape, lass from 5 to 15 days, depend-
ing on environmental conditions Thtsovcrripcmng
period mist be characterized by an alternation
between" short humid periods (3 -4 days), favoring
conRlia) germination, and longer dry periods
(about 10 days) pcmiiliiig gtape concentration aid
chemical transformations lo occur
For quality noble mi development, the pheno-
menon mist be rapid and occur near maturity, but
Ihc berries mast reach this stage intact.
Many years of observation in Santcncs vine-
yards (France) have shown that ihc llrsi symp-
toms of attack appear 15 -20 days before matu-
rity Regardless of climalK conditions. B. i
development issktw nntil maturity Al thiss
The Grape and iCi Maturation
Tabic 10.11. rn j,.
pal aMKli fecal io*. uf cbcmltal |!npc ion
Mimic* by i noble rot mack
lo«iucB
IV i lie* of muu
l'c> 1000 bertie*
II (a 1 by Noble rotted
Heal by Noble Mted
beny henj
Wefchi ( g )
—
_
Vokimenl muu<ml>
pH
3JJ
3
SuprocKCMMkiXpl
217
317
Glyeemt(g>
7
Alkalinity aafaOEtp
33
81
Total acidity ImEa.)
i::
II?
Taitar*.- nil ImEti)
"i
33
Malic acid (mEq)
81
117
C«ricackt<mEql
2.7
3
Acetic Bcid<mEq)
Vi
6
Gluconic ackl<mEu.>"
10
85
56
Amino »ckM*|tl
1282
1417
ProieuHlmg)
28 IS
3795
Ihc parasite sprc:»is rapidly and lis grow ih t, explo-
sive. At a ccrciin moment, a high percentage of
N.i]"- simullaaci in.-,!;, a-.", li ihe paurri plein slagc
iFigmc 10.27).
Crape maturation al a vineyard, in a panel or
even on ihc bk grape cluster. is never absoliicly
synchronous (Section 10.4 I ). As sooa as a berry
approaches maturity, it is con tarn inaled by coaidia
l-'ift 10.27. Evolution of a *>bk ml attack
Saute rocs \ 'iacyanl |l nui (jveage of ID yean o
pcrimcniation oa iliflciei* pinch)
from a neaiby rotten berry. This asynchionisni
makes sacccssivc sorting necessary during the
harvest (successive sorting* is a local term which
means that successive handpKking is used to
ensure that only noble rolled grapes arc harvested!
The nsc of dilTcntnt varieties with varying dcgiccs
or precocity is beneficial in practice: for example
Muscadclk. Scmillon and Sauvignou Blanc in
Sautcrncs (Prance) or Firm in I. Harslevcltt and
Muskat Otloncl in Tokay (Hungaiy).
10.6.4 Changes in (he Chemical
Composition of Noble Rot
Crapes
Physical daci t Juice volume, berry weight) cITcc-
tivcly cxpicss the grape dcsKcalion phenomenon
Grapes can be concentrated by a factor of 2 to 5.
depending on climatic conditions.
The enotogkal prolilc of must obtained from
bolryti/cd grapes is specific (Table 1011). This
Juice Is very rich in sugars but its acidity is similar
to that or juice obtiincd from healthy grapes. The
tartaric acid concentration is often even lower and
Ihc pH higher (from 35 to 4.0). attesting to the
Handbook or Fnokigy: The Microbiology of Wine anil Vmificaiions
concentration or oilier substances such as potas-
sium ions. Compounds not preseni or in negligible
concentrations in hcalthygrapcs arc encountered In
considerable quantities, especially in ptnirris r&is
grapes. For example, glycerol and gluconic acid
can reach concentrations of 7 g/l and over 3 g/l in
boiryti/ed must, respectively.
But Ibis concentration phenomenon masks pn«
found chemical constitution changes resulting from
the biological activity of B. einereu. As Mulkr-
Thurgau dcmonsliaicd as early as 1KXS. these
changes affeel sugars and organic acids
II einereu uses Utile pcckiccllulosic cell wall
residue for let development As a result, the con-
taminated grape becomes rich in galacmronic acid
derived from Ihc degradation of pec tic compounds.
Il prefers lo .tssimilatc glucose and fructose accu-
mulated in the pulp cells, and Mfi of the sugars
are lost in lie production of these noble rot wines.
Nfctabolic slndies in vitro have shown thai
the young mycelium of B. cinerea possesses the
enzymes of the Embdcn - Mcyerhof pathway, the
hexosc monophosphate shunt and the tricarboxylic
acid cycle (Doncchc. 1989) It dinxlly oxidises
glucose into gluconic acid. The taller, according
lo a process identical to the Enlncr-DoudonrolY
pathway, permits the young mycelium lo syn-
thesize substantial quantities of cellular material.
However, when the fungus is partially deprived
of oxygen, mycelial growth B tow and the com-
plete oxidation of glucose Is accompanied by the
liberation of glycerol in the environment
Thus the initial fungal development under
the grape skin rs marked by considerable glyc-
erol accumulation (Figure 10.28). When B. einereu
emerges on the outside of the grape and reaches
its stationary phase, remarkably, it can no longer
assimilate gluconic acid. This acid, which accu-
mulates in the grape, is a characlcrislic secondary
product of significant sugar degradation The glu-
conic acid concentration depends on the duration
of the external development of the fungus, varying
from 5 to 10 mBq/bcrry.
The glycerol concentration of contaminated
grapes abw varies according u the duration
of Inc respective internal and external fungal
development phases. The glycerol concentration
sunr
I'lfi ltl.28. Suvt.ir ivMBiLiitiB and moiHbry pmikKt
lorauiion during a noble n« atuik (Doncchc. 19871:
:: . glicovc + fnKicncL f. glycerol: ■. ft..-. ■ ■ ■ acid.
Supci of InlccikimO) hcafchy hem: i2) ipotied berry
lipa iti.imtici leuihan S am*); fJ)»»oltcd berry (*po>
damcicr gitulci Una S mm'): • J ■ lull', nxicd berry
(completely spoiled): (5) anpeunnce of mycelial hyphnc
on ike beny lurfacc: (0) pmrti rati
Is between SO and 60p.mol per berry al the
pmrri ' plein stage Despite the concentration phe-
nomenon, only 10-40 |tmol exist per berry at the
pmrri rM stage. Pari of the accumulated glyc-
erol is oxidized by a glycerol dehydrogenase in
the course of the external development phase of
B. cinerea. Musts obtained from bolrylircd grapes
normally contain 5 to 7 g/l of glycerol.
The concentration ratio of glycerol to gluconic
acid represents the length of internal and external
development phases of the parasite It constitutes a
noble rot quality index (Figure 10.29) In vintages
with favorable climatic conditions (for example.
1981. 1982 and 1985). rapid grape desiccation
from the pairri plein stage onwards leads to an
elevated glycerol lo gluconic acid ratio.
During a botrytis attack, other polyots Imanm-
lol. erytnriiol and meso- inositol) arc formed and
their concentrations increase in ihcgrapelBcrUand
elal.. 1976). B. cinerea also produces a glu-
cose polymer, which accumulates in contaminated
grapes, let concentration can attain 200 mg/1 in
must Thisglucan is often al the root of subsequent
wine clarification difficulties (Dubourdicu. 1978).
The i ir.i|v and in Maturation
289
llfi 10.19. Influence nl vinapc on ■iHk n* qmliy.
cxtrcucxtby I be ul..cnil.'aki«.-ni. acid rain IDancchc.
1987 J
i development is always accompan icd
l<y lie degradation of the principal acids ol' Ibc
grape This btotogKal dcacidllication lowers Inl-
lial grape acidify by 7M on average. Tartaric acid
degradation R progressive Id Ihc course of ihc
infcclion prixess. and it stimulates sugar assinv
i ..iii.Ti MalK acid is generally less degraded: il
occurs especially alihe end ofa botrytlsaitickand
corresponds to a sirong energy demand iu order lo
accumulate reserve substances Iu ihe developing
con rim
The other acids arc less degraded This some-
times leads lo their increased concentrations in
ptnnrh rotis grapes Cilric acid is an example and
can be synthesized by certain B einerea strains
I'ni. In spite of the conccutratlon phenomenon, its
couccilnilion rarely exceeds 8 mEq/1 Acetic acid
behaves similarly.
Mucic acid has abo been observed Id accumu-
late It is a product of Ihc oxidation of galactur-
onic acid tWurdig. 1976). This acid is capable of
precipitating in wine In Ihc form of calcium salts,
bul this phenomenon is rare and seems limited lo
northern vineyard wines. More generally, il also
affects wines made from grapes that were insnffi-
cicntly ripe when Botrytissct In
/' einerea development is also lo the detriment
of grape nitrogen compounds. The fungus degrades
grape proteins and liberates the nitrogen in amino
acids with the help of proteases and amino oxi-
dases dill used in the grape. B. einerea then assim-
ilates this niirogcn and synthesizes Ihc metabolic
proteins necessary for its growth. The grape thus
becomes rich in cxoccllular fungal proteins Musts
obtained from pmrris r(ni% grapes contain less
ammonium and more complex forms of nitrogen
than musts from healthy grapes
Like many other fungi. B. einerea produces an
cxocclluku License: /•■diphcnol oxygen oxktoic-
duclasc (Dubcrnct rial.. 1977) This enzyme oxi-
di/cs numerous phcnolK compounds II is involved
In Ihc pathogenetic pioccss and its synthesis is
Induced by two groups of substances. The Hrst
group comprises phenolic compounds (gallic and
bydioxycinnamlc acid), most likely loxic lo the
fungus The second group consists of pcclK cell
wall substance degradation products (Marbach
ei al.. 1985). The fungus adapts Ihe molecular
structure of this cxoccllular Uncase to Ihc pH of
the host tissue and the nature of the phenolic com-
pounds present. The quantity of the enzyme pro-
duced Is also regulated
Laccasc transforms the principal white grape
phenolic compounds (cafcK and p- con marie
acids— both free forms and forms esicrificd by
tartaric acid) into quinoncs (Salgucs el al., 1986).
These quinoncs lend to polymeria:, forming brown
compounds These compounds arc most likely
responsible for Ihe characteristic chocolate color
of paurrit pleins grapes.
Towards the end of development, the fungus
produces less taccase and this enzymatic activity
lends *> decrease at the poitrri rati stage. Botry-
ti/cd grape mosis are less sensitive to oxidation
than supposed.
Aromatic substances arc greatly modified dur-
ing a bolrytis attack. GlycosMtascs produced by
B. einerea hydroly/c Ihc Icrpcnlc glycosides The
fungus oxidizes the free terpen ic compounds,
which seem u have fungicidal properties. Into less
Handbook of Fnokigy: The Microbiology of Wine anil Vinifkations
Fift 10.31). Samba
odorous producMBockcf at., 1986). Even though
aldehydes art icdnccd lo their corresponding alco-
hols. / '. cinereti dc vclopnici i is more c harac Icri .vA
by the accumulation of furfural, benraktchyde aid
phcnylacctaldcnydctKikashlirf til .. 19B3). Accord-
ing to Masada ?t til (1984). sololon (hydroxy-
3-dimcthyl-4.5-2(5H) furatoHc) (Figure 10.30) is
one of the principal compounds involved in the
characteristic rdfi aroma of botryti/td grapes, but
■iKh rescacch is still needed to discover the exact
componcnLs of this specific aroma
After a bolrytis attack, grapes and mast con-
tun polyosides with phytotoxic and fungistatic
activities. B cinerea also produces divers antibi-
otic substances; botrytKlial. norfaotryal acetate aid
botrylactonc Sonte of these substances can be the
source of fcrmcntttion difficulties
10.6.5 Cray Rot and Other Kinds
of Rot
Large qaantilicsof a variety of epiphytic microflora
(bacteria, yeasts, and fnngal spores) arc present oh
the grape skin sarfacc. The development or icpto-
daction of a given mKrooiganisnt is. above all.
determined by environmental conditions (tempera-
ture and free water).
When healthy grapes ripen under dry condi-
tions, the low level of water activity on the skin
sarfacc promotes the proliferation of osntophilic
mK morgan isms, especially yeasts (Rousscai and
Donee he. 2001 ). Average water activity (from con-
densation or fog) R required for fnngi » develop,
while bacteria need latgc quantities of free water,
generally from heavy rainfall, before they can mul-
tiply (Figure 10.31).
Furthermore, the various microorganisms inter-
act (antagonism and competition for nutrients).
Indeed, a number of yeast strains capable of
ViH 10. J 1. Minima
..■i t -.-..-.i
nhc^apc
restricting the development of B eiaerea are used
in organic disease control
Noble rot is a regular phenomenon dcvckiplng
nniformly throughout the vineyard. Gray rot at-
tacks, however, are usually very heterogeneous.
Partially or totally infected grape clusters are oltcn
encountered on one plant whereas the grapes of the
neighboring vincslock arc totally unuached
The gray rot infection process by B. einerea
Is identical to the noMc rot process previously
described, but early fungal development is difficult
to delect oh red grapes. The external development
of the fungus Is ccreunly the most characteristic
trait of gray rot. The conditions leading to the
death of the fangus in the case of noble rot do not
occur. A mycelial felt* forms oh the surface of
The Grape and its Maturation
291
grapes. Tbc contamination or neighboring grapes
is facilitated by the intense biological activity of
this mycelium. All Mtlmlitir.il factors increasing
grape cluster compactness and maintaining a high
amount of moisture on the grapes thus favor the
spreading of tic disease.
The chemical composition of grapes R greatly
modified in the coarse of a gray rot attack. All of
the intermediary products between noMc rot and
gray rot can be encountered
II cinerea consumes grape sugais while accu-
mulating glycerol and gluconic acid Contrary h>
noble tot. sugar concentration by grape dchydra-
tloi remains low in comparison with sugar degra-
dation (Figure 10.32). Consequently, the sugar
concentrations of musts obtained from grapes in-
fected by gray rot rarely exceed 230 g/1 The fun-
gus also accumulates large quantities of gluconic
acid daring Its external development phase (more
than 10 (cEq/bcrry or more than 3 g/l of must)
Malic and tartaric acid degradation is more
significant than in the case of noble rot Up
Id 'JtH* of the initial concentrations present in
healthy grapes can be degraded. The fungus also
accumulates higher amounts of citric and acetic
acid in the contaminated grape, but the acetic acid
concentration rarely exceeds S p.Eq per berry.
The differences between the two kinds of rot arc
even more pronounced when considering phenolic
i'u in i.» i- >.i I ui;j:l- .*.-•!! Air in
I'ift U'.il. fohiuiuhipbciwecauitariktrrubiinnind
beny dchydrukinatttinUntfkiibciypcof ml (Donccfac.
1992)
compoands. These are much more oxidised by
laccasc. especially in red grapes whose skii B rich
in phenolic substrates. Laccasc activity increases
as mycelium grows and indicates age of gray rot
The risk of color breakdown, known as oxidasic
cassc. is considerable when the must is exposed to
air after crushing (Chapter 1 1)
In contrast to noble rot. which gives sweet
while wines their specific qualitative aromas, gray
rot often causes aromatic flaws The grapes and
wines obuined often arc marked by characteristic
mold or undergrowth odors. The responsible
compoands arc culicular fatty acid ( l-ocKn-3-onc.
l-oclcn-3-ol) or terpenic compound (nn identified)
derivatives formed during pellicular maceration by
the mycelial biomass (Bock Hal., 1988 1
Other fangi arc often simaltineoasly present
with B cinerea. As a result, the rot lakes on
varied colors: bfctck (Asperfjlhts ni&er). white,
blue or green IPenicillnoii sp.. Clmk&porhm sp )
These fungi develop less mycelial biomass than
B. <7/n>Jwr, glycerol and gluconic acid accumulation
is less substantial. The conEiminatcd grapes arc
often extremely bitter and possess aromatic fttws
originating from amino acid and skin phenolic
compoand transformations These compoands give
to wines phenol and iodine odors (Ribcrcan-Gayon.
1982). CtiMbriptrnmt possesses a much higher
laccasc activity thanff cinerea: in addition, laccasc
synthesis by B. cinerea increases in the presence
of Aspergillus or PeniciUhmi (Kovac. 1983). All
fungi have similar water reqaircmenfs and the most
decisive parameter in their selection is. certainly.
Icmpcralare For this reason, the loxinogcnic strains
of Aspergillus arc most widespread in hot-climate
vineyards
Some grapes have a strong damp earth* smell
due to the accumulation of gcosmiu This com-
pound. derived from the biosynthesis of terpenoids,
is formed by several strains of PeniciUhmi. in the
presence of B cinerea Research is in progress to
identify the factors behind this phenomenon, which
affcclssomc vineyards on a regular basis
A second category of microoiganisats exists
on the surface of grapes Differing from fungi.
Ibis category generally docs not possess cell wall
hydrolysis cn/yincs and therefore cannot penetrate
292
Handbook of linology: The Microbiology of Wine anil Vin ideations
grapes with intact skins This group is cssci Hilly
made apofoxidauvc yeasts and acclic acid txtcleria.
B. cinrrea frequently exerts a powerful antag-
onistic influence and hinders Ibc multiplication
of these microorganisms. b»t in want condition
acclic acid bacteria proliferate, nllli/lng sweet J nice
thai escapes Iron) Ike fissures created by the enter-
gence of B. cineiea at exterior of the grape. The
evolution of the grape from the pourri plein stage
onwards rs this different and leads to sonr rot
tpairiinoe aigiY) (Figure 10.33) fScclion 122.1).
These bacteria transform the glycerol, formed
before-hand by B. cinereti, into dihydroxyaccionc.
Among these acclic acid bacterid. Glueonabarier
species oxidize glucose with the help of membrane
dehydrogenases Sugar degradation Ls thus substan-
tial and is accompanied by the accumulation of
glKonic. kcti>2- and kcto-5-gluconic and dikcto-
2.5-gluconic acid in the grape. The production of
these kc tonic compounds substantially increases
the combining potential of the must with sulfur
dioxide.
The development of these acetic acid bacteria
R also characterized by acetic acid prodKtion.
The musts obtained from grapes infeclcd with sour
rol can contain more- than 40 g of acclic acid
and up to 25 g of gluconic acid per liter. Since
these bacteria only slightly degrade grape acids,
the musts obtained have extremely low pHs. This
r> tic worst form of rot.
Fig 10. J A Evotuiou of gluconic- acid >
In t' n ( t "' 'luring devckipn*!* of B. c
B. (fins-en tallowed by dcvclapMcnt c
Bacteria
Yeasts may also be involved in grape contamina-
tion, cilhcraloac orassociaicd with acetic acid bac-
teria. In fact, yeasts have been identified as respon-
sible for certain acid rol attacks in Mediterranean
vineyards. This disease is caused by oxidative
yeast development fCaiubhi. Kloeekem. Hunseia-
taponi). It is known that certain pbylopathogenK
strains arc likely to cause lesions in plrni tissue.
Tartaric acid of the grape is not attacked The for-
mation of gluconic, acetic and galacturoaic acid
greatly increases acidity. These yeasts produce a
small amount of ethanol and the must possesses
high concentrations of ethyl acctxlc and ethanol.
Damaged grapes cannol avoid this alteration
in development Grape skin lesions can occur al
any stage of dcvckipmcnt. The causes arc diverse:
bursting due to a rapid water flux, insect biles or
skin degeneration due to overripeness.
Exceptionally, B. cinerea can be observed lo
develop exclusively, forming a crescent-shaped
mycelial mass to obstruct the fissure- (Donccnc.
1992). Changes in the chemical constitution of
Ibc grape arc this also charactcrraic of a gray rol
attack.
Usually, however, the sweet Juice seeping oul
of the damaged grape favors the multiplication
of oxidative ycasl and acclic acid bacteria. These
microorganisms arc generally transported by the
insects responsible for Ihc lesions. The grape thus
inevitably evolves to wards vulgar (Ot.
Crapes infeclcd with vulgar rot tsour or acid)
cannol be used to make wine. Their presence in
the vineyard mast be delected as soon as possible
and the grape clusters should be eliminated lo Until
the spreading of the disease.
10.6.6 Evaluating the Sanitary State
of the Harvest
i development, alone or associated with
other microorganisms, lowers potential grape qua-
lity. The cnological consequences arc serious in
wines made from altered grapes: oxidations, degra-
dation of color and aromas, and fermentation and
clarification difficulties. The objective mcasarc-
ment of Ibc sanitary Stale of the harvest therefore
presents an obvious interest.
The Grape and us Maturation
293
Pur 11 kmg line, only visual evaluation methods
were available b> Ike vine-grower (or jink-mi 1 the
extern of a contamination This technique tikes
only external fungal development on Ibe berries
ilk) account. Yet. I! aiwrea has already purllally
altered the grape before emerging on Ihc striate
of the grape. To complicate Ihc nailer further, the
infection spots are diflicilt to see on Ihc surface of
a red grape.
At present, a grape crop scleclioi criterion is
based on moaikHing Ihc laccasc activity wilhii the
grape. sccrcKd early on by B. einerea. Due to the
natural prance of another oxIdorcdKtasc ( tyrosi-
nase) in healthy grapes, a specific sabslrak: must
he used lor measuring the laccasc activity in must
Tut) measurement methods exist.
The first method is based on a palatograph k
measure of the must oxygen cotsamplion in the
presence of a laccasc-spccilie substrate This me-
thod is not very sensitive and Ihc elimination of
must phenolic compounds is not indispensable. It
has the advanttge of tiking into account all of the
oxidasic activities likely K> exist in must obtained
from contaminated grapes, but this method may
not be able lo differentiate between slightly con-
■aminakrd and perfectly healthy grapes A machine
based on this method has been developed which
automates this analysis (Salgics el til., I9B4).
The other process includes a cokirimclric me-
asurement that makes use of syringalda/inc.
a laccasc specific sab&lraic (Harkin and Ohst.
1973) ThLs colorless orthodiphcnol is stable with
respect k> chemical oxidation as well as the
presence of tyrosinase and polyphcnoktxidasc. in
healthy grapes. The quiuoac formed in the pres-
ence of laccasc has an intense rosc-mauvc color
(Figure 1034). The speed at which it appeals
is measured by spectrophotometry (DibourdMu
el ill.. 1934). The reaction must be carried out
on a non-salfitcd mnsl The phenolic compounds
must also be eliminated by percolation on a
polyvinylpolypyrolkkmc (PVPP) colimn lo avoid
their interference in the analysis. The results arc
expressed in laccasc activity units per milliliter of
mnsl. A laccasc activity nnil isdclincd as the quan-
tity of the en Ante capable of oxidizing a nauontolc
of syringalda/inc per minute in analysis condi-
tions. Manual analysis by this method is relatively
quick (5-10 minutes, depending on the percola-
tion time for Ihc sample). It is simple lo use since
rcady-to-usc klK exist, including PVPP cartridges
and ready- uvnse react in is as well as a colored
chart indicating Ihc corresponding laccasc nni&.
A semi-quanbtalivc determination is thus possi-
ble in the winery without using calorimclry An
automated analyzer also exists based on the same
Vip. 10. M. Oikkukin mcibanf \>nnj.ldi/ine by Uci
294
Handbook of Etiology: The Microbiology of Wine anil Vindications
principle ami adapted lo luigc- volume operations.
The rcsulls. obtained in 2 minutes, are given in fcic-
cascunio.aswlih the manual colorinicirK method
A strong correlation has been demonstrated
between visually determined grape conbimiuatron
levels, and lactase activity (Table 10.12). hit Red!
and Koblcr 11992) emphasized that the laccase
concentration docs not penult the estimation of
the iota! phenolic compound decomposition level
of rot contaminated grapes Notibly. there is no
condition between the laccase activity and the
totd phenolic compound index, dclcrmincd by
spec Irophotomc try at 280 nm.
Cagnieal and Majarian (1991) developed an
immunological method for detecting B. cineieti.
Polyclonal antibodies arc used which recognize
the presence of spec ilk polyostdcs. secreted by
the fingus. Other microorganisms present on the
grape, such as Ayiergiltus. Peak/Ham. Cliulospti-
riiun. acetic acri bacteria and oxidative ycasfci.
do not inlcrfcic with this immunotogkal lest.
Thanks lo its sensitivity, the fug us can be detected
20-30 days before the harvest, providing adequate
time for applying fungicides. This method is also
capable of differentiating between wines made
from botryli/cd grapes and healthy grapes at any
given stage of the wincmaking process (l-iegoni
el nl.. 1993).
In future. Fourier transform infrared spectropho-
K'nteiiy will be an invaluable tool lor assessing
the condition of harvested grapes This method is
already capable of detecting the presence of rotten
grapes, but there is no close correlation with the
;nt of laccase activity.
lull!.- Mi:, i;,- Lh. ...-..% in j. betneen (be kvel «( M
determined by vnuul inspection and lacca.tc acliviv
aacawiicd by the oi«Ial»» of ivnifiUvlic (Rcdl aad
Kobkr. 1992)
Level
Lai
0.39
0.78
225
656
8.12
IS86
10.7 CONCLUSION
In conclusion, progress in vine-growing and dis-
ease prevention has greatly improved wine qual-
ity, not only by diminishing wine flaws but also
through permitting the harvest to be delayed until
optimal maturity
New risks have amen from this progress. Im-
proved vineyard practices can result in excessive
plant vigor. Above a certain level, increased veg-
ctitivc growth is always detrimental lo the blo-
chcmkal maturation processes. The start of these
processes is at least delayed and thus may occur in
unfavorable climatic conditions. Excess production
Is another risk: diluted grapes are obtained, produc-
ing wines with little structure, color and aroma.
The grape is more than a reserve slock-
ing organ. At harvest time, it still possesses
an Intense metaholk activiry. Particular attention
should therefore be given to grape handling, espe-
cially when a percentage of the grapes arc rot
infected In this case, the grapes contain many
additional enzymes of fungal origin
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Abbal P.. Boutd ir. aad Mniaouoet M. (1992). /
biitvn. Sri. \fffir Uit . 26. 231.
Arnold R.A. and Ilkdux A.M. (1990). Am. J. Bitot.
Uric. 41, 74.
AmJOnlfei M.M.( 1980). Budc dc I ialUieocc de ccntrim
fciiiiun vuc k* compo»c* phcoolBpm ill rania cl du
vin. DoctoM d'Uancahc, Bonkauv-
Balk) I. II . I I9SS.I. Thr Eiolunon o/Om Sititv Friths.
MacMilbn Co.. No' York.
BaibcauG..Au,elinC. and Mortal R.<l998)ftt<tf. OIV.
SH5-S06. p. 247.
Bayonovc C. ( 1993). In Isi ivquiiaiion* Recmtts m
Claomiioff-fbir du Mil led. B. Donccac) Tec &
Doc Lavobier. Pari*.
Bayonovc C Richard H.awlConloaakr R.( 1976). CR
Ac.nl. Sri.. SfiriV C. 2K3. 549.
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11
Harvest and Pre-Fermentation
Treatments
11.1 InliulKlion
112 Improving grape quality by overripening
1 1 J Harvest date aid opctuiions
1 1 4 Ackliry adjustment of the harvest
1 1 5 Increasing sagar concentrations
1 1 .6 Enzymatic transformations of Ihc grape aflcr its harvest
1 1 .7 Use of commercial enzymes in wincmaking
299
51 II
11.1 INTRODUCTION
The definition of maturity. Ihc biochemical trans-
formations of grapesduring maluralion and related
subjects have been described in Chapter 10 Grape
mammy varies as a rcsalt of many patamckris and
is nol a precise physiological slat
In certain conditions (for example, dry while
wincmaking in warm climates), grapes ate some-
times harvested before complete malarity. In other
conditions (in temperate climates, for example),
the natural biochemical phenomena may be pro-
longed when unfavorable climatic conditions have
disrupted normal maturation kinetics, and this
has become a tradition in certain northern vine-
yards( laic harvest .beerentunlese, etc .). It Other
regions. Buinlis cinerva in the noble nM form
causes overripening (Sections 103 and 142.2).
All on- vine overripening methods increase the
ratio of sugar to acid. Gtupcs accumulaic sugar
while breaking down malic and/or tareuic acid. In
all cases, this natural drying process lowers crop
volume due to water loss.
Similar tesalK aic sought by exposing pick-
ed grapes to sunlight or storing them in ven-
lifctled bnildings. These tcchnK|nes arc nscd in
.;:.■
Handbook of Etiology: The Microbiology of Wiie anil Vinifieai«>ns
grapc-giowiag regions as different from one
another as Jerez (Span) and Jura (Prance).
Must quality can abo be improved afler ihe
»»MM bui these wine adjustment, whether phys-
Kal or chemical, should not be made simply
lo compensate for basic viticullural inadequacies.
These various processes arc strictly regulated, to
avoid potential abuse and to avoid their becoming
standard practices with the objective of replacing
the wo tk of nature.
The grape remains a living oiganism afler
it is picked Many enzymes maintain snlticicnl
activity H> ensure various biochemical processes.
Ein/y malic activity is regulated by cellular com-
parimcnlation. which continuously limits avail-
able substrates l-n/ymatic activity Is higher in
rot infected grapes, in which case grapes should
be main tuned intact for as long as possible.
Crcat caic should be given to their harvest and
transport
Pic-fcrmentiiion practices at the winery destroy
the structure or grape cells. Enzymes arc placed
in dirccl contict with abundant substrates, result-
ing in explosive enzymatic reactions Laccase
activity Is the best- known rcaciioa: secreted by
B. cinerea. it alters phenolic compounds In the
presence of a sufficient amount of oxygen Not
all of the enzymes picscnt arc harmful lo qual-
ity — pcctinalytic cu /vines, for example, favor
must clarification by parietal constituent hydrol-
ysis. For several yeats. cuoiogtsfe have sought to
amplify these favorable reactions in using morc
active, industrially produced enzymes
Thanks to an incrcascd fundamental understand-
ing of giapc constituent and their en zymotic trans-
formation mechanisms, manufacturers have greatly
improved equipment design, treatments and tech-
nological processes Many methods for maintain-
ing, increasing and. if necessary, correcting grape
quality arc currently available to the cnologist.
Enology will likely bring about other improve-
ments in future years, notably with respect to phe-
nolic compounds and aromatic substances
Nevertheless, two stgniticaut constraint, pcr-
sm International trade imposes increasingly stricl
regulations, encouraged by the consumer's desire
for natural products Also, certain technological
methods require costly equipment and thd level
of investment is restricted to large wineries.
A bridge between viticnltural practices and
wincmaking methods, pre- fermentation treatments
demand great carc front the cnologist.
11.2 IMPROVING GRAPE QUALITY
BY OYER RIPENING
Ovcrripcning Is a ii.iinr.il prolongation or the
maturation process, but dilfcts from maturation
on a physiological level. The maturing of stalk
vascular tissues progressively isolates the grapes
from the rest of the plant. As a result, crop volume
generally diminishes since evaporative water loss
is no longer compensated for by an influx from
the roots. Ovcrripcning is abo characterized by an
increase in fcrmcuutivc metabolism and alcohol
dehydrogenase activity (Tcrricrc/ ill.. 1996). Noble
rot is also a process which ameliorates grapes by
overripeness; it is described in Sections 10.6 aid
1422.
11.21 On-Vinc Grape Drying
The grapes arc left on the vine for as long as pos-
sible with this natural drying method —sometimes
even afler grape cluster peduncle twist. The berries
progressively shrivel, losing their water composi-
tion They produce a naturally concentrated must,
richer in sugar and aromatic suhstinccs. Acidity
docs not increase in the same proportions and
can even decrease by malk acid oxidation. Other
biochemical maturation phenomena also occur
notably, the skin cell walbt deteriorate. This meth-
ods should therefore be used only with rela-
tively thick-skinned varieties to limit the risk of
i development
11.12 Off- Yinc Grape Drying
In certain regions, this method can be limited lo
simply exposing grapes to sunlight for a variable
length of time. In the Jerez region. Pedro Xintcncz
variety grapes arc exposed to the sun oi straw
man for 10-20 days before pressing (Reader and
Dominguc/. 1995). The grapes arc tuned over
Harvest aid Prc-l-crnvcntation Treatments
»1
regularly and covered at nighl lu prolccl Ibcm
from moisture During ihls process, locally called
jiA'd. mast density regularly attains 1.190 b>
1210 bnl sometimes exceeds 1235. TV juice
yield is very low (250-300 l/tonnc) and only
vertical hydraulic presscsare capable of extraction.
The resulting juice is extremely viscoas and
dark, with pronounced grape aromas The Pedro
XiincK/ vancty is particularly rich in organic
acids The heat of the AndalusEin san provokes the
formation of a significant quantity (50-75 mg/1|of
bydroxymcthylfaifaral from fructose.
In this same region of Spain, as well as in many
other Mediterranean vineyards (Greece. Cyprus.
Italy. Tarkcy. etc.). this sun-drying method is
applied to nmscat grape varieties! Alexandria Mus-
cat, for example) More than simply concentrating
grape sugar, sun-drying in particular incirases the
typical aroma of the mast These niu.sts attain high
free and odorous tcipcnic alcohol concentrations.
In the Jura region, healthy grapes arc sotted on
the vine and the different varieties are judiciously
gathered (in particular. Savagnin). The selected
grape clusters are hung to dry- spread out on
wooden grids covered with straw or suspended
on wires in strongly ventilated storage rooms.
This drying method results in extensive waste, not
only by desiccation but also by ml Contaminated
grapes are removed rcgalarly. This operation lists
2-4 months. The grapes are generally pressed after
Christmas, producing musK containing 310-350 g
of sugar per liter (legal minimum = 306 g/l) with
a higher than normal volatile acidity. The yield is
approximately 250 l/ionnc.
11.13 ArtiBcblOvcrripcnin;!
Nataral grape drying is a difficalt operation to
master, dac especially to the risks of rot-indaccd
grape alteration. Since the beginning of the 20th
century. otologists have been trying to replace this
natural process with an adapted technology. The
principles of an industrial overripener are simple.
The equipment consists of circulating hot and dry
air over the grapes, which arc placed in small boxes
inside the healed compartment. The ventilation
system circulates 2500-5000 m' of dry air ( below
l.i'" relative humidity) per hour, al a temperature
varying front 25 to 35*C.
According to recent experiments which confirm
earlier tests I R ibcrcau-Gayon el id.. 1976). the
apparatus reduces the grape crop mass by 10- \5'i
in 8-15 hours and increases the alcohol strength
by 1 5'i volume in potential alcohol. The decrease
in acidity, by the oxidative degradation of malic
acid, varies according to air tempcratare
Other biochemical phenomena probably accom-
pany this artificial overripening Wines obtained
from these treated grapes are richer in color and
tannins and arc always preferred al tastings. This
Ucalmcnt is exclusively for red vvincmaking.
since the resulting increased phenolic compound
concentrations are detrimental to quality while
wine making.
liqiiipmcai costs and utilization constraints Until
the use of this technique, but its effectiveness is
proven.
11.3 HARVEST DATE
AND OPERATIONS
I-irsi and foremost, the grapes should be protected
from attic ks and contaminations such as Eadcmis
and rot. right up to the harvest.
Optimal enologlcal mammy depends on grape
variety, environmental conditions and wine type
(Section 10.4.1). Thus a perfect knowledge of
reiiiiam conditions and half-Wvuvnw dales will
permit the vincgrowcr to ontani/c the harvest
according to Ihc various mammy periods. Maturity
analysis monitoring complement* this information
(Sections 10.4.2 and 10.4.3).
The giapc crop should be harvested under
favorable climatic conditions After rainfall. Ihc
grape cluslers retain water that is likely to dilute
Ihc must The giaprs should therefore be allowed
lodrain. al least partially The most recent research
measuring water activity on the surface of grapes
has shown that it needs to remain there for at
least two hours. Morning fog can also cause musl
dilation Harvesting should begin alter the sun has
dried the vines, bul at the same time the prolonged
maceration of harvested grapes in Ihc juice of the
302
Handbook of Fnokigy: The Microbiology of Wine anil VinUkations
inevitably burs! grapes during the wannest hours
of the day should be avoided
Mechanical harvesting facilitates the rcali&ttion
of ike above recommendations Progress In harvest
machine technology has helped to avoid bciry
alteration and excessive vegetal debris. Thanks K>
its speed and ease of use. the harvester permit,
a rapid harvest of grapes at Ibclr optimal quality
level and at the most favorable nionicnl Manual
grape-picking can be even more selective aid
qualitative, but its cost is not justifiable in all
Whatever Ike harvest method. Ike vincgrowcr s
principal concern should be Ike maintenance of
grape qnalily.
11.3.1 Gr
: Ha
From ancient times k> recent years, harvest
methods have barely evolved — ruber than slight
improvements in tools for gtupc culling and
gathering.
In certain appellations (Champagne, for exam-
ple I and vineyards, quality concerns prohibit
mechanized harvesting In noble rot (Section 103)
vineyards, it cannot re implcmcnlcd because the
harvester is not capable of selecting grapes thai
have reached the proper slage of noble rot.
Everywhere cLsc. since the beginning of the 197*.
mechanical harvesting has undergone spectacular
development as a result of increased production
costs and the disappearance of manual labor
(Viomandl. 1989)
Lateral or horizontal strike harvesting techniques
arc easily adapted to traditional vine training meth-
ods The grapes arc shaken loose by two banks
of flexible rods which straddle Ike vine row The
bunks of rods transmit an alternating transversal
oscillation to Ike vincMFtgarc I I.I). This move-
ment transmits a succession of accelerations and
decelerations to the grape clusters which results in
individual grapes, partial grape clusters or entire
grape clusters fulling. The adjustment of these
machines is complicated and requires a complete
mastery of the lev unique The number, position and
angle of ike rods or rails in Ike banks must be cho-
sen in accordance with Ike training and pnning
system used. Finally. Ike striking frequency must
be adapKd h> the foiwanl speed of the harvester.
The frequency is adjustable from to 600 strikes
per minute on conventional harvesters, sonic more
recent models attain up to 1400 strikes per miuulc.
Harvesters should be adjusted to allow for not
only the variety i.ihc case of grape diskxlgcmcnl
depends on the variety! but also Ike pnning
method employed and the canopy density at the
lime of the harvest. For example, a harvester with
an insuflkicnt striking frequency in thick foliage
does not harvest the entire grape ciop. On the
contrary, an excessive oscillation amplitude and
striking speed will transmit a kit of kinetic energy
k> the rod bunks, so that the rod-strikes hurst the
poorly protected berries.
New striking methods are responsible for most
of the recent improvements in mechanical harvest-
ing. Oi conventional luirvcslcrs. the rod ends arc
nol attached, thus their inertia is not controlled.
In more recent models, manufacturers have elimi-
nated this free tod end Their solutions vary with
rcspcci to the various machines I Figure 112). In
some cases. Iwo rod ends arc connected by an
articulated link and these flexible rails strike by
bending In other cases, striker shafts at Ike front
and back of the machine oscillate semi-rigid rods
at different amplitudes
The dislodged grapes arc gathered on an Imper-
meable nvobilc surface made up of a scries of
overlapping plastic elements in general, two lines
of plastic elements shaped like fish-scales. These
elements yield to vine-Jocks and Irellct posts by
moving on their rotation axis. Lateral grill or per-
forated belt transporters then drain the grapes as
Harvest aid Prc-l-crmcniatlon Treatments
Fig 11.2 New lii.-t.il Mule hirvcMcr
rii.ir... 1989): ■.. ■ Ik.H.- M.-1..-I. ih. ■ .1
mikcr. (c) dilfcicatHl rod bank Miikci
imirfc. (Vro-
hdmc r^kl
they arc conveyed. Upon reaching the extremity
of the machine. Ihc grapes are transferred lo a
shclfor bucket elevator (Figure 11 JlThR fypcol
machine permit high harvest speeds, to Ihc detri-
ment of crop quality. Juice losses may represent
np io li»< of total weight.
Striker mechanisms inevitably cntiain leaves,
leaf fragments and other vine parts <MOC —
material other than grapes) with the grape clusters
ike latvcMcilViu
The Mix; should he eliminated as quickly as
possible. preferably before becoming covered in
juke. It Is usually removed by Mowers Extractor
fans ptuccd above the conveyor belt arc effective.
When these extractors are properly adjusted and
combined with destemming screens, they are
capuHc of reducing Ihc rale of miscellaneous
rubbish k> 051*. The crop is generally stored
temporarily In one or two hoppers wilh a capacity
of 8 -20 hi. These hoppcts are capable of dumping
Ihc cropdireclly into the transport con tuners.
R ibtreau-Gayon el at ( 1976) had already indi-
calcd thai rigonws comparative studies between
man b.i I and mechanical harvest quality were
practically impossible The two methods would
have lo be examined on a sulficKnlly kirgc and
homogeneous parcel, and the harvest reception and
wincmaking equipment would have to be capable
of Klcntically handling a Latgc immediate grape
supply from mechanical harvesting or a progres-
sive grape supply from manual harvesting .
An experienced cnologisl Ls capable of examin-
ing the grape crop visa. illy to compare mechan-
ical harvest and manual harvest quality, includ-
ing the proportion of buislcd or rotten grapes and
Ihc presence of leaves and leaf-stalks (intact or
lacerated).
Recent research has continued those initial
observations (Ckiry el ill.. 19%). Prudent har-
vesting wilh a coirecily adjusted harvester pro-
duces similar results u classic manual harvesting
Manaal harvesting, however, continues to permit
Handbook of Etiology: The Microbiology of Wive anil Vinifhations
more cxlcisivc bin
(Scctwn 12.2.3).
t expensive grape sorting
11.3.2 Harvest Transport
Choosing harvest transport equipment Is a complex
Issue. Il is linked lo lie organizing of harvest wort
and Ihc winery rcccplion installation and is subject
lo cenain c no logical aid economical constraints
Fn>m an cnological viewpoint, grapes should
arrive at tbc winery intact Mote precisely, the
container should transport Ihc grapes in the physi-
cal or biochemical state obtained after picking and
transfer them to the reception bin The exaggerated
bruising and crushing of grapes can be avoided by:
• using shallow transport containcis (not exceed-
ing a depth of 0.8 m|:
• using easily cleaned material tt ensure proper
hygiene;
• limiting the number of grape transit - is and the
kxkl and dumping height.
Vinc-growcis in certain viticultural regions (Cham-
pagne, for example) mc requited to follow these
strict rules Small perforated containers, preferably
plastic, arc used for grape-picking to ensure grape
qaality from the first st;p of the harvest. These
containers are slacked on an open Iruilcr and gently
emptied at the winery.
Mechanical harvesting produces a different
grape supply rate compared with manual picking:
• Hourly crop volume is considerable tfmm 4 to
10 tonnes per hour) and Ihc daily duration is
often 12 hours.
• The harvest is partially destemmed and
crashed — sometimes with a kit of Juice ( 10-
30"# of total juice)
• The harvest is fnll of MOG (leaves. Icaf-stalks.
shoot fragments) and sometimes small animals
Mechanical harvest transport does not follow
the same rules The harvested grapes should be
brought rapidly to (he winery after the jnice has
been separated from the solid parts to the extent
possible. Sulfnr dioxide should not be added lo
the nnsepaiuled harvest: it favois the maceration
of tbc solid material during transport. Similarly,
carbon dioxide only protects must from oxygen
when separated from (he grapes (Jacqnct. 1945).
Transport equipment can be grouped into two
categories (l ; ig«ic 11.4):
1 . Removable containers are placed on a transport
chassc — sometimes several at once, depending
on their sin; (02-10 hi). In some cases, the
containers have large capacities (10-150 hi)
and are used for transporting (he grape crop
over long distances This operation is not
recommended from an enological viewpoint.
2. Gmpc recipients should not be too latgc. lo
avoid crushing the grapes and to reduce (he
number of times they need to be transferred
3. Fixed containers may be used, correspond-
ing u a transport unit. Within (his category,
damping containcis instantaneously empty (heir
cntiic crop into (he reception bin. and contain-
ers equipped with screw-pumps progressively
empty the crop. Thus they can adapt to any
reception installation (Eignrc 1 1.4).
The transport container for harvest trailers is
permanently altiched :•• Ihc chassis The vineyard
therefore requires additional trailers for odicr vine-
yard operations. These trailers are distinguished by
their capacity and dumping method The smallest
(from 20 to 30 hi) are able to p*a bclwecn vine
rows, thus eliminating intermediary crop transfers.
They are gravity-tilt trailers and often require a
costly recessed reception area. High-capacity bin
trailers are shallower, which limits grape bruising
and crashing, bnl they arc too large to pass between
vine rows. These containers empty the grapes into
the reception bin using a hydraulic lift system.
Elevator bin trailers also exist and are capable of
lifting their contiiners up to 15 -2 m before emp-
tying, depending on the model. This system docs
not require a recessed reception area. The hatvesl
ran be directly fed to the first step of the wine-
making process (destemmer— crusher or press).
Similarly, screw-bin trailers equipped with pumps
ll-igure 11.4) eliminate the need for a rcccplion
Harvest and Prc-l-crmcntalion Treatments
M£ 111 Dilknni hir.tM inn* pan comaincn (Rfccicau-Gayaa n U.. 19771 Portable cuMalocn: ») 20-90 1
*«labk- txue: <b)0D-1001 flank oi wooden wnuioca: <c)600-SM)l handling bin: 14)1000-2000 1
biph-tapuciiy cuMiincn.. m a* pa it at by duck: (c) 15-20 bl portable ml> Taller*: (ft 15-25 hi gravty duaping
bio; (pi 20-30 hi mcchankalduapirifr bin: I hi cfcntoi liimpiap bm: in Hrcw-drbcahin: (]| *crtwa*d punp-drivea
but; (k) fumpcrlufc
installation. Although these systems arc very prac-
tical, the nice luui tun or some screw-pumps is Kx>
brutal ami can decrease crop quality.
Mechanically harvested grape crops, especially
white, require rapid draining. This is most ol'm
cffeclcd with grills foming a double lloor In the
bottom of bin trailers.
11.3.3 Cleaning and Sorlm*
the Crape Crop
These operations Include eliminating MOG (leaves
and stalks) and damaged, nnripc or rotten grapes.
During mannal harvesting. MOG is eliminated
(at least for red grape crops) at the same lime
as the crushing and stemming process The sane
equipment is used for cleaning mechanically
harvested grape crops, but the different aspect of
these grape crops would justify adapted machinery,
which will certainly be developed in the future
Sorting the harvest to eliminate had grapes Ls
only possible with intact grapes This operation is
difficult with machine- harvested grapes. Cutlers,
however, may precede the barvcsKr to eliminate
most of the damaged, spoiled or nnripc grapes
.;::<,
Handbook of Enokigy: The Microbiology °f Wine anil Vindications
However, heller res ■lis have been obtained using
automatic sorting systems with vibrating screen
Manually harvested trapes have Ike undeniable
advantage of being able li> undergo an effective
sotting. Tkls can be carried out as the transport bin
Is being filled At present soiling bibles arc used
and workers, placed around these tables, remove
bud grapes.
Among the various sorting bibles proposed by
manufacturers, there arc models adapted to the
back of the tractor that directly feed the bin
trailer. Usually, however, the sorting table is an
independent, detachable unit installed between the
reception area and the first pKcc of wincmakiig
equipment A sorting bible essentially consist, of a
conveyor belt on a metal chassis. The belt is driven
by rollers powered by an electric motor The belt
speed should be slow (less than 5 m/min) Id limit
workers' eye fatigue. The belt is often made of
food-quality rubber and is sometimes perforated.
An articulated plastic bell is also used The sorting
tables arc often slightly inclined <5- lOtt) to
facilittlc draining. Only incict grapes should be
supplied u the sorting tiblc. Screw-pumps often
damage grapes and therefore compromise sorting
effectiveness. Ideally, small-capacity contiincrs
should be emptied directly on the bible, in which
case tfcc grapes arc spread out as the containers air
emptied Vibrating tables are now used to spread
the grapes out. although they arc rather noisy
11.3.4 Crape Selection and Selective
Must Extraction by Low
Temperature Pressing
Generally, in a homogeneous panel containing
only one grape variety, maturation intensity can
■- -u >■ I mi i one grape cluster to another and even
from one giapc in another on the same cluster
(Section 103). During manual grape- picking, and
even more so during machine harvesting, the level
of maturity of grapes is difficult to distinguish.
A best, the incoming grape crop can be sorted
accoiding to its sugar concentration and sanitary
stats.
In noble rot regions, grapes and grape cluster
fragments are selected by successive (sorting) har-
vesting. Only the grapes having reached the rfiri
stage arc picked (Sections 103 and 14.2.2). Even
in this case, the grape-picker cannot always pre-
cisely evaluate the degtee of grape concentration.
Furthermore, the state of the harvest docs not nec-
essarily make the appropriate choice possible.
In while winemaking. a method called controlled
Icmpcraturc pressing currently permits selective
must extraction from grapes richest in sugar.
These grapes freeze al a lower Icmpctaiurc than
those less rich in sugar This modem technique,
which is authorized as a harvest selection method
by European legislation, originates from a tradi-
tional grape-picking method in certain viticoltural
regions In the northern vineyards of Germany.
Austria and Canada, the wincmakcr benclits from
Ike severe climate by harvesting and pressing while
grapes while they are partially frozen A must par-
ticularly rich in sugar is thus obtained and is used
to make the highly prized ice wincs(mt<W») This
pressing method strongly accentuates the concen-
trations of sugar and aromatic substances of these
overripe g tapes.
The method of selective cryocxtraction* con-
sists of cooling grapes until only those richest
in sugar remain normal. The others arc frozen
solid and arc not compressible. The grapes arc
then immediately pressed. Only the must from the
grapes richest in sugar and therefore highest in
quality R extracted (Cnauvct el erf. 1986).
The ik I hod Pi carried out differently depending
on the harvest method. With manual harvesting,
grapes in small containers are placed in a freezing
chamber. After cooling, the boxes are manually
emptied Into the press. When grapes are harvested
mechanically, this method is less effective The
grapes musl be drained before cooling and are then
frozen in bulk by a freezing device. LkiuKI nitrogen
is olun the cooling source.
Noble rot grape winemaking has found partic-
ularly Interesting applications for Ibis technique
(Scclioa 14.2.4b) hut it can also be applied lo
healthy grapes for making dry while wines. In
addition to sugars, other elements of grape chemi-
cal constitution arc modified. Even though the total
acidity and malic acid concentrations are higher
in the selected must, the tirctric acid concentra-
tion generally decreases The increased patissium
Harvest aid Prc-l-cruvcniation Treatments
307
concentration resulting from cryocx traction makes
a portion of ihisucid insoluble. Phenolic compound
concci (nation* mum stable, indicating thai Ihc
cryocx traction acts essentially on the pulp wilhonl
altering Ihc skit The nines produced aic richer
and mote complex
Supra- extraction (Section 13.3.6) is derived di-
rectly from cryocxlraction. It consists of subjecting
Ihc grapes u frecic-dcfrost cycles. Ihea pressing
■hem :n room temperature The Ke crystals icar
Ihc cell walls, so pressing extracts even moie
com poinds from ihe grapes.
11.4 ACIDITY ADJUSTMENTS
OF THE HARVEST
Generally, in temperate climates and in traditional
winemaking regions, tegular grape sampling and
maturity assessment and the cancel choice of
grape varieties ensute a proper level of harvest
acidity In the Bordeaux region (France), the low
acidity of Menbl is often compensated by the
higher acidity of Cabernet and possibly Malbcc.
planted together in the sane vineyard. But acidity
may need to be corrected when making single-
variety wines or in extreme climatic conditions.
Acidity adjustments consist of cither increasing:
(acid additions) or decreasing (acidity reduction)
total must acidity Various products are used for
■his purpose. Contrary Id ovcrripcuing techniques.
acidity adjustments are strictly regulated in Ihc
European Community and in other countries that
icspcclOIV (Office International dc la Vignect dn
Vin) recommendations
11.4.1 Acidification
In hot winegrowing regions and during excep-
tional ripening years in IcmpcraK Ames, a con-
siderable amount of malic acid is degraded during
maturation. In order K> maintain the freshness and
firmness of wines desired by consumers, especially
in whites, the acidity should be increased by adding
an acid. High acidity also enhances the protective
effects of sulfur dioxide.
Strong inorganic acids, snch as hydrochloric,
phosphoric and sulfuric acid, may not be added to
wine aid arc prohibited in all countries European
legislation and Ihc OIV only recommend tartaric
acid, bul this acid lends to harden wines and should
be added with caution.
Foe a total acidify comprised between 3.0 and
33 g/l as H.SO, (4.6-5.4 g/l as tartaric audi. 5" g
of tartaric acid should be added per hectoliter
If the total acidity of Ihe grapes is below 3 g/l
as H.SO,. then 100 g of tartaric acid should be
added per hectoliter. In any case, legislation lim-
its this acidification to 15 g of tartaric acid per
liter tTable I I.I). The need for acid additions can
also be determined with respect to must pH. For
example, they arc necessary for pH above or equal
to 3.6. In bow acidity musts. Ihc production of
succinic and lactic acid during fermentation lends
to increase acidity in greater proportions, and this
should be considered when determining the need
for an acid addition.
In both red and white winemaking. tartrate
should be added before, or. preferably. Unvard the
end of fermentation Allowing for precipitation,
the addition of 100 g of tartaric acid per hectoliter
increases the acidity by I g/l expressed as H.SO,
1 15 g/l as tartaric acid) Acid additions, however,
should never be calculated to bring acidity up
to normal levels As adding tartaric acid in
sol utilizes potassium, it tends to have more effect
on pH and flavor than on total acidity levels
Harvesting a portion of the crop before full
maturity or using grapes from secondary flowering
can provide natural acMliticalion. bnt these methods
are not recommended as they are detrimental to
quality.
The addition of citric acid is not a useful solu-
tion, since Ihc total citric acid concentration limit
is set at 1 g/l as citric acid This limit, imposed by
European legislation, docs not noticeably increase
the total acidity. Furthermore, lactic bacicria can
break down this acid during winemaking. increas-
ing volatile acidity.
Calcium sulfate I plaster or gypsum) was tradi-
tionally used in certain viticullnral regions i.lcrc/
in Spain). Its addition at doses of 1.25-2.25 g/l
lowered must pH by precipitating calcium tartrate
This method has now been practically abandoned
Although effective, it is not advisable. ■
I landbook of linology: The Microbiology °f Wi»e anil Vindications
Tubb 11.1. Gmpi
acidity o
1 .Y.'lL.i. 11!
.' i.- i he i Unipci
in Co»mu»hy (ctccpt Portugal)
Scheauik delink;
ioillUFJ
j»n»
Ackli»cai«>ir"
1 l..|
ililihtiilKin
So—I.
can E.ceptionalycar*' 1
U) Viacyaid* of Bclptua. luvemhuBi.
Holland. CJumi Britain. Anuria anil Ger-any
(except ihc Baden regno)
lb) Vincyaids of Bade • and the umber* half
ol I raon ILoi<e Valley included)
(C Ij| Vjacyaid* of i he m** hers kill' of France.
except Meridional, and noRh-»cu uf Spain
(C lb) Alpine vineyanfc. of ■onnem hah
(C II) Meridional vineyard* of Fiance except
*>ne<C III): vineyanfc of ihc nonbern half of
Spain: Italian vincvanfc except mac* (C Hand
<C III)
(C HI) VincyanUoftbceauern Kyieaeci. Vat
and Conica in Fiance: vineyanfc of Ihc
louthcrn half of Spain: Italian vineyanfc of
Ba%Ulcatc. Fouillei.Cabbria. Sicily and
Sinlinia: the vineyanfc of Gnrcce
i j pa
-
Atxhoriittl
Authorised
Authorized
■a*. \5fA
Author) fed
\i' b ■ H..-.1
mat. 13(7/1
—
Authorized
■urapcm
v namallY
I "Mil J.
• im acai
i cxcq*Mt
•rapr nun aiu ik«
pamllco 1 tj) to ! " i I
iLs use dim be mentioned on Ihc wine label
tBcnitcje/irf. 1993) (\blumc 2. Section 12.4).
Catk* exchange treatments In must and wine aic
authorized in mine ion nines When used for stabi-
lizing tartaric precipitation, these treatments acid-
Ify lie resulting prod net; bnl in normal treatment
conditions. Ihc pH is not lowered by more than 02.
Eliminating potassium by electrolysis (Volume 2.
Section 1251 afco causes a slighl decrease in pH.
dcacidificd This practice is authorized in many
countries and varioas product* are available
The two main compounds, calcium carbonate
and potassium bicarbonate, react according in the
same mechanism (Blouin and Pcynaud. 2001).
When they combine with tansuic acid. H.V. the
caibonalc is broken down inn carbonic acid,
released as Co.. and the calcium or potassium
forms an insoluble salt with Ihc tartrate, which then
prccipilaKs on I.
11.4.2 Dcacidificalkm
When grapes do not reach complete maturity in
northern vineyards, grape acidity can be consid-
erable In these conditions, malic ackl concen-
trations are almost always greater than those of
tumric acid When the biological degradation of
malic acid is not desired due to Ihc oiganolcptical
changes that it causes, the juice must be chemically
— wilh calcium carbonat?
fCaCO)
1M. V
-CaT |+COi J+HiO
— with potassium bicarbonate
H;T + KHCO» KHT | + CO, \ + H,0
150 g 100 g
Harvest and Prc-l-crnvcntalion Treatments
309
Theoretically, in both cases. I g/1 ofdeacidH) Inj:
agent neutralizes 15 g/1 of H>T. giving a decrease
in acidity of 20 nicq/1 or I g/1 expressed in HtSOj
(or. of course. 1 5 g/1 expressed in tartaric acid). In
practice, the reaction is less efficient, especially in
He case of KHCO,. where higher doncs arc rec-
ommended hi achieve the same level of dcacid-
ilication (Table 11.2). so this product should be
reserved for minor acidity corrections. In any case.
itshoikl always be borne in mind Inat these dcac id i-
tying agents ac I cxclusivclj' on tartaric acid, so they
should not be ascd to try to adjnst acidity to nor-
mal levels, which woaldncccssi tile the elimination
of excessive anion its of tartaric acid and catsc an
unacceptable increase in pH.
In the case ofcak'inm cartMnalc. dcacidilicalion
resale, directly from thcsaltschcmical reaction, so
maximum dcac id ideation is rapidly achieved aid is
relatively predictable. However, if. for any reason,
the wine has a high calcium content, there is a
risk of f inner prcetpitiitoi at a titer date, even in
boltfc.
However, the dcacidilkalkin mechanism involv-
ing potassium bicarbonate is more complex. Fol-
lowing an initial decrease in acidify dnc to Ihis
sails reaction with tartaric acid, the formation of
potassium biranraK npscls the ion equilibrium and
precipitates, producing a secondary dcacRHficalion
that is not. theoretically, predictable It is advis-
able Id condnct laboratory trials u determine the
appropriate dosage (Han:*}'. 199B).
For all these reasons, it is advisable to use cal-
cium carbonate lo deacidify musts with excessive
acidity IcveLs before orduring fermentation. Poras-
sinm bicarbonate should be reserved for slight cor-
rections (after laboratory trials*, during the final
preparation phase ll Is. in many eases, preferable,
to implement dcacidilicaliou in two stages.
Tabic 11.2 PnxhHI >1 -..-■■ for (he dcjckli licit nil of
Inanl taul K-iiU>
IS H,«J.,/I>
leu. i ban 7.0
From 7.0 lo 73
Viou 7.S lo 8.0
GiuictilunKfl
CiCOi
(gAI>
KHCO,
(BVhll
Another aalhori/cd prodnct. neutral poLtssimn
tannic. Is rarely used, due lo its cost and lis low
dcacMlifying power. To lower the total acidity by
I g/l as snlfnric acid, requires 23-3 g of neutral
potassium tartrate per liter. This prodnct dcac idifics
by precipitating putassiim hydrogen tartrate, which
possesses an ackl function.
All of these products act uniquely by precipitat-
ing tartaric acid, since the poussinm and calcium
sails of malic acid are soluble. Yet insufficiently
ripe grapes contain excess malic acid.
VYucncrpfcnnig (1967) recommended a dcacid-
ilicalion technique in Germany based on the pre-
cipitation of a double calcium ualale and tartrate
salt, insoluble above pH 45. With Ibis method, a
fraction of the mnsi lobe treated is completely nea-
trali/cd by calcium carbonate, containing a small
amount of calcium malatc and ttrtralc seed crys-
tals After pree ipituion of the double salt, this
strongly dcacidilicd volunK is fillcrcd before being
blended back inu the untrealcd fraction. This pro-
cedure was developed and perfected by Hanshofcr
(1972). Where M (in hit is the (oral volume of
mnsi *> deacidify. the fraction to be treated (.Mji
by calcium carbonate Is calculated by asing the
following formula:
M d =
<*i - h)
M
(11.11
7*i is the (i Datable mast acidity in grams of tartaric
acid per lilcr: 7; is the desired acidity after the
treatment. At a must volume .'..',,. a quantity of
calcium carbonalc is added, given by the formula:
0.67 (T, - T t W.
When there is too much surplus malic acid in
the initial must, pan of the calcium carbonalc
added lo the trealcd volume produces soluble
calcium malaic. At the bmc of linal blending, the
surplus calcium reach with the tartaric acid of
Ihc ii n treated mnsi. The deackliticalion thasoccars
in two steps and acts essentially on the tartaric
acid. The tirtaric acid concentration therefore
has lo be adjusted in order lo decrease main
acid concentrations substantially, by double salt
formation (Usscglk>Tomassct and Gusli. 1992)
This rcsull can be obtained by using a mixture
of calcium carbonate and cakium tirtralc. also
iio
Handbook of linokigy: The Microbiology of Wit* anil Vinificaiions
containing a small amount of ihc double calcium
salts of italic and (ailuric acid u favor calcium
tartnmalai? crystal I i/a tun The dcacidiltcalion
powcrof ihismixiuic depends on the proportion of
cakium tiitr.iic used. In this case. Ibc use of Ibis
dcai id! Illation piocess becomes complex. It only
■caches final equilibrium aflcra long linte.
In while wincmaking. this dcacklificalion should
be effected after masl clarification bul before
fermentation Aromain ester production by yeasts
rs facilitated by a modcralc pH Conversely, this
trta Intent permit! a more prcii.se dcacidificatiou of
red wines when perforated al the end of alcoholic
fcrmenuUon.at Ihc lime of running off. Hem also
help 10 trigger nialokK ik fcrmcntatkin
European legislation docs nol impose must
dcactdificalKii limits, but then- is a limit of 1 g/l
of the kttal acidily as tartaric ackl for wine. Tabic
wines mnsi have a minimum total ackliry of 45 g/l
as tartaric acid. In any case, this treatment musl
be declared and cannot be combined with ai
acidification.
11.5 INCREASING SUGAR
CONCENTRATIONS
Certain regions have diflicnlty in pioducing quality*
wines. Chapter 10 discussed the climatic and soil
conditions which excessively favor vine giowlh
and grape development These conditions lead
to must> with high sugar concentrations, bul
the resulting wines lack finesse and aromatic
complexity
In the bcsl leini'rs of northern vltcyynls. unfa-
vorable climatic conditions during difficult vin-
tages often hinder maturation. Many parame-
ter deduced pnotosyn theses, continued vegetative
growth, excessive crop yields, etc ) limit grape
sugar accumulation: thus, adjusting lie natural
sag ar concentration can be useful. Of course, these
adjustment masl be limited and are not capable
of replacing a complete maturation. In particular,
their use should not Incite premature harvesting or
exaggerated crop yields.
Vilnius snbtractivc techniques increase sugar
concentrations by eliminating part of Ihc water
found In g tapes— similarly to natural ovcrripening.
The crop yield is consequently lowered. Although
some of these techniques are still in the experimen-
tal sctgc. they are largely preferred by international
authorities over additive tcchik|acs(Tinlol. 1990).
Additive techniques, despite their ktig length of
use. always have Ihc inconvenience of Increas-
ing crop volumes by the addition of an exogenous
product— sugar or concentrated must (Dupay and
dc Hough. 1991).
11.5.1 Snbtractivc Techniques
These techniques, similarly to drying and cryocx-
Iraclion. consist of eliminating pun of the water
ion laiied in grapes or in must. Two physical pro-
cesses can be nscd: water evaporation, or sclcc-
nvc separation across a semi- permeable membrane
( reverse osmosis! European legislation has set the
limits for these tieatnicnts: a 2lfi maximum vol-
ume decieasc and a 2'i volume maximum alcohol
potential Increase.
Quite apart from installauon costs, tnc consid-
erable ctop yield loss icsnltlng ftom the use of
these methods hindered their proliferation, and
chap tali /at ion was preferred for a king lime In
recent years, a fixas on Increasing wine quality has
renewed an inleicsl In these methods In ted wines
in particular, tannin conccnttalions ate simultane-
ously increased Of course, these methods should
never be used with Ihc intent of correcting exces-
sive crop yields
Heat concentration Is a longstanding method
often used In Ihc food industry Hoc mote than
40 years, il has been used to make concentrated
masl. bat potential wine quality must not be com-
promised during the heatiig process The denat-
u ration of the ntosensi live musl constituents and
Ibc appearance of organoleptic Haws Ihydiox-
ymcthyl furfural, for example) must therefore be
avoided. Earlier equipment, operating at atmo-
spheric pressure, lequited relatively high tempera-
lures, whkh produced off-aromas. Ear Ibis reason,
certain Eiench appellations prohibited their use
Today, vacuum cvapoialots have lowcicd the cvap-
oiutioi Icmpcialme to 25-30'C and interesting
1 1 Dalit i .i' .- tcsults have bcei obtained Even lower
evaporation tcmperatutesaic possible but the must
delivery rate becomes too low.
Harvest aid Pic-l-crnvcn(a(ion Ticatmcnis
ill
In addition, horizontally gioupcd tubular ex-
changers permit continuous high-speed irca invent
of must ins syskrni i.iun:- Hil risk of heal
hinds — (he prolonged contact between a lr.it Hon
of ihc m ns i and (he hoi exchanger surface. A thcr-
mocom pressor, acting as a heal pinp. extract, pan
of Ihc musl vapor and mixes il with (he vapor
prod need by the sKam generator. The system has
Ihc twofold advantage of lowering the treatment
temperature and saving encigy (by favoring musl
evaporation) Cnrrcnt equipment can trcal from 10
hi 80 hi of must per hoar, with an evaporation
capacity of 150- 1200 l/h In controlled appellation
nines, this treatment should be effected in a closed
circuit directly linked to the fcrmcnicr. The evap-
oration occurs at a low temperature (25-30"C>
and in these conditions the conccntralion facior
is always less than 2 iBcigcr. 19941. The sugar
conccntralion of must treated by the concentrator
remains practically constant during iK operation
cycle. Iron and malic acid ate concentrated to ihc
same dcgice as Ihc sugars, but polassium and tar-
taric acid conccmrationsarc lower. due to ibeir par-
tial prccipiuuion during the treatment (Tabic 1 1.3).
This Krchmqne. however, is nol reconimcnded for
concentrating musts made from grape varieties
with marked varietal aromas.
Pcynaud and Allatd ( 1970) nsed reverse osmosis
to eliminate water f torn grape must at ambient tem-
peratures. The results obtained vvctc satisfactory
from an cuotogKal viewpoint, but problems in
icgencraling (he cellulose acetate membranes
stopped (his technique from being developed
further The usefulness of icve
Tabic 11.3 MiM c
ralor<Cjhcmel Smi
figH
'»»• *«» * vacuum cvnpa-
■. Bonleaui. 1992)
(..■Mine til
Ininl Canccn- ConkKed
muu (rated vapors,
pll
k.ntg/1) IT9 204
Total ackli) (g/l ll.Vi.l
Millie jekKg/l)
Tartaric 4* .1 1 p/l)
Pb<a»iua tu/1)
ln>n(aui/l)
obtaining wines of superior quality wasconfinKd
by Wuchcrpfcnnig (1980) bul il was the devel-
opment of composite membranes thai gave rise
to new experiments with ie verse osmosis (Guinv
bcrtcan el of., 1989).
r-tgurc 113 illustrates the principle of reverse
osmirsLs An appropriate membrane is used to
sepatulc a concentrated saline solution (A) from
a mote diluted soluiioi (B). The difference in
chemical poicnlial Knds to make Ihc water puss
from Ihc low potential compartment (o one with
the higher Ihc potential I direct osmosis): (he laltcr
1 '
; i '
1>'«£ U.S. Principle of it
««f.. 1989)= (aldiiccl
libriim (OP = ™m<HK pi
(P = |i kv.--i.iii: .:a-.ik-.- I Iiipi
.mint. Kiuimbcitciu
.: (b) osmotic equl-
312
Handbook of linology: The Microbiology o( Wine anil Vmificaiions
is drilled. The diffusion of walcr stops when
(he interna) pressure of compartment A losmotic
press ■ rclcountcrbalanc :cs Ihc press arc inat diffuses
(he water across Ihc membrane If a pressure
greater than Ihis osmotic pressure is exerted on
conipartmeii A. Ihc direction of ihc dilTis*on
of ihc walcr R reversed (reverse osmosis) aid
solution A is coaccnlraicd.
A prcssarc of ai least mice the osmotic pressure
mist be c\cncd 01 Ihc iniisi to force Ihc walcr of
Ihc iniisi lo cross Ihc membrane. This concentrators
variois musl cousti Incuts. During walcr liansfcr.
mofcciks and ions letaincd by the membrane art
apl lo accumulate on its surface, increasing the
real misl concentration lo be trealcd aid this
ihc required picssiic To limit Ihis concentration
polarization phenomenon. Ihc filtering side of
ihc membrane must be cleaned to minimise the
thickness of ihis accumulating limit layer aid
U facilitate ihc rctrodiffusiou of the retained
soliics. Reverse osmosis can be placed in the
same category as tangential hypcrfiltralion. The
equipment is comparable and only differs by the
nature of the membrane.
Several models exist, depending on membrane
Liyont. Plate ntodilcs. derived fmm filler- presses,
■scd lo be the lirsl used (Figure 116). The fluid
st be treated circulates between Ihc membranes
of two adjacent plates This assircs the mechani-
cal sipport of the membrane and the draining of
Ihc permeate. The systems currently in use arc
equipped with spiral or tubular ntodilcs. Mem-
brane surface is often maximized lo compensate
for Ihc low delivery rate of these systems. Sev-
eral ntodilcs must be installed in parallel lo have
a satisfactory delivery rate.
Dnc lo the extremely thin flow stream, the must
undergoes an intensive clarification. Depending on
the equipment. Ihc musl should be settled, partially
clarified or perfectly limpid before Ihis process In
practice, for red wine grapes, the musl is Liken
from a vat. cooled, clarified by settling or filtration
K a turbidity of aronnd 400 NTU (concentrated
by reverse osmosis, and returned lo its original
vat. In certain cases, to avoid the formation of
potassium hydmgentartrate cryslabi. the use
mcLitartrx acid has been suggested Depending
of
I'ift 1 1.0. Pule membrane module for
m: l.its<bcB.ili< dilgnm olcaiir aMKhlk: IhlilcUil
of cell. (I) Mint la be licMcd: (2)concci*niied *mM:
|3| fcnncatc:(4) iMcrmcdnry plnc:<5> memhnne sup-
pott; (A) membrane
the modules, the working piessuic varies front 60
to 120 bars, ihc lempcraliic is regulated between
15 aid 25 U C and the permeate delivery rate (the
quaitiry of walcr removed over a period of time)
is from 05 to 5 l/n/ m J of filler surface.
Several findings have been continued in exper-
iments over ihc last 10 years (Bctgcr. 1994) The
membranes have a solute retention rale of over
995'*. but fosses increase when Ihc number of
modules in parallel is increased in order to alttin
elevated continuous treatment delivery tales. In
this case, traces of sugars and minerals arc fond
in Ihc permeate (Table 1 1 .4). Tastings rcveal lhal
the permeate sometimes gives off silfnrois odors
reminiscent of low tide. The rclentatc or enriched
mist is of a good oiganolcplKal quality. Generally.
Ihc cokci (rations of malic acid, metals, phenolic
conipoinds and macromolcculcs sach as proteins
Harvest and Prc-l-cra>cniation Treatments
Tunic 1 1.4. Cow
Sugar
(ximxttmikij
■ (■(A)
fH
:.[,.) Kkttv i-i H
ihU.)
Malic
«UHp/|)
limn
eaeidtpm
/aicrcliainaicd
< permeate)
awl polyosidcs increase proportionally with the
sugar concentration Salification phenomena cause
a 1i m i led modification of the concentrations ii tar-
taric .H i'l ami ]- >rassium ami the pH.
Subiraclivc techniques increase tannin couccn-
iralions by decreasing Juice volume wiih rcspcci
Id pomace volnnic and arc therefore more often
applied to red wincntaking than while In this man-
ner, they act like lank- bleeding (Section 125.8).
but without the loss of sagar These enrichment
methods have the distinct advantage of being
self- limiting for technical iconccnlralKHi of bad
tastes and flaws) and especially economic (oper-
ation costs and volnnic losses) reasons. They rep-
resent a considerable cost: in equipment invest-
ment, operation costs and crop yield loss The
lasl inconvenience docs not exist if the crop
yield R above authorized limits. The main Ink-r-
est of these techniques is to treat must made
from grapes soaked by heavy rain during the
harvest.
1 1.5.2 Additive Techniques
Contrary Id subtractivc techniques, increasing
the sugar concentration by the addition of an
exogenous product is not directly limited by tech-
nical constraints. These techniques arc therefore
very strictly regulated. Such strut regulations have
led the technical branches of rcgulaury otgail&i-
lions Id develop sophislicalcd analytical methods
Id verify that the regulations arc indeed being fol-
lowed: magnetic resonance of the deuterium of
ethanol. the isotopic ratio of "C by mass spec-
trometry, etc. i Martin Wo/.. 1986) Must sugar
concentration can he increased by directly adding
pure sugar, concentrated musl or rectified concen-
trated must. It is generally legally limikd to a 2*1
volume increase of the alcoholic strength — more
under ccrtiin conditions. In practice, however, this
increase should be limited to 1-1 5'i volume lo
avoKI discquilihialing the wine by an excessive
vinosiry which would mask wine Ira illness. Within
responsible limits, sugar addition is an effective
Tabic
11.5. Ar
inlcd m
,.!■ i ►.
billed (Hnwiii.nl I9S7.I
x-h«c muu and ihc uac
Com it
Concentrated »mi
Rc.iihcd iiiHcnaactl
Dc*t>-(3TC)
Su|8>re»ncci*Mion(|i/l)
rWntnlak«h»lf; vol)
T*al ackihv lg/1 H.SO.,1
Iron (mti/l)
Copper (mg/I)
Aaha (•p/kg ot «*ta>M
Ik. I p he nob i=i"'li' "I xupai
314
Handbook of Enology: The Microbiology of Wine anil Vindications
means of Increasing ihc gustalivc quality of wine
by .1 Hi. 1 1 1 l ir.uu body anil harmony.
Sugaring, bcllcr known ;ls chaptuli citron since
(he end of the 13th ccnlnry. consists of adding
refined white saccharose lo must. The sacchanise
mast be at least W> pure but can be derived
from any pfctnl (sugar cane, sugar beet. etc.). The
quantity of saccharose required to increase a wine
by i'i volume alcohol varies from 16 to 19 g/l.
depending on the yeast strain, must oxygenation
and the initial sugar concentration European
legislation has established precise doses IS g/l in
red wincmaklng and 17 g/l in while wincmaking.
The saccharose is dissolved in a fraction of
mast. This operation should be effected during
the first one-third of alcoholic fermentation and
daring a pnmping-ovcr The saccharose dissolves
more quickly In warm must and the simultaneous
aeration stimulates the fermentative activity of the
yeasts The augmentation of alcoholic strength by
chaptali&tlton modifies some of the constituents
of the corresponding wine The total acidity
decreases by 0.1 -0.2 g/l as H,S0 4 for 1% volume
alcohol added. This diminution is caused by
an increased potassium bitartralc piccipitillon. In
red wincmaking. phenolic compound extraction
increases by approximately 511 forcacb additional
l'i volume of cthanol. The Increase in volatile
acidity R negligible as long as the chaptali/attoa
is not exaggerated. For alcoholic strengths greater
than li'i volume. It increase?! by approximaicly
0.05 g/l as H2SO4. Glycerol and dry residue
increase in lesser proportions than cthanol For
a long time, lie differences In concentrations of
the varioas wine constituents, expressed in ratios,
were the only methods available for enforcing
chaptiliftition regulations. Today, rsotopii methods
can distinguish the sugar and thus the cthanol
torn ted in ernisof its botanical origin Igrapc.sagar
beet or sugar cane).
Varioas atoms have Isotopes: deuterium 2 H for
hydrogen 'H and "C for carbon "C. for example.
In a multiple atom molecule of natural origin, a
very small but variable quantity of these atoms
can be repined by the corresponding isotopes
The cthanol molecule possesses several hydrogens.
An isoiopomcr is formed If one or more of its
hydrogens, for example, are replaced by deuterium
The proportion of these different isokipomcrs
depends on the origin of the fermented sugar.
Indeed. Ihc vines photosynthesis mechanism is
different from that of other crops (beets, sugar
cane) and the dcukrium content varies accord-
ing to latitude Isotoponcrs are determined by
nuclear magnetic resonance I NMR). This method
Is capable of detecting the addition of sugar beet
sagar(chaptali/ation) by comparing a sample wine
with control wines made in the same geographical
region during the same vintage. In order to evalu-
ate a mixed addition of sagar beet and sagar cane
sagar daring fermentation, this fust analysis must
be complemented by a carbon isotope determina-
tion using mass spectrometry
The addition of rectified concentrated must
IRCM) is similar to chaplali/alion. This col-
orless liquid contains an cqulmolar mixture of
glucose and fructose. The rectified concentrated
mast is obtained by dehydration All compounds
other than sugar arc eliminated by ion exchange
resin treatment (Table 113). European legisla-
tion has precisely defined the characteristics of
this product The RCM must have a rcfractomct-
ric index greater than or equal to 61.7'i The
RCM. diluted lo 25' Brix (Section 10.3.3). must
not have a pH greater than 5. an optic density
greater than 0.1 at 425 nmora conductivity greater
than 120 ^ S/cm The legal limits are set at less
than or equal in ihc following concentrations: titra-
tion acidity. 15 mEq/kg tof sugar). salfurdioxide.
25 mg/kg: total cation concentration. 8 mEq/kg:
and bydtoxyme thy I furfural. 25 mg/kg.
RCM is used in the same manner as pure sac-
charose and leads to the same chemical constituent
modllicatious in the corresponding wines. It has
several Inconveniences with respect lo saccharose
First of all. RCM is more expensive. Equally
important, it is not In crystallized form, therefore
lo parity and stability over time depend on prepa-
ration and storage conditions. Attempt! have been
made to separate the glucose and frucusc from
the must in order to crystallize them separately,
bnt lis production costs arc Wo high Otherwise
RCM increases the dilution of the product. Finally.
Harvest and Prc-l-crnvcntation Treatments
Tabic 1 1
■ .uliilL...-.
»• (value.
cxficMcd in * vol.)
■■:■. p.- In. ... in iklcnnincd i
--"
* ■"
»**«■* o
Iiin>rcia viHulmral
A
B CI
i. ■ II
C III
(■) tbl
alcokol before juldiiu*
Ikh in paealul alcohol fS .oil
An honied vokinn
-By*
a similar product prepared by lac hydrolysis of sac-
charose can be used Id adallrralc and Ktmctlmcs
counterfeit RCM. The only advantage of RC'M is
that It is derived from grapes. This reference to
Its origin is secondary, since lac purest possible
prodacl rs desired and obtained through appropri-
ate treatment
Must constituent modifications an: even more
pronounced when non-punlicd concentrated must
Is used. These concentrated musls are usually
obtained by indirect healing. All organic and min-
eral elements in the musls arc concentrated They
have very low pHs (oflcn lower thai 3 ) despite the
insoliblli ration of a portion of the tartaric acid in
lac form of potassium and calcinm sails. In Tact,
snllur dioxide is ascd in high concentrations Tot
conserving the mnsl before concentration The SO?
Is oxidized into sulfates during the treatment. Con-
centrated musts arc also very rich in iron and arc
apt to cause iron cassc. They arc always very dark
ia color, dac to the decomposition of sugars and
nitrogen compounds in this hot and acidic envi-
ronment The resulting produce intensify lac color
prodnccd by phenolic compounds. The minimum
concentrated must sugar concentration is 582 g/l
(51 Brix). Concentrated mast greatly modifies
the composition of the wine obtained — especially
while wines; a slight incicasc in »tal and volatile
acidity and a more significant increase in glycerol,
dry extract and phenolic compounds in red wines
Concentrated musl oflcn comes from the same
grape varieties as the musl n» be enriched and
sometimes from the same geographical zdbc.
Due lo the divcisiry of its villcultural /ones,
the European Community has developed very
elaborate legislation on this sabject (Table 116)
In many appellation /ones, notably in France, the
legislation is ever more restrictive.
11.6 ENZYMATIC TRANSFOR-
MATIONS OF THE GRAPE
AFTER ITS HARVEST
When conserved iatact alier its harvest, the grape
still maintains an intense physiological respiratory
activity — which is utilized daring drying. The
lack of oxygca and the depletion of available
respiratory sabsiraKs rapidly provoke the initiation
of fermentative processes in the berry.
The ccllutur destruction of grapes during prc-
fermentation treatments results in oxygen dissolu-
tion, despite the precautions taken Two cnryBK
categories, oiktoicductases and oxygenases, arc
responsible for many grape constituent transfor-
mations. They oflcn harm grape quality
316
Handbook of Enology: The Microbiology of Wine anil Vindications
Depending on wincmaking techniques, thedura-
lion of giupc solid maceration in musl varies. Dur-
ing this period, hydrolase- type cn/ymes acl on
grapes and mnst These enzymes arc responsible
for the hydrolysis of diverse macromotccalcssuch
as proteins, polyosidcs. hetcrosidic derivatives aid
varioBs esters Their action often improves ihc
grape/must mixture This maceration phase shoald
therefore somciimes be prolonged.
11.6.1 Hydrolysis Enzymes
The active prolcosynihcsis lhal characterizes mat
a ration Is responsible for Ihc high protein concen-
tration ii malarc grapes. In must, prolcins often
represent 5(fi of ihc total nitrogen. In white wine-
making, part of these proteins arc Insolablc aid
arc eliminated during clarification Endogenous
grape proteases are apl lo hydrolyzc these pro-
Kins into soluble forms These forms arc morc
easily assimilated by ycasBt during fomentation.
The cn/ymes catalyze the hydrolysis of the pep-
tidic bond between two amino acids (Flgirc 1 1 .7).
The grape posscsscsa low and constant protasic
actlviry daring in herbaceous growth phase. From
wraium onwards, this activity snougly Increases.
In a ripe grape, the ptotcasK activity is essentially
tocakrd in the pulp (Table 11.7). Bnl the proteases
arc generally bound *> cell structures Healthy
grape juice thus has relatively few proteases (iff A
of iota! proKasK activity).
liihfc 11.7. Dbirbutbn of fioicjnit aclivay In ihc
diftcica aicjn of a healthy piiipc (cipicucd «i '• of
laulaclivcy prhem ><C<iidonBicraiul Dug*!. 1908)
All physical treatments of grapes (mechanical
harvesting, stemming, crashing) increase the prop-
ortion of soluble proteases. The higher free amino
acid concentration of these musts attest to this
(CantagreUfn/.. 1962).
Crape proteases arc acidic, with an optimum
pH icar 20 In the pH range of must 40-609
of Ihc potential protcasK activity exists. Protein
hydrolysis activity during the pre- fermentation
phase varies greatly, depending on grape malarily
and harvest treatments This certainly affects
fermentation kinetics but the relationship has never
been established. A slight sulfur dioxide addition
(around 25 ntg/l). however, has been confirmed
to slimatrlc piotcasic activity. This explains, al
least partially, its activation effect on fermentation
(Section 8.7 3>.
Finally, botrylizcd grapes also contain fungal
proteases. Contrary to grape proteases, these arc
solablc and pass entirely inu Ihc must. BtXrytis
cinerva aspartate proteinase has an optimum pH in
the vicinity of 35 Whatever their origin, proteases
arc thermostable, they increase soluble nitrogen,
even daring thcrmovinificalion.
Among traits, the grape is one of the least rich
in pec tic substances These substances arc predom-
inately located in skin cell walls (Section 102.6).
Must consequently contains a small antoanl of
these compounds in Ihc soluble form (05-1 g/l
expressed in galaciaionic acid) Depending on har-
vest treatments, some insoluble skin compoands
may be extracted Must pectic substance concen-
trations can thus attain 25 g/l. They arc prin-
cipally associated with cellular debris and musl
sediment Most are rapidly hydrolyzcd by pec-
tolytic en Ames of the grape. Ethanol sabscqucntly
precipitates Ihc test of them at the end of fcr-
mcntitlon Wine is therefore practically devoid
of must peclK substances (l_"sscg)»>Tomassct.
1978).
Mil /it H
_l_ l_l_l_ ,
l, — C — COJl ' HjN — C — K;
I I
n co,n
Fifi 1 1.7. 1'iacMC mode of uf ion by hydinK>n of peptide bond*
Harvest aid Pic-l-craicntiiina Treatments
il"
Thc ripe grape Is rich in pectin methyl esterase
tPME) awl polygalacturonases. Ripe grapes have
high pectin methyl esterase anil polygalacturonase
couvnts. bat conttla no pectin lyase. PMH is
not a hydiolysis cn/ymc but rather a sapoal-
Ikalion c»ni« ]i liberates the acid fuKlions
of galaclironic units, resulting in the accumula-
tiai of methanol in the mnsl (Figure 112). Grape
PME is thcrmosGiMc and has an oplimam pH
of 7 lo 8 Ir activity is reduced at the pH
of must, but its action beforehand in the grape
icsalls in a significant decrease in the degree
of cslcriticalion of liberated pectic compounds in
the must. This action Is essential, because the
polygalacturonase can only act on the free car-
boxylic fa notions of the galactnroiK units Two
types of polygalactaronasc exist in the grape: exo-
pDlygatacturonascs exert their bydrolytic action
sequentially, beginning at one end of the poly-
galacturonic chaia. cudo-polygalactaiDnascs act at
random on the interior of the chains, la the lat-
ter case, althoagb the pectin chain bydrorysR is
very limited, the cndo-polygalacturoak activity
leads to a rapid and significant decrease in must
viscosiry. The viscosity is reduced by one-half
when fewer than 5'* of the glycolytic bonds arc
broken. Grape polygalacturonases retain a signif-
icant activity' at the pH of must (optimum activ-
ity pH is berweca 4 and 5). The homogalactur-
onaae /ones are susceptible to rapid hydrolysis.
The rhamnogalaclaronanc Aiacsare more resisciat.
due to the presence of side-chains of arabinosc and
gafackne.
When the Bmryiis cinereti fungas infectsgrapes.
it synthesizes cellalase. pectinasc. and protease
en aiiics that break down the cell walls. ISec-
lloa 10.5.2). In addition to PME and polygalactur-
onases. Botnlis ciaeieu produces a lyase that cms
pectic chains by f elimination (Figure 1 1 Hi. This
endolyasc activity is not influenced by the ester-
ilication level of the carboxylK functions of the
gabicturonic umis. All pcclolytK cn/ymes of this
fungas have an optimum pH near 5 and arc there-
fore very effective during a bolrytis altick Must
made from contaminated g rapes conseqaently con-
tains very few pectic substances. A polyosldc. gla-
can.sccrcicd by Batrytis litieieu is mostly rcspoa-
slblc for their viscosity (\blnmc 2. Section 3.7 2)
°^£x
'^<t>L'
Kifc U.S. Modcofa.
(R =H orCHi)
anoldiRcKU pcctulyik
J 18
llandlxwk of Etiology: The Microbiology °f Wiie anil Vindications
The pcciolylic cn/ymes produced by ibe grape
or inc fungus arc (airly rcsc&tnt Id sulfur dioxide.
Their activity is. however, icdnccd at temperatures
below 15 'C and above 60*C.
In muscat- type aromatic variclics. a considerable
proportion of their arumalic potential is in the
font of icrpcnic hclcrosides — not-cxlonms in ripe
grapes (Scctioi 10.28). During prt- fermentation
treatments, enzymatic hydrolysis of these com-
pounds incrcascs ninsl aromatic intensity (Figure
11.9). This phenomenon Is enhanced by macer-
ation of grape solids because of the high con-
centration of bound tetpenic compounds in skins.
Fi& 11.". Dcaonuriiinn of the envyautk
ot tetpenic glycoside* (Bnyoaotc. 1WJ)
These compounds, called diglucosides. arc com-
posed of a glKOsc associated with another sugar,
ie rhamnosc. arabinosc or apiosc The hydrol-
ysis of these hetcmsides requites two sequential
enzymatic activities. A 0-i.-rhamnosldasc. an a-
i.-arabiaosidasc or a Jt-i>apkisldasc must act on
the molecule before the 0-i>glucosidasc Is able
to exert its acta* (Figure 11.10). In practice, this
hydrolysis is relatively limited. Grape glycosidascs
have an optima) activity at a pH between S and
6. and they only retain pun of this activity at
the pH of must. These glycosidases arc very spe-
cific and arc tot active on certain tcrpcik het-
cmsides. notably tertiary alcohol derivatives, such
as linakil Moreover, ff-glucosidasc is stn>ng)y
inhibited by free glucose (Bayonovc. 1993). In
coutiminatcd grapes, the glycosKlases secreted by
Bimylis comma arc more active. Bnl the fungus
totally degrades the aromatic potential of the grape
i Sec l Mm 103.3). Furthermore, the j)-glucosidasc
of Biitryn's cineiea is inhibited by the gluconofcic-
tone It produces
11.6.2 Oxidation Knzymcs
A green leaf-type odor or herbaceous note is
produced when vegetal tttsuc (especially leaves.
Fig II. ID. En/;, miii, bydniUiii mokinitm otrcipcnk (Jhciakki IB;
(2) |! i>- j pn.iKl.se: (3) a l-rkimmtiUuc: (4) B D-pluci»,fcfa*e
Harvest and Prc-l-crnvcncition Treatments
319
bnt oho I'niitt is crashed. This phci
shown id cxst in grapes by Rapp el ul. > 1976).
Fair enzymatic activities arc sequentially involved
tFignre II. Ill First, an acylhydmlusc frees the
mi;;- acids from mcmbianc lipids. Next, the
lipoxigcnasc catalyzes the fixation or oxygen on
Ihcsc C"ih insaturatcd fatly acids. This enzyme
prcfcicnlully forms hydroperoxides ii Cd I'mm
linolrk ami linolcnic acids. The pciuxidcs obtained
are then cleaved mm C« aldehydes Sonic of thcni
are ttduced to their corresponding alcohols by
the akohol dehydrogenase of the grape (Cronnrt,
1986) These alcohols an- responsible for their
corresponding odors Sine the cleave enzymes
are linked n membrane fractions, the aklchydc
conccitra lions ate proportional lo the intensity
of solids maceration To limit their concentration
during while wincmaking. a sufficiently clear must
should be obtained asquKkfy as possible < less than
2a)NTU><Duboirdietfr<if.. 19861
Grape ccllukir structure breakdown during pre-
fermentation treatments R also accompanied by
other enzymatic oxidations. Oxygen consumption
speed thus vanes from 05 to 5 mg/l/min. depend-
ing on must origin. This vanation r> caused for Ihc
most port by the oxidation of phenolic compounds
Ripe grapes contain an orthophcnol oxygen
oxidorcductasc. also known as cresolase. catechol
oxidase and tyrosinase lt> activity is extremely
variahlc. depending on the grape variety and
degree of ripeness. <Duberncl. 1974). Tytosinasc
consBo of a group of Coenzymes differing in
inductor nature and catalyzed activities t Mayer and
Haicl. 1979).
t'ili 11,11. Ennaaik fctimjlbn mcclaat'
KHfii. < I , |AcyI|h>tlnfaac: (2) lipnypcM
ikkviimgctmc
ial.kh_.ik* aide, iknhnb. itiptmihk inr^ci", Ojvdb. i'Hiai.
the pioencc "i»i._vt (.1) peroxide .kj.jp canmc; (4) alcohol
J20
Handbook of Fnology: The Microbiology of Wive anil Vwifications
In white grape must. Ills enzymatic activity pref-
erentially oxidizes tartaric derivatives of hydiox-
ycinnamic acids 1 1 1, majority phenolic compounds
in grape pulp (Figure II 12). Tk quinoncs pro-
duced (2) arc unstable and likely to enter into two
different reactions (Figure 11.13). First, these very
reactive quinoncs can condense with other pheno-
lic compounds (fUrvonoids). forming polymerized
products. Their color evolves fiom yellow *i blown
according to the degree of condensation (Single-
loi. 1987). The quinoncs are also upl to icact with
a strongly icdnctivc molecule such as glutathion.
This reaction produces a colorless derivative. S-
glueilbionyl-2-fmHS-cafcoy I tartaric ackl. known as
the Grape Reaction Product or GRP (3) (Salgues
el al . 1986) This derivative is not oxidizablc by
tyrosinase and thus docs not modify the color of
the must
Must browning depends of course on the
fktvonoid concentration and consequently on
mechanical treatments of that favor grape stalk
maceration. These operations arc also involved in
the solubilization of the tyrosinase bound to the
chloroplasl membranes.
Vet the trapping of quinoncs by ylutithion limits
oxidation phenomena Musi browning is therefore
also dependent on the glutaihioa concentration.
The tyrosinase of grapes is active bnl unstable
at the pH of must (optimum activity at pH 4.75).
Temperatures above 55 : C or the addition of
more than 50 ntg of sulfur dioxide per liter arc
necessary w denature this enzymatic activity.
Lower sulfur dioxide concentrations only modify
oxidation rates. In fact, the bisulfite ions regenerate
the potential enzyme substrates by reducing the
quinoncs formed Finally, treating must with ben-
tonile reduces the soluble fraction of tyrosinase.
Phenolic compound oxidation is much more
dangerous when the grapes have been attic ked
by BfHtyn's. Bolryti/cd grapes contain a /'-phenol
oxygen oxidotcductasc known as laccasc (Dubcr-
net. 1974). Contrary to tyrosinase, this fungal
enzyme is stable at the pH of must and is more
resistant to sulfur dioxide. It is also able to
oxidize a greater number of phenolic substrains
and molecules belonging to other chemical fam-
ilies. Laccasc is thns capable of oxidizing the
phenol-gin lath ion ( * I complex lo quiione (4) The
ko.
Fig 11.11 Mode <i
acliviy (•: couuuri
idaM t>f pope tyuBHUuc on hdwvycinmuikr a
Kid. b: cafcic acid) (2) CalcchoUvc activity: la) a
i iMayc. and Haiti. 1979). <l)Cic
icacid;lb)quii»nc
Harvest and Prc-l-crn>cn(atlon Treatments
and I- .(.-. M.-r.t gaft :if t b) hoiaK (Sj||U^.
glntathion. therefore, can no longer trap quinonc
t Sals* iics el irf . 1986). More brown condensation
prod nc Is arc formed from Ike same iiilEU pheno-
lic compounds by kiccasc than during oxRlalion by
If the gluralhiou conccnlralkin Is elevated, the
qniBonc (4) can be partially reduced to phenol
with the fixation of a second glurathion molecule.
This new derivative is no longer oMdi/iiblc by lac-
case. Oxidation phenomena and the corresponding
browning arc thus limited This second reaction is
not likely in bolryti/rd grape niusc^lSalgucscr of..
1986). Tic oxygen consumption rate is not higher
than in healthy grape must (Section 8.7.2). but the
action of sulfnr dioxide rs slower (l-ignic 11.14).
The contaminated grape contains many other oxi-
dases that also consume oxygen (glucose oxi-
dase, amino oxidase, etc ) A Icmpcralnrc of 50'C
destroys kiccasc more quickly than tyre
thermal dcnaturalion is the only possible treatment
as adding hcnkmtr only very slightly decreases
laccase activity.
Peroxidases have king been proven to exist in
crapes (Ponx and Ournac. 1972). This enzymatic
activity Is essentially located In grape cell vac-
uoles. It most likely plays an important role in the
oxidative inctir.il ism of phenolic compounds dur-
ing maturation (Caldcron el ill.. 1992). During pre-
fcrmcntalion trcatmcnls. the activity of thiscn/ymc
seems to be limilcd by a pemxide deficiency. A
low sulfur dioxide concentration Is sufficient to
destroy these peroxidases.
An increased understanding of these oxidation
phenomena has spurred the development of a pre-
fermentation technology called white mnsl hyper-
oxygcnaiion<Mtillcr-Spath. l990)|Secllon 13.4.1).
Handbook of Etiology: The Microbiology of Wine anil Vinifications
t"'ifc 11.14. Ellen of sulfa, dbiklc on oiype« con-
sumption in musis miiilc fiom ncathy «*1 mllco grapes
(I) Time &cic*s.ir. In MopotYgcn loasumplun iker (he
MkUrua "i sul&r .l»..i.k In mu*i auk fnim beUby
grapes. (21 Time accessary lo slop oxygen consumption
»ficrihe»dUiionofsutiufdioKkk In must m*k fro*
fnim hcihhy unfn; ■ . otygen coasumptkin in mini
mauf fnim itMica grapes
A sufficient and controlled addition of oxygen as
soon as cellular structures arc degraded provokes
Ibc denain ration of tyrosinase during Ibc oxidation
rcac lions thai it catalyzes. The disappearance of the
enzyme and the depletion of oxidictblc phenolic
sabslralcs tats nuke the mast stable with respect
lo oxidation The condensation products responsi-
ble for browning should be cliniinaicd before fer-
mentation. Dne k> iLs possible impact oi aromatic
elements, this vchntqac seems better adapted to
certain cnllivars. It is not applicable lo botryti/cd
grapes, due to the resistance of laccasc
11.7 USE OF COMMERCIAL
ENZYMES IN WINEMAKING
The beneficial action of diverse hydrolysis en-
Arm-s from grapes is often limited by must
pH or an insufficient activity due to the limited
duration of pre- fermentation treatments. Manufac-
turers have developed better adapted enzymatic
preparations, essentially from diverse species of
fungi {Aiperplba. RtBjppu and Trichotlrruuiy
Research in this field is very active (van Rcns-
bargand Pretonas.2000). The enzymatic profile of
currently available commercial preparations is still
unclear and users mast develop their own experi-
men ration Many countries permit the use of these
preparations. They arc added as early as crashing
to increase juke extraction, or to finished wine lo
improve filtcrability. These methods can also be
used to improve color extraction and must qaality
(settling, fermen (ability and aromatic intensity) in
red and while wiacmaking. respectively.
11.7.1 Juice Extraction
The addition of pccioiytic enzymes in crushed
crapes can improve Juice extraction for certain
varieties very rich in pectic substances (Muscat.
Sylvancr. clc.). Commercial preparations contrin
diverse enzymatic activities which arc active al
a low pH: pectin methyl esterases, polygalactur-
onases, pectin lyases and hcmKcllulascs. Atacon-
CHtratin of 2~4g/hl. 15* more juice can be
obtained daring a settling period of 4- 10 hoars:
even a shorter settling period (I -2 hours) increases
the ptoponion of free run (Tabic 1 1 8). Effective-
ness varies according to the nature of the grapes.
These peciolyiK preparations can also conuin
diverse glycosidascs(Cordonnier^/(rf.. 1989) and
proteases (Schmitt elal.. 1989). responsible for
secondary transformations Their degree of parity
mast therefore be assured
11.7.2 Must Clarification
Pccioiytic preparations lower while must viscosity
and thas accelerate sedimentation (Eigurc 1 1.15).
In less than an hour, the colloidal equilibrium is
destabilized, resulting in a rapid sedimentation and
increased must limpidity. A more compact must
deposit, facilitating static settling, is rare. This
treatment can lead to excessive juice clarification,
lis use shoakl be determined according lo must
Tabic 11.8 En/yaMtic liW»
iKaibiVa vanclv. Hungiiy:
mbed gapes
>.„„.,„
e 2 g/all (CaMH-Uubeic
. I9K9)
like
Com ml
Wihca
,=c
free nin
PlCU
t>yr,
3T&
93'
Harvest aid Prc-l-crnkcntatiou Treatments
323
lift 11.15. EfTc« o( pcclolytic cno-" •"■ 'he *ili-
ncnuikm %pccd uf »b*e muu lee* (Caiul-Lbuberc*.
1989)
composition. The en /ymatic ilqz radation of pectic
compounds B subsequently demonstrated byadis-
tinci improvement in the fillcrability of ibe musts
anil wines obtained These wines arc often belter
prepared for tangential nitration (Volume 2. 1 1.9).
In red wincmaking. these preparations arc used
in particular lor press wines aid bcal-trcalcd
grapes and must In the lalicr case, the must
is very nch in pcctK compounds and devoid of
endogenous grape enzymes. TTtcsc arc destroyed
hy beat (Marlinicrc and Ribcrcan-Gayon. 1973).
Pcefcdytie enzymes can also he used at the time of
running off after a UudilkHial maceration.
In botrytized grapes, the pectic compounds ate
degraded for the most part and replaced by a fungal
polymer, glucan (Section 105J). A ghtcanasc is
indnslrially prepared from Tsiclttxleimti sp fnngus
cultures (Dubourdicu <•( id.. 198 1 ). The en /vine is
preferably added ( I -3 g/hl) after fermentation, lis
action takes from 7 to 10 days and must occur
at a temperature equal to or greater than IOC".
Higher doses arc required in red wincmaking
since phenolic compounds partially inhibit the
glncanasc. Industrial glucanasc also affeco the
yeasl cell walls and improves the wine's ioik>id.il
stability.
grape maturity, length of maceration, number of
pumping- overs, temperature, etc. (Section 123)
Adding peclolytic enzymes at the start of macera-
tion can facilitate' this extraction (Table 1 1 9) The
resulting wine is richer In tannins and anlhocyanins
with a higher color intensity and redder tint.
This treatment also improves the organoleptical
charackrs (notably slruclurc) of the wine (Canal-
Llanberes. 1992). It apparently favors color stubi-
limiron by forming polymcri/cd pigments < Parley
ei al.. 2001 ) Further research is needed u evaluate
the stability of these changes during aging These
preparations also con tun |?-i>glucosidasc. likely to
hydrolyzc anthocyanin glycosides (van Rcnsbnrg
and Prciorins. 2000).
11.7.4 Freeing of An
The glycosidascs contained in commcicial pec-
lolytic enzymes arc capable of partially hydroly?-
ing lerpenK glycosides (Table II 10). Tie first
Tabic 11.9. Influence uf pectohlic ciamo <in cok>r
curartinn in led nine ma ling (Mental in Bordcaui.
Prance. 19*8: Vim nine 3 g/hl m illing) (Ciml-
l.taubc*». 1990)
Coniiol unl Enzymes!
AbMubjncc n
280 n
04
M
Tnnnin* (g/l)
Aahacyim (mg/l)
Cokii intcmiy
708
1 .58
38
893
108
Tim
::-J
0J0
AhMinSunce i
iiO n
27.8
202
AbMubjncc I
520 n
■ o>
03 .0
05J
AhMinSnncc a
A2D n
n(»
9.2
83
TnUe ILW Uhcniitin of tcipcnnb by ennnwli
hydroUiU (Gewiin ra miner 1985: Sovofcr- 12 <
13 ml/fcl.l aiimhinvubniionat K CMCinal-l.bubeie
1990)
1 1.7.3 Color Extraction
and Stabilization
Red wine color results from maceration of grape
solids (skins, pips and sometimes stalks) during
alcoholic fcmicntttion. Phenolic compound extrac-
tion thus depends on many factors: grape variety.
i. in j i. i
324
Handbook of Etiology: The Microbiology o( Wive anil Vinificalions
tsis of these enzymes wrc conducted on dry
winn because or the inhibiting effect cxcrlcd by
glacoscon lie ^-glucosklasc. These en /ymes may
also aci on olher aromatic compounds prcscni in
the form of odorless prcciisois in ccnain grapes.
This treatment ts intcaded K> coniplclc ihc
etpenic coaiponnd transfonnalions effected by
yeast, diring fermentation. However, n releases
all Inc Kipcnic alcohols mo rapidly. The plcasant-
smcllitg monoKipcnes. such as linakil. ncrol. aid
gcianiol. may be convened into more stable forms
daring aging, including icrpiucol. which has a
less attractive aionia (Pari. 1996). (Volume 2.
Section 12)
In any case, care shoald be taken lo avoid
enzyme piepatallons containing cinnamalc decar-
boxylase as il may lead lo the development of
clhy I- phenols with a highly unplcasunl musky odor
(Volume 2. Section 8.4J).
Enzymatic preparations shoukl never contain
cinnamalc decarboxylase. This en /vine can lead
u Ihc formation of ethyl-phenols with a very dB-
agrccablc animal odor (Chapter 2).
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Hemic- J.G . Gramtal-DebBido M.M. and Manin ID
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Kibcicui-" layon J.. Pcynaud K . Rlicicau-Gayon P- awl
Sudani P. ( 1970) Amclionukin dc la vcadange. In
Sinti.rt ri Tediaiqiiei du \iii Vol. 3; llm'ii.Divn
iriHsfotmtioits du tin. pp. 3-28. IXinod. Bonlat.
Pare,.
!.:.mii.: - ! ■■. :,". i \ . v .,.,„■: I M .. ■! '.;. .. i . .,„., M
( l990)Mccanc.mci.d »i><blkiodn polypbcMih<bm
let mouubbnc*. Rtv. Fr.OZiml.. 124.27-31.
S.iktict M.. Cheynkf V.. Guana Z. awl Wykfc R.
11980) O.ydaikin of grape aike 2-S-nbtaiabnyl
calTcoyl lanark acid by Boiri/ii rinereii becaac
and .li.iiu.icri'idiiii of • no tianunce: 23-di-5"-
pariathki-nylcaffeuyl lananc acid. J. Food Sri.. 51.
1191- 1194.
S.limil A.. Kuhkl II ., MiUeabeiper A. andCuachwann
K.(1989)Vcimk-i .urn einui/ pelioiyiitchercnnme
mil pnMcuUiuchcracbciuLiivkai.SMsinAir llh'n/uw.
11.408-414.
Siagktoa V.L. ( l"S7 ) < hygen with plico.lt and lelaicd
icacibn* in muu. uinei. awl model sytiemv ob*er-
vaibm. jad pracikal implkaiba*. Am. J. Eaot Mf£c,
38.69-77.
Terrier N.. Sauvapc P-\. awl Romku C. (1990) Ab-
ICOCC ik i rite mpiiatolit. imliKiimi dc I'aclivac
akool .ksbydnipc iu.se el dimiwjba dc I acklic
<acuolaiickio.de b maruiaiiiindu rj it in In (En-togie
95. Com pic Rcadu Seme Symposium Intcmaibaal
d Qnobgk. pp. 24-28. Tec A Doc UvobKr, Pails.
Tinlot R. (1990) la tiimton momlnk de lenrkh-
iMcmeni. \i$ici H«. S. SI-S4.
lu.epUi-romai.tei L (1978) Acqubabw, rcccntc* iui
kt phenomcao, couVktati* dam le* mouK, el kt vim.
Aim. Jatrnol. AgrU: . 27. 201-274.
I'ucplb-Tomauei I., and Bosia P.D. ( 1992) la dcucr
dificaibn dct moul* *ckm b met node alkmamk. Bull.
0/l'.73l-7J2.5-l3.
Vm RcnOHiip P. and Pnioni> J.S. (2000). S A#i. J.
Eiuil. HnV.. 21.52-73.
Vmwandl G. (1989) Doukr tcwbntNxact. U(i 137.
83- 103.
Wucbcrprcane K. (1907) Tendantct dans I cvolu-
i»n du iiavail de* vim. en Alkaupac. In CoiNptc
Raida 2erte Sympouani biiirmiiiauJ ffEnototft
pp.4ll-433.INRA.
Wucbciplcanrj K (1980) Pot.tHIHCt d'uilitjiion des
piDicitji mcmbianaiict dan lindutincdn buiuoas.
Bull. 01V, 583. 180-205.
12
Red Winemaking
12.1 Generalities
122 Mechanical harvest Ucuiatcnfe
12 J Filling vats
12.4 Controlling alcoholic fermentation
125 Maceration
12.6 Rubbibjz off and pnrKsIn^
12.7 Malolacbc fermentation
128 Automated ail wincmaking methods
12.9 Carbonic maceration
329
334
339
111 GENERALITIES
Red wine Is a macerated wIk. The extraction of
solids from gape cUstcrs (specifically front skins,
seeds and possibly stents* accompanies the alco-
holic fcmicnlation of the juice. In conventional led
wincmaking. extraction of grape solids is by means
of maceration, which occnis during must fermenu-
tio» Other methods exist that dissociate fermenu-
i ■ '- .iii'.l maceration. such as Ihermovinifkation.
The locali&ilion of icd pigment exclusively in
skins, at least in the principal varieties, permits a
slightly Haled or white wine to be made from the
colorless jiiic obtained from a delicalc pressing
of ted grapes Wines for the elaboration of
champagne arc a good example. The designation
None ile bttinc was created to distinguish white
wines derived front while varieties and those from
ted. Finally, varietal nalnic Is not sufficient for
charackri/lng the origin ofa red wine. Maceration
Intensity is of prime Importance
The length and intensity of maceration arc
adjusted acconling to grape variety and the type
of wine desired. In fact, maceration Is a means
by which the wlnemaker can personalis the wine
Primeur wines arc made to be drunk young: their
.7—l.y I
i >.* ir.;,.. .'-.■*..■ ..
! Ja.-Luii.^
Handbook or Etiology: The Microbiology o( Wine anil Vinific.uions
i aid fruitincss greatly oatweigh phenolic
compound concentrations, hui premium wines
rcqnircasafficicnt tannin concentration to develop
propcily daring aging.
Grape quality directly influcnccsgrapcskin mac-
eration quality in icd winemaking and is thus of
the greatest importance In fuel. Ibc grape skin is
■voce affected lhan ihe juice by cnllivallon tech-
niques, maturation conditions and sanitary stile.
Vincigc and growth rankings are therefore much
■tofc clearly delined with icd wines lhan whiles.
In Ihe Bordeaux region, anthocyanin and tannin
concentrations in Ihe same parcel can vary by as
■inch as a factor of two. from one year to another,
according to maturation conditions. Musi acidity
and sugar concenlmlionscan tluciaatc by 50% and
15*. respectively These numbers arc not snrprR-
ing. since Ihe plan! rcquircsa lot of energy to syn-
thesize anthocyauins For this reason, ihe northern-
most vineyards produce only while wines In any
case, when phenolic compound concentrations are
examined in relation to environmental conditions,
their nature, piopertiesand localization in the tissues
mast also be considered. Enologisls readily define
good* tanninsasthchc that give wincsadense stric-
ture without aggressiveness, and bad* tannins as
those characterized by vegetal and astringent herba-
ceous savor*. The natnrc and chemical properties
of these varices phenolic compounds arc covered
in i baptt r '■ of the second volume of this series.
This highlights the need lo wail until the grapes
reach fill phenolic malarity. which nay occur later
than physiological ripeness Similarly, high levels
of uicthoxypyra zincs in insufficiently ripe grapes
ofccitiinvaricticstcspccially Cabernet Sauvignou)
arc responsible for a hcrbaccoas. green bell pep-
pcrcharaclcr In must and wine that is considered a
defect above certain levels (Aolnme 2. Section 7.4)
Grape composition and qaality variability result
in heterogeneous grape crops. Grape selection can
compensate for this heterogeneity and tanks shoa Id
be filled with a homogeneous single- variety grape
crop that has the same sanitary state and level of
maturity lemar. quality, vine age. roocMock. fruit
toads. and ami in her of other factors should betaken
into consideration. Appropriate vineyard manage-
ment methods arc increasingly being applied to
achieve the low yields essential lo ensarc perfect
grape ripeness and high quality This batch selec-
tion, effected at tilling time, must be maintained dur-
ing the entire winemaking process, antil the dctim-
tt vc stabilization after malolactic fermentation. The
best batches arc then blended together to make a
wine of superior qaality. The complementary char-
actcrlstics of the virions hatches often produce a
blended wine that is superior in qaality to each of
the baKhcs before blending
The grape crop should also be carefully sorted lo
eliminate damaged or unripe grapes. This operation
can he effected in the vineyard during picking or
In the winery at harvest reception At Ihe winery,
the grapes arc spread out on sorting tables. A
conveyor bell advances the crop, while workers
eliminate bad grapes A concern for perfection in
modern winemaking has led lo the generalization
of such practices. Their effectiveness (seven more
pronoanccd when they are applied to grape crops
of superior qaality
Red grape crop heterogeneity requires specific
winemaking techniques to be adapted according lo
the crop. Much remains lo be learned in optimizing
the vartoas grape specifications
The generalization of uiatoLiciic fermentation is
another characteristic :of red winemaking This phe-
nomenon has been recognized since the end of the
last century bat. antil the last few decades. It was
not a consistent component of red win- making . For
a long lime, a slightly elevated acidity was consid-
ered to be an essential factor in microbial stability
and thus contributed to wine qaality Moreover, red
wine must acid ilicatioa was a widespread practice.
1 1 has currently disappeared for the most part, since
It Is only justified in particular situations. Today, on
the contrary, malolactic fermentation Is known to
produce a more stable wine by eliminating malic
acid, a molcenle easily btodegraded.
It was in temperate regions that malolactic fer-
mentation (MLF) first became widespread. These
wines, which arc rich In malic acid, arc distinctly
improved, becoming more round and supple MLF
was then progressively applied lo all red wines,
even those produced in warm regions already hav-
ing a low acidity. This type of fermentation may
not be advisable in all regions and another method
329
of stabilizing red wines containing malic acid
should be sought
The classic steps in icd wincmaking aic:
• mechanical harvest treatments (crushing, destc-
mming and tank filling):
• vailing (primary alcoholic fermentation and
maccralionl:
• draining (separation of wine and pomace by
dcjulcing and pressing):
• liial fcraici til ions i exhaustion of (he last grams
of sngar by alcoholic fermentation and maloiac-
be fermentation).
There arc currently many variations on each
stage in traditional winentaking. but the opera-
lions described in Ibis chapter constitute the basic
method for producing high quality red wines. II
docs, however, require considerable tank volume
capacity and many constraining manipulations. In
consequence, other techniques have been devel-
oped The standard order of certain operations has
beencbanged to make a certain level ofailomation
possible— for example, in continuous vindication
and heal extraction (Section 128).
Finally, fermentation wilb carbonic maceration
tikes advantage of Ihc special aromatic qualities
produced by fermenting whole grapes nnder anaer-
obic conditions (Section 12.9). ThR special fer-
mentation gives these wines specific organoleptic
c ha racers.
12.2 MECHANICAL HARVEST
TREATMENTS
12.2.1 Harvest Reception
Diverse methods, adapted lo each winery, arc used
k> transport the harvest from grapevine to winery.
In workl- renowned vineyards, small-capacity con-
tiincrs arc caicfully manipilalcd by hand. In mosl
vineyaiUs. (he harvest Is transported in shallow bed
trailers or trucks. Whatever the container capac-
ity. Ihc grapes should be transported inlacl without
being crushed. Transport containers should also be
kept clean. If Ihc transport time is long the grapes
should be transported during the cutler hours of
Ihc night.
Red grapes arc certainly less sensitive lo macer-
ation and oxidation phenomena than white grapes,
bul microbial contamination is likely lo occur in a
partially crushed harvest. Icf I in the vineyard, espe-
cially in the presence of sunlight. These risks musl
he avoided.
During mechanical harvesting. Ihc grapes arc
transported in high-capacity containers Speed and
hygiene arc even more important In this case, since
Ibcgrapcsarc inevitably partially crushed with this
method
Small-volume containers arc emptied manu-
ally. More generally, a dumping trailer is used,
which empties its kvid into the receiving hop-
per (Fig lie 12.1). In bigh-capaciry installations,
the bins arc placed on a platform which dumps
the grapes sideways— this avoiding excessive
truck and tractor maneuvering. If the winery is
equipped, the grape crop may pass on a sorting
table (Section 113.3) before teaching Ihc receiv-
ing hopper. Manual sorting is only effective If
Ihc grapes air whole. II R almost impossible to
combine with mechanical harvesting: at best, obvi-
ously damaged grapes can be removed from the
vines before the harveslcr arrives. Olhcrwise. sort-
ing may take ptuc in the vineyard, immediately
after the grapes have been picked, or when the
gtupes arrive at Ihc winery In Ihc latter case, the
grapes should be transferred to Ihc sorting tabic
manually lo spread Ihcm evenly Transfer screws
should not be used, as they crush the grapes and
make sorting impossible.
Two sorting tables may be necessary at wineries
producing high quality wine The grapes arc ini-
tially sorted when they arrive from Ihc vineyard
The second sorting operation, alter deslcmming.
removes any small fragments of stems and leaves,
etc. that were missed during Ihc first sorting
operation, and is followed by crushing. There arc
now Increasing numbers of machines, based on
various techniques, available to do the* operation
automatically.
Receiving hoppers arc available in various de-
signs In small wineries, they may be installed
directlv above Ihc crusher- summer and filled
Handbook of Etiology: The Microbiology of Wine anil Vindications
rift 12.1. Hvampki of to
coiumuflK'atiuu). (a)Gi>ndi>b w
itljiMahk (limp height. (C)Ga<
(ccii deuemmer— conker, (e) Si
coovc>oc(2)»bc Inifcrwhn
KH.Malionuiy hopper feeding the ikuct
ihc timber by gravky; (8) ruling tabic
iv duap pondol
Ring ubk bctu.
rlcviini *yi*
.Imp (So
'. giaviy dumping ft>) EIcvmo
(d)Ctaviy duap pontlob. ck
• mllci ind dcMcancr— cn»d
i; (3) dumping taikr. (4) dole
P. bcifiei. Bonlcaui.
pundob will sure.- c
Hot huppci designed l<
r. Key: (l)laikr wun
.her. IS) go
.- . ii-. i.v >■■ 'i,i i ;. ■ ' i .1--. ..-.-. i hopper n ah idjimabk ln.-i.-lu t>
directly from Ihc transfer vehicle. In general, a
pcipeiual screw in ihc boitont of ihc hopper rcgu-
fcilcs throughput and it should (urn sJowly to avoid
excessive crushing of Ihc grapes. Throughput may
be Increased by using a laiger-diautclcr hopper.
When buying grapes according lo weight and
sugar concentration, these valuer mnsl be deter-
mined al the lime of reception. Crape crop hctnv
gencity complicates the dclcniiinalion of the sugar
concentration. The sample should therefore be
tikcu after crushing and bomogeni/atiou The san-
itary condition of the grapes may also be assessed
al this stage by analyzing their Uncase activity
I Section 10.6.6).
Al the outlet of Ihc crusher— destcmntcr. a
pump distributes the grapes to a given tank. Sulfur
dioxide is aided at this time (Sections 8.7. 8.8.1)
and any necessary addition
Crape handling should be mininii/cd. limiting
transfer distances and maximizing Ihc use of
gravity Rough handling is likely lo shied or
lacerate stem tissues, so that sap R libcrakd
front vcgctil tissue and later found in wine.
The suspended solids concentration simultaneously
increases: in fuel, this measurement may be
used lo evaluate equipment quality The most
quality-oriented solution consists of sorting and
destemming the grapes by hand, then crushing
Red Wine making
tlcn. if incwii). through a wooden screen. Ihns
eliminating ihc iced to crush inctn mechanically
Finally, die must Is transferred wilboul pumping.
Or con inc. only ihc most prestigious csutcs can
affonl lie high cost of Ihcsc Kchniqucs.
In partially bolryti/cd grapes, a brutal mechan-
ical acliui on grapes disperses a glucidic colloid
Iglucanl in Ihc musl. Clncan Is produced by Boiiy-
lis ciaereti and R located between ihc pulp and ihc
skin inside ihc berry. The wine obtained Is diffi-
cult lo ...nil;. When Ihc same grapes are CURrfullv
handled, the wine Is clarilicd ninch note eas-
ily Wine clarification difticDliies with botryti/cd
grapes arc always observed in Ihc saiK wineries.
The lypc of equipment Bscd is often responsible
12.2.2 Crushing
Crapes arc iradilionally crushed to break the skin
ii order to release the pnlpand the (nice. This oper-
ation is probably one of the most ancient harvest
treatments. Partial crushing can be obtained by the
traditional technique of treading the grapes. High-
speed cenlrifigal crusher— dcstcninicrs assure an
energetic crushing. Then: arc also many othcrsys-
lems bciwccn these two extremes.
The consequences of crushing arc as follows:
1. The juice is aerated and it is inoculated by
yeasts The fermentation Is quicker and the
temperature higher. In ccrtiin circumstances, a
slower fermentation speed and lower temper-
atures can be obtained through not crushing
(carbonic maceration. Section 12.9.4)
2. Aeration can be harmful. In partially rotted
grapes, it can piovokc an oxidasic rum*
3. Crushed grapes can be pumped, and snlnling is
more homogeneous
4 All of the juice is fermented, at Ihc time of
running-off. the press wine diKs not contain
sugar
5. Crushing has a significant effect In facilitating
maceration and accentuating anlhocyanin and
tannin dissolution. An energetic crushing inten-
sities this effect. Tannin concentrations propor-
tionally increase more rapidly than the color.
l'*t 111 Canhc. mllcr drsiga: t.ii%pinil ribbed mi-
ke.: ib'ttmovcd ioIIcd. i»Hh I «c nuance* lug plonk*
This increased maceration can be an advantage
In certain cases but it tends to increase the
herbaceous aslringcncy and disagreeable tastes
of average varieties.
Premium wine grapes arc traditionally lightly
crushed to burst the berries without lacerating
Ihc solid parts Crushing is used to facilitate
fermentation and avoid residual sugar in press
wines Methods other than crushing should be used
lo incrcasc maceration tvaltlng lime, pumping-ovcr
operations, temperature) They better respect wine
quality. Even when carbonic maceration is not
strictly used (Section 12.9). wincmakcrs may wish
to avoid crushing Ihc grapes for great wines with
long Mining periods, to avoid bruial damage to the
plant tissues.
Two kinds of crushers exist Roller crushers
t Figure 12 2) arc coated with plastic; Ihc opposing
rollers turn In opposite directions and their spacing
Is easily adjustable. This system works well but
delivery rales are limited. High-speed perforated
wall crushers! Figure 12 .'I can he cither horizontal
or vertical. A beater projects the grape clusters
against a perforated wall, and Ihc burst grapes pass
through Ihc perforations. These machines simu-
ltaneously dcslcm the grapes. As they arc rough on
Ihc grapes, they arc not recommended, especially
In making high quality wines.
1113 I KM .-in mm-
This operation, also known as desialking. is now
considered indispensable (after much discussion of
its adjutages and disadvantages for a long time)
Dcstcniming has a number of consequences:
I. A primary and lin.mci.illy important advantage
of this operation is the reduction of the required
Handbook ol' Knology: The Microbiology °( *'l»c anil VnifkatioBS
1 ' •--'■ .
Fig 1 2. J. Opciaikin principle oil. i., horimaul dc*-
icnntct and (h) u vertical ceirtrihnial dcntuuKr. Key:
(I) hop-pet: <2>>, tat "in am and faddk>,;<3t peribnt-
tcd cylinder. (J) item outlet: (5)dcstcmmcdpiapc mi let
tmk capacity by Mfi . In addition, the pomace
volume to be pressed is greater wiih a stemmed
grape crop. Although Ibc stem facilitates juice
extraction during pressing, a higher-capacity
press is required.
2. FcmicnGil»ns in the presence or sicms aie
alvrayst|UK*kcrand more complete* Figure 12.4).
The sir in facilitates fermentation not only by
ensuring the presence of air bat also by absorb-
ing calorics. limiting temperature increases. Fer-
mentation difficulties arc raiclycnconnte red with
stcmiKd grapes
3. The stems modify wine composition They con-
uii waicr and very little sugar, thus lowering
alcohol content. Moreover, stem sap is rich
in potassium and not very acidic. Destcmmiig
therefore increases must acidity and alcohol
content.
1'ift 114. Intoeoce of Mca on alcoholic fcimcaiMioa
i !., . i. i'-' in; n a <rf.. 1970). I: dcMcamcd -ii|il-.
II: aotrdcMcniatcdgnipci III: anning-off lUie
4 With botrylizcd grapes, stems protect wine
color from oiidasic cassc The laccasc activity
of Batryiis cinerea is most likely fixated or
inhibited
5. Dcstciiming most significantly affects unnin
concentrations. Table 12.1 indicates the approx-
imate proportion of phenolic compounds sup-
plied by the various parts of the grape cluster.
In this experiment. 54'4 of the total tannins
conic from grape skins. 25'i from seeds and
21'* from stems. Results may vary according
to grape quality and grape variety. More pre-
cise details «■ the nature and concentrations of
phenolic compounds from the various parts of
the grape cluster arc given in Chapter 6 of the
second volume
Table 12 2 recapitulates Ihc principal modifi-
cations of wine constitution caused by destem-
ming. Despite Ihc increase in total phenolic
compounds in the presence of stems, color
intensity diminishes This long-observed fact
is interpreted as Ihc adsoiption of grape skin
anfhocyauins on Ihc ligneous surface of the
stems This iutcrprcuiion has been continued
in a model solution containing anthocyaninsand
tannins: cither a stem extract or Ihc stems them-
selves is added. In the first case, the tannin
concentration increases considerably, white the
Tabic 111. Indicate of ilitTcrcnl fia\ »f ibe (f-apc chnlcr un pkcnolk
ooapou«k indwiac coIomMjIkc. Icrmouibaai 2S C mtiog lime 10 ifays)
(KihcicaiMiivo. «■.•«'. 1970)
Tint
AnhcK^anliH
Tannlm (o/l)
Ton I p he noli
(pcn-uapai
ale inkk)
■■umt.lv = a
lOI)l)la«(
>DS»=op
048
I'.SS
3.25
color intensify slightly Iicirascs. In the second
case, i he tannins incicasc bin lie color Intensity
decreases Tannins play an important n+z in the
color of niaiaic wines. Although wines made
from slcnimcd harvests have less color when
young, they become ntorc colored than their
dcslemnicd counterparts in the couise of aging.
6. The increased cmnin and phenolic compound
concentration or wines made from slcnimcd
harvests can increase wine qualm in certain
cases, e.g. for young viics and wines wiln
insufficient strnciure without the sleits. Yet
grape Mems air likely id give vegetal and
disagreeable herbaceous tastes to wines. In
general, when finesse is favoied. dcslcmming
R indispensable. In any case, the decision of
Tabic 12.2
(Rt>c<cau-G»y<i
mpmiu
'l...|,.|. mi, --ill r. vol
Total acidly imEu/1)
VoLlilc Jt ■.!■*. (■EUi.'li
Total phenol*-
I pcrmin^inJIC lailct I
Cotoi*:
Inteuiy
a total or partial dcslcmming mnsi lake inlo
account slcm quality*, which is rclaled lo variely
and maturity level
In (he pasi. the grape crop was dcslcmmed by
band directly in the vineyard or. more generally,
in the winery, by ribbing the grape clusters wiln
rakes against a wooden hurdle Today, ibis oper-
ation is carried onl mechanically (Figure 123)
Desk* nunc is comprise a pedbrated cylinder, wiln
a shaft equipped with paddle-like arms running
through its center. When the shaft turns, il draws
in (he grape clusters and expels the sicnis out Ihc
outer end. The Juice, pulp and grape skits pass
through ihc perioral ions The continual quest for
higher output has lead K> increased rotation speeds
and replacing Ihc paddle-shaped rods wiln heal-
ers The beaters apply saffKicnt force to burst Ihc
grapes without the need of a crusher. Vertical shaft
dcstcmmcrs( Figure 123) can beat 20-45 mciric
ions per hour. They operate al 500 rpm and ibe
ccnlrifagal force evacuates Ibe stems by Ihc lop of
Ihc machine. These machines have a brutal action
on fbc grapes and produce line suspended solids,
imparting vegetal and herbaceous tastes b> wines
Their use shoukl be avoRlcd. al least for the pro-
duction of premium wines (Scciion 122.2).
Crushing aid destcmmlng ate generally effected
by the same piece of equipment, but in cer-
tain cases il would be desirable lo have Ihc
option of nol dcslcmming For a long lime, wild
334
Handbook of linology: The Microbiology of Wine anil Vindications
conventional crushcr-dcstcmmcrs. custui; pic-
ceded dcslc niniing. Today. there is an increas-
ing number or machines thai eliminate ibe sums
before crushing the grapes The stems do nol pass
vi'" ■ Ihc crusher rollers In ibet manner, ihc risk
of shredding the stems is lowered. This order of
operation increases must quality, since stem shred-
ding liberates vegetal vacuolar sap. which is hitter
and astringent.
A quality dcslcnimcr should not leave any
berries attached K> the stem Reciprocally, the slcm
should not be impregnated with juice The skrm
should ;dso be cntiicly eliminated, with no broken
fragments remaining Tie Ulceration of ligneous
slcm IIssk by the machine can seriously affect
quality and in these iasEinccs destemming should
be avoided.
AttcmpLs haw been made to eliminate residual
slcm waste after the destemmer and before the
crusher. Quite Luge quantities arc removed in Ibis
way.
113 FILLING VATS
113.1 Filling Vats and Related
Operations
In Ibe case of fermentation with carbonic macer-
ation (Section 129). vats must be filled directly
from the top with uncnishcd grapes, which obvi-
ously requires a wry complex system. Otherwise,
grapes arc usually received at a single winery loca-
tion anil transferred to the fermentation vats after
destemming and crushing Transfer pimps must
do as little damage as possible to grape tissues
and distances should be kept Ri a minimum, with
as few bends as possible in the hoses. This opera-
tion can be carried oil manually, without pumping.
As the must increases in volume during fermen-
uoon. about 2>*i empty space should be left in
each vat. Ifan ti- foaming agcnis( Section 3 .2 5 1 arc
■scil. less headspace is required
A considerable volume of gas Is iclcascd during
fcrmcntiUoi. approximately 50 I of carbon dioxide
per liter of must fermented [Insuring that a flame
sEtys alight inside the fermentation vessel before
going inside helps check for oxygen, in view of
the danger of asphyxiation from carbon dioxide.
The grapes must be sullited adequately and
homogeneously during transfer to the vat (Section
88.1).
Sewral operations may be carried out during
transfer of Ihc grapes/must, or in the following
tew hours Firstly, they may be inoculated with
a fermenting must (a few percent corresponding to
10* cclLs/m) or dried active yeast (LSA). 5. cere-
visine. chosen from among Ihc various commercial
strains (owr 100). The main qualities required are
the aptitude lo complete fermentation successfully
and heat resistance The impact of yeast strains
on the character of red wines is less marked than
in the case of white wines (Section 13.7.2). Winc-
makcis must still ensutc. however, that the strain
selected Is suitable for the type of wine being
made. Recommended doses of 10-25 g/hl corre-
spond lo inoculation with 2 10*~10.10* cclls/m.
Indigenous ycas& must be inhibited by appropri-
ate doses of SO? to ensure effective seeding. Dried
yeasts must be reconstituted prior u use. by mix-
ing them ink) a mixture of must and water (1:1)
at 40'C. The reconstituted yeast must be spread
evenly through each vat.
Acidity can be corrected (Section 11.4) during
the Initial transfer into vat or at a later time. If
sugar levels need to be increased (chapulioition.
Section 115 21. this is best done when the must is
warm at the beginning of fermentation The sugar
dissolves mote easily and Ihc subsequent aeration
stimulates fermentation, while relatively kiw sugar
levels promo*.- multiplication of the yeast cells dur-
ing the growth phase
If an assay (Section 3.42) indicates a nitrogen
deficiency, ammonium sulfate 1 10-30 g/hl) may
be added as soon as the vat has been tilled, or.
preferably, once fermentation has started.
Adding tannin during fermentation had been
abandoned for a long time, but the qualify of the
producLs now available, particularly those made
from white grape skins or fresh grape seeds, has
revived interest in this procedure. These producLs
arc nol only considered capable of improving body
and tannic structure, but also of stabilizing color
by promoting condensation of anlhocyanins and
tannins I Volume 2. Section 6.3.10).
Red Wincmaking
33S
ll is ihiis useful to add tannin eaily in the
fermentation process, when lie lannius have nol
yet Ixcn extracted from the grape seeds, so lhal
Ihcy can read with Ibe aaiaocyanins released
eaily ii vatling According to sonic authors. Die
icsslis arc aacven dac *< Ihc low solubility of
the tannins aad ihc difficult}' of mixing Ibcai into
Ihc must Ulloaiu and Pcynaud. 2(KII). so ii is
preferable to add the product after running-off.
High doses (20-50 g/hl) arc required to raise the
iaitial lanain levels by approximately I"'
Another operation currently attracting some
iatcrcst R the addition of pecutytic engines to
promote extraction of phenolic compounds i Sec*
I was 1 1.73; 125.1). for the purpose of obetining
wines with a higher lannin content, bat less
astringency aad biltcraess iBIonin and Pcyaaud.
2001).
Clyeosidascs amy also be ascd to promo*:
extraction of Icrpcnic aromas— particularly useful
in making Muscat wines Care must lv taken
ia traditional red wincmaking to avoid producing
off-aromas. The asc of enzymes in wiaemakiag
requires farther scientific research.
Some jaicc caa also be bled off at this stage
(Section 12.58). mainly toclimiaatc rainwater and
jaicc that has nol yet absorbed compouads from
the skins. Decreasing the quantity of aiast facili-
tates concentration of the phenolic compounds dur-
ing vailing. Tins operation is generally carried oul
after the vat has been lilted and the juice has been
separated from the pomace. Water is climinalcd
tci' by reverse osbkis-s or vacuum evaporation)
(Section 1 1.5.1) at the saatc time. The results arc
very similar but these methods maintain the natural
grape sugars. These techniques are capable of con-
centrating the aiast by 5 - 10-4 . or even as much as
2(/» Excessive coKCBlraliou of the must changes
the flavor balance of the wine completely and il is
preferable to adapt vineyard management methods
and reduce yields on the viae to achieve similar
resale..
12.3.2 Principal Vatling Systems
Various types of fcmcnlor exist They arc distin-
guished by the aeration level supplied to ycastsand
the modulation of skin contact AcralKin helps to
ensarc a complete fcrmcaialion. and skin contact
modulation inflaences maceration and phenolic
compounds extraction.
Fermentation releases gas within the mast.
The babbles rising toward Ihc surface of the
fermentor entrain solid particles, which unite and
agglomerate, forming the cap The skin cap is
maintained at the top of Ihc fermentor by the
pressure of the released gas
Pomace plays an important rote. First and fore-
most, during maceration, il yields its conslitacnls
(anlhocyanins and tannins) These compounds arc
indispensable components of the character of red
wine. Yeasl multiplication is also particularly
intense within the pomace: 10-50 x 10* cells/ml
have been observed in the jaicc at Ihc bottom of
the fcmicnior and 150-200 x 10* cells/ml in the
juKc impregnating the pomace.
Although no longer recommended, open floa-
ting-cap fermentors are still used in small-scale
Installations. They were used in the past becaase
Ihc extended contact with air pcrmilied success-
ful fcrmcati lions, even in musts containing high
concentrations of sugar Moreover, temperature
increases arc less significant Ycl. the inconve-
niences arc undeniable. Alcohol tosses can attain
and sometimes exceed 0S'4 (Section 12.6.1: sec
Table 12.10). The rfik of oxidasic cassc with botry-
tized grapes Is also ccrctin. Additionally, as soon
as the active fermentation slops, the pomace cap
surface is no longer protected from aerobic germs
development Bacterial growth is facilitated and
con timi nation risks arc high due to Ihc large sur-
face area of this spongy surface. As soon as the
fermentation slows, the pomace cap slum kl be reg-
ularly immersed lo drown Ihc aerobic germs. This
operation, known as cap punching (pigcagc). can
only be carried out manually in small-capacity
fermentors. If necessary, it can be mechanically
effected with a jack or another ptccc of cqaip-
ment. Subatcrging the pomace cap also con tributes
to the extraction of its consliiacnls. Il also aer-
ates the mast and homogenizes the temperature
Bat this type of fermentor does nol permit a long
maceration. The Ginks mast be run off before the
carbon dioxide slops being released Afterwards,
spoilage risks in Ihc pomace cap arc certain and
336
Handbook of linology: The Microbiology of Wine and Vindications
Ihc resulting press w)k would nave an elevated
volatile acidity. Manually removing Ihc nppcr layer
of Ihe most contaminated pari of Ike poniacc cap
is nol sufficient, nor is covering the ttnk wilh a
tupuulin after fcmcnGiuou
To avoid pomace cap spoilage and lo eliminate
ihc laborious work of regularly punching down
the cap. systems have been developed thai main-
tun Ihc cap immersed in ihc musl— for example.
under a wooden hnrdkr filled K> ihc Link after till-
ing Tie hum in contact with air Is permanently
renewed by Ihc released gas. Acetic acid bacte-
ria have more difficulty developing In this envi-
ronment The compacting of the pomace against
Ihc wooden hurdle does not facilltite the diffusion
of its constituents, and several pumping-ovcrs an?
therefore recommended lo improve maceration.
Today, mosl ted wines arc fermented in tanks
thai can be closed when the carbon dioxide release
rat falls below a certain level The complete
piolcclion from air permlo ntacctalion tines lo be
prolonged, almost as long as desired. The lank can
be hermetically scaled by a waler-filled tank vent
(Figure 125) or simply closed by placing a cover
on the ttnk hafch In the latlcrcasc. the COj which
covers the upper pun of the lank disappears over
lime and the protection is nol permanent. The Gink
should therefore be completely filled with wine or
a slight pumping-ovcr operation should be carried
out twice a day k> immerse the aerobic germs.
Fift 12.5. Hcmak waicrfilkd u>k vent alkiwiniMhc
ftteiic of CO- from lank during fcimcnutua ■> Iiho-.i
ilrcnicrliuj
For a long lime, the major i
Ihc closed fcnticntor were a considerable temper-
ature increase and the absence of oxygen. As a
result, fermentations were often long and difficult,
and stuck fermentation occurred frequently. Today,
these two inconveniences are mitigated by temper-
ature control systems and pumping-ovcr operations
wilh aeration, permitting Ihc dissolution of ihc nec-
essary oxygen for a successful fcrmcntition.
In conclusion, this fcrmcnlor design avoids al-
cohol loss by evaporation. Press wine quality is
greatly increased, while the laborious work of cap
punching is eliminated This kind of tank has also
been empirically observed to facilititc malotaciic
fermentation.
12.3.3 Fcrmcntor Construction
Red wine fcrmcniois have been successively made
of wood, concrete and steel, and on occasion
plastic.
Wood is a noble material and wooden tanks
have long been part of the tradition in great
wincmaking regions. New wood releases aromatic
compounds into the wine during fermentation but
Ibis property is attenuated alter a few years and this
phenomenon nolongcroccursduiing fermentation.
Disadvantages are lhat wooden link maintenance
is difficult, lhat old wooden tanks arc a source
of con tun mat ion and bad ttstcs. and thai wooden
tanks are not completely hermetic. They musl
sometimes be expanded with water before use.
wilh all of the corresponding risks of microbial
contamination. In addition, the flat ceiling of a
truncated tank is rarely hermetic— this kind of Gink
is not suitable for prolonged wine conservation.
Wood is also a poor heat conductor. Wooden
tanks are subject to considerable temperature
Increases that musl be compensated by appropriate
refrigeration systems, yet when the fcnucntition is
completed, they retain the heal generated fora long
lime, favoring a post- fermentation maceration.
Concrete permits the effective use of available
space, since the tanks arc manufactured on site,
but the ackls In wine attack concrete. The inner
lank walls musl therefore be protected The Ginks
can be coated with a Hf* solution of tartaric acid
applied Ihrcc lines al intcrvab. of several days In
Red Wincmaking
;;-
these conditions, the inner lank walls arc caftd with
calcium tartrate. The wine in the tank contributes to
maintaining Ihc coiling. Il is. however. preferable
in coal Ihc inner tank walls with an innocnons
anil chemically incrl lining sach as cpoxy resins
or araMitc Whatever lining is ■sal. ihc coaling
ot these Links requires continuous maintenance
Concrete is a better heat conductor lhai wood, bul
icfngcration systems arc slill indispensable. These
Links arc complclely hernKlIc and can be used Tor
wine storage.
Steel. partKularly stainless slccl. is Ihc mate-
rial most often used today for manufacturing
fcrmcnlors. Two categories of stainless slccl exist:
one contains molybdenum, the other docs not.
C ironic -ikkc I -molybdenum slccl is morc rcsis-
tint Id corrosion and it is necessary for the
long-term conservation of sulfilcd white wines,
especially in partially filled Links in the humid
atmosphere above Ihc wine, sulfur dioxide gas is
conccutrakd and Ihc condensation formed on Ihc
lank walls Is conosivc For red wincmaking and
sloragc in completely filled tanks. Ihc less expen-
sive . in - ' ■mi mi. ki I sKel is sufficient
Stainless steel tmks have the significant advan-
tage of being he rate lie and easily filled wilh vari-
ous types of equipment. Their internal and external
maintenance is ;Uso facilitaicd; Iheir inner walls
arc impregnable. Stainless steel also has a good
thermal exchange, avoiding excessive temperature
increases. In certain cases, red wine fementa-
tkms can occur al 30*C wiihonl cooling. In any
case, cooling is simplified: a cool liquid is cir-
culated within the doable wall of the Link or in
an integrated thermal exchanger. When a saffi-
cicnl amount of cool water is available, raaning
water over the exterior of Ihc tank can be safH-
cicaf la Ihc 1960s aad 1970s, stainless slccl tanks
represented a considerable advance in Icmpcralurc
control compared with wooden and concrete tanks
and this superior control was much appreciated by
wincmakcts. Today, however, i' has been observed
that these tanks insufficiently wumi Ihc fermenting
must when the ambient temperature is loo low.
This phenomenon is acccntaated in cases where the
tanks have been placed on hide w lower Ihc cost of
investment- Assooa as fermentation sups, the tank
lempcralaic rapidly decreases to Ihc ambient tem-
perature: as a result, maceration phenomena, which
arc influenced by tcmpcralatc. arc slowed. To mas-
ter red wincmaking. Icmpcralurc-coniiollcd (heat-
ing and cooling i stain less steel tanks arc necessary
However, in any cases, as heal inertia Is limited, il
is difficult to obtain homogeneous temperatures in
the post- fermentation phase. Recent dcvclopa>cnts
in cooling equipment have led to renewed interest
in wooden or concrete fermentation vats, when:
homogeneous temperatures arc easier to maintain.
Tank capacity must also be considered when
designing a winery. High-capacity Links arc of
course economical, bat tank siar should not be
exaggerated and should be adapted to the win-
ery (50-350 hi). It is difficult to control Ihc var-
ious steps in wincmaking in vals containing over
350 hi Tanks of limited capacity permit superior
IxiKh selection and skin extraction due lo inc leased
skin con tic i. The lank should be filled before the
start of fermentation and for this reason the filling
trme shoald not exceed 12 hours.
Tank shape is important for red wincmaking
The exchange surface between the pomace and
the juice shoald be saflkicnt. The dimensions of
sheet steel used during manufacturing sometimes
result in tanks that arc too high with respect
lo their diameter Reciprocally, tanks should not
be too wide. In this case, pomace leaching
is greatly reduced and pumping-ovcr operations
lose Iheir effectiveness: air contact can also be
excessive. Tank height should slightly exceed
lank diameter Nigh- performance pumping-ovcr
systems can compensate for disproportionately
high tanks to a certain extent
12.3.4 Equippiog Fcrmcnlons
Basic red wincmaking tmks should be equipped
wilh the following:
• Two juKc evacuation taps on the tower part
of the tank, placed at different heights lo
facilitate nicking (elimination of sediment), wilh
an orifice at Ihc lowest point of Ihc tank for
emptying and cleaning
• One or two doors: one a hit higher permits the
lank to he emptied after draining. Ihc second.
Handbook of Etiology: The Microbiology of Wine anil Vnifkatkws
lower door is less essential baican he useful tor
cleaning the tink
• A gauge to indicate the filling height
* A tasting spigot for Liking samples.
• A thermometer.
* A hermetic lid at Ihc top of the lank. A walcr-
lilled Link veil (Figure 123) makes the talk
completely airligbl. while allowing Ihc IHjaid K>
expand.
This basic conligarauon has oflcn been com-
plcmciicd wilh addilional. more spccilic add-ons.
Steel Links lend Ibemsclves particularly well to
additional equipment. Nowadays, several mannfac-
turers offer va& spccilkally designed for ferment-
ing red wines They arc equipped with complex
allachmcuK that turn them into complete syMcms
for monitoring and controlling fcniKnlalion. For
example. Selector System (GimarTccno 15040.
Occimiano. A.I.. Italy) has an automated vat clean-
ing system, programmable pnmping-ovcr with or
without aeration and/or spraying the pomace cap.
temperature control, and management of fermen-
tilion kinetics accoiding to changes in specific
gravity. so that the entire fermentation cycle can be
prog rammed and controlled directly by the system.
Temperature control systems are generally the
fiat add-on. Automatic temperature monitoring,
sometimes continuous. Is standatd. Tcmpcratnte
probes, which mast be properly placed to ensure
correct measurements, are often part of a more
elaborate temperature control system. Initially,
these systems simply consisted of flowing cool
water over the exterior of the Link Today, cooling
fluids (waieror a dilate glycol solutions) are often
circulated through a doable wall in the tink or
an internal thermal exchanger The latter Is more
efficient but makes tank cleaning more difficult
Tanks not only need to be cooled, but also heated
on occasion An identical system is therefore used
to circulate a healed liquid (hot water). The liquid
can be sent through the same thermal exchanger,
or preferably another pipeline. Since the liquid (the
juKc) and the solid (the pomace) arc separated in
the tank, a pumping-over operation is required, at
Ihc same lime as cooling or healing, to homogenize
the temperature.
In high-performance installations, when a tank
reaches a maximum preset temperature, a pumping-
over operation is automatically performed to
homogenize Ihc temperature. If Ihc temperature
remains loo high after this first operation, the cmk
is cooled. Automated temperature control systems
have been designed that rcgatale the temperature
throughout the entire fermentation process
Automatic pumping-over systems have also
been sought, to facilitate skin extraction (Section
125.5) and permit the aeration of fermenting must
(Section 12.4.2). In Ihc past the carbon dioxide
released dining fermentation and the resulting
pressure were ascd to pump the fermenting mast to
an upper lank, after cooling if necessary. Opening
a valve releases Ihc pressure, causing Ihc must to
cascade on the pomace cap. Due to the complexity
of this system, the pump shonkl be specifically
adapted to the Link This pumping-over operation
can be done wilh or without aeration in the
tink located below the fermentor. Aeration could
be better controlled by injecting a predetermined
quantity of oxygen in the lines. Pumping-over
frequency and duration should of course be
adapted to the must. Pumping-over too often may
make Ihc wine excessively hard and astringent
Various systems arc available to improve pomace
leaching and intensify skin contact
Injections of pressurized gas at 3 bars (nitro-
gen. CO?, or even air) can replace conventional
pnmping-ovcr operations A spec ially adapted pipe
Inject the gas throagh the piping of Ihc lower pari
of the Link Resale* that would normally tike over
an hour with a trodluonal pumping-over operation
( rotating Irrigator. stream breakcr.clOare obtained
in a few mlnukrs. This system has been combined
wilh standaid pumping-over at Ihc beginning and
end of maceration, using food-grade gas.
Other methods complementing or replacing
pnmping-ovcr operations can be employed to
improve pomace extraction. Hydraulically control-
led pistols have been developed which immerse
Ihc pomace cap. replacing the traditional cap
punching method '/vxtx/jo ). Various systems break
up Ihc pomace cap inside Ihc link, particularly
Kit; lift Sclf-empiyiap tub: (a, b)i
coavcyor B. iniiiponicil i*o Ihc ikutc
evacuation: (el aydiaulk- dump unl
i-empiyi»g by u»viiy: (el *ciew-conY«
Ihc noltoa of (be laak); (d) rualinp 1
self-cm ply i*g unl (m
cake break-up awl pom
nit i ling cylindrical tanks Dnc to Ihc continuing
evolution of these systems, a more detailed
description k difficult (B kuinand Pcynaud.2001>.
bul (heir use is covered in Section 125 8.
Another piece of equipment in nigh demand
is an automatic pomace removal system >vhKb
c vac nates Ihc skins from the lank lovvanl Ihc press,
replacing Ihc animus lask of manually emptying
Ihc unk.
A number of self-emptying Links have been
proposed. Models with inclined floois 120" slope)
improve poitacc cvacualioi. The worker removes
Ihc pomace with an adapted rake without having
k> enicr Ihc lank Automatic self-emptying tanks
arc aLso available and several mulct, have been
proposed (Figure 126). The mosl simple arc
cylindrical with an extremely inclined floor (45 :
slope) ending in a huge door. The slope of the
lank fktor and Ihc door dimensions should be
appropriate to the nature and viscosity of Ihc grape
.!■']■. for example, long valting times dry pomace,
making evacuation more difficult With this rypc
of evaluation system, the cntiic lank contend
should be emptied in one go: a sufficiently laigc
receiving system linked to Ihc press mnst therefore
be placed bekiw the tank. Progressively emptying
Ihc Gink may cause Ihc pomace to get stuck: it
forms an increasingly compacted arch, which is
very difficult to break.
Hydraulic systems cvacnatc pomace by inclining
the tank. Screw conveyor systems inside tanks
pcrmila controlled and rcgilar pomace evacuation
(Figure 12 61. Ihcy are nscd in rotating tanks
114 CONTROLLING ALCOHOLIC
FERMENTATION
12.4.1 Effect of Ambient Conditions
In Ihc past, red wincmaking methods in warm
and cool climales were differentiated In certain
variable-climate vineyards, warm and cool vintage
wincmaking techniques were also distinguished
Problems linked b> fermentation temperature
MQ
Handbook of linokigy: The Mictobi«l«¥y of Wine anil Vlniftcalions
control and grape composition were responsible
for these dlstinc in ms These differences aic less
important holnv . The necessary conditions for
successful wlucmaklng air known ihcy anr
adapted to the nature of Ihe grape crop and air
■ni difficult to carry out. as long as Ibe appropriate
equipniciil Is available.
A cool year or cool climate Is characterized by a
bile and often insufficient maturity Crape acidity is
elevated and ihe musts air thus relatively ptuKctcd
against bacterial attack. However, their is a risk
of ho try lis attacks and the formation of oxidasic
cassc. since cool climates often correspond to
rainy climates In addition, grape crops arriving
at the winery are often characterized by relatively
low temperatures in cool years As a result, the
initiation of fermentation can be difficult, even
■kmt so when the grapes arc washed by rain: the
natural yeast Inoculation can be insufficient
Feirf (1958) observed in Bntgundy region vinc-
yanis that fcrmcnuiion was activaicd in 12 hours
at 25"C. in 24 hours at 17-18'Cand in 5-6 days
at I5'C; it was nearly impossible at IOC. These
numbers arc of course approximate and depend on
many other factors, in particular the yeast inocu-
kilion concentration. Tanks should not he left at
insufficient temperatures. The resulting fermenta-
tions are often slow and incomplete, with a risk
of mold development beforehand. Tanks are also
Immobilized foe prolonged periods, which can cre-
ate problems In vlncyaids that use each unk sev-
eral times during Ihe harvest.
The must should therefore be warmed as quickly
as possible la 20'C. If the fermcntalkin docs
not begin shortly after wanning, the Icnipctalure
rapidly drops down to its Initial value. A simul-
taneous yeast Inoculation is required to avokl this
problem. It also accelerates the fermentation and
thus provokes a more considerable Icnipetainre
Increase. If Ihe Icmpcratnre becomes too elevated,
cooling may be required after these ope unions
which accelerate Ihe fermentation. Aeration also
remains nscfnl. as long as the harvest is not sus-
ceptible to oxidasic cassc.
In contrast with acoolycar.a warm year or warm
region produces a forwaid harvest. The resulting
must Is rich in sugar and so a complete fermen-
tation can be difficult to obtain. The low acidity
also increases bacterial risks and requires adapicd
snlfiting (Section 88 1 1 All of the harvest condi-
tions combine id produce an elevated fermentation
Icmpcratnre. and Icnipcralnre control systems are
therefore indispensable. Insuchasltualion. the risks
of a stuck fcrmcnuiion and consequently bacterial
spoilage are maximal. Paradoxically, the highest
quality wines can be made in these wlncntaking
conditions. In temperate climates, the greatest vin-
tages have long been known as the most difficult
ones to vinify. but wlncntaking methods adapted to
these conditions are relatively recent. Temperature
con mil in particular has been essential Although a
moderate icmpcratnre (20) is necessary to initiate
fermentation correctly, the temperature should not
be excessive Yeasts in their growth phase are par-
ticularly heal sensitive when the initial temperature
Is between 26 and 28 C. the increase in temper-
ature during the yeast growth phase makes stuck
fermentation more common and Increases the risk
of producing excessive volatile acidity. Initial cool-
ing of the grape crop Is therefore recommended.
ristablishlng the temperature during fermenta-
tion Is dependent on many fac tors concerning ler-
mcncition kinetics and skin extraction by mac-
eration. Stuck fermentations are likely to occur
when ihe temperature exceed 30'C Slightly lower
and relatively constant temperatures l25-28 u O
are advised for musts with clevalcd sugar con-
centrations and In difficult fernKncition condi-
tions Premium quality wines capable of aging
require a maceration permitting considerable phe-
nolic compounds extraction (Section 125 5 1 Ele-
vated kmpcratures play an essential role in this
phenomenon After a successful fermentation, the
Icmpcratnre can be raised to above 30'C Id
Increase this extraction. Crape quality shouM of
course also be considered before prolonging skin
conuct (Section 125 8) Primeur wines, however,
are made to be drunk young and respect the fruity
character of the grape; lower fermentation temper-
atures air recommended for these wines (25 "C).
In difficult fcrmcnuiion conditions linked to
excessive temperatures, several palliatives were
formerly recommended. Limited crashing slowed
Red Wincmaking
the IV mien Lit ion process ami (bus produced krss
neat. Another method consisted of simultaneously
tilling several fcrmcntois over several days In the
hope that the regular addition ol fresh grapes
would moderate the It ran- illation process In the
killer case, sulliting is noi saflicicnt lo avoid
tie increased risks and resulting consequences of
stack fcrnicniation and hydrogen sulfide produc-
tion Finally, elevated temperatures justify early
draining. This process. which scparaKs Ihc juice
from the pomace, towering bacterial risks, is
employed in warm regions Long vatting times,
however, arc practiced in more temperate /ones.
Today, musl aeration or. more specifically.
aeration of ycasc^ during their growth phase
iScclioo 3.7.2). along with tcmpcralnrc control, is
Ihc most effective way of helping difficult fermen-
tations. It Is earned out dnring pumping-over oper-
ations, or. possibly, by means of microoxygena-
twn Other processes capable of facilitating com-
pletion of fermentation (eg. adding nitrogen or cell
bulls. Section 36 2.) are described in Chapter 3.
114.2 Pumping-over Operations
and Musi Aeration
The disadvantages of open fcrmcniors have already
been covered (Section 12.3.1): alcohol evapora-
tion, baclcrial spoilage risks, etc. These tormentors
do. however, permit air contact and therefore a bet-
ter fermentation Fortunately. Ihc same effect can
be obtained with a closed fermentor Pumping-over
can assure saflicicnt air contact with Ihc must, sup-
plying the needed oxygen. This operation consists
of letting fermenting masl flow in contact with air
and then pumping it back into ihc upper part of
the tank. The effectiveness of this method has been
known in the Bordeaux region since the end of the
I9ih century. In-depth research was earned ont in
the I95ffc Dnc to its simplicity, its use has been
widespread .
The numbcis in Table 12.3 (Ribireau-Cayon
elal., 1951) demonstrate Ihc effectiveness of
pumping-over operations for improving the fcr-
mcncuKan process They also show that ycasR bet-
ter resist elevated temperatures, when aerated, bul
there is a certain amount of confusion as to Ihc
Tabic 1
13. Effect
ofacmbn
hv Mi*pinu-<»
cion fee
menial *
>n ktneik*
(Rfccieau-Gayao a rt.. 1951)
Time
Tank a
riatcd by
Non-ncwcil lank
pumpiap-ovcr
-nli... (.mi
p B&H Vt It
Tcapcmi
ire Dcmiv
.«-.". J," .-.,■11 ll-
Dcmiv
fC)
fC)
Day 1
22
LOSS
23
IDS!
Day 2
26
1084
20
lOU
Day 4
32
1 14-
29
1.073
Day
:i<
0590
27
1045
Day ID
—
—
27
1020
Day 2D
20
1002
most opportune time to aerate. The same authors
demonstrated that correct timing of oxygenation Is
essential. Harly aerations at the beginning of fcr-
mcnttlkm help to prevent stack fermentations the
ycaslsarcin their growth phase, and oxygen is nth
li/cd to improve iheir growth and produce survival
factors (Section 3.72). Early pumping-over oper-
ations have the additional advantage of avoiding
alcohol loss by evaporation.
An aeration carried out on the second day of fcr-
mcnttlioa is the most effective. The effectiveness
of later aerations, in the presence of fermentation
difliculties. is greatly diminished (Figure 12 7).
sometimes lo the point of being non-cxBtcnl In
the final stages of fermentation, the yeast docs
nol make use of oxygen, since clhanol and olhcr
toxic metabolites hinder iis nitrogen assimilation
A nitrogen addition in the final stages of fermen-
tation, therefore, docs not help lo re-cs&tblish fcr-
mcnttlion activity, even after aeration
Pamping-ovcr with aeration Is only beneficial at
certain moments, bat the pumping-over operation
in general has other effects. It homogenizes
Ihc temperature, sugar concentrations and yeast
population of Ihc fermentor. compensating the
effects of the more active fcrmentilKw in and just
bckiw the pomace cap (Sccudn 12.3.11 Above all.
ibis operation I'acililaKs extraction of compounds
from the pomace (anthocyanins and tanninskind
enhances maceration (Section 123.4).
The pumping-over process is schematized in
Figure 128 The fermenting musl flows from a
lancet located at Ihc lower pari of Ihc fermentor It
should be equipped with a tillering system inside
Handbook of Enology: The Microbiology °f Wiie anil Vinitications
(*>*>
Fift 12.7. Kflcd of noacMar acialkin (by pump-
injMivcr npciai»n al dillcrent limn) ud (cinnniJiHm
liiKtio I: open lank. pcnniKnt ucnibaaU — ill w£ji
In avnt |SI| rv fermented. II: ckned lank. JCOlbd on
2nd day bv puntplngMivci — ihc fomentation n accel-
erated, ufeh icipcn In total ,i nac nibiiu is. a ad complete .
HI; ckned rank: aciaiba on 6th day— ibe aceckoibn
of fcracnialkia n ta-enllkanl- IV: timed lank it total
lial»on<S2) j.-vcnluo SI
ihc Link lo slop seeds and. skins from blocking ihc
orifice, since the obstruction of this orifice parents
a serious problem ii early pimping-overs befonr
skin tup formation. The musl Hows fiom a ccrciin
height inlo a container wiih a capacity of scvciul
hundred liters The pressure of the falling juice
produces an emulsion which facililatcs oxygen
dlssiiluiion. Running Ihc must over a fkil surface
R also recommended, to Increase air contact.
Specialty equipped faKcts Intensify Ihc cmuLsion.
The acraled musl is Ihcn pimped back to the apper
pan of the fcratcnlor. soaking the pomace cap.
Aeration may be eliminated by pumping must in a
closed syslcm. diicclly from fanccl lo pump to the
■ppcr portion of Ihc fermenur. Pomace leaching
may also be avoided by placing Ihc pipe, al the
■ppcr pari of ihc fcratcnlor. below the pomace cap
into Ihc liquid
AdmiiKdly. the syslcm in Figure \2 8 and its
■sc are based oa empirical data Tic quantity of
oxygen dissolved in mnsl with this system can-
nol be even approximated. However. Ihc quan-
tity of oxygen dissolved in musl exposed to air
is of Ihc outer of 6-8 mg/l and varies according
lo temperature The quantities necessary lo avoid
stack terminal nn arc approximately 10-20 itg/l.
which can be obtained by pumping-ovcr with aera-
tion twice. 24 hours apart. Aclnal dissolved quan-
tities during pumping- over operations arc probably
lower. Experience shows that this amount is safli-
cicnt Ncvcrlhclcss. a syslcm pcrmiiiing controlled
oxygen addition (from a compressed gas botllc.
for example) would be preferable Indeed, this is
Ihc aim of process known as mlcrooxygcnation.
The precise amonnl of oxygen necessary mnsl also
be determined. Of ionise, oxygen is added simply
fc> assure yeast growth and survival, and a quan-
tity* greater than Ihc optimum dose has no adverse
effects on ycasK Nevertheless, enzymatic oxida-
tions in must may occur, despite ihc protection of
carbon dioxRIc during fermentation. Tannins pro-
tect healthy red grape juice from excessive oxida-
tion For this reason, they better tolerate aeration
than while grape musts, which arc not generally
pumped over tScctlon 13.7.3). With more or kss
rotten red grapes, oxidasic casscs arc easily trig-
gered and the amount of oxygen added should be
limited. If not nil.
Certain technical requirements mnsl be met for
pomace extraction lo be effective (Section 1253).
Unfortunately, they are nol always satisfied in
practice This process docs not circukilc mnsl lo
assure the direct dissolution of pinnace constituents
by leaching, bnt it docs replace the saturated mnsl
impregnating Ihc pomace cap with mnsl taken
from Ihc bottom of the fermentor. Approximately
two-thirds of the pomace cap is immersed in the
fermenting musl and one-lhinl fkuls above the
IKinid All of Ihc mnst should be pumped over
and the entire pomace cap should be soaked lo
obtain satisfactory results These conditions can
be difficult lo realize In narrow parallelepiped
fenne mors, especially if Ihc lid is not located
in Ihc center of Ihc tank The same limited
mnsl fiuction participates in this pumping-ovcr
operation. Draining (possibly with aeration) a third
to a half of the tank volume and then bratally
Kift UK l\impIn|i-a>c(iipciiikin.«.hDW
iclcasing Ihc msl from Ihc top of Ihc Link permits
Ihc immersion of ihc entire pomace cap (rack-
and-rctum). During this process. Ihc pomace cap
descends Id ihc tank. Various systems (cables)
iciim: Ihc cap nt he broken up aid reformed
Various types of routing irrigators cxbi. They
must be placed al Ihc center of the fcmiciior
and assun: ihc thorough soaking of the cnllic sur-
lace. To be fully effccllve. Ihclr pump delivery
rale ihiim be sufficient Increasing ihc (tow rait
during a pnmping-ovcr opcralK>n can suffice for
modifying tannin concentrations and consequently
wine slylc. Even In ideal conditions, the liquid
may pass through preferred passages in the pomace
cap. Depending on operating conditions, pumping-
over cffcclivcncss with icspccl to pomace extrac-
tion is cxUcmcly variable Close moniloring is
indispensable
144
Handbook of Etiology: The Microbiology of Wiac and Vinific.itions
A volume of JiKc corrcspoadiag lo OK-Ibint
t> one-half of the tank volaaK should generally
be pumped over. The number of pumping-ovcr
opcr.it ions should be increased but nor their dura-
lion li any case, the ficquency of pumping-ovcr
operations should be modalalcd This opciutioa
contribaics 10 ihc tannic slruclaic of wine aid
fa\«xs Ihc extraction of Ihc highest qaaliry tan-
■its. making wine rich and supple, tat an exces-
sive tannin concentration can lead lo bard, aggres-
sive, disagreeable wines. Other tcchnN|acs leg.
pinching down the cap) also give similar results
1 Section 1255).
Dnc lo in simplicity and lis favorable effect*,
pamping-ovcr is an essential operation in red
wincmaking. Inspired by the recommendations of
Pcynaad ( IS8 1 1, the following steps air applicable
u Bordeaux- style wincmaking:
I. As soon as the cuk is filled, a homogcni&ilioa
pumping-ovcr operation blends the different
grape ciops and evenly distributes the sulfur
dioxide. Yeasl may be inocnlalcd at lhat lime,
together with any other additives, but aeration
Is ■■necessity.
2 Pumping-overwilh aeration is essential as soon
as fermentation starts, as well as the following
day and. posably. the day aflcr that
3 During active fermentation, pumping-ovcr oper-
ations are clTccicd for extraction of phenolic
compounds. The aumbcr of pumping-ovcr opcr-
alKms shoakl be adapted H> wiac type and grape
quality The fermenting must should be pumped
ovcrcvcryonc to two days. Free-ran' wine aid
press wine arc also homogenized daring this
operation.
4. Aflcr fermentation, pumping-ovcr operations
shoakl be discontinued in hcniKlK. closed
cmks lo avoid oxygen exposure In open
tanks, pumpiag-ovcr operations ;uc coatinued.
Extended maccralions in partially filled taaks
require a short pamping-ovcr operation twice
per day to immerse aciobK germs.
5 Pumping-overs rarely snflicc *i restart slow or
stuck I'ereic ii unions
12.4.3 Monitoring (he Fermentation
Process— Determining its
Completion
Monitoring icmpcraiarc and density during fcr-
mentation kinetics has already been described
(Sections 322 and 32.3). It P. indLspcnsablc in
wincmaking. Cither controls arc aba recommended
lo complement Ibis data.
fermentation rales have been observed lo vary
under apparently identical coadiliOBs (temperature,
sagar content. aatoant of yeast inoculated, etc.).
Sluggrdi fcniKntationsmay be completed s
fully, hut they arc always a cause
Besides speciDc factors in the must, ok explana-
tion R that scvcml yeast strains arc invulvcd aid
fernicntilion kinetics may be affected by antago-
nism between them (Killer effect. Sections 1 .7 and
38.1).
At the end of alcoholic fermentation, malic acid
concentrations should be determined and moai-
lorcd if necessary. Malolaclic fermentation (MLF)
normally occurs after Ihc complete depletion of
sagars An early inilialioa of Ml.l- is generally
linked to alcoholic fenncntation difficulties aid
iasaflh'icnt sulriiiug. In certain cases, the two fcr-
mentations Cike place simullancously. even thoagh
Ihc antagonistic phenomena between yeasts and
bacleria tend to inhibit alcoholic fenncatuton.
Volatile acidity concentrations can be monitored
to identify fiKten.il coa lamination. It R indis-
pensable when malolaclic fermentation is initi-
ated daring slow akoholic fermentations, before
Ihc coaipfcte depletion of sugar (Seclioa 3.8) On
rare occasioas. apparently normal alcoholic fcr-
mcnciliOBs prodacc excessive vohtik- acid con-
centrations In this case, the fermenting aiast has
most likely been contaminated by the fcnncakir
or poorly maintained equipment. Spoifcigc bacte-
ria can also produce volatile acidity before the
start of alcoholic fenncntation. After fermentation.
iK origin is mote difficult lo identify Renovating
Ihc winery and replacing ohl equipment gcacrally
eliminate this contamination risk.
Yet the production of volatile acidity does nol
always indicate the presence of bacleria. Yeasts
may aLso produce volatile acidity. In certain.
Red Wincmaking
.•4i
as ycl poorly defined, ca>
of volatile acidity arc produced (0.4-06 g/1 in
H ? SO t . or 05-0.7 g/1 in acclic acid). By deter-
mining ihc concentrations of the Iwo lactic acid
isotKislU+Haciic acid and W-Uac tic acid), tic
origin of ihc acetic acid can be identified. Dur-
ing fcnucntilion. yeasts produce a few do/en mg/l
of the former and less than 200 mg/1 of the lat-
Icrfl^fon-Lafourcadcand Ribc'rcau-Cayon. 1977).
Higher values indicate the involvement of lactic
acid bacteria in Inc production of volatile acidity.
hm the possibility of high volatile acidity produc-
tion levels by yeasts should always be considered.
Moreover, the sumdaid methods used to protect
against bacterial spoilage have no elicit on yeasts.
Additionally, wine acidification tends to increase
yeast-based volatile acidity production. Certain
yeasts have an increased capability for volatile
acidity production which attains a maximum in the
couisc of fermentation, tending to decrease uward
the end. In red wincmaking. an excessive tem-
perature (28 -O at the initiation of femicntalion
contribntcs K> elevated volatile acidity levels.
The linal stages of fermentation should be
closely monitored. When Ihc density drops below
I (XX). this measurement is no longer sufficient to
measure precisely the evolution of the femicn-
talion. Moreover, the relationship between possi-
ble residual sugar and density is complex When
fermentation is complete, wine density can vary
between 0.991 and 0.996. accotding to alcohol
content. In addition, free-run wines always have
a lower density than press wines, which are rich
in extracted constituents
The completion of fermencilion Is verified by
chemically measuring the sagar concentration. For
a long time, the reducing property of sugais was
exclusively used to determine their concentration,
but methods based on this characteristic also mea-
sured other substances in addition to fermentable
sugais (glucose and fructose) Due lo this inter-
ference', fermentations were considered complete
when these methods indicated less lhan 2 g of
sugar per liter. This value actually signified the
presence of less lhan 2 g of reducing agents
per liter, including, among other substances, glu-
cose and fructose. Due in an increased quantity
of reducing agents in press wines and wines
made from rotten grapes, fermentations arc con-
sidered complete at approximately 3 g/l in these
cases. Today, more and more glucosc-and frnctosc-
specific analysis methods arc available Their over-
all concentration should not exceed several hun-
dred milligrams per liter when the fermentation
is complete. This is nccessaiy lo avoid spoilage
due lo Ihc development of contaminant yeasK
tBreiiiinnttiycfs) during barrel-aging iVolumc 2.
Sec lions 8.45; 8 9.6).
125 MACERATION
125.1 The Role of Maceration
Red wines arc macerated wines Maceration is
responsible for all of the specific cbaractcrptics of
sight, smell and taslc that differentiate red wines
from while wines. Phenolic compounds (anibo-
c van ins and tannins) arc primarily extracted, par-
ticipating in the color and overall structure of wine
Ycl aromas and aroma precursors, nitrogen com-
pounds. polysaccharides l in particular, pectins) and
minerals arc also liberated in the must or wine dur-
ing maceration.
The corresponding chemical elements come
from the skins, seeds and sometimes Ihc stems
Each of these oigans supplies chemically and gus-
latorily different phenolic compounds. The gusta-
tory differences are continued by tasting wines
made in Ihc presence of one or more of these
otgans. Mi ii'- give wine herbaceous ftavois and
seeds contribute to harshness Skins contact alone
prodnccsa supple but incomplete wine that is loo
fluid in structure Skins and seeds contact makes a
more balanced wine The phenolic compounds of
each oigan also vary according to variety, matura-
tion conditions and other faclois. Furthermore, in
the same organ (for example, in the grape skin),
herbaceous, vegetal and bitkr substances along
with leafy and grassy substances arc located along-
side phenolic compounds favorable u wine qual-
ity. Fortunately, the latter substances arc extracted
before the others.
Consequently. Ihc maceration should be mo-
dulated and fiuciionalcd. Only useful giapc con-
stituents should be dissolved — those positively
346
Handbook of Enofcgy: The Microbiology of Wine anil Vindications
contributing hi wine flavor and aroma. The ex-
traction or these desirable substances should be
maximal, if nol total.
The concentration ofsubseinccs in grape (Issues
dclrinicnial to wine quality incrtascsasgnrpc qual-
ity diminishes. This phenomenon can be verified by
chewing a grape skin alter the pulp aid seeds have
been removed by prosing the berry between the
thamband index finger. Initially, mild savorscvotvc
toward mellow tannins. Afterwards, vegetal sen-
sations become increasingly hitter and aggressive.
The ir.insr. ■ m rale in ■m plcasanl to disagreeable
scnsaliois varies according to giapc quality. The
evolution uf tinnin quality can be evaluated dur-
ing maturation in this manner. The same experience
effected on seeds leads to similar result. Harshness
and aslringcncy diminish diring maturation, while
sensations of body and harmony increase
An abundance of pleasant- lasting substances
esc fa 1 for wine making and a lack of unpleasant
ones characterise the grapes of top- ranked growths.
These characteristics typify matarc years, i.e. great
villages Such wines arc capable of undergo-
ing the most intense extractions and prolonged
vailing times The resulting high tannin concen-
trations arc necessary to ensure their long-term
aging. Lesser quality red wines, made for imme-
diate consumption, have relatively short macera-
tions — more flaws than qualities wouki rcsnllfrom
longer macerations.
The extraction of pomace consiitucnLs during
maceration should therefore be modulated accor-
ding log tape variety and quali ty and also Inc style of
wine dcslied (sec Volume 2. Section 6.6). Yet each
grape ciop is capable of producing a given type of
wine, depending on natural factots (the temiirt.
Premium wines require a tannic siruclnic which
should not compromise finesse and elegance.
These wines arc difficult K> produce and require
grapes of superior quality benefiting from great
lemurs and great growths. Light, fruity red wines
arc relatively easy k> obtain —grape quality Is nol
essential, but if grape quality* (variety, maturity,
sanitary slate, clc.) is insufficient, tannic ted wines
rapidly become heavy, coarse and without inarm.
Short vailing times and limited maceration lessen
c of disagreeable characteristics
A number of methods arc available to the v
maker to adjnsl extraction k-vcK during i
ton They essentially influence tissue destruction
and favor the dissolution of phenolic compounds.
Techniques arc continually evolving and engineers
icgnlarly propose new solutions. Current methods
will be described titer in this chapter, each one
probably having preferential eflei is on one oi more
groups of extracted substances For example, bru-
tal crushing promotes Ihe extraction of Niter and
herbaceous substances. Percolation of must, on the
contrary, favors supple and full-bodied tannins.
Constituent cxUactability of various organs varies
with several factors (variety, maturity level, etc.).
Enzymatic reactions, activated by grape enzy-
mes, arc involved In cell wall degradation. They
favor the dissolution of their vacuolar contents
(Section 11.7.3). Commercial enzymatic prepara-
tions have recently been developed lo activate these
phenomena: they have pecfinasc. ccllilase. hcml-
ccllakisc and protease activities of diverse ori-
gins (Aniranhjoutei. 1993) These enzynves seem
to favor the extraction of skin tannins over skin
authocyanins They act on the tannins linked to the
polysaccharides of the cell wall, giving Ihe enzy-
matic wiac a mote full-bodied character than the
control wine.
Tou&iil etal. (1994) obttined encouraging re-
sults from an enzymatic pool produced from
Botiyiis cineiru cultures, not containing laccase.
This preparation aittcks cell walls and favors the
anthocyanin extraction over tannin extraction. It
coukl therefore be interesting for making primeur-
style wines, to be drunk young, frail)* and rich in
color but not very tannic
Future research will be required to determine
the selective effect of this enzymatic extraction
on the various phenolic compounds, according lo
conditions, lis effect, with respect to standard prac-
tices for regulating niaccralion. also needs to be
explored. Regardless of Ihe mechanisms Involved,
tasting has confirmed the interest of using enzy-
matic preparations for maceration during red winc-
making.
These various result* demonstrate Ihe Utility of
a belter chemical and gustatory undcrstinding of
the molecules involved in maceration phenomena.
Red Wine making
i-T
Thc cximction of ;i specific combination of these
molecules could thus be obtained according to ihc
maceration Iccbniquc used. Certain practices may
be beneficed « »nc quality in some situations hul
nol in others Only fundamental research on ihc
chemistry of phenolic compounds will be capable
of giving definitive answers These studies arc
complicated by the extreme complexity and the high
reactivity of the molecules involved (Volume?.
Section 6 J).
In traditional wlncmaking. maceration occurs
dnting vailing tetmiison). white the pomace souks
in the juice. Alcoholic fermcii union occurs in the
juice, producing clhaaol and raising the tempera-
iiic Both ethanol und temperature panicipatc in
the dissolution of pomace constituents
12.5.2 Different Types of Maccralion
There Is a current Ircnd to distinguish between the
various types of maceration, other than standard
extraction during fermentation:
I) High- temperature extraction prior to fermenta-
tion used in thcrmoviuiiication i Section 12.8J).
cither followed by normal fermentation, or sep-
arate fermentation of Inc juice.
2> Cool- temperature extraction prior u fetmenta-
tion. aimed at enhancing aromatic complexity.
The start of fermentation is postponed by main-
taining km* temperatures and an appropriate
level of SO?, as well as by delaying inoculation
with active yeasts
A more elaborate form of this Iccbniquc con-
sists of cooling the grapes to around 5'C. by
injecting liquid CO; or dry ice. and maintain-
ing this temperature for 5- 15 days. The tem-
perature shock bursts the grape skin cells and
releases intensely colored juice (Blouin and
Pcynaud. 2001 l Once the mnst has been healed
to normal temperature, fermentation proceeds
as usual. The purpose of this technique Is to
obtain wines with high concentrations of phe-
nolic and aromatic compounds. The results of
this rather laborious method are not univer-
sally appreciated, further research Is required
to Identify the conditions required to produce
deep-colored, aromatic wines without any res-
tic or herbaceous character. Satisfactory results
have been achieved with Pinot Noir(Ranzy.
19%). producing finer, fraiticr wines In any
case, the results are better than those obtained
with cokl maccralion. following stabilization
with considerable doses of SO?, which gave
deep-colored wines that were lacking in vari-
etal character and tended u dry out on the end
of the rulaic ilcuilLit. 1977).
3) Post- fermentation vailing is required by the best
premium quality red wines to prolong skin
contact after the end of fermentation, some-
times combined with an increase In temperature
(final, high- temperature maceration. 1233).
115.3 Principles of Maceration
The rxissagc of pomace constituents, particularly
phenolic compounds lanlbocyamns and tinnins).
Into fermenting juKc depends on various elemental
factors The results constitute overall maceration
kinetics The phenomena involved are complex
and do nol cause a regular increase in extracted
substances In fact, among these various factors,
some lend lo increase phenolic compounds, while
others ktwer concentrations. Moreover, they do not
irily always act in the same manner on the
s constituents of this group.
Maceration is controlled by several mechanisms
(sec also \«lumc 2. Section 6.6 I):
I. The extraction and dissolution of different sub-
stances. Dissolution is Ihc passage of cell vac-
uole contenh from the solids phase into the
liquid phase. This dissolution depends first of
all on vine variety and grape maturity levels
This is especially important for anthocyanins
In ccmin cases, strongly colored mnsfs are
obtained Immediately after crushing. In other
cases, a period of 24-43 bouts Is required
Tissue destruction through enzymatic pathways
or crushing facilitates dissolution. The more
Intense the crushing. Ihc more dissolution is
favored. Finally, dissolution depends on the var-
ious operations that participate in tissue destruc-
tion sulliting. anacroblosis. ethanol. elevated
temperatures, contact time.
:-.■
Handbook of Enology: The Microbiology of Wi»e anil Vinifkalions
2 Diffusion of extracted substances Dissolution
occurs in ihc pomace, anil ihc impregnating liq-
",.l rapidly becomes saturated with extracted
substmtcs. exchanges thctcfoie slop. Furlhcr
dissolution is dependent on the diffusion of
the extracted substances throughout the mass.
Pumping-ovcr or punching down the pomace
cap renews tic juice impregnating lie pomace
cap This diffusion is necessary for mi i table
pomace extraction. It homogenizes the fcrmcu-
i"i" and reduces the difference between the phe-
nolic compound coKcnlratious of free- run wine
and press wine.
3. Relaxation of extracted sibstances on certain
subscinces in the medium: stems, pomace,
yeas*. This phenomenon has been known since
FcnC's 1 1958) obsetvalions (Section 12.2 3)
4 Modulation of extracted substances. This
hypothesis still requires farther theoretical intcr-
prctutons Anlhocyauins may temporarily be
reduced to colorless derivatives (Rib&cau-
Gayon. 1973). The reaction appears to be
reversible, since lie color of new wlncsexposcd
to air for 24 hours increases, with the excep-
tion of those made from rotten grapes. Antho-
cyniiB-Fe lt mm complex formation may be
involved in this color increase in the pres-
ence of oxygen. Ethanol may destroy tan-
nin -anthocyan in associations extracted from
the grape (Somcrs. 1979). In the same envi-
ronmental conditions, free anthocyaninsarc less
colored than tannin -an fhocyauin combinations,
which arc formed again daring aging and assure
color stability.
The quantity of anlhocyanins and tannins found
in wine depends first of all on their concentration in
the grape crop. Ripe grapes arc the first condition
for obtaining rich and colored wines. However,
only a fraction of the phenolic compound potential
of the grape is found in wine. Their concentration
depends not only on the case of phenolic com-
pound extraction but also on the extraction meth-
ods used. The phenolic compound concentrations
of varioas components of grape clusters and wine
nave been computed. Approximately 20-30*' of
the phenolic potential of grapes is transferred to
wine. Tic loss is significant and dibits have been
made to improve this yield but. due to the com-
plexity of thct phenomenon and the molecules
involved, a simple solution is difficult to find.
Finally. Needing" a valt Section 125.9) Is a way
of raising tannin levebt by reducing volume. Elim-
inating water by other techniques (ScclkiB 115. 1)
achieves similar results, but keeps all the sugar in
the mast.
In red wincmaking . maceration mast be adapted
to suit Ihc grapes constilalion (Volume 2. Section
662).
125.4 Influence of Maceration Time
(Vailing Time)
The dissolution of phenolic compounds from
solids into fermenting must varies according to
maceration time, but no proportional relationship
between maceration time and phenolic compound
concentration cists. Color intensity has even been
observed to diminish after an initial increase
during the first 8- 10 days (Fcirf. 1958; Sudraud.
l-'ili 11". Coknintcwiv «*1 phenolic compound con-
ibaiimclRihciciu-C&yuarw.Y.. 1970). Color i»cmity
n defined » (be mid of* Ibc op Hal dcnbritci .1 420 ial
520 am ai I ma thick mm.. (CI = OD 420 -t OD 520).
"Ib<al phcmlk compound* aic determined hy the pci-
m.iii^m.iic Indei
Red Wincmaking
349
1963). The graphs in Figure 12.9 depict Ibc
evolution or ;i maceration extended well beyond
normal conventions. In this laboratory experiment.
Ihc color uik'nsity passes through a maximum
on Ihc eighth day :ind Ihen diminishes. The
evolution of wtd phenolic compounds Is different:
during an initial phase Listing a leu days, ihclr
conccn tmilon increase Is rapid and Incn slows
afterwards This behavioral difference Is due to
tannin concentrations tskins and seeds) in Ihc
grape crop being ID times grcatcr than anlhocyanin
concentrations. In both cases. Ihc concentrations
incicasc during the tirst lew days. Aftcrwaids.
tannin losses arc proportionally less signilicanl and
an overall Increase is always observed Certain
varieties have very low tannin concentrations.
In this case, tannins evolve similarly Id color
(Figure 12.9) Other experiments have shown that
Ihc nature and properties of tinnins vary in func-
tion of maceration tunc
Similarly, the various grape organs (skins, seeds
and sic ins) contain specific phenolic compounds.
Their extraction varies according to diverse condi-
tions Skin ailhoryanins arc cxiracfcdficsi. ethanol
is not required for their dissolution Skin tan-
nin extraction begins soon after, facilitated by Ihc
increasing presence of ethanol during fermenta-
tion A relatively long maceration is necessary for
seed tinnin extraction The presence of ethanol is
required to eliminate lipids. The skins contain the
most supple unnins. but they can become bitter
if grape maturity is insafHcicui. Seed tannins arc
harsher but less bitter
Evidently, these notions pertaining to the evolu-
tion of color and anthocyanins in terms of macer-
ation time primarily concern new wines — aniho-
cyanins arc in fact the esscnli.il cknicnts of
their cokir. As wine matures, the role of tannins
becomes increasingly important. Extended vailing
times produce moic cok>ied wines, even if ihc
resulting new wines initially appear to confirm Ihc
contrary
The causes of this drop in color intensity, after
several days of vailing, have been examined and
intctprcKd. Stems have long been known u dec-
rease the intensity of wine color i See lion 12.2.3):
this phenomenon is the result of their adsorbing
anthocyanins. While grape skins have also been
shown to adsorb anthocyanins when pktccd in
red grape must A yeast biomass in a fermenting
medium adsorbs both anthocyanins and tannins
Chemical reactions also diminish color inten-
sity Giupc tanmn-anlhocyanin combinations arc
destroyed and anthocyanins arc rcdnccd to color-
less forms during these reactions (Section 125.1).
These facet lead in an important conclusion
On approximately the eighth day of maceration,
wine color intensity is at its maximum and tinnin
concentrations arc limited, permilting fruity sensa-
tions to be conserved. This vailing method is best
adapted to wines for catly drinking. In contrast,
long vauing times produce rich tannic wines capa-
ble of extended aging, bit these clevaKd tinnin
concentrations require grapes of high quality.
12,5.5 Influence of Pump trig- over
and Cap Punching (Pigeage)
Pimping -over Is important in red wincmaking. at
least for certain varieties such as Mcrlot and Caber-
net Sauvignon Section 12.4 2 describes Ihc objec-
tives and steps of this operation In addition to
introducing oxygen, il playsa major role in extract-
ing compounds from ihc pomace and homogeniz-
ing the conkrnls of the vat It plays a major role
in pomace extraction and lank homogenifation. At
skin and Juice separation, the drawn-off wine and
press wine have more similar tinnin concentra-
tions when pumping-ovcr operations ate catricd
out The numbers in Table 12.4 show the dual
effect of pumping-ovcr ope rations Increasing the
number of pumping-ovcr operations accentuates
these effect*. An effective pumping-ovcr should
thoroughly leach the pomace cap. Precise exper-
iments have shown that phenolic compound con-
centrations and color intensity vary significantly
according topnmping-ovcrconditions. Rack-and-
return is recommended for this purpose and con-
sists of running-oil pari of the fermenting must,
then rcin trod ncing il all at once over Ihc ba>kcn-up
pomace
Pumping-ovcr has an additional advantage that
is more difficult to evaluate. This operation
docs not affect tissue integrity and promotes the
350
Tunic 124. In Hut
Handbook of Fnology: The Microbiology of Wi»e anil Viniliiations
na cokii and tannin dbaoluibn i* open ferae vcn. (Sudcuul. 1903)
n(> <hy*
n 10 days
083
087
1 1 H
Coin
extraction of higher-quality tannins. :u least )■
some grape varieties. It iiii|tiris a rich anil lull*
bodied structure to wines, without bitterness and
vegetal characteristics.
The liming of pumping-ovcr operations influ-
ences lie selective extraction of skin and seed
tin n ins. Skin tannins arc more easily released
and aic saffkicit for making prmmir wines,
bat seed tannins arc necessary *> obtain a pre-
mium wine. Pumping-ovcts in the final .stigc of
fcrmentition arc used to extract these compounds.
The advantages of this technique have led to
iK general use. However, in certain cases it is
abused, producing aggressive and disagreeable tan-
nic wines: the resulting press wines arc thin and
■■ usable
Gas injection systems have been proposed
lo replace traditional pumpings (Section 1242.
Figure 128) for the extraction of pomace con-
stituents These systems consist of introducing a
specially adapted pipe into the lower part of the
sink. The pipe injects carbon dioxide, nitrogen or
filtered air into thefcrmentor. The lank intents arc
churned up briskly. This operation Is much morc
rapid than traditional methods, and a duration of I
minute per 100 hi of grape crop at a pressure of 3
burs Is recommended. The technique should not be
•sal during the final stage of fermentation (when
the density falls below 1. 010). Continuing injec-
tions beyond this value would lead lo the definitive
disintegration of the pomace cap.
In certain cases, punching down can replace
pnmping-ovcr. As its name saggesK. punching
down consist, of plunging the skin cap completely
into the liquid. This immersion results in the
disintegration of the cap and increases maceration
phenomena The process promotes seed tannin
extraction and thus increases the tannic structure
of wine. For certain varieties (eg Pinot Noir).
punching down the cap produces belter results than
pumping over, but there Is some concern that it
may give a slightly rustic character to Cabernet
Sauvignon and Mcrlot wines, especially if used lo
excess. Various types of tanks can be equipped
with automatic punch-down systems (Bk>nin and
Peynaud. 2001 ). This equipment Is better adapted
to small- capacity Links
12.5.6 Influence of Temperature
Heat is a means of degrading llssacs. It Increases
the dissolution of pomace constituents and accele-
rates maceration. The technique of heating crushed
red grapes has been nscd for a long time and
Is particularly important in thcrmovinificalion
(Section 12.8.3).
In addition to this extreme case, temperature is
an essential factor in standard maceration. It should
be sufficiently high u assure satisfactory extraction
of phenolic compounds. The experiment (Sudraud.
I%3) in Table 123 clearly illustrates its impact
Both the average and maximum temperature
affec I cxtrac lion . The results of a laboratory experi-
ment in Tabic 12 6 indicate the simultaneous influ-
ence of maceration time and temperature. When
maceration is prolonged, an elevated temperature
351
Tabic 12.5. I •Science »f fcrmcMaiba Icatpcoiuic u
diuoUbnol phenolic : ...nifouah (SullMKl. 1963)
Temperature Toial phcnoUc Color
■ pC llll Ill I'.. ' UC
index)
20'C U 0.71
25*C 48 0X7
30"C 52 0.96
20-37"C (avenge 29.5) 52 1.21
25-3TC (avenge 32.61 60 Ui
tun exacerbate ibc drop In anthocyanln «
(rations ami color intensity. An clcvaicd i
Hot Kmpcraturc also favors the extraction of ycasl
manuoprolcius. which participate in the production
of soft and full-bodied wines.
Mode rat maceration temperatures (25'C) are
preferred for the production of primeur-style
wines These are made to be drunk young and this
approach gives them a good color while conserving
their fruity aroma characteristics. Moderate tem-
peratures arc also recommended when there Is
a risk of fermentation difficulties (elevated sugar
concentration) An elevated temperature (30'C)
extracts the tannins required to produce a premium
wine capable of long aging; higher temperatures
would promote farther extraction but would also
compromise yeast activity— they should this be
used with caution
In stainlcss-slecl vans, especially if they are in-
stalled in Ihc open air. excessively low fermenta-
tion temperatures may cansc problems in certain
years in some climates These temperatures do not
permit a sufficient maceration. Moreover, as soon
as the fermentation stops, such tanks can rapidly
drop in temperature, no longer producing calo-
ries The temperature in wooden ranks develops
differently
Tank temperature control systems have permit-
led an almost perfect regulation of the maceration
icnipcralarc during fermentation. Cool grape crops
and excessively cool fermenting )ukc can be
Tabic 12.'-
LHUCIKC of
■uaaufhu
lempcniiurt on dnwilutkin of phcit
>lk.
ompouacb (Rkeiemi-COYon <* <rf.. 1970)
Time and ti
rmpcraiurc
Tim"
Color AM lot;, -Jain
imcuMty* <s/l)
Tanain Tola I phenolK
(g/1) can pound.
1 permanganate index)
It m pc mlvi
B
20 ' '
034
2S°C
032
30"C
038
MacentKH
iK da)*
Tcmpcraiu
b
20 i '
0JS
2S°C
03ft
30°C
03ft
Maccniioi
i 14 dnv»
1 c m pc racu
B
20 C
033
2S*C
031
30 6 C
03ft
MaceniKH
i 30 <hv%
Tcmpcraiu
20 '.'
03ft
25 C
0.67
W i '
0«0
352
Handbook of Etiology: The Microbiology °f Wit* anil Viniflc.uions
warmed in ;i suitable Icmpcrataic. bat since ai
integrated neat exchanger wants inly a portion
of ibe twk contents, a pumping-ovcr operation
Pi required to homogenize Ibc lank. Temperature
eonirol must of coarse be set according k>
fcrmcntitioa kinetics. For example. Ibe lalilunoa
of fermentation requires a moderate Icmpcralnic
<20''C). sine Ibe yeasl is relatively beat scasitivc
daring Its growth pbasc (Section 3.7.1 and .VS.li
and the re is a greater risk of slnck fermentation ai
thai stage.
Glories at of. < JSB I ) developed a lechniquc
called healed post- fermentation maceration. The
objective of this method is to separale the fcr-
ntcnlalion phase al a moderate tcmpcralnrc from
(be maccralioa pbasc. Afcrfcrmcntalioa. tbe laik
content! air warmed lo between 35 and 40"C for
several days. In Ibis process, oil)* the wiic is
directly healed, the pomace cap being healed indi-
rectly. TV wine is healed lo between 50 aid 60'C
daring the entire maceration period. This process
has inn been observed to allcr Ibc tistc of wiac.
In addition, matotactic fermentation tikes place
normally. As long as this method is effected in
tbe absence of residual sugar. Iherc Ls no risk of
bacterial spoilage, rcsulliag ia Increased volatile
acidiiy. The method most significantly affects phe-
nolic compound concentrations and color intensity,
which arc grcally increased by hot maccratiOB alter
fcrmcntitioa.
In ibc experiment in Table 12.7. lasting reveals
wine A to be diluted because of an elevated crop
yield and insillicient malarily. Wine 8 Ls more
full-bodied and has more lastc bnl no vegetal
character. Wine B is grcally improved when com-
pared with wiac A. which is clearly lower in qual-
ity. Applying heated post- ie rmc niailon m.tceraiion
to wiacs thai arc already mm rally rich grcally
Intensities their concentration —which could be
iBKiprclcd as an improvement, except that Ibis
concentration is oflca al Ibc expense of gustatory
finesse: the tannins rapidly become bard and rus-
tic, aad this tannic ^stringency lends to increase
during aging. The repealed pumping over required
n> maintain Ihc temperature during this process
can magnify these Raws. Suspended solids arc
often produced and press wines often become
unusable Tbe above observations emphasize the
need lo consider wine quality befoie using this
method.
Temperature drops in steel tanks shoakl be
avoided allcr tbe completion of fermentation. A
post- fc rate n la lion maceration al 3Q ! C daring sev-
eral days often favors wine quality This approach
emulates Ihc thermal conditions ia wooden Ginks
following !c riven (a tun High- temperature, post-
fcrmcntallon vailing mast nol be confascd with
thcnnovinilkalKva (Section 12.8.3).
Temperature control, in addition fc> pumping-
ovcr operations and valting time. Ls another means
of modulating extraction during maceration Tem-
perature regulation can profoaadly modify the
tannic structure of wine Bui there Ls a risk
of also increasing the rustic character of wiac.
The nature of Ihc grape crop and Ibc type of
wine desired arc criteria which should be con-
sidered when determining optimum temperature
tegulation.
Tubfc 12.7. Ellen of heated fmt-le
c ""'°"
-
TrndMonal win
(Wine A)
ali-J!
Hcate
d f..u-lcimc.
Kin (J <U>9, a
(Wiac Bt
1 JS'C)
Color int.
■.-....
D.4S
067
Tint"
0.B2
0.75
fatal phc
nalkc
■npuiat*.
29
38
(Folin
Index)
ninfa
!*!,']>
2->
329
12.5.7 Effect or Crape Sulfitinjl
and Alcohol Produced by
Fermentation
The impact ol must salriting on pigment extraction
is covered in Scclion 8.75 Sulfnr dioxide destroys
cell lissac and promotes Ihc dissolving ot pomace
constituents, but in iradilional wincmaking with
ripe, healthy grapes, pumping over, temperature
and valting time have a greater impact All things
considered. SO. has little influence on the cokir
inlcnsity and phenolic compound concentration of
normal red wines.
However, the dissolvent effect of SO; B mani-
fest in rose wincmaking. since the phenolic coat-
pound concentration Is low in this case In fact.
SOj can be detrimental u while wincmaking. This
dissolvent effect may ab>o affect red grapes if they
are insufficiently ripe, and pigment cxtractibility is
poor In that case, snlliting facilitates anlhocyanin
extraction in the early stages, especially during
cool- tern pc rain re maceration (Section 12.52).
With rotten grapes, sulfiling docs not improve
the extraction of pigments, instead, it prevents
the laccasc activity of BtMrytis einerra from
destroying them. The numbcis in Table 12 8 show
that high snlfnr dioxide concentrations increase Ihc
total phenolic compound concentration and cokir
intensity in the case of highly contaminated grapes
The elevated lint valicin the control sample is due
to a yellow component. caaracKrRtic of oxidasK
The impact of Ihc cthanol produced by fer-
mentation seems to be complex. According io
Somcrs (1979). it is involved in Ihc decrease
Tabic 128. Influence of sulhlin^ botiyiiJcd prapcb on
phenolic coapouKb. uf ihc •CMitinp a lac* (Sudnul.
1903)
Sullllin^
I'm
(PC.
il phenolic
■UHJlMtC
lade.)
Cokir"
baoiy
Tint*
Com ml
32
033
0.70
Sullitcd *
In
g/hl
41
003
042
Sullited «
Hi
g/hl
55
0B3
043
in color intensity observed during vailing The
mechanism seems to correspond to the dcslruc-
tioa oftauuin-anlhocyanin com hi nations, resulting
in the liberation of free anlbocyanins. which arc
less colored. Al the same time, alcohol is con-
sidered Io purticipak: in tissue destruclion and.
as a result, in Ihc dissolution of pomace con-
stituents (Tabic 1291 li large wineries contain-
ing many fermentors. with relatively homogeneous
grape crops, the wines with Ihc highest tannin coa-
ccnlratioas and color intensities arc often observed
to have the highest alcohol strength
115.8 Impact of Various Mechanical
and Physical Processes Acting
Directly on the Pomace
(Flash- detente)
The impact of crushing grapes has already been
covered in Section 122.2. Energetic crushing
increases Ihc diffusion of solid Ussac compo-
nents, bul. accoidiag to a general rule, the corre-
sponding tissue destruction pmmoics the extraction
nccof alcohol
n ( 10 d»>* of n
un of pomace phenolic compound*
it 20"C. pH 3.2><Canb**. 1971)
Alcohol
Tannin*
lotnl phcaollc
AmhocianiiB.
Cobr
(gl)
Ipcrnia-jMn-c
index)
<mp/ll
intent Iiv''
O'-f vol.
DM
12
109
1.95
V, vol
OA
10
214
300
10'r vol.
IJ2
20
22~
0.35
354
Handbook of linology: The Microbiology o( Wine anil Vnifkatkms
of inferior-quality tannins. These tannins im|xui
vegetal and herbaceous (asKs lo tic must aid
resulting wine Unfortunately, no measures arc enr-
■cilly available to confirm this fact. Yet when
complied with wines Diadc from energetically
crashed grapes, pumping-ovcr operatious have
been observed to favor the extraction of soft and
more agreeable crnnins. The extent of the negative
effect caused by excessive crushing Is according to
the grape variety and its degree of vegetal charac-
ter. However, all things being equal, quality grape
crops arc much more sensitive to crushing intensity
than ordinary grapes.
Similarly, the breaking np aid punching down
of the pomace cap has long been used to increase
maceration while piotccling the wine from bac-
terial development. In the past, these operations
were common and carried out manually in small-
capacity open fc coventors They arc no longer
possible in today's large fermentors. but tanks
can now be equipped with mechanical devices
(screw, helix, fack and piston-based) which assure
the breaking up. reshaping and pinching down
of the pomace cap (Blouin and Peynaud. 2001).
The pomace cap can also be broken np by a
mechanical claw, which is also used for devat-
ting. When the cfctw Is not functioning, the rank
is ekised by a removable cover. The operation
of these various systems is fairly complex but
they arc effective in tcrmsof extracted substances.
They can be applied to certain varieties (Pinot.
Section 125.4). but with other varieties, these
methods can rapidly lead to excessively hard and
disagreeable tannins
Rotating cylindrical fcmientors have similar
constraints. A fixed internal device inside the lank
breaks np the cap and is also used for devalling
the fermentor This kind of fermentor is relatively
expensive. It has the same advantages and incon-
veniences as the equipment described In the above
paragraph. Crape crops with tow concentration?, of
phenolic compounds arc ex Uuctcd rapidly. In other
situations.;! satisfactory extraction is also obtained
rapidly, permitting the fermentor to be used sev-
eral times during the same harvest. This system
does not necessarily give satisfactory results for
producing premium wines.
Another rccently-dcvck>pcd process. (TMECA
DIF. 34800 Clermont- L Hcraut. Prance) aimed at
intensifying maceration of pomace In grape must,
is known as Flash-ileieiile (Bon let and Escudicr.
1998). (1 consist, of bringing the crushed grapes
rapidly to a high temperature, then chilling them
almost insian tmconsly in a high vacuum. It may
be considered a variation on thcrmoviuifiralion
(Section 12.8.3). Under these conditions, the skin
cell tissue structure Is completely degraded and
components essential lo wine quality (phenolic
compounds, polysaccharides, aromas, etc.) are
rapidly released during later vailing.
The grapes arc dcslemmcd and part of the juice
separated out As soon as the crushed grapes
come into the beating chamber. It is brought up
to a high temperature (70°C-90°C> very rapidly
by direct injection of saturating vapor at MX) i".
produced using the reserved juice The must is then
transferred by means of a positive-displacement
pump ink) a high-vacuum chamber connected to
a vacuum pump coupled with a condenser. When
the hot must enters the vacuum chamber, the water
it contains Is vapori/cd. cooling it rapidly lo the
boiling point of water under the vacuum conditions
nscd. i.c. 30"C-35*C.
According to Boulct and Escudicr (1998). this
sequence has three consequences: the crushed
grapes arc cooled in less lh;rn one second, the grape
cell walls arc broken, and there is only a very low
concentration of rcsklual oxygen in con tic t with
the mnst.
The water recovered from the vacuum chamber
is condensed, and represents 7—129 of the total
volume of the must. Part of this is nscd lo produce
the hot steam nscd in the system, in addition lo
that made from the separated juKc. The rcst may
re added back into the must, making it more
concentrated. This is only possible If permitted by
legislation. This technique does not dilute the must
as the steam used lo heat the must is made from
grape juice, but may concentrate it. as pari of the
water IS eliminated .
If the treated grapes arc pressed immediately,
the resulting wine is similar lo that produced by
fermentation combined with sundard techniques
for heating the mnst (Section 12.8.3). If. however.
Red Wincmaklng
i-vS
Ihc must Is left on the skins al a suitable (civ
pt ■ iMin a- aflcr Flttsli-tleit'nte treatment, the pomace
extraction kincticsare much faster lhan ■■ a normal
wincmaklng situation, teaching maximum antho-
cyan in and polyphenols levels alter .1-4 days
This high- temperature treatment destroys lac-
case, soil is extremely suitable for grapes affected
by Bmiytis einerea Pcctolyuc enzymes arc also
partially destroyed, so the must becomes viscous
aflcr Flusli-t<nle and is difficult to drain Prcss-
iig is facilltulcd by mixing treated must with a
sufficient quantity of nn treated juice.
Generally speaking, according to Boulcl and
Escndicr (1998). wines treated by Flaih-dftem
contain Ml-tffi more polyphenols than controls,
even aflcr the Iraditonal .'-week vatling time on
the skins.
The resulting wines have more intense color
and a hcltrr tannic stricture. They have different
aromas, but do not lose their varietal character
This xviucmaking KchnKjnc is obviously better
Mined 10 some grape varieties lhan others
These mechanical and physical extraction tech-
niques will become more widely used when the
substances extracted from different grapes under
various conditions and their properties arc better
known.so that the processes most likely to enhance
quality can be applied in each case.
A better understanding of Ihc nature and proper-
lies of the substances extracted in dlffcrcnl macer-
ation conditions will lead lo the development and
use of mechanical techniques enhancing quality
maceration phenomena. In Ibis sector, as in many
others, empiricism has preceded research. There is
currently no theoretical knowledge of these phe-
nomena thai pe nn its the explanation and prediction
of the results observed
12.5.9 The Maceration Process: <;rape
Quality and Tannin
Concentrations in Wines
(a) Grape Quality
In Section 12.1. Ihc importance of g tape quality
was emphasised. It directly influences Ihc conse-
quences of maceration The quantity and quality of
phenolic compounds are directly related to grape
variety, lerwir. maturity level, disease status, clc.
Proper maturation conditions are essential to
the ace u mi tu lion of phenolic com pounds. The
climak: plays a major role in phenolic compound
production, since this requires a considerable
amount of energy. Vine culture fcchniqics also
affect maturation Moreover, phenolic compound
accumulation is limited in young vines: therefore,
relatively old vines arc necessary for premium
wine production
Crop yields also greatly affect Ihc accumula-
tion of tannins and anthocyanins. but this fac-
tor mist be interpreted carefully. In some cases.
Ihc same climatic criteria that favor quality can
also favor quantity. In vintage-dependent, temper-
ate vineyards, the best quality years arc sometimes
also the most abundant. Reciprocally, low-yield
vinlagcsdo not necessarily produce the rest quality
grapes When considering crop yields, ptinl den-
sity should also be Liken into account must sugar
concentrations have king been known to dimin-
ish when per vine production increases. Vineyards
with a king tradition of quality choose to main-
lain their plant density at H)t«X> vines /ha in poor
soils In Ibis manner, a satisfactory production is
assured while respecting grape quality li richer
soils, lower plant densities decrease cultivation
costs (as low as 2000 vines h . and sometimes even
lower). As a result, to have the same production
per hectare, higher yields per vine are required. In
satisfacKiry climatic conditions, the grapes on these
vines ripen normally, producing a rckilivcly large
harvest, bnl li less satisfactory maturation condi-
tions, crop volume R more apt to delay maturity,
with low plant densities as opposed lo high plant
densities
The relationship between vine production and
maturation conditions (in particular, phenolic com-
pound concentrations) is complex and difficult to
interpret Practices that increase vine vigor (fcriil-
i/ing. rooWock. pmiig. etc.) arc known to delay
maturation. Phenolic compounds are Ihc first sub-
stances affected When production is excessive,
wines rapidly become diluted and lack color. Some
gtupc varieties (eg. Cabernet- Franc) are more
susceptible to flavor dilution due lo excessively
356
H-imllxx'l or Etiology: The Microbiology of Wine anil Vindications
high yields than others (eg. Cabernet Sauvigaon).
A carefully measured equilibrium between ai
acceptable selling price and optimal wine qual-
ity will deli- ran hc imp yields and consequently
the futute of premium red wines The future of
great red wines no doubt depends on maintain-
ing a batuncc between producing enough wine
*> achieve reasonable profitability and keeping
yields kiw u optimize quality. A few Bonlcaux
wincgrowcts have serried keeping their yields
extraordinarily low— about half the normal level
(20-30 hi/hectare)— w prodncc extremely con-
ccnlralcd wines that find a market at unusually high
prices.
Excessively vigorous vines and excess rain,
leading to berry swelling, also cause abundant har-
vcsls. Various techniques arc available to mitigate
the resulting delects 'lie litst of there is cluster
thinning, which consists of eliminating a portion
of the gtapc clusKis between setting and vt'r.vuw.
Cluster thinning should preferably be carried out
near r^raium. At this time, grapes manifesting
physiological rclardation can be eliminated; the
vegetation isalso less affected Thisgrecn harvest,
however, is diflkult work and its cffcclivcness is
Untiled The retained gapes swell lo compensate
for the thinning. When MM of the grapes arc
removed, crop yields generally decrease by only
15'* . Vine vigor and pruning should ptcfciably be
regnlalcd K> assure a crop yield corresponding to
quality grapes.
Eliminating a fraction of the must can also
increase tannin concentrations of dilute grape
mast. This method increases the ratio of skins
and seeds (pomace) to juice A few bonis after
tilling the feratcntor. as soon as the Juice and
solids can be separated, some of the Jnicc is
drawn off (approximately 10-20* of the tool
juke volume). Tits operation significantly affects
tin n in concentrations and color intensity The
method should be used with caution, as excessive
concentration of the must can lead lo exaggeratedly
aggressive tannins. The volume of must to be
drawn off depends on skin qualify, maturity, the
absence of vegetal character and on grape disease
status The diawn-off juice can be used to make
rose wine. It Is not advisable to throw away the
excess must or rose wine to avoid pollution. In
any case, it consulates a loss of production, which
must be compensated by producing a betlerquality
wine i ..[ii >k of fetching a higher price.
Sicgrisl and Lcglisc (1381) have obtained datt
illustrating the importance of the solid part of
the harvest In their study, a Pinot Noir must
containing (*fi juice and 4i.fi pomace Is higher in
quality than a similar mnsl containing &< '• jnicc
and 2lfi pomace.
Instead of drawing off juice, it is now possible
to eliminate water directly from the grape must
(Section 115.1). Two methods currently exist:
the fiist circulates the must across membranes
which retain water by reverse osmosis (Degrc-
monl. be.); the second evaporates water in a low-
hrmperatarc (20-24'C) forced vacuum (Kn tropic.
Inc.). These techniques have the additional advan-
tage of increasing the sugar concentration, thus
eliminating the need for chaptali/alion in some
In addition to phenolic compound concentra-
tions, the nature and properties of these substances
also play an essential role in the maceration pro-
cess and its consequences
The potential dissolution of skin pigment varies,
especially according lo maturity level. Phenolic
maturity corresponds to a maximum accumula-
tion of phenolic compounds in the berry. Cellular
maturity Is defined with respect to the level of
cell wall degradation (Volume 2. Sections 65.3:
65.4). Extraction of phenolic compounds me leases
with this degradation level (Amrani Joutci. 1993).
Augustin (1986) defined the anthocyanin extrac-
tion coefficient (Ar.) as follows:
^___ wine anthocyamns
mature grape anlbocyanins
This coefficient varies from one year to another
For example, the following values were obtained
for Mcrkil and Cabernet Sauvignon: 465 in 1983.
265 in 1984 and 39.0 in 1985 Moreover, these
values correspond fairly well with the maturity
level, expressed by the ratio: (sugar coKcntra-
tioni'itotil acidity). The same coefficient varies
less for tannins (268 in 1983. 23.2 in 1984 and
Red Wincmaking
15"
30.7 in 1985). Il docs not correspond to juice
maturity.
The oiganolcplical quality of tannins is directly
related H> in; i lu ration conditions KnotogisK have
defined ibis quality in terns of good* tunnins
and hud* tannins. The chemical understanding of
these phenolic compounds bus made il possible li>
make a belief choice of wincmaking techniques
■hat optimize (he quality of various kinds of
grapes. A perfect stale of phenolic maturity nol
only supposes a maximum tannin concentration: il
also corresponds to soft, non-aggressive, aon- bitter
tannins.
Environmental conditions (femur and climate)
and grape variety determine this phenolic maturity,
which can be illustrated by Cabernet Sanvignon.
In cool climates, its insufficiently ripe tinnins take
on a characteristic vegetal not. The same flaw can
occur in excessively hot climales: the rapid sugar
accumulation tones harvesting before the tannins
reach their optimum maturity. A haraiouious matu-
ration of the various constituents of the grape char-
acterizes great terrms and great vintages When
conditions pcrniil. grapes should never be har-
vested before complete phenolic maturity Harvest
dates based on sugariacid ratios shoikl be delayed,
when necessary, so that tannins may soften. Ti>
ensure this maturation, several more days are
sometimes needed before harvesting. During this
period, grapes should be protected against Botry-
lis attacks in certain situations, in other situations,
excessively high sugar concentrations should be
avoKlcd by ckisc monitoring
lb> Wine Tannin Concentration
By taking into account the previously mentioned
notions, general red wincmaking principals can be
improved for Ine bcller control of maceration time
and intensity.
If grapes have low anihocyanin and lannin con-
cc Dilutions, only light red wines should be made
These wines, however, should be fresh and fraity.
A limited concentration of grape phenolic com-
pounds nevertheless merits an explanation. Il can
be a varietal characteristic . which must be taken
into account Vine cultivation conditions, favoring
crap yields over qnaliiy. can also be responsible.
Adapted wincmaking techniques arc necessary in
these cases. Techniques for compensating a pheno-
lic deficiency arc palliative and are not a subsume
for perfect grape maturity
Grapes rich in phenolic compounds arc capable
of making premium wines. Tannins play at least as
important a role in wine aging potential as alcohol
or acidity. Their rote is at least as important as that
of alcohol and acidity. However, tannin quality
also contributes to aging potential. For example,
common varieties, incomplete maturity and poor
sanitary conditions contribute aggressive phenolic
elements Their addition in wine shoikl be limited,
if not totally avoided Vilicultural traditions have
led iodic establishment of the longest vailing times
in Ihc best temars Reciprocally, ruse wines should
be made from grape crops whose quality docs nol
improve with maceration. Intermediate techniques
can .ils ■ be used.
In the 1970s, the great Bordeaux wines were
considered k> have insufficient tannic structure
The young wines did not taste well and there
was concern that they would not age as gracefully
as older vintages There were certainly significant
changes in vineyard management practices dur-
ing this period, leading to higher yickls and less
concentrated mist. However, at the same time,
progress in wincmaking improved management
of the fermentation process. The residing clean,
frniiy wines no longer needed many years' aging
for certain delects to be attenuated. Nowadays,
thanks to rcccnl developments in vlieyard man-
agement and wincmaking techniques. Bordeaux
wines have good structure and arc already enjoy-
able immediately after vindication.
The extraction of phenolic compounds should
also be modulated according to Ihc anticipated
aging potential of wine. Some experts believe that
rcccnl premium red Bordeaux wines lack tannic
aggressiveness, they arc thought to be mo easy to
drink when young and not capable of long aging
According to snch expects, the tannic aggres-
siveness of past vintages has contributed to their
present quality and extended aging potential, but
this line of reasoning is highly debatable. First of
all. in past vintages (fot example, from the begin-
ning of this century), the fermentations were less
Handbook of Etiology: The Microbiology of Wine and Vinificaiions
pure and Ihc grapes were less healthy, even though
crop yields were low and the wine concentrated As
a result, these wines were agg tcssive wbci young.
The harshness of Ihe tannins was reinforced by ihc
elevated acidily (less ilpe grapes and no matofciciic
fermentation). Many years were repaired to soften
the tannins, la certain limited cases, great vintage
wines sesallcd. Today, wines arc mote pleasant
*> drink at the end of fermentation because of
improved wincmaking and viticultural Kchniqacs.
It Is possible to Judge these wines and evalu-
ate their quality when they are Mill young The
commercial value of these wines is often estab-
lished withinafcwycaisof their production, when
offcrcd to the market A disagreeable- tasting young
wine would be difficult to sell in today's market
by simply arguing that it should improve with con-
side table aging
Despite the agreeable taste of prcscnl-day pre-
mium wines immediately following fermentation,
they are still capable of long-term aging Addition-
ally. Ihc number of well-made wines ismnch higher
than in Ihc past. Yet aoiall wines lend themselves to
long- term aging. Terwir and viniagcalso participate
in a wine s aging potential. Vine cultivation condi-
tions leading tohighcropyicldsalso limit the poipcr
development of grape constituents Truly great vin-
tigc wines, however, arc fruity and enjoyable when
young, allhongb they have sufficiently high levels
of good-quality tannins to age well for a remark-
ably long time Although as plcasan t as lighter new
wines, these wines ate capable of long-term aging.
They arc made from the grapes thai best support
extended maceration, resulting in a harmonious tan-
nic struct! re
Thus, in Boolean in Ihc 1990s, winegrowers
have reverted to more qaality-oricnted vineyard
management practices. In particular, yields have
been reduced to produce wines that arc both mote
complex and more intense, as well as fruity and
well-balanced. In certain cases, wincmakcrs aim
for extreme concentration, by keeping vine yields
very low (20-30 hl/hcctarc) and emphasizing on
extraction (bleeding off. pumping over, and long
vailing times). The resulting rich flavors arc
reinforced by marked oakincss. Of coaisc. these
wines must be sold for sufficiently high prices to
jastify these expensive fcchniqacs. A ■amber of
ihcsc wines have been commercially successful,
indicating that their quality has been recognized.
These wines arc appreciated for their deep color,
their rich aromas, featuring oak as an essential
clement, and their powerful structure and complex
flavors They stand onl from other wines in blind
tastings and arc real competition wines". As
accompaniments to a meal, however, they are less
enjoyable due *> their aggressiveness, which may
dominate to the point of being barely acceptable
It is easy to understand the variable appreciation
of these wines.
Another consequence of this type of production
is a standard isi lion of quality that is mote due
to vvincmaking Icchniqacs than natural factors. In
general, these wines are made with noble grape
varieties from well-known winegrowing areas.
However, successful wines have been produced by
these methods from lerwirs that had never been
recognised as top quality, as well as from others
that certainly had been recognised. Einally. there
is no information available as yet on their aging
potential ll is understandable thai there should be
some doubt concerning the long-term future of this
type of production and n.-- attendant prestige
In conclusion, only Ihc best grape varieties
grown on the best lemurs produce wines that
combine the high tinnin content indicative of aging
potential with aromatic finesse and complexity.
On tasting, these wines arc not only superbly
concentrated, bal also well-balanced and elegant.
Wincmakcrs today are aware that excessive tannin
extraction tends to mask a wine s Iran and that
perfect balance is Ihc sign of a well-made wine.
12.6 RUNNING OFF AND PRESSING
116.1 Choosing Ihc Moment
for Running Off
Choosing Ihc optimal vailing lime is a compli-
cated decision with many possible solutions. II
depends on the type of wine desired, the char-
acteristics preferred I tannin intensity and harmo-
nious structure arc not always compatible) and the
nature of the grape. This dccgiion also depends on
Red Wincmaking
359
wincmaking conditions For example, only closed
fcmicniors pcraiil extended vailing limes. Ii open
fcmicniors. ihc must, in foil contact wiih uir. fer-
ment easily, bul (he risks of bacterial spoilage and
alcohol loss m:ikc short vailing limes necessary
tSccuon 12.3.1).
In I he 1950s in France, vailing limes tended
Id be .shortened from ihc traditional 3 or even
4 weeks. The goal of this approach was w pro-
duce more snpplc and less tannic wines, bul Ihc
major reason was ihc preoccupation wilh avoiding
bacterial spoilage, l-crrc (1458) was a principal
advocate of short Killing: times for quality wines:
Vailing limes can be reduced to 5 or 6 days
without affecting wine quality: v ailing times longer
than X days should be avoided, if only to reduce
lie alcohol loss occurring in open fementors ' The
data in Table I2.10aic important: they indicate the
amount of alcohol loss thai can occur
More recently, new nx :hniqucs (aeration through
pumping over. Icmpcraiurc control, ck.) nave
made it possible to piolong valting in closed v.as
without risking spoilage. Wincmakcis also aim
to achieve greater conccnlralion in many types
of wine. Today, premium wines oflcn have vai-
ling bines of 2-3 weeks. Extended vailing times
arc chosen to increase tannin concentrations but.
according to analyst, the Ibiid week docs nol
significantly increase this conccnlralion The pro-
longed valting time nevertheless has a maturing
effect' on Ihc Lib bus This maturation softens
Ihc Einnins and improves Ihc gusdlivc quality of
wines The chemical transformations during this
: not known precisely, bul they
can be appreciated by lasting macerating wines
between their Slh and 2<)lh day of valting The
oxidation of tannins is a possible explanation of
these transtorm.il ions The oxygen introduced dur-
ing pumping- overs would be responsible for this
oxidation. Controlling this phenomenon would rep-
resent a considerable advance in wincmaking.
Certain vineyards macerate their wines for only
2-4 days. The wines produced arc ordinary. This
technique is often nscd in hoi climates, because
short vattmg limes climinaK the risk of significant
bacterial spoilage. Additionally, longer vatling
times land thus greater extraction) nsk increasing
gustatory flaws u the dclrimcnt of finesse. In
fact, maceration intensity should be established
in accordance wilh grape quality Maceration
is shortened for ordinary varieties and in poor
quality ternary, improved grape varieties in qualify
v itKiiliiir.il regions allows extended maceration.
Adjusting the vailing lime is a simple method
for modifying Ihc maceration and it is theicfote
one of Ihc most variable characters of red wine-
making from one region to another. Ik duration
should be chosen by the wincmakcr according
Id grape quality and cannot be generalised: it
varies fn 'in one vineyard lo another, one year to
another and even one fcnucnlor lo another, since
grape quality is never homogeneous. This qualify
depends on the maturity level of the grapes (result-
ing lioin vine exposition and age) and their dis-
ease slate Wincmaking equipment should never be
the determining factor for deciding vailing times,
but unfortunately loo many wineries do nol have
sufficient lank capacity. Wincmakcis are therefore
Tabic 12 III.
Alcohol to ac
...nlin,- u. ■..illmi.M.n
ic in an open unl (1'enc. 1958)
<<U)*>
Dciuiy at
ai tfcvjlling
..k..7..h. .K..||... l,*v
Mienphof *ioe <fi vol.)
(tt vol.)
_;,.,,
Handbook of Fnokigy: The Microbiology of Wine anil Vindications
sometimes forced to tun off wine prematurely In
order to free up kink space In such cases. vailing
limes can be too short
Three types of vault): techniques are summa-
rised below.
I Running off before the end of fermenta-
tion — Ihe wine still contains sugar, and the
must density Is between 1 .020 and I 010. This
short Mining time of 3 -4 days is generally rec-
ommended for avcragc-qtality wines coming
fiom hot climates. This method is adopted for
producing supple, light, fruity wines for early
drinking, but it can also be used to attenuate
excessive tannin aggressiveness due to variety
or lermir.
2. Running off immediately alter fermentation,
as soon as the wine no longer contains
sugar — approximately Ike Nth day of macer-
ation. In these conditions, a maximum color
intensity with a moderate tannin concentration
Is expected (Section 125J. Figure 125). The
gusEilory equilibrium of new wines is opti-
mized. Their aromas and fruilincss are not
masked by an excessive polyphenol concentra-
tion. This vailing method is recommended for
premium wines which arc to be rapidly com-
mercialized. The resulting wines are not harsh
or astringent and can be drunk relatively yonng.
When the grape crop is exceptionally ripe and
thus very concentrated, premium wines may
also be made in ihrs manner, finally, open fcr-
mcnlors must be run off immediately following
the end of fermentation
3 Running off several days after alcoholic fer-
mentation. Vaiiing times may exceed 2-3
weeks. This method is often used to prodncc
premium wines The tannins assuring the evo-
lution of the wine arc supplied during this
extended maceration (Figure 12.9) Alter sev-
eral years, free anthocyanins have all but dis-
appeared. Wine color is essentially due Id com-
binations between anthocyanins and tannins.
When making premium wines, successful wine-
making requires a compromise On the one
hand, the tannin concentration must be suffi-
cient to ensure long-term aging. On the other.
the wine should remain fairly soft and fruity.
These criteria arc important, since wines are
often judged young
In fact, vailing times do not follow precise rules.
They depend on the kind of wine desired and on
grape quality.
12.6.2 Premature Kernicntor Di
due to External Factors
Sometimes fcrmcuurs must be drawn off before
the ideal tannin concentration has been attained.
This operation is recommended for stuck fermen-
tations (Section 38 ll. For reasons already men-
tioned (Section 38.3.1. these is the risk of devel-
opment of lactic acid bacteria in sugar-containing
musts with inactive yeasts The volatile acidity
would consequently increase dramatically. Draw-
ing off the juKc is a means of eliminating the
majority of the bacterial population located in the
pomace Sulliting can be effected at the same lime
(3 g'hh This operation may. of course, delay mal-
olactK fermentation, but the sulfur dioxide concen-
tration should be calculated to allow the alcoholic
fermentation to restart while blocking bacterial
activity.
Various vine diseases alter grape ciops. As a
irsalt. disagreeable tastes often appear in wine.
Early draining may help to lessen the severity
of these alterations. Gray rot tBiHnlis cinema)
is a typical example. Certain vincyaids arc sus-
ccptiMc to gray rot. since the maturation period
coincides with the rainy season. Fortunately, the
pesticides currently available have greatly reduced
the frequency of this disease. Botrylts has multiple
elicits on grape constitution and wine character
(Chapter 10). Their Impact influences maceration
decisions
First of all. the various forms of rot impart
mushroom- like, iodine-like and moldy odors to
wine A short vatting lime avoids their concen-
tration.
Moreover. Biirylis secretes taccasc. This
ui ■■.mi has a very high oxidative activity
tSectin 1 1.62). and it can rapidly alter a red wine
exposed even briefly to air In this case, lactase
TUhlc 12.11. !*ci
c dmojtinn (ciprcucd I
■l'.Ii.-,' in '.til In . Hi- (fm-iua
Carropondlot)
IniUI bocuc
U«*icaH
airily alter
SO, added
Img/1)
fore SO,
(■g/1)
ih)»
15 day*
Wine no. 1
0.16
11.10
0.16
50
10
0.16
0.02
D DO
Mil
28
0.16
II. (in
DJH
Woe DO. 2
0.13
0.13
0.12
50
is
0.13
0.10
om
100
34
0.13
0.03
oai
Wine no. 3
0.16
0.10
0.16
50
28
0.16
0.11
DJM
100
56
0.16
. N
0J)3
analysts or. niorc sin ply . an oxidative cassc tcsl
Is advised tcforc running-off. This test consists
of filling a wine-glass halfway anil leaving il
in contact will air for 12 hours I he wine Is
cousMlcred ii) risk a cassc if: Ii) il changes color:
(ii) it is turbid: (ill) iheic is sediment in the glass:
(iv) its till b lag brillnnt; (v) Hen; is am iri descent
film on (he surface: (vl) ihc color becomes a
brow nish yellow.
If Ihc results ol' Ihcsc tests arc positive, an
extended maceration is not necessary. A prolonged
vatting tinK would In fact intensify Ihc flaws due
lo giupc rol. In i in- case, the wine should also
be salfilcd at the line of running-oil the wine.
Malolactic fcmicniallon will of conisc be mote
difficult, bnt all oxidative risks arc avoided during
diuining.
Ccruln measures should be Liken when wines
made from rotten grapes ait run off. Hirst of all.
snlfurdioxidc hasa high combination tutc in these
wines The sulfur dioxide concentrations must
therefore be relatively high (5 g/hl. or more). In
Ihc presence of SO?, enzymatic activity is insttnlly
inhlblKd. bnt the complete dcstrnclion of laccasc
activity is slow. At concentrations of 20-30 mg
of free SO. per liter, several days ait required
lo destroy Hits en nine completely Fortunately,
during this time, the sulfur dioxide protects the
wine against oxidasic cassc After the complete
destruction of laccasc. the protection is dclinillvc
and independent of the presence of free SO,
The data in Table 12 11 demonstrate ihc effec-
tiveness of SO. in destroying laccasc. Thrcc dif-
ferent wines receive 0. 50 and 100 mg of sulfur
dioxide per liter The second column indicates the
combination rale, which is particularly elevated in
wine I. This wine also has Ihc lowest residual
free SO; concentrations. The following columns
Indicate Ihc decrease in laccasc activity (expressed
In arbitrary unitsi after salfiting. In wine 3. the
en nine has noi totally disappeared within 15 days
of sn Kiting at a high concentration ( 10 /g/hl).
Figurc 12.10 indicates the role of sulfiting in
protecting against oxidasic cassc In wine. The
wine contiins laccasc and is exposed to air
Figurc 12.10a corresponds to the aeration of a uon-
snlfilcd sample. The laccasc activity diminishes
according to line but decs not disappear. In the
first phase, the red color component (OD 520) and
the yellow color component (OD 420) increase. In
the second phase, the oxidasic cassc appeals with
an increase in the yellow color component and a
decrease in the red color component. In phase 3.
Ihc oxidasic cassc causes a precipitation of colored
matter.
Figure 12.10b corresponds to the evolution of
a sample of the same wine, exposed to air.
after being sulfiled at 36 mg/1 The free SO;
Handbook of Etiology: The Microbiology of Wine anil Vnifkatioas
llm- iliuivi
Kifi 12.10. Evohafon of laccusc activity, red cubr
tOD 520). yellow cobr (OD 420) and free SO, coocen-
lralinnupo«aircomilttlDuberi>et.l974):(a>l«ili-iulfitcd
waple; lb) uaplc uilfccl ai 16 n^/L it) wmpte «il-
fcccl at 55 BgJl. I: bciii»c activity. II: OD 420 (opti-
cal .k»i : . ,n 420 na. 1 ma ihickae«l yellow.
Ill: "I) 520 (optical dcatiy at 520 to. 1 ma ihlck-
•CHl.red- IV- free SO,
disappear* after 24 hours, bnl Ibc laccasc activity
Is not entirely destroyed As king as the wine
contains Tree sulfur dioxide, it Is protected againsl
oxidasic cassc (the red color component and the
yellow color component increase). The oxidasK
cassc occurs afterwards, lowering the red color
component.
Figure 12 10c Indicates the evolution of the
same wine exposed loair.aficrsulfilitgal55 itg/l.
When the free SO- concentration falls to ■.■ i > alter
48 honrs. the taccasc activity has been completely
destroyed. The wine is this definitively protecKd
from oxidasK cassc. The yellow cotor component
and especially the red color component increase
with exposure » air
12.6.3 Ru
The i
njjOIT
ining-off operation consist, of recovering
the wine which spontaneously flows out of the
fcmiciloc by giavlry The wine is then placed
in a recipient where alcoholic and makitaciic
fermentations ate completed
In the traditional, quality-orientated European
vineyards, the drawn-off wine was collected In
small wooden barrels. The wooden fcmicniors
were not hermetic enough to protect vine from
contact with air. Concrete and stainless steel tanks
have been recommended since their development,
for wine storage during the completion phase of
fermentation This completion phase precedes bar-
rel aging. The tanks must of course be completely
full and perfectly .i.n - ■ i
When wines are barreled down directly, without
blending beforehand, the wine hatches may he
heterogeneous Yeasts and bacteria participate in
these differences and they govern the completion
of the fermentations. Asa result, wine composition
(residual sugar, alcohol and tannm concentrations)
may be affected. The less the grapes arc crushed
and the fewer the puntping-ovcr operations, the
greater the difference between barrels of wine.
Temporarily putting Ihc wine in vats, a Kch-
nkiuc that came Inn general use in Bordeaux In the
1960s, offers four advantages. First, it presents an
opportunity m blend Ihc wines. Second, yeast and
bacieria cells may be evenly distributed; fcmcnla-
uoasaic thus more easily completed Third, abrupt
Red Wincniaking
363
temperature drops occurring in small containers arc
avoided (they can hinder the completion of fcrmcB-
tat ion). Fourth, the dally analyse* of fermentation
kinetics, including the completion of alcoholic and
malolaclic rctnicn ration. r> easier and more rigor-
ous with a limited number ot large tanks than wilh
a great umber of small barret
New wines nevertheless evolve differently ac-
cording to the storage method used Slow final
fermentation stigcs up to several months) acccn-
liatc evolitioii differences with respect to stor-
age conditions. Wine clarification in tanks occirs
more slowly and is more difficult to obtain than
in barrels. Carbon dioxide concentrations ate also
main tamed over a longer time, negatively impact-
ing wine tisie. Tanks arc also known k< generate
(eduction odors from Ices, snch as hydrogen sulfide
or me nap ta us
Since the late 1990s, it has become increasingly
popular lo run the wine oft into banc! immedi-
ately (Section 12 7.2). as malolactic fermentation
in wood has been shown to enhance aromatic com-
plexity as well as the fiiesse of oak character
In fact, it is not known whether this undisputed
improvement Is due to the clfccl of bacteria on
molecules released by the oak. or the fact that the
new wiic is still warm when it is put into barrel.
The fact remains that, if red wines are *> be band-
aged, they shoild be run off into barrel as soon as
potable. We now have all the necessary techniques
to avoid the problems thai led to the abandonment
of barrel-aging in the past: blending, temperature
control, analytical moniuring of fermentation in
individual barrels, etc.
12.6.4 Pressing
After the wine B rnn off from the fermenur. the
drained pomace Is emptied from the tuik and
pressed. Sell-emptying and automatic dcvatling
fcrmcnlms are capable of executing this operation
.mt- mi. in,. i..;- (Section 12.3 J). Dcvatling can also
be carried on manually, but this is laborious
These automatic alternatives do not always
respect quality criteria. In fact, fermented skins
arc more sensitive lo the shredding and some-
times grinding effect of mechanical solutions than
fresh grape crops As a result, suspended solids
arc formed and press wines are lirbid. bitter and
sometimes colorless Furthermore, pressing must
be rapidly cfTccted. due to pomace sensitivity lo
oxidation phenomena Finally, in some cases, the
addition of a fraction of press wine to run-off
wine can improve overall wine quality The goal
of obtaining quality press wines is therefore com-
pletely justifiable.
To simplify dcvatling. a method was developed
consisting of energetically mixing fcrmcilor con-
tents to disperse the pomace and homogenize the
lank A pump transfers the mixture lo the press:
the jnicc and skins arc then separated in the press
cage. This method, however. Is detrimental to
wine quality: the brutal mechanical action on the
pomace induces vegetal and herbaceous tastes: fur-
thermore, these daws arc not limited to the press
wine— they arc distributed to all of the wine.
To assure wine quality, dcvatling should be car-
ried on manually and the pomace extracted care-
fully. A screw or. even belter, a conveyor- bell sys-
tem is used to transport the pomace out of the tank
Clearly, a worker must be inside the tank lo feed
the pomace transport system I the absence of car-
bon dioxide mast be verified before a worker enters
the tank). Ideally, the extracted pomace should fall
directly from the transport system into the press
The press should therefore be mobile and capable
of being placed in front of each ctnk. This, how-
ever, is not always possible. Moreover. Ibis kind
of pressing operation affects winery cleanliness
For this reason, pomace pumps arc used to transfer
drained grape skins to the press through a pipeline
The press is immobile and generally located out-
side of the Link room, favoring winery cleanliness,
but this set-up jeopardizes wine quality Since the
appearance of these pumps on the market 20 years
ago. their operation has been much improved.
Current models have less of an impact on tis-
sue integrity, especially with short pipelines, but
■he high pressure required to displace the pomace
through the pipeline, especially through its bends,
is detrimental to wine quality This system there-
fore always affects the quality of press wine. The
addition of wine lo the pomace to facilitate its
transport further diminishes overall wine quality
364
Handbook of Etiology: The Microbiology of Wive anil Vnifkatioas
When ihc press cage cannol be pfciccd in front
of ihc biik door, a conveyor bell system can be
■scd as long as Ihc lank and ihe press are no)
loo fur from each other. Another possibility is h>
till several IO&YI con misers dircclly in front of the
link These containers can Ihen be transported to
ihc fixed location press aid emptied into it.
Oxidation should be avoided during all pomace
handling (dcvalling. transport and pressing). All
material aid receiving tanks should also be
perfectly clean. Good hygiene avoids the possible
development of acetic acri baclcria. In fact, these
bacteria may already be present in Ihc pomace, if
the vailing time was long and the tormentor not
completely hermetic.
Presses currently used arc illustrated in Figure
12.11 They arc also used in while wincmaking.
But Cemented skins are pressed more easily than
fresh skins. In fact, a smaller press capacity is
needed for red wincmaking When Ihc pomace is
pressed, the solids must be broken up between each
pressure increase-decrease cycle so that morcjnicc
Fig 12.11. Difterc* lypcsuf pie** (Source: f lacquel. Pimkam. pcnoaal commuoicaikio). O) Vesical hydraulic
prcu. The ihuH (II. driven by in electric moior(2). ones ihc ■ot.ilc prcu rxMoa (3) and in.* the pomace
in the bukci 14) againu the fHed bcad<3). ihi Moving- head prcu (ion heads) (I). (I) la Ihc prcMing phase, the
hndkudi **ciogkr>>ardcach other wihihc movement of the ihicadcd axle (2). itoklc the cage (3). picuihe pomace .
(ii)Duriag head inrac<K>a.ihe chains a«dihc hoop* (.^brcak up ihe cake, while ihc cage is being n»aied<c) Btadder
preu. (i)The iijccia>n of comprcucd air) I) n responsible for pressing by inflating the bbddcn2i again*) ihc
pm cage Hi. (ii) Alter dccompreukin. ihc ml* bo ol ihe cage i.mahliioi break* up ihc cake, (d) Coirtinuou*
prcu: ( I) hop per. i ?i pcifoniicd cj Under, like ring ihc juice: I?) micm : <■!) jnti-iMJlion v. sic" pin cmiaji ihc poauce
Imm nxatiog »ahihe*crcw:(S)"cooiprc**«ii chamber. (6) rcMrkinu door; (7) ■oior.Two or lhre* leveh. of juice
select bod. ii.iiilarc poufctc along lac length of Ihc acrcw
Red Wine making
365
can be obtained. Bach of (hcsc operations has un
in pel on Ihc quality of Ihc press wine, so K should
be kepi in separate IxiKhcs
Vcrlical hydraulic presses were of ihc okl-
cst design. They produced good quality press
wine, but loading, unloading, and breaking up the
pomace between pressure cycles were awkward,
labor- in IcbsIvc operations. There has recendy been
renewed interesl )■ (his type of press, as il can eas-
ily be moved in front of tbc vat door for tilling.
Breaking up ihc pomace has been simplified, or
even eliminated altogether, by inserting efficient
drains through the pomace, which makes il possi-
ble to extract a laigc volnmc of good-quality press
wine in a single operation without applying exces-
sive pressure
Moving bead presses have Ihc significant advan-
tage of being automatic and sometimes pro-
grammable Chains and press rotilion arc used to
break up the cake aflcr decompression. This oper-
ation produces suspended solids and can lead to
olfactory defects.
Pneumatic presses comprise a horijontal press
cage and an inflatiblc membrane. Air forced into
Ihc membrane crushes the pomace against the
cage Aflcr decompression, cage rotation breaks
up Ihc cake The hick of a central shaft, as
opposed in moving head presses, increases the
press capacity of a cage of the same si/c. This
type of press produces the highest quality result*
Another option is a pneumatic press continually
fed by an axial pomace pump, but Ibis practice
decreases wine quality and is not recommended.
Part of ihc advantages of apneumalic picssaic lost
with this technique. In any case, the pomace should
be transported as short a distmcc as possible with
a minimum number of bends in Ihc pipeline
Quite a few years ago. continuous screw presses
were fairly popular, due to their case of use
and high pressing speed. Ycl. even with a laigc-
diameter screw turning slowly, these presses have
a brutal action on pomace; piess wine quality
is affected. Screw presses always produce lower
quality wines than other presses Due to pressing
variations along the length of the screw press, the
press wine receiving lank shoukl be divided for
separate collection of the huKbcs corresponding
with ihc first pressing, second picssing. etc. of
discontinuous presses. The small volume of the last
buicb is generally very low in quality: it should be
eliminated and distilled.
12.6,5 Composition and IV
of Press Wines
The wine impregnating ihc pomace constitutes
Ibc press wine, lis volume during wincmaking
depends on the level of pulpiness of the grapes. In
Ibc Boidcaux region, il represent! approximately
15'* of ibc finished wine on average. Press wine
contains an interstitial wine. This wine b. easy to
separate from Ibc skins and relatively similar to
free-run wine, when ihc I'cmicnlor has been well
homogenized by pumping-ovcr operations and the
grape correctly crushed. Il is. however. ;Uso made
np of a wine which saturates the pomace tissues
This wine is very different from free-run wine
and much more difficult to extract FoUowing this
principle, two kinds of picss wine are generally
separated Tbc first press wine (approximately
Iffi of ibc finished wine or two-thirds of tbc
press wine) is obtained through a direct pressing
When pomace handling and picssing arc correctly
effected, the firs! press Is of a good quality. The
second press wine (approximately 5'i of ihc total
quantity of wine and oac-third of ihc press wine)
is not as good in quality, as it is obtained at high
pressure after the picss-cakc has been broken up
This damages grape (issues (hat have become more
fragile during fcrmcuuiiou. releasing subscinccs
with bitter, herbaceous overtones and accentuating
Ibc characteristic astringcucy of press wines, due
lo their high tannin content.
Grape quality primarily aflccfe press wine
quality. Ordinary quality varieties and grapes
ripened in hot climates can produce press wines
contiining a high concentration of aggressive
and vegetal tannins Press filling conditions and
pressing methods also affct'l press wine quality.
)e. both Ibc number of pressings with cake break-
up and ihc maximum pressure Finally, a slow
and regular pressure increase, even between two
pressings, is beneficial lo press wine quality A
single pressing, without pomace cake break- up. is
366
Handbook of linoUigy: The Microbiology of Wi»e anil Vindications
recommended for premium wines, olio
assuring a slow and rcgutir pressure i
Ki Ihc maximum This method produces less press
wine bnl of a superior quality.
All elements, cxccpl fot alcohol, arc concen-
trated In press wine. TaHc 12 12 gives an example
of ihrs phenomenon. The alcohol content decreases
b>' 4W.>, and in some cases b>' even more The
presence of reducing agents Is most likely respon-
sible for Ihc highcrsugar concentration Unerushcd
grapes may also liher.Hc un fermented sugar dur-
ing pressing. The volatile acidity of press wine is
always higher than in tree-run wine — indicating
an increased KKicn.d risk in the pomace Toeil
wine acidity is generally also a little higher, bnl the
higher mineral concentration also increases ihc pH
of press wine. More phenolic compounds (antho-
cyanins and lannins) are present, reflected in the
extract values. Press wines also contain more mito-
gen compounds. In certain hot climates, high matu-
rity levels krad *» extremely conccnlralcd grapes:
the resulting press wines air so rich in tannins
that their listing can be too bitter and astringent to
market These xvincs should be distilled. Although
there is little analytical data on Has. press xvincs
aLso con lain polysaccharklcsandolhcrcolloids thai
add body lo Ihc overall fktvor.
Press wine quality also depends on wincmak-
ing conditions. Repealed pumping-oxcis. elevated
maceration tcmpcralures and other techniques thai
increase maceration will deplete the pomace of
qualitative phenolic compounds. The resulting
press wines tick body and color and are dominated
by astringent and xcgclal savors These inferior-
qnality wines cannot be blended wilh lire-run
wines to improve overall wine structure and qual-
ity, and in usi sometimes be distilled or elimi-
nated, representing a considerable loss in wine vol-
ume. Press wine quality most therefore be ensured
by avoiding excessive maceration and extraction
techniques.
The decision to blend press wines wilh free-run
wines is complicated. |i not only depends on both
free-run and press wine quality Nil also on the
type of wine desired In general, press wines are
nol added when making primenr-style wines for
early drinking, except when Ihc press wines are
excessively light Moreover, press wines should
nol be incorporated into wines made from ordinary
and rustic varieties. Premium wines made from
very concentrated gcipcs ate oficn very tannic,
even aficrshort veiling times. Ihc addition of press
wines docs not improve their overall quality When
possible, press xvincs that arc nol used for blending
should be distilled.
Press wines arc. howexcr. required most of
Ihc time for making premium wines in temperate
climates In this case, press xvincs generally have
higher tannin concentrations than free-run wines
and arc often excessively astringent Dnc to their
colloidal structure, adding a small pcrecntagc of
press wine makes a fuller and more hom<^cncous
finished wine. Bnl even press wines without flaws
generally havcahcavyodorwhKh masks the fruity
character of new wines Immediately following
Ihc addition of press wines, wine aroma is less
Tabic 1111 CoBfmlkxi of liec-mn
(Rfrcnrju-Cvon « <o\. 1976)
Component
Ficc nin
I'ltVN WOK
Alcoholic wicn|3b fi vol.)
120
lib
Rcduci*pui^iB(g/l)
\9
26
Euewt fg/l)
21.2
24J
T«Ul acid*) Ivvl 11. SIM
3.23
337
Vobtik aciduv <g/l H,SO,)
OJS
OAS
Tola! nkiDpcn (g/l)
028
0J7
Tout phcaolK compountb
33
68
(fcntuat-MnMc index)
AMhocvanitt, lgl\t
0J3
■ ' J
Tannin* If fit
1 73
330
Red Wincmaking
n: lined and fruity, bui this rfciw tends *> disappear
wiln aging. The wine Is. however, fuller and more
balanced and harmonious— capable of long-term
aging In certain qnalily wine regions, press wines
arc generally considcicd indispensable lo wine
qnalily.
Delaying the addition of press wines so thai
tic clarification process may lake place can be
beneficial lo wine quality The press wines may
undergo fining or pcclolylic en /vine trcamcnls
(03 g/hl) at the time of draining . before nialolacilc
fermentation. Too long of a delay (for example,
until the spring following the harvest! causes
the free- ran wine to evolve As a result, it may
nol blend well with the typical savors of press
wine. The best solium is to decide whether
to add a certain proportion of press wiic soon
after the completion of nialolacilc fermentation.
The press wine pciccnctgc (5-lOSfc) must be
determined according lo the anticipated aging
potential of the wine. When the ideal blend
i'.m. ' is obtained after laboratory trials, these
proportions arc used for blending the various
hatches in the winery At Ibis sCigc. a certain
level of unnic aggressiveness should be sought.
These tannins improve Ihc barrel and bottle
aging potential of the wine The press wine may
also be piogressivcly blended during the months
following fermentation, to compensate for thinning
i which always accompanies the first sdgc of wine
maturation). With this process, the wine is al its
optimum quality during the pcrwd when il is
judged and sometimes sold.
117 MALOLACTIC
FERMENTATION
12.7.1 Hblory
Research on nalolaclk fermentation of red wines.
its role and its importance have greatly influenced
the evolution of posi-Paslcurian cnology. Various
concepts have been developed leading lo con-
tradictory wincmaking methods. Several decades
were necessary for the establishment of a general
doctrine in all viiiculliral legions
Ribtfrcan-Gayon and Pcynand < 1961 1 wrote:
For twenty or m> yean, a belter undciUiiiKlintr
of Ihc *nk>Uclic l(inui><p phenomenon. Us
D)X*u. ii mcchuunm und ii.t tattoo his pec
milled coiuidcahlc pnficu in matobaic fer-
of ohscnwiou and Mudin in concerned nine-
miiliitj! ic|i»n hive iho paakipaicd in i ba-
icrundciuandiiniofthu. pniccu. Ycl viicukual
icpbm nie »low lo apply lh» informal bn. hi
pnillic *ccm* lo ipic.id fctowly tm« one itjlitin
In inoihcrind b diKcul lo enabled. The com-
plH-nionof uincnukini mctnnib by Ihcic noon
wiinpaltoacnteiniunphntiotkmin hu cic-
aied * ««uin amount of roniincc. The need lo
modify outdated. bui generally accept cd.dociitnct
hu ibo ilaacd pmgicu. Il a MirpiUing (hu
wl.lh cMablbhcd indwideh coniiracd ihMhik
lave eacouaicfcd \o mnny obMacln.
Malokictic fermentation is bolh relatively simple
and extremely important in practice, and all sen-
sible wincmaking and ted wine storage techniques
lake its cxiMcncc and laws inlo account. It is an
important clement in premium wines, even incom-
plete maturity years. In addition, il rcgulaKs wine
quality from year to year. The less ripe the grapes
and therefore the higher Ihc malic acid concen-
tration, the more malolaciic fermentation lowers
wine acidity. The differences in acidity of nines
from the same legion arc much smaller than those
of the corresponding musts.
Another less readily accepted consequence of
malolaciic fcrmcntitwn is an improvement in bio-
logical stability caused by bacteria thai eliminate
bigblv unstable malic acid, which results in an
.ncreasc ii pH.
The cxBtcncc aid importaice of malolaciic fcr-
mcnuiKm were not easily recognized. Il occurs in
variable conditions which make proving lis exis-
tence difficult. If it takes place during or imme-
diately following alcoholic fcraicitalion. ilean be
com pie led without being noticed, bit il can also
occur several weeks or months after alcoholic fer-
mentation Since Utile carbon dioxide is released.
Ihc phenomenon is sometimes almost impercepti-
ble. The decrease in total acidity observed can also
be interpreted as a potassium bydrogeno tartrate
precipitation Additionally, the chemical analysis
Handbook of Enology: The Microbiology of Wine and Vindications
of malic acid, especially in Ihc presence of tartaric
acid, had always been difficult. The dctcrminaOon
of italic acid concentrations by paper chiwmatog-
rapky was ihc first simple and significant method
IRibcrcan-Gayon. 1953): it could be ascd ii the
winery and permitted the diminution of malic acid
to be moiitorcd. It greatly contribuKd to the cslab-
lishmcni of the notion of malolactic fermentation.
Malolaclic fermentation Is nevertheless a wine-
making tradition. II occurred incgnlady but did
exist in past red wines. The data in Table 12.13
arc significant in this respect. Il was not until the
decade from 1963 to 1972 that malolactic Fer-
mentation became systematic A better control of
microbial spoilage simultaneously permitted the
lowering of volatile acidity concentrations, essen-
tially affecting maximum values. Bordeaux was
the forerunner with this systematic control of mal-
olactic fermentation, which occurred much Later
in many viticuliura) regions throughout the world.
Although not pertaining directly m this chapter, the
figures in Table 12 13 concerning the alcohol con-
en I arc interesting: they show thai chaptali nation
has permitted the alcohol content of recent vintage
Bordeaux wines to be regulated in comparison with
past vintages, but maximum values have remained
similar over the years
The fiisl ohsc nations of malolactic fermentation
date back to the end of the 19th century i« Switwr-
land and Germany and to the beginning of the 21Hh
century in France. The data in Tabic 12 14. per-
taining to wine making in 1896. give characteristic
examples of malolaclK fermentation. Researchers
at that time were not capable of correctly inter-
preting the informations: they focused in partic-
ular on the volatile acidity increase, following
Ihc bacteria population increase observed under
the microscope. They attributed the lowering of
total acidity to potassium hydrogcnEirtratc precip-
itation— the disappearance of malic acid was not
even considcicd. This situation was thought to be
the beginning of a serious microbial contamination
that should absolutely be avoided
Past researchers also noticed that sulliling of
must resulted in higher acKlity wines This phe-
nomenon was interpreted as a greater dissolution
of ihc acids of pomace in the presence of sulfur
dioxide. The idea thai bacteria were inhibited and
that malic acid was not degraded was not even
considered.
Tuhfc 12.11 AmIyk*
(Rfccmiu-Gayoa. 1977;
of dilfcKi* vinigc* of led w
1
oca, McdiK und Grave* viacyu
d*(i
luulpe
^"ormed
in I97B:
Period Nu«btfn
IM»fcl
f Level* Akoholie
Micnmh
tr, vol.)
Total Volatile
•catty aektty
lg/1 H,SO.) (g/1 H.SOO
Tol*l
SOi
<mp/lp
Milk
ncid
St«nr
3.72
033
5.59
i."i
AM
0.71
392
0M
SJ9
I 10
4J2
0.75
3A3
0.15
5.29
1.05
tm
0.70
3.19
0.45
5.29
0.72
3.55
0.57
3.09
0J7
351
0.50
3J8
0J2
Tuhfc 12.14.
Alcohol* and*
alolactic fcnncatarl
u )• lank: re*
iks obtained la 1896 <Cavon. 1905)
Tank no.
Duration of
(day*)
Kcuiniap
ncfclhy
(«/IH r SOil
Vobtilc Numbcrnfbaciern
ackiny under (nkoiwope
<g/l H,SO.)
4JS
0.12
425
0.12
425
0.13
1-2
] 19
021
25 Ml
! 13
0.35
30-35
3.15
0.36
15 4C
425
i.i'.i
H
425
0.10
4.47
0.12
J :::
0.17
4-5
3J5
023
20-25
:a::
i..;
50-60
The general existence of the. pbcnoi
established from 1922 to 1928 il Burgundy by
the research of L. FcrrC ( 1922} and from 1936 to
1938 by the studies of J Ribtrcau-Gayon il Bor-
deaux (both authors cited in RibCrcau-Gayonrt ill .
1976). The importance of this .second fcmicitilion
was demonstrated lobe an essential step n making
premium red w incs. Yet the appaicilly simple cor-
responding notions weir difficnll to accept. For a
k>ng time in cuologica) works. malolaclic fermen-
i.i" i: was described in the chapters covering dis-
eases and spoilage. Certain cnology scbooLs con-
tested both the existence and especially the value
of this second fcrnien tilion .
The imporctnee and nlililyof malolaclic fcmicn-
tation wen: slow u be established because of the
involvement of lactic acid bacteria, considered to
be contaminating agents (Table 12.14). Their fre-
quent presence in red winemaking was thought
to correspond with the beginning of spoilage that
should be avoided at all costs. Pasteur once said:
Yeast make wine, bacteria destroy it II seemed
pretentious attnc lime lo go against the beliefs of a
great scientist. Finally, the idea that tnc same bac-
teria could be benclicial when they degrade malic
acid and detrimental when they attack other con-
stituents was difficnll to accept
Furthermore, a slightly elevated acidify win con-
sidered in the past to be a sign of quality. A low pH
effectively opposes back: rial development and thus
am limit the production of volatile acidity How-
ever, malic acKI is a highly biodegradable molecule
and iLs disappearance results in a biological stabi-
lioiliou of the wine, even though the pi I increases
When a red wine containing malic acid is bottled,
there Is always a risk that malolactic fermentation
will sun in the bottle after a few months, result-
ing in spoilage due lo gassincss and an increase in
volatile acidity
The diagram in Figure 3 9 (Chapter 31 summa-
rizes the principles of correct present-day red wine-
making Lactic acid bacteria shoukl only be active
when all of the sugar has been fermented inter-
ference between the two fermentations should be
avoided (Section 3 8.1). It compromises the com-
pletion of alcoholic fermentation and can result
in a considerable increase in votililc acidify, if
the bacteria decompose the remaining sugar When
there is no longer any sugar, the bacteria develop
mainly by degrading malic acid, the most easily
biodegradable molecule. In this case, the bacteria
have a beneficial effect. The high biodcgradability
of main acid requires its elimination. The bacteria
arc thus beneficial during this process.
As soon as the malolaclic fermentation Is com-
pleted, the same backrria can rapidly become
detrimental and certain precautions are necessary
lo avoid this unwanted evolution. The bacteria
arc apt to decompose pentoses, glycerol, tartaric
acid. ck. These transformations ca
370
Handbook of Fnology: The Microbiology of Wine anil Vnifcatkws
wIbc diseases (tulle disease, aneiiwm. bra,
etc.). which increase volatile acidity aid kiciic
acid concci (rations to a variable degree. Totil
acidity Is Ihns also increased. The second lei-
■.Hi' i in Table 12.14 gives an example of ibis
phenomenon. Between (be Kin and l.l(h day. ihc
bacteria populalioi. (oul acidity and volatile acid-
ity ik reuse considerably. These increases indicate
thai the transformation is no longer a pure malo-
bctic fc mien ration.
Fortunately, lactic acid bacteria have a prefer-
ence for malic acid— otherwise, prcscnt-day pre-
mium wines would not exist — but care mast be
tiken h> assure useful miciobial transformations
while avoiding harmful ones. Despite its acidity
and alcohol conicnl. wine is alterable, bnl luckily
not too alterable.
The errors committed in certain French winer-
ies during the lists due to wincmaking princi-
ples at Ihc time are understandable Wine must
was massively sullilcd to be absolutely sure of
avoiding bacterial contamination. On the one hand,
the wine did not benefit from the advantages of
malolaclic fermentation. On the other hand, since
the wine was not sored in sterile conditions, it
remained susceptible H> sabscqucnl coutunination.
An untimely and uncontrolled malotictic fermen-
ution could therefore occur at any moment.
In view of the gradual decrease in total acidity
observed in many vincyaids today, there may be
some doubt as to Ihc absolute need for malofctc-
tic fermentation In future, sfcrps may be taken to
prevent it in certain, specific cases. Of course, for
the moment, that is only a hypotheses as. according
lo our present understanding of these phenomena,
malolaclic fcroicntiUon Is still an indispensable
stigc in red wincmaking.
Current technology should lead to the develop-
ment of stabilization methods preventing uncon-
trolled malolaclic fermentttions. The Mrs! step is
to avoid excessive contamination, even though
absolute sterility rs diflicult (if not impossible) lo
obtain. Physical methods such as heat treatments
arc Ihc most effective methods for eliminating lac-
tic acRl bacteria. Various sterile bottling techniques
exist that make use of cither filtration or beat
treatments Among chemical methods, sultiting is
effective dnc to Ihc antibacterial effect of bound
sulfur dioxide. Lactic acid bacteria inhibitors also
exist: egg white lyso/yme (Section 95 2). fnmaric
acid and nrsin. Their use need to be authorized.
These substances are not always completely effec-
tive, nor arc they perfectly stable In any case, the
high resistance of certain strains in wine should
be Liken inw account, especially when wine pH
is high.
117.2 Wine Transform a lions by
Malolaclic Fermentation
This section provides further details on the chenv-
ical and flavor changes that occur in wine dur-
ing malolaclic fermentation (Section 6.3.3). The
mechanism reactions involved ate described in
Chapters and the overall reaction of this phe-
nomenon rs shown in Figure 12.12 This reaction
is a simple decarboxylation, explaining the lifts of
an acid function. In practice, at the pH of wine,
malic acid is partially neutralized in the form of
dissociated salts, thus in an ionic form, but the
overall phenomenon described remains the same.
Bach lime that a molecule of malic acid, in the
free acid or ionic form, is degraded, a free acid
function is climinatd. Only a limited amount of
ik* io.li ■ a
Fig 1111 Mikibciie Icrmeatxie
Red Wine making
371
carbon dioxide is released, but il is perceptible if
Ike cellar Is quiet. Il can. )■ fact be Ihc first sign
of the initiation of malolaclic fcmiciialion.
The decrease in acidity following maloktctic fer-
mentation varies according h> the malic acid con-
centration and Ihus grape maturity This decrease
ii acidity can be from 2 g/l in II. SO, to some-
limes 3 g/l 13-43 v'l in tutaric acid) Total acidily
decrease from 45 -6.5 g/l in HiSO, (6.75-9.75 g/l
in tartaric acid) to 3-4 g/l H2SO4 (4.5 -6 g/l in tar-
taric acid). Tic fermentation of 1 g of malK acid
per liler lowers Ihc lofal acidily by approximately
04 g/l in II. SO, (06 g/l in tartiric acid).
The preceding rcaclion docs nol explain acelK
acid production, but volatile acidity always increa-
ses during malolaclic fermentation. Thet production
is dnc. al leasl in pan. Id citric acRI degradation
i Sec ii -ii 4.3.3). Although a molecule of citric acid
produces two acclk acid molecules. Ibis degra-
dation is always limited because grapes do nol
.■ hi -mi large quantities of this acid.
Bacteria also produce volatile acriity from tnc
degradation of pentoses In fact, these sugars might
be used as energy sources Malic acid degradation
does nol seem sufficient *» ensure cell energy needs
(Hcnick-Kling. 1992).
Observations show that volatile acidify increases
at Ihc end of this phenomenon, when malic acid is
almost entirely depleted Moreover, this increase
is even greater when malolactic fermentation is
facilitated (low acidity musts, for example)
Table 12.15 shows Ihc main chemical transfor-
mations in wine during malolaclic fermentation. In
lhiscasc.it is incomplete, as the wine slill contains
05 g/l malic acid thai has not been degraded.
The results imEq/ll assess Ihc consequences
The tactic acid formed corresponds to half of the
malic acid transformed. The diminution in fixed
acidily corresponds approximately to the difference
between the loss in malic acid and the gain in laclK
acid.
The chemical transformations of wine by malo-
lactic fermentation arc much more complex in real-
ity Malolaclic fermentation also produces ethyl
lactate, the formation of which contributes to
the sensation of body in wine (Hcnick-Kling.
1992) Additionally, olhcr secondary products
haw been identified, the most important being
diacetyl. produced by bacteria la few milligrams
per liter), that belongs lo a complex pool of pro-
duction and degradation mechanisms At moder-
ate concentrations, this secondary product con-
tributes lo aromatic complexity, but above 4 mg/l
the characteristic butler aroma of this subscmcc
dominaKs.
Another transformation attributed to lactic acid
bacteria is the decarboxylation of histidinc into
histamine, a toxic subscutcc. This reaction does not
occur often and is earned onl by certain bacterial
strains in specific conditions ll is responsible
for elevated nistuiiinc concentrations 1 10 mg/l or
higher) sometimes found in certain wines.
Wine color modification always accompanies
malolactic fermentation. Color intensity decreases
and the brilliant red tinl diminishes. This modifi-
cation is dnc *' the decolor) /atkin of anlhocyanins
when Ihc pH increases, but condensation reactions
between anlhocyanins and tannins arc probably
also involved These reactions modify and stabilize
wine color.
Tabic 1
(Rtocicj
2.1S AMh» al i >
M-GuyaaM ,.i . 1970)
■dc before
i*l iter i
mi Mad K-
fermentation
U«U
„„„.,„,
oiu. (g/lC
Cant
-»»'"■
(■Eayi)
Before
Alter
Before
A Her
DilJe rencc
Total an id Iv
Volatile acidly
r'iicd acidily
-23
-40
• 1"
372
Handbook of linology: The Microbiology of Wiae anil Vinificaimns
The organoleptic character or Uk wine is also
greatly in proved. Pimi. wine aromas arc moic
complex. Wine bonqucl is incnsilKd aid Ibc char-
acter and firmness of the wine arc improved, as
kiag as tic tactic notes an; not excessive. Malo-
tactic fermentation conditions I hac trial straits and
cnviroamcntiland physical factots) certainly influ-
ence rcsulls. and this fact Is illustrated by effecting
malolaclK fermentations on while wines, which
ate. of course, simpler anil thns moic sensitive to
changes brought a boat by malolaclK fcniicnlation.
These transformations merit further study Harmful
aromatic flaws may ocenr. especially with difficult
malolaclK fermentations and towanl the end of this
The OsK of the wine B also considerably
improved. The role of dcacidifkulion becomes
more important when Ibc initial malic acid concen-
tration of wine increases. The sofkrning of wine is
die fiist of all to a decrease in acidity The sub-
stitution of the malic ion by Ibc lactic ion also
contribiKs In fact, malic acid corresponds to the
aggrcssivc. giccn acid of nnripe apples Lactic acid
R the acid found in milk: if has a much less
aggrcssivc tislc. Additionally. Ibc association of
the flavor of malic acid wilb the astringcacy of fan-
lias is not hanuoaioas This phenomenon permits
red wines to lose their acid and bard character.
They become soflcr. filler and fallcr — csschieU
elements foraqaality witc.
Attempts have been made to determine the influ-
ence of bacterial straias and operating conditioas
oa oiganolcptKal changes in red wines broagbt
about by malolactic fermentation At prcscat. ao
definitive rcsilis have been foaad.
Accotding to certain rcccnt theories, rcd wine
qaalily is evea morc greatly improved when
aialolactic fcrmcnciuua takes place ii IxirrcLs
f Sccrion 12.6 J i Wine aroma is moic complex aad
fine and the oak character moic inKgralcd. taaaias
arc filler and morc velvet)' These diffcrcnecs
arc alicady present at the end of malofciclic
fennenutioa. but they arc often less flagrant at the
time of bottling. When this technique is applied
correctly, if has no detrimental effects, but rcqniics
a lot of extra work in the cellar, especially la
handling and inspecting laigc numbers of bands
liually. concerning the transformations in wine,
the degradation of the malic acid molecule leads
In a biological stabilization even though the pH
iac rcascs (Sec tioa 12.7.1).
12.7.3 Monitoring Malolactic
Fermentation
It is esscBiEil to determine Ibc initiation of mal-
olaclic fcrmcatalion aad in monitor the diminu-
tioi aad complete depletion of malic acid ia each
tank. For a long time. Ibc malic acid concca-
tration was difficult to determine chemically — it
coukl only be extrapolated through compiling
MHal acidity beforc and after malolaclic fcrmca-
talion. In Ibis case, simultaneous poussiim nydro-
gc ii tin rale prccipitatioB could also lower fcwal
acidity, falsifying the estimate of malic acid
concentrations, la due course, paper chromatogra-
phy appealed ( R iMrcaa-Gayon. 1953). This ana-
lytical tool, which permitted a simple, vtsaal
method for monitoring the dimiaatioB of malic
acid (lugurc 12.1.1). rcprcscnlcd a considerable
advance and grcally contributed to the general use
I'ifi 11.1.'. Scpimioii d( ixpjan ink (mini is nine
by papcrchmnutogaphy <Rfecreau-G»yon. I9S3). I ctl:
wine ttai tun m* undcipi>nc ■.ikiU.li> fcracnniion
Ri^hi:ihc %amc ulnc uScinulo tut kfc imcm ition. Ta jI
acidity * exposed in g/l H,SO.
Red Wincmaking
.".;
of malolactic fermentation in wineries. Altboagb
nol a vciy precise method for analysis or malK
acid, il also permits the gustatory characteristics
of a wine lo be compared according to ihc stage
of malolactic fcrmcnttlioa.
Paper chromatography is easy lo use and is
always widely employed in wineries lo monitor
■his second fermentation. though Hit method Is
sometimes slightly varied. The Bordeaux region,
for example, produces 4-6 million hi red wine per
year, representing some 40.000-60.000 vats thai
have to be teslcd at least twice, requiring a con-
siderable number of analyses. Today, there is also
a more accurate enzymatic method for assaying
malic acid The reagent* arc expensive but the
analysis may be automated and is especially suit-
able for checking the completion of malolactic
fermentation They ait also well adapted to ver-
ifying the completion of malolactic fermentation,
but the rcactives arc expensive
Mabfac tic fermentation is normally moniloicd
after the wine has been nn off— in other wonls.
after the completion of alcoholic fermentation Yet
malolactic fermentation may begin prematurely,
when misstate insufficiently silfilcd or inoculated
with bacteria before alcoholic fermentation. In this
case, the two fermentations may be slow but com-
plete The overlapping of Ihc two fermentations
can lead to a stuck alcoholic fermentation and in
this situation the bacteria arc also apt to produce
volatile acidity from sugars (Sections 3.8.1 and
38 21 This must be closely controlled, to avoid
a scrions accident, and the monitoring of both the
malic acid concentration and volatile acidity is rec-
ommended. When the alcoholic fermentation is
nol complete, the sugar concentration shonld also
be closely followed. If bacterial spoilage begins
to occur, the wine should be immediately sullied
(3 g/hl).
Experience has shown that, even in sugar-
containing media, lactic acid bacteria during their
growth phase do not produce acetic acid and
decompose only malic acid (Section 38.31. The
complete depletion of malic acid, however, greatly
increases the risk of serious alterations when the
wine still contains sugar. For example, malolactic
fermentation sometimes occurs before Ihc wine has
been runoff, during the post- fcraicntation phase. In
this case, the wine may still contain some itsidnal
sugars, especially in the case of slightly crushed
grapes
A ft r skin and jnicc separation and pressing, the
tanks arc completely filled with wine The malo-
laclK fermentation process is then monitored daily,
using paper chromatography. This second fermen-
tation generally lakes a few days to a few weeks
(maximum). An excessive delay of its initiation is
generally due to wincmaking errors: too much sal-
filiug or Wo low a pH or wine temperature. In the
past, certain vineyards and sometiniescntinr viticul-
uii.il legions claimed that malolactic fermentation
was impossible. A wincmaking error, most often
excessive saltiting. was generally responsible.
If malolactic fermentation does not commence.
Ihc fear of an increase in volatile acidity decs not
justify prematurely su lilting the wine In fact, in
Ihc absence of sugar, lactic acid bacteria begin
to develop by degrading malic acid into lactic
acid. Yet an excessive delay of the initiation of
the fermentation docs require Other precautions to
be taken. To avoid excessive oxidation, the wine
should be sultilcd. Although the wine sediment
contains bacteria, il may be necessary to remove
il to prevent reduction odors. Maloktciic fermen-
tations have then been observed (especially in the
post) to occur normally in the spring or summer
following Ihc harvest.
The diminution of malic acid is also monitored
in order lo choose the ideal moment for definitively
stabilizing Ihc wine. Sulfiling at 3-8 g/hl (press
wine) permits this stabilization. Before sulfiling.
the wine is racked to eliminate a fraction of ihc
bacteria with the coarse sediment. This operation
should folkiw the complete depletion of malic
acid. After its initiation, malic acid degradation is
normally completed in several days. In rare cases,
bacteriophages destroy baclcria(Scction 65): they
stop malotactic fcrmenttlion and several hundtcd
milligrams of malic acid per liter may remain
nndcgiadcd In this situation, the wine must be
Rrinocutilcd to restart the fermentation.
The residual malic acid content should be under
100 mg/1 when Ihc wine is stabilized by sulltt-
ing and it would be dangerous lo bottle a wine
374
Handbook of linokigy: The Microbiology of VVInc and Vindications
annulling over 200 mg.'l At the very minimum.
In tit acid bacteria should be allowed Id continue
■■III concentrations have fallen below 200 mg/l.
*> prevent future bacterial alterations. This stabi-
:■.,'" '" should not be excessively delayed: bacte-
rial spoilage, such as increased volatile acidity, is
most likely to occur li the final stages of malotac-
lic fermentation. The possibility of straits highly
rcsnGinl to silfur dioxide, especially at high pHs.
should also be considered, bit they air ml a sig-
■i In ant problem if the wine no longer coutilns
sigar. malic ackl and citric acid and if its con-
servation lempcraliRT Is relatively low. The evolu-
tion of these wines shoikl nevertheless be closely
moa lured
12.7.4 Conditions Required for
Ma lo lactic Fermentation:
Influence of Acidity,
Temperature, A era t km
and Sulfiting
Bacterial development conditions are described ii
Chapter 6. Diring the first hours after the fermen-
wr is filled, the bacteria originating on the grape
develop rapidly As soon as alcoholic fermenta-
tion Is initiated and ethaiol ts formed, the bacte-
ria pop u la i ion greatly decreases and environ men-
til conditions become increasingly hostile — oily
the most resistant slratis arc capable of surviv-
ing Diring separation and preying, the contact
of the wine with contaminated cquipmcit may
increase the population of these resistant strains.
These resistant strains, rental! latent fora variable
period, then start italolaclK fcmicitalion when
the population reaches levels of the order of 10*
cells/ml.
In good wiiemaklng practice, the latent phase
should be sufficiently long to avoid the ■■de-
sirable overlapping of the two fc mien tit tons
(Section 12.7 J). This phase shoikl also be short
enough so that the malic acid may be degraded
within a reasonable anoint of line.
Techniques may be employed u influence mal-
olactic fermentation but the ideal conditions for
this phenomenon remain ill-defined. Malobctic
fcrmc nubility of wine varies according to the
region, vineyard and year Wine also tends to
ferment better in large containers than In small
ones. These facts arc difficult to Interpret but
Lur-nvTuiiLur.il and nutritive conditions of bac-
teria appear *> play an Important role Bacte-
rial growth is limited by alcohol and acidity
in wine: in addition, bacteria arc incapable of
synthesizing certain essential substances (mito-
gen com poinds, amino acids and growth fac-
tors! Specific deficiencies may therefore make
certain uudoLuMic fermentations difficult Yet. In
practice, modifying wine composition docs not
improve malobctic Icemen lability to any signif-
icant extent, except by increasing Its pH Each
bacterial strain probably has an optimum nutritive
medium.
Optimum growth conditions for grape origin
bacteria arc described in this section Inocitallon
will be covered in Section 12.75.
Alcohol is the first limiting factor of makiLu-
tic fermentation Malic acid concentrations often
decrease fastest in tanks containing the lowest
alcohol concentrations. leucimmiiK oentn (now
known as OenociKais veni) B predominantly
responsible for makilactlc fermentation In red
wines and it cannot grow in alcohol couccnlra-
oots exceeding I4'i volume. Some tactobacilli
can resist 18-20".* volume alcohol and arc apt to
cause spoilage in fortified wines BesKlcs alcohol
production, the wine yeast strain responsible for
alcoholic fermentation affects bacterial growth and
malolactic fermentation. It yields macromotccilcs
(polysaccharides and proteins) lo the medium.
The en/ymalic systems of the bacterial cell wall
li>ili- '.;■ '•■ these substances.
The following factors participate In the control
of nudoticlic fermentation: acidity (.Section 62.1).
tempcratire (Section 6.2.4). aeration (Section
625). vailing lime and sulriting (Section 6.2.2).
(al Influence or Acid Ih
As acidity increases, a growing number of bac-
terial species is inhibited Malolactlc fermentation
becomes increasingly difficult but. simultaneously.
It is Increasingly pnre. Malic acid is predomi-
nantly degraded. The degradation of other wine
Red Wincuutking
.v5
components R slight. thus limiting the increase in
volatile acidity.
Luc lie acid bacteria growth is opiinium al a pH
between 4.2 and 45 In the pH range of wine
(3.0-4.0). malolactic fermenution speed increases
with Ihc pH. The pH limit Tor growth is 2.9
rnii even at 3.2 bacterial growth is very limited.
Malolactic fermentxbon becomes possible at a pH
of 3 J or higher.
Malolactic fcrmcnutKw is necessary with insuf-
ficiently ripe grapes bit the high malic acid con-
ccnirauons in these grapes hinder this fermenu-
uon With very ripe grapes having a low acidity.
Ihc impact on wine taste is Icsssigniticanl bat mal-
olaclK fermentation occurs easily and the risk of
bacterial spoilage is much higher.
When the pH is excessively low. wine can be
dcacklificd in faciliUfc the initiation of malolactic
fcmicitition: for example. 50 g of CaCOj per
bcclolifcTcan be added to the wine (Chapter II).
The role of this dcacKlificalion is to rectify the
pH without removing an excessive amount of
tartiric acid. This operation must take into account
the decrease in k*tl acidity broaghl aboat by
malolactic fermentation, according to the malic
acid concentration in the wine. This operation
should be effected on a fraction of the utal volume
(20-304. for example) This dcacidilicd fraction
is used to initiate the natural dcacidilicalion
reactions (malolactic fermentation followed by
potassium hydrogen tartrate precipitation).
i |i| Influence of Tempera tire
The effect of temperature is twofokl First, an ele-
vated temperature (above 30'C) during vatling can
affect bacteria. Second, buclerial giowth and the
initiation of malolactic fermenution require a cer-
tain temperature range. The impact of temperature
on hack* rial growth depends on the alcohol con-
tent of the wine ForO-4'i volume of ethanol. the
optimal growth krmperaturc is30 : C. as opposed to
18-25'C for an alcohol strength of 10-144 vo»-
umc(HcnKk-Kling. 1992) In practice, the optimal
temperature for malolactic fermentation Is between
20 and 25 C The fermentation is stowed outside
these ranges.
New wines should therefore be maintained
at a temperature of at least I8C Temperature
is Ihc simplest means of influencing malolaciK
fermentation. In the past, the wine eclfcus were
not temperature-controlled and the cold autumn
air -ias Ihc principal factor blocking malolactic
fermentations.
The fermentation of malic acid is slow at
15'C. whereas it is complete in a few days at
20 l C. When initiated at a suiGtblc temperature,
malolactic fermenution is generally completed,
even when the temperature drops to IOC. Ik
initiation in winter is unlikely if the temperatures
arc unfavorable: fermenution will most likely
occur the following spring, when the temperature
rises naturally. Sulliting should be carefully tinted
to avoid blacking this phenomenon.
The fermentation should be conducted at as
low of a temperature as possible (18-20 : C. for
example). The low temperature makes malolactic
fermentation slower but limits the risk of bacterial
spoilage— in particular. excessive volatile acidity
production due k> the transformation ofsuhsunces
other than malic acid.
tc) Influence uf Aeration
Each bacterial species has in specific needs. In
practice, these different needs are not known
Makilactic fermenution is possible for a large
variation of aeration. This factor docs not seems
to be preponderant. Wine oxygenation by contact
with air generally accelerates the initiation of
malolactic fermenution. but saturating wine with
pure oxygen dcuys or completely blocks it In
practice, a moderate aeration is often beneficial to
malolactic fermentation (Peynaud. 1981).
td) Influence of Sulfiting
Bacterid arc known to be highly sensitive Id sul-
fur dioxide (Section 8 6.3). They are much more
sensitive to it than yeasts (Section 8.7.4). Mod-
erate concentrations of sulfur dioxide assure a
pure alcoholic fermenution without bacterial con-
tamination— always dangcroes in the presence of
sugar. Both free and bound sulfur dioxide have an
effect on bacteria Ovcrsevcral months of storage.
376
Handbook of Enofcgy: The Microbiology of Wine anil Vinifkalions
wine is regularly sullilcd: this operation i
the total SO> codicil t r.n ion anil malolaclic fermen-
tation becomes dlflicult. II not impossible, even
wiifc a low coKCitRiIion of free SO;.
Sulfiling particularly affects malokictk fermen-
tation] In two circumstances: sulfiting the crushed
grapes during lank filling and sulfiling wine at skin
and jukc separation.
in norntal wincmaking. Ihe wine should nol
be sulfilcd al running oil h> avoid compromising
..'..Ji'Lin fcnucnfatii ■!! There arc two exceptions,
corresponding Id accklcntal factors: contaminated
harvests and slnck fermentations c Section 12 6.2).
In the first casc.a light silfiting (2-5 g/hl) protects
agaitsi oxidask cassc: in Ihe second case, it avoids
Lie di disease In boih cases, the situation is serious
enough u justify making malotactic fermentation
more difficult Aflcrsulfilingat25 g/hl at running
off malolaclic termed la lion has been rcporied K>
be delayed until the following summer. Sulfiting al
5 g/hl can definitely block fcniKntalion. In these
exceptional cases. Inc wine should be massively
inoculated with wine thai was nol salfitcd al
running off wilh a normal malolaclK fermentation.
New red wines should not be sullied immedi-
ately. This can pose a problem if the initiation of
malolaclic fermentation Is slow. Baclcrial spoilage
is. of course, inlikcly because lactic acid bacte-
ria initially degrade malic acid, bit oxidation can
be detrimental u> wine qialily. In general, pre-
mature sulfiling can make malolaclic fermentation
impossible.
Adding snlfur dioxide when the mist is pnl
into vat also has an impact on malolaclic fer-
mentation. The fermentation may be delayed b>
a variable extent, depending on the concentration
of sulfur dioxide iscd and the way it is mixed
ink) Ihe must (Section 8 8.1) and may. in extreme
cases, even he permanently inhibited. The concen-
tration chosen mist be sufficient to retard malo-
laclic fermentation, to avoid its interference with
alcoholic fermentation and Ihe associated risks.
(Ml not so excessive lhat Ihe malolactic fermenta-
tion cannol be completed within a reasonable time
period
The action of sulfur dioxide depends not only
on the concentration chosen but also on grape
composition. The pH and disease stale of grapes.
in particular, influence the SO? binding rale The
ambient temperature is also a factor. In Bordeaux
region conditions. 5 g/hl has little effect on
delaying malolaclic fermentation. 10 g/hl clearly
slows it and at 15 g/hl or higher it becomes
impossible. In northern-climate vineyards. 5 g/hl
can be sufficient to stop it. but in hoi regions,
malolaclic fermentation may still accural 20 g/hl.
Determining harvest sulfiling levels is difticilt
Sulfililg. however. re mains a particularly sensitive
method for modulating Ihe malolaclK fermenta-
tion process. Proper sulfiling permits a complete
alcoholic fermentation, while avoiding spoilage
phenomena, without compromising or excessively
delaying the fermentation. The use of lyso/ymc
(Sections 95.1 and 95.2) has been recommended
k> supplement Ihe effect of SO? in delaying the
development of indigenous bacteria and. thus, the
son of malolaclic fermentation.
117.5 Malolactic Fermentation
Inoculation
The optimum conditions for obtaining malolac-
lic fermentation in new wines were recommended
in Ihe last section. Mosl often, this fermentation
begins within a reasonable amount of time, tut if
is nol always initiated spontaneously Al optimum
conditions in a winery where alcoholic fermenta-
tion has already occurred, malolaclic fermentation
often occurs in al least a few of Ihe fcratcnlors.
If can therefore be propagated throughout the win-
ery by massively inoculating the other fcrntenlors.
For example, a third of the volume of a fermennr
with a completed malolaclic fermentation can be
mixed wilh Iwo- thirds of Ihe volume of a fcrmcnior
with a difficult makifciciic fermentation. Pcrmen-
kxs arc sometimes inoculated wilh fhc lees from a
nearly completed malolaclic fermentation vat The
fermentation is thus (hopefully) completed within
a few weeks instead of a few months The elim-
ination of malic acid no longer poses significant
practical constraints. Better control of winemaking
conditions, especially Icmpcralirc control and sil-
liting. have led to the progressive resolution of past
difficulties.
Red Wine making
Mixing wines *> Inoculate other fcrmcnlors.
however, may oppose ihc legitimate desire of
selecting hatches according lo grape quality.
Dcliniiivc blending is generally carried oui several
weeks after maktlactic fermentation, when tasting
pcrmlK a more exact judgment of wine qualm*
The temperature must he maintained white
waiting for spontaneous malolactic fcrnieitallon
and this can become costly Finally, indigenous
bacteria arc not iccessarily bigber in quality than
commercial straits: an inoculation with selected
strains could be preferable
As a result, research has been focused for a
long time on developing commercial, selected
bacterial strains which can he Inoculated into wine
to ferment malic acid Hie possibility ol implanting
genuine mabtactic fcrmenttlKw static is in wine
Is definitely Interesting bul It has also posed
many dlflicultics. which have been progressively
resolved, though these solutions arc aot definitive.
Such implanting is genctal practice In many
wineries in the world but is not used systematically
in all of them la general. Oenoeocaa t#ni strains
are used and this bacterial species Is best adapted
lo malolactic fermentation.
For a long time, direct inoculation of new wines
after alcoholic fermentation constantly failed. The
bacteria intnxluccd could not develop, due lo the
environ menu! conditions tpH and alcohol content)
encountered In wine. Bacterial populations were
observed to regress rapidly, resulting from cell
death The interpretation of this situation can be
summarized simply. To have a saflicicnt biomass.
commercial bacteria are initially cultivated in an
environment promoting thcirgrowib and arc there-
fore adapted lo these specific environmental condi-
tions When placed in wine, a much less favorable
environment, they must adapt It order to multiply
and initial: malotaciic fermentation. This adapla-
twn becomes increasingly difficult, the more the
composition of the two media differs. Indigenous
bacteria from the grape, however, undergo a pro-
gressive selection according lo their ability *) adapt
in changing environmental conditions. They more
easily ensure malolactic fermentation than com-
mercial strains. The difficulty It using commercial
strains led to the experimental development of ihc
techniques described below, even if they are no
longer in nse.
• inoculating must before alcoholic fermentation,
when the alcohol-free environment Is most
favorable lo bacterial growth:
• using a sufficiently large nou- proliferating bac-
terial biomass in degrade malic acid without
cellular multiplication:
• inoculating wine after alcoholic fermentation
with a commercial biomass which has under-
gone a reactivation phase Just before use:
• inoculating wine with a commercially prepared
biomass which is already adapted lo wine.
tat Inoculating Musi before Alcoholic
Fermentation
In traditional wincmaking. bacteria from the
harvest multiply In the sugar-con tuning must
before the initiation of alcoholic fermentation
(Section 63.1). From this initial population, a pro-
gressive selection, during alcoholic fermentation.
results in a reduced population, which is. however,
relatively well adapted to environmental condi-
tions. This reduced population capable of carrying
oul malolactic fermentation can be increased by
Inoculating Ihc must with Oenttcaccta aeni before
the initiation of alcoholic fermentation
To avoid Ihc risk of inhibiting yeasts by a bacte-
rial inoculation, it is advised to Inoculate simulta-
neously with yeast, and bacterid. Current commer-
cial preparations, fitcc-dncd or fro/en. contain
10" -10" viable cells An inoculation of 1 g/hl
corresponds lo 10*- 10' cells/ml Bacteria can be
directly added to Ihc must without preparation
beforehand
Proposed since the 1960s, this method seems
lo be a satisfactory solution lo the problem of
inoculation for malolactic fermentation. It has
even been used lo obtain makitactic fermentations
In harvests snllilcd at 15 g/hl. as long as the
bacterium slarlcr is added at the time of the
Initiation of alcoholic fermentation — for example,
during the first pumping-ovcr when the free sulfur
dioxide has disappeared.
Handbook of Etiology: The Microbiology of Wine anil Vindications
The rcsulLs. however, arc not always as satislac-
wry as supposed. In practice, intcc filiations can
I. After a significant population decline, bacterial
growth occurs toward the cad of alcoholic
fermentation: mabtactic fermentation initiates
simultaneously and completes rapidly. This is
ihe ideal situation
2 The populaboi decline k;«±s to their complete
dl.'vippearancc. Malolaclic fermentation kinetics
are not improved. The inoculation has no effect.
3. A difficult alcoholic fermentation, accentuated
by antagonistic phenomena between yeasts and
the high hac trial population, leads to a stuck
alcoholic fcrmcii union and premature growth
of lactic acid bacteria in a sugar-containing
medium. Volatile acidity is produced. Oemi-
cocoa twin Ls the best adapted bacterium tor
maktlactK fermentation It rs. however, a hef-
crofcrmcnGilivc coccus which forms acetic acid
from sugars The production of volatile acidity
Ls a serious accident.
It Is almost impossible to establish the ideal con-
ditions for consistently obtaining Ibc first situation
for every wine, in lerms of i is composition (alco-
hol content. pH). These conditions arc influenced
by the selection of an adapted homofementatrve
strain and the respective yeast and bacterium inoc-
ulation concentrations
Considering the serious dangers of this tech-
nique, inoculating with Oenoeoccus twni before
the initiation of alcoholic fermentation is not
advised. Even when simsltincoasly inoculating
with active ycasR. the risk of stow and some-
times stuck fermentalloKs Ls too great. The sugar-
containing medium would be lefl lo lactic acid
bacteria. This technique Ls. nevertheless, still used
regularly in some wineries. The risk of an increase
in volatile acidity has probably not been accurately
More recently, another at tempi m inoculate must
before alcoholic fermentation was made using
a Uicttibticiltiis planttmm starter tPrahl el til .
1988). making use of non- proliferating cells.
lb) Inoculating with Non-proliferating Bacteria
Having witnessed the difficulty of obtaining tac-
tic acid bacicriagrowth in wine. Lafon-Lafouitadc
(1970) studied the possibility of obtaining malic
acid degradation by using a biomass sufficiently
abundant and rich in malolaclic cn/ymc so
that the reaction can occur without cellular
multiplication.
When the evolution of an inoculated bacteria
population in wine is studied, an abrupt drop in the
number of viaHc cells Lsobscrvcd in the tirst hours.
Aftrwards. the decline Ls slower. Aflcr sc\cral
days. h.icK-nal growlh may occur, but this growth
Is too uncertain la be used as a technique tor initiat-
ing malotactic fermentation. Yet. during the decline
of the population, the malotactic cn/ymc supplied
by the bacteria induces the partial degradation of
malic acid. In Ihis case, the bacteria do not acl
as a fermentation sCirlcr bul rather as a potential
enzymatic support
Despite efforts lo cstibllsh the Kcessary con-
ch lions, a OeiwctKi'us twin btomass inoculated in
wine Ls not capable of degrading all of the malic
acid present. The complete reaction con only be
obtained by massive inoculations ( 1 -S g/l). which
arc not feasible in practice. In general, when the
population has completely disappeared, the reac-
tion stops, leaving malic acid. In addition, ifac mal-
olaclic activity of commercial preparations rapidly
diminishes during conservation, even at low tem-
peratures.
The kinetics of the reaction could possibly be
improved by Axing cells or even enzymatic prepa-
rations on solid supports. The resulting protection
with respect to the medium could increase the
average duration of the enzymatic activity Wine
would circulate in these reactors to be demar-
cated At picscnl. this research has not lead lo
practical applications
The reaction would of course be easier in
the must before alcoholic fermentation, bul this
technique is not feasible with bctcrofcrmcntilivc
OentKoecut strains. The risk of these bacteria
developing in a sngar-contaiuing medium cannot
be taken, since volatile acidity production would
be significant (sec below).
Red Wincuiaking
379
However, a iiicuriHxiltus pUmtim
could be introduced it ihc must This homofennen-
talivc strain uniquely produces lactate Iron) sui>-
b& a » : el ul. 1 1988) demonstrated that a .',..■■■.-
bacillus plinmmmi preparation could be inoculated
into ihc must al Ihc ilmc or filling Ihc fcmiciior
in degrade malic acid. Tkc preparation conuins
5x 10" viable cclls/g. A concentration of 10 g/hl
Is used, corresponding lo 5 x I0 1 cells/ml MalK
acid dcgnidalion is iiilialcd rapidly, ii Ihen cob-
tinncs slowly and is completed during alcoholic
fcrmcitilion. Tbesc baclcria are nol icslslanl tt>
cthanol As a icsalt. Ihcir activity progressively
diminishes; sagar assimilation is negligible and
no volatile acidity production is observed. This
method is simple and has no adverse otganolcp-
Uc cITccls but its use is limited. dK to the risk
of the bacteria population completely disappearing
before the end of the reaction. Furthermore, the
bacteria are sensitive to free sulfur dioxide. For
these various reasons, the general application of
this technique Is not possible for the moment.
to Inoculating with Corn mere til OeHocaeaa
otni Preparations after Keaclhatlon
Qenocvccus oeiti Is the hcst-adaplcd strain for
malolactic fermentation in wine. It is involved in
practically all sponlancous fermentutioas- Due b>
the presence of cihanol. adding this strain in wine
after alcohol K fcmcnlaliou results in a significant
decline in us population. Pari of the malic acid
may be degraded bnl Ihc cellular multiplication
necessary for assuring a complete malolactic
fermentation docs not consistently occur.
Lafon-Lafourcadc el «tf 1 1983) were the first to
show that bacteria survival could be improved dur-
ing Ihcir transfer lo wine, as long as Ihc population
is brotgbl Id a suitable physiological stale before-
hand. These authors proposed using the expression
reactivation li> designate this operation In tact,
this is not a simple piecullivation. The population
increase that accompanies this operation Is a ben-
eficial side effect, bul is nol Ihc primary objective
sought.
Many authors have used Ibis idea of reactivation
Although many different procedures have been
proposed, thai of Lafon-Lafonreadc el irf 1 198.1 \
is the most used Noa-sultilcd grape Juice is
diluted io half its original conccniraiion <8(>g/l
of sugar per lilcr): a commercial yeast autolysate
is added (5 g/ll: and Ihc pH is adjusted lo 45
with CaCOi Afler several houis. comnKicial
biomasscs inoculaicd at 1(1* cells/nil at 25'C
produce fermentation surfers rich in ntalolacltc
en Antes These Miners arc also more resistant
in wine than non- reactivated starters. Populations
increase lo 10*. in" and It) 1 cells/nil after 2 hours.
24 hours and 6 days of reactions, respectively.
The starters prepared in this manner are inoc-
ulated into wine afler alcoholic fermentation
Table 12.16 attests to the effectiveness of this
operation In all cases, wine is inoculated at
II'' cc!b.inl By the end of 12 days, ccllularmulii-
plicalioa has occurred and malolactic fermentation
is nearly complete, if the seiner has undergone a
reactivation of 24 hours or 6 days. A 2- hour reac-
tivation is insufficient. Without reactivation, the
population declines and malolactic fermentation is
still nol initialed afler 12 days.
Table 12.16. Ellcci '
Ciidcr.V. IWC3)
>i bndcui
mciivMiiin
condaionj
i on nulohcik; fcrmcnuitkin 1 1 a(tin-l jimr-
Mt-jMiirmcn
on 12th <by
Non-icMi
lini— ii i->i
anted
ulaikin
Inoeubibahy asaothatcd biimju
ducuunuf ncih'Mba
2 noun 24 boun b <Uy*
fbrulMbn tccll/ml)
Malic acid depaded (p/l)
10'
(1
1 x 10* 44 x 10' 9.4 x Itf
2.3 3.7 36
Handbook or Enotogy: The Microbiology of Wine anil Vinificattons
In practice, the reactivated starter preparation
added to wine should not exceed a concentration of
1/1000. since the yeast autolysaic is highly odor-
ous. To obtain a cellular concentration of Hi*- lo 1
in wine, its coacenlralioR nasi be between 10"
and 10* cells/ml in ihc reactivation medium wiih
a icacllvabon lime of 48-72 hours. Coin menial
sGuicr preparations contain 10 , *-10" viable cells
per nmm Tic rcaclivalion medium musl therefore
be inoculated al 10 g/l.
This method is effective, bnl it docs require
a certain knowledge of microbiological met-
hods — not always possible in wineries. This con-
straint limits its development Many wineries
prefer spontaneous malolaciic fermentation, even
thing h it requires IH>1C time.
Id) Inoculating with Commercial
Otnococeus oeni Preparations* wit
Requirlnga Kcacthation Phase
For a long time, attempts to inocuk
cial biomasscs directly into wine after alcoholic
fermentation failed. Bacteria populations had dif-
ficulty adapting to the physKochemical condiliois
of wine.
The reactivation procedure previously described
could be assumed to confer an indispensable char-
acieristic lo bacteria. II would therefore be very
difficult ill not impossible) to obtain commer-
cial preparations ready for use in wine. How-
ever, since 199.1. Chr Hansen's Laboratory Dan-
mark A.'S has marketed a stirtcr. under the name
Viiii flora Ocnos. that can be inoculated directly
iuuwinc immediately after alcoholic fcraicnlation.
Experimental results obtained with this preparation
in Ihc laboratory and in the winery have shown
that bacterial growth and malolactic fermentation
can be obtained 15 days in advance, with respect
to a control (Figure 12.14). No organoleptic flaws
arc observed.
The effectiveness of this preparation is based
on selecting a suitable strain, in terms of ils
rcsistantc lo alcohol. pH. SOt and other various
limiting factors in wine It also depends on the
particular preparation conditions of the commctcial
biomasscs This preparation includes a progressive
vldnvvi
1'ifi 12.14. UHblDn of ukiUcik lir mental »n in
red wine (Cahcmei Sauvip*>B. Grave*. 1992) by
direct inovubiun uih a liccc-dikd f Kpamua (tftfi-
flora amos . Chi. H-nucn'r. Laboratory. Daanurk ll/S).
Tempeatiire = 20C; »H = 35: alcohol = ll* r r vol.;
ion] Si), =S mg/l: uhicnc = 0J0 t vl. fwcimc =043
uyi. Milk acid Cn/l): — #— tioocuhled mcdlim);
— ♦— (coHidI). Vnhlc cclK (ID*/ ml)- — O— (inoc-
ulated medium); (Kl'.ml) — Q— (cob roll
adaptation lo the limiting environmental conditions
of wine.
These preparations have nol yet been proven
to initiate recalcitrant malolactic fermentations.
Accelerating Ibis phenomenon by several days is
an advantage, but is not essential.
118 AUTOMATED RED
WINEMAKING METHODS
U.S.] Introduction
In red wincmaking. the complexity of Ihc oper-
ations linked K> controlling skin extraction lends
itself to the development of manufacturing pro-
cesses permitting Ihc automation of wincmaking
steps. Equipping fcrmentors to provide a certain
level of automation has already been discussed
(Section 12.33). In the 1960s. Ihc development
of two particular wincmaking techniques focused
on automation continuous wincmaking and thcr-
movinifkation (healing the harvest). The Traite
Red Wincmaking
Sciences el Techniques tin Vfn (RibCrcau-Cayon
el al.. 1976) was edited white these techniques
were being developed. II gave a dcuilcd descrip-
lioi of these methods (approximately 100 pages).
They wen- subject to ibe sane popularity thai all
imovatioDs were causing at this liuie. Some tech-
niques weir being gcncralivd that, in reality, were
besl salted to specif*: applkations. Today, tbc use
of these techniques is on the decline: Ihcy arc still
worth mentioning bnt no longer Justify u detailed
description.
t Wincniakin;
ll.X.l i ..iiim
Inilially. the development of continuous wincmak-
ing was based on the advantages of continuous
fcmicntation. This method was adopted in cer-
tain industries nsing fcmienttlion because of its
rapidity and regularity .Continuous fermentation is
generally conducted in a communicating fcrmcu-
tor baiter)' 1-resh mast enters at one cxtrcmiry and
the fermented product flows out from Ihc other.
In these conditions. Ihc multiplication of yeasts
is con milled and their population and activity arc
at their maximum The same conditions may be
reproduced by regilarty supplying a single fcr-
mentor with must at Ihc botum and extracting the
fermented product from the top al Ihc same rale as
Ihc sapply.
In continuous red wincmaking . fermentation and
maceration aic sought simultaneously, For Ihis
reason, continuous fermentation cannot provide the
full be net its of traditional wincmaking techniques.
Continuous wincmaking permits rigorous opcra-
lioi control and good work organization It Is besl
applied I" ii.'l-'.Mlunii' '.' .n.'ii].i..:-i ol the s-i-*'
qualify and style wines.
Continuous tormentors (Figure 12 15) comprise
a -100 to -KKX) hi Stainless slccl lower. A -KXX> hi
system can handle 130 metric tots of harvest per
day anil it can produce approximately 23 OCX) hi
of wine in 3 weeks. An annual wine production
of -KXKK) hi is necessary to justify the cost, of
snch a system. These fcmicntors permit Ihc daily
reception of fresh grapes and the evacuation of an
equivalent amount of partially fcraicnird wine and
skins In the upper part, a totaling rake n
1>*«H 1215. Coallauni
i2)adjuMaMc-*.|«cd ><
inkl nlve;(4) pontic
(&1 expansion dome fc
conduit: (H) |B— cc .
etii.. 1976.)
■c«CR(l)Kcdcvuuil«ii:
uki valve: (J)giape mvw
anlkm: (S) cooling »uh;
e Mo rage; (7) pjmp lag-over
lion rale. (RfccRau-Qiyo*
skins toward a continuous press A quarter of the
total volume of the lower is renewed each day. This
corresponds with a 4-day average maceration time
The seeds which accumulate al the bottom of the
tank arc regularly eliminated: seed maceration for
long periods in Ihc presence of alcohol can confer
herbaceous flavors aid excessive aslringcncy to
wine. The weight of the seeds thus eliminated
depends on lank dimensions and sometimes attains
I metric ton per day
The wincmakcr has all of Ihc equipment
required for controlling operations at his dis-
posal Before being transferred to the fcrntcntor.
Ihc harvest is automatically sulliKd by a dosing
pump. Pre- fermentation adjustment, snch as mod-
ifying acidity and c haptall onion can be performed
Handbook of linology: The Microbiology of Wk and Vinificalions
Temperature control ami pumping-ovcr operations
aic automated. The daily supply or fresh grapes
minimizes Icnipcraiirc increases: in similar con-
ditions, the temperatures in continuous fermen-
wis arc 5-7'C kwver than in ir.Hliinii.il batch
fermentors
In continuous wincmaking. cuvin>nmcutal con-
ditions arc favorable lo yeast growth The yeast
population is approximately two times greater than
in traditional wincmaking. sometimes reaching 2 x
10* cells/nil For this reason . fcraicntation is rapid.
It Is further accelerated by the introduction of
oxygen Wines flowing out of the fcrnicntor still
contain sugar but arc satnralcd with yeasts The
completion of alcoholic fcrmcutaton Is facilitated.
The principle of continuous wiacmakiig favors
the most cthanol- kklcrant yeasts: apicukilcd yeasts
arc ell in (natal The alcohol yield is consequently
slightly higher (0.1-0.2'.* volume). The glycerol
concentration simaltancously decreases by 1 g/1
oa average. Finally, the decrease in pcciorytic
enzyme activiry in an alcoholic mcdiini decreases
methanol concentrations
Maceration is regulated by the daily supply
of fresh grapes. lis conditions mist be perfectly
controlled. The maceration starts in an alco-
holic environment and at an elevated tempera-
lure — conditions that promote extraction of pheno-
lic compounds The maceration R relatively short
bit it can be increased by pumping-over opera-
tions. The concentration of phenolic compounds
in the wine Is related lo the frequency of pimping
over and addition of fresh grapes
When this method is correctly applied, the
resulting wines have no significant orgaaolcptical
differences with respect to traditionally made
Continuous fermentors present a particularly
high risk of bacterial contamination Their operat-
ing conditions lend themselves lo tactic acid bacte-
ria development, and auilolactic fermentations can
be initialed since the fcrnicnior is continuously
supplied with fresh grapes In this sugar-containing
environment, lactic disease may occur inside the
fcrmenkir To avoid this dangerous contimina-
tion. a homogeneous sultiling is recommended.
The SO; concentrations should be slightly higher
than in traditional wincmaking. Lactic acid n>
mcr analysis is particularly el lee live fur detect-
ing bacterial conttmination in continuous fermen-
tors 1 Set-lion 12.43) tPcynaud elid.. 1966). Con-
tamination by laclK acid bacKria can thus be
detected (well before the detection of bacteria
under the microscope) through monitoring acciK
ackl production and using paper chromatography
to observe the evolution of the concentration of
malic acid. Excessive microbial contamination can
require the immediate slopping and draining off
continuous fermentors.
There arc several advantages lo (his method.
The quality* of the products isal Icasl identical, if
in ■! superior, lo that from traditional wincmaking:
space, labor and material air saved: temperature
increases arc less significant: malolactlc fementa-
lion is facililaKd: and the control of the operations
is grouped together and therefore more eflicieni.
The first inconvenience of this method K the
risk of bacterial contamination. k> which the
wincnakcr should be alert These fcmiciiors
also need a continuous supply of grapes, even
during weekends, regardless of the frequency and
speed of the harvest. For this reason, continuous
and traditional wincmaking methods should be
employed simultaneously lo adapl lo varying
conditions.
The primary disadvantage is Ihc need lo mix
grapes of different origins and quality. Grapes
cannol be selected, nor can their diversity be
expressed in the wine: a single lypc is produced.
This approach is contrary lo current wiacmakiig
concepts— the diversity °' grape origins is now
emphasised. For this reason (at least in France),
after a period of development. Ibis kchniquc losl
popularity
In the first half of the 20th century, various
wincmaking methods using continuous feraventa-
tionwerc studied, in particular in the Soviet Union.
The first industrial continuous fermentors appeared
in Argentina in 1948 and were tiler developed in
Algeria and in the south of France (Midi). The
largest expansion of this method was in Ihc 1960s
and 1970s, when about 100 of these ptmis wc re-
built Today, their use is on the decline.
383
118.3 Tbcrmovinification: Heating
the Harvest
Healing whole or crushed grapes promotes the
diffusion of phenolic com pounds from (he skins.
Colored mustsmc Ibus obtained This phenomenon
has been known (or a long lime: il was referred to
even in the 18th ccatary. Attempts have k»ng been
made to Increase red wine color by heating.
Until fairly recently, heating methods remained
very empirical. Only pari of the harvest was
healed: it was Ihca blended wilh the rest of the tank
and underwent traditional wincmaking methods
The idea is not new but. during the List 30 years,
industrial healing processes have developed. They
permit large volutes of grapes k> be hcakrd
rapidly to high temperatures (65-75T). Various
techniques are used, although healing the grapes
directly with steam has been almost entirely
abandoned (Pcynund. 2001). Deslcmmcd. crushed
grapes may be heated directly in a tubular hcat-
exc hanger, heated by slcam or. preferably, hot
water, or plunged into juice that has been scparaKd
from the solids and hcakrd
The pressed juice may be cooled before fcrmca-
tilion. but if the mnsi is to be fermented on the
skins, the solid and liquid components must be
cooled together, which is a much more complex
operation, requiring special equipment.
Produce based ou this method were developed
with two distinct objectives In one application, the
method was integrated into traditional wincmaking
to increase concentrations ol phenolic compounds,
especially an Ihocyan ins i colon In the other, ilwas
used loautomalc ted wiacmaking. thus decreasing
the cost of labor
Heating the grapes to extract more color is not
currently in lavor. at least in appellinii>n<fnrifjne
coittnWe vineyards. Hist of all. fcrmcniors arc
now preferably equipped wilh tempctulnrc control
systems, which permit a more flexible use of heat
lo promote the extraction of phenolic compounds
Excessive heating of the entire crashed grape crop,
combined with a traditional maceration, might
cause excessive tannic bitterness— Ihc wine is
consequently without liacssc The increased must
color ob tuned through heating the crushed grapes
has also been shown to be unstable, disappearing
during fermentation (Tabic 12.17).
In addition, even if new thcrntovia Ideation
wines arc more colored than traditionally made
wines, they progressively lose this advantage
during maturation.
The miovini lieu Iwa liaes were developed wilh
Ihc goal of automating wincmaking The destcm-
lin l . crushed grapes are heated to between 65 und
75 "C. then transferred to a vat for up to an hour
The res u lis depend on the temperature used and
on the length of time Ihc heal is applied. They are
then cooled and pressed. The highly colored juice
is then fermented During this time, il krscs part of
Tabic 1117.
Evoluion
at anthocyani*
■■d
cokir ioiciAty
in a healed
udpi
:«Cil ami.
com rated w
■ h irad«»
ul uiacmakiag
, dui
linn aaoofcilio
fciincnuikin
lR*Krt*ii-<i.ivi>n
«<*.. 19701
Duation
of
TnKlhbMl
■.. mi
-akin?
Thcraov
inifccutkin of red
ICr-CB.ll
(kmo
)
gi
■pen
Rl
Aatawy-™
Color
Anlhocyaa
Color
Img/I)
lmcM.lt>'
tmgd)
iawnr.«y
252
037
Bid
J08
3
218
OJS
si ■
258
a
244
047
824
3JS
10
2IH1
0J8
930
3 JO
24
ZOO
051
595
123
72
ill?
n 81
508
100
90
41'.
Lid
540
120
l\«l nl fciincnlJiKin
41 »
0.7S
470
0.92
Handbook of Enology: The Microbiology of Wine anil Vlnific.nions
its color. All of ibe opcr.iilon.\ can be automated,
wbicb results in substantial savings in tabor cosr.
Moreover, this system significantly decirascs the
amount of fermenur volume needed. This alone can
Justify the installation of a IhcrniovinifKation line
Whatever the heating method used, it H recom-
mended that the must or crushed grapes should be
cooled before the initiation of Icrnicn ration, which
must take place at approximately 20'C. Excessive
productioa of volatile acidify by yeasts can hope-
fully be avoided
Tasting results are not always homogeneous and
depend on grape composition, and on healing and
maceration conditions. The participation of these
factors is poorly understood In certain cases, the
wines obtained have more color and arc better
than the traditionally made control wlacs. They
can be rounder and fuller bodied, while still hav-
ing a fruitincss giving them personality In other
cases, they have abnormal tastes, an amylk domi-
nant vegetal aroma, a loss of their freshness and a
bitter aflcnasic.
Figure 12.16 shows that the temperature should
be higher than 40'C for 15 minutes u obttin
significant color ex traction, but the extraction R nol
increased for temperatures above SOT. Identical
results are observed for ibe tannins. For this reason,
a temperature of 70 C for 10 minutes corresponds
u a standard thcrmovinilication treatment.
Healing grapes destroys the natural pcciolyiic
cn/ymes of the grape and so sponcmcous clarlti-
calion of new wines is difficult. This circumstance
intensities potential gustatory lltvvs. Adding com-
mercial pcclolylic enzymes can resolve this prob-
lem, but their effectiveness varies.
Destruction of oxidases and protection against
oxidations arc favoraMe consequences of thcr-
mov initiation. Rotten grapes benefit the most
from this treatment as they contain taccasc. which
has a significant oxidizing activity. However,
enzymes arc only destroyed al temperatures over
60'C. while their activity increases with tempera-
ture up to that point, so the must has lo be heated
very rapidly. Enzymatic activity actually increases
al temperatures below 60 C The Increase in lent-
pcratute during this process must therefore be
rapid Finally, it rs accepted that heating Cabernet
T—F-
Kip, 12.10. Amh<K>ini* curwibn i«l i-.ok.-m of
color 1 Me ia "a v uvunl i «p t o Ic m pc otitic { R be tcau-Giyti n
a U.. 1976). Cobdnteudiy (OD S3) + OD 1!D|
h .n ■ in--
Sauvignon must attenuates the green bell pepper
character produced by nicfhoxypyrazincs in insuf-
ficiently ripe grapes
Healing also affects fermentation kinetics. Vcasl
activity continues al temperatures thai ycasR gen-
erally do not support Al temperatures well above
those thai kill yeasts, heated musts ferment easily.
However, this healing destroys nearly all of the
yeasts originating on the grapes. A second natural
inoculation occurs during the subsequent handling
of juice and skins and this new population rapidly
becomes signilicanl Thus manual Inoculation the
harvest is unnecessary Heating is therefore not a
viable me thod for killing Ihc indigenous yeasl pop-
ulation, which should be eliminated when using
selected yeast strains. This activation of Ihc fcr-
mcnuiion is nol due lo a natural selection of fhcr-
moresistuil yeasts: it is most likely caused by the
dissolution, or at least dispersal, of activators in
the grape must belonging lo Ihc slctoid family.
Red Wlncmaklng
3SS
These aclivatots come 1'n'in ibc g rape skins. Flash-
paste uri/al ion. nipid healing t, > a nigh temperature,
has also been suggested as a means or restarting a
stick fermentation (Section 38J).
Nitrogen compounds may also be involved in the
improvement of fermentation kinetics Hailing the
emshed g tapes increases not only the total nitrogen
and amino compound concentrations, but also the
consumption or nitrogen during fermentation
Healing grapes v 111 -'- many complex chemical
and mkiobiolog ical phenomena into play. Yet until
these phenomena are belter understood, separat-
ing the maceration and fermentation phase has
no distinct advantage In addition, the perfor-
mance of Kmpcraturc control systems nscd in tra-
ditional batch fermentors is contlmally improving.
These systems often produce higher quality wines.
For this reason. Ihermovinlficaiion techniques no
longer present the same interest as they did not so
long ago.
12.9 CARBONIC MACERATION
12.9.1 Principles
Like all vegetal organs, the grape berry has an
aerobic metabolism. Respiration produces the see-
cssary energy to ensure its vital functions In this
complex chain of reactions, the grape makes use of
oxygen from air to decompose sugar Into water and
carbon dioxide Vet when many ptantsarr deprived
of air. they adopt an anaerobic ntelabolism and
prodncc clhanol from sugars. Sacclummyces ctre -
risiue is Ihc classic example of this phenomenon
The anaerobic metabolism is significant because
this yeast Ills a gixxl tolerance k> clhanol.
The whole, uncrushed berry also develops an
anaerobic metabolism when placed in a carbon
dioxide atmosphere. During this phenomenon,
various chemical and physKochcmical processes
occur, especially clhanol production. They ate
linked to Ihc functioning of ihc cells in the whole
berry bit. in contrast to yeasts, grape berry cells
are not vcr> tolerant of clhanol Flhanol production
is therefore limited: it varies from 1.21 K> 1.89**
volume for the Cangnan variety, regardless of
the must sagar concentration, when between 184
and 212 g/1 (Flan/)* euil.. 1987) The Intensity of
anaerobic metabolism is in accordance with ihc
variety, the vintage, and maceration temperature
and duration.
Enzyme systems in the grape cells, particularly
alcohol dehydrogenase, cause Ihc phenomena that
give carbolic maceration wines their specific
c ha racier.
Anaerobic metabolism occurs whenever the oxy-
gen conccnltalion is low. in either a gaseous
or liquid environment, but in a liquid environ-
ment Ihc Intensity of Ihc phenomenon diminishes
tFlan/y end.. 1*17). Whole grapes immersed in
mnsl undergo a less intense anaerobic metabolism
than the same grapes placed in a carbon dioxide
atmosphere This diminution is due to exchanges
between the berry and the ambient environment,
which are greater In the liquid phase than in a
gaseous atmosphere. The diffusion of sugars, phe-
nolic compounds aid malic acid across Ihc grape
skin toward the solution lowers the concentration
of anaerobic metabolism substrates in Ihc berry
In addition, the diffusion occurs in both direc-
tions When intact berries air placed in a medium
containing alcohol, their ethanol conccnltalion is
increased, thus inhibiting anaerobic metabolism
This observation demonstrates the impoitincc of
■be condition of the grape crop for carbonic mac-
eration. The higher the proportion of uncrushed
grapes, the more effective is carbonic maceration.
Pasteur Is credited with first noting the taste
modification of whole berries during fermentation
He confirmed his observations by placing grapes
in a bell jar filled with carbon dioxide. These
grapes took on a vinous odor and lastc reminiscent
of fermented grapes. He concluded that crushing
grapes has a dominant impact on red wincmaking
This is the basic principle behind fermentation
with carbonic maceration, initiated by M Flan/y
in 1935 and studied in detail by C Flan/y (199S)
Before mechanized crushing, when grapes were
still emshed by fool, many berries remained
whole. A ccrciin degree of carbonic macera-
tion occurred in Ihc fermentor. At the same
time, the juice of uncrushed grapes was pro-
gressively released by Ihc weigh! of the harvest.
Ihns fermenting more slowly. Consequently, the
Handbook of linoUigy: The Microbiology °f Wit* anil Vinificalions
(ermenmr Icmpcraiaic was moderate la warm cli-
mates, wiucmakcrs directly benefited from Ibis
phcnonKnon ■■ Ihc post
Carbonic maceration comprises two steps
• The fcrmcttor is lilkd with whole grapes aider
a blanket of carbon dioxide and kept at a moder-
ate Icmpcralnic (20-30'C) for 1-2 weeks. The
atmosphere of the fcrmcutor is then saturated
with CO? for 8- 15 days ThR is the pure car-
bonic maccraUon phase Anaerobic metabolism
tractions modify grape composition. Subscinccs
from the solid tissue disintegrated by anacrobkv
sls arc also diffused in Ihc JuKe and Ihc pulp.
• The fcrnicnlor is emptied aid the pomace is
pressed. The juice is then ran off. the pomace
pressed, and the frce-rui and press wines air
usually assembled prior n> normal alcoholic and
malolaclic fermentation.
In fact, il is impossible to fill a fcrmenur with
only whole berries. Some ate crashed and their
juice undergoes a normal alcoholic fermentation.
During maceration, addiuoual grapes continue fc>
be crushed as the processes during anaerobiosis
weaken cell tissue. The fcrmcnuiK* of completely
crashed grapes and pure carbonic maceration occur
simulcineously *> varying degrees. The condition
of Ihc grapes influences Ihc amount and intensity
of carbonic maceration. In practice, during the
first step of wincmaking. yeasl-based fermentation
always accompanies the anaerobic metabolism of
the berry. The wincmakcr shoald cikc slcps to
c ihrs interference.
12.9.2 Caocous Exchanges
During the first hours of anaerobiosis. Ihc berry
tissues to absorb carbon dtoxMlc. Metabolic path-
ways make asc of this dissolved CO>. Using CO;
marked with "C. il has been demonstrated thai
the gas Is integrated not only into various sub-
strates, malic acid and amino acids, bat also ink)
sagar and akrohol. The volume of carbon dioxide
dissolved into Ihc berry in this manner Is Kmpcra-
ture dependent It represents Hf> of berry volume
al 35"C. 3Cfl at 20'C and SCfA at I5 J C (Flan*y
The berry metabolism simaltancoasly releases
COj which eventually attains an equilibrium with
Ihc amount absorbed. In a closed system, the
equilibrium is established in 6 hours at 35°C,
24 hours at 25 ; C and 3 days at 15'C (Flan/y
et al., 1987).
The initial CO? concentration contiols Ihc inten-
sity of the anaerobic maceration phenomena,
reflected by clhanol prodaction. In certain experi-
mental conditions, for a given lime and Icmpcra-
larc. Ibis production can vary by a factor of Iwo.
depending on Ihc COj coaccnlrailoa in ihc atmo-
sphere (20- 100*).
12.9.3 An
obic Metabolisi
Il has king been known that the giapc berry is
capable of producing ctbanol. This production is
always low and depends on Ihc variety. According
lo different authors, il varies from 12 10 25'i
volume or from 0.44 to 2 20* volume The speed
and limiti of clhanol production ate governed by
Icmpcraturc (Figure 12.17). Maximum production
Is obtained earlier al higher temperatures than
mfCfjiim UI..A ■ « ,V
Red Wincmaking
ai lower temperatures, bui a highci
obtained at lower temperatures
Temperature P. consequently a major factor in
the Intensity or anaerobic metabolism. Raising the
temperature of excessively cool grape crops is
therefore recommended Healing conditions also
have an impact.
The yield of the transformation of sngar to
alcohol is difficult to determine It seems to be
similar to the alcoholic yield of yeasts — IS 5 g
of sugar per \'i percent volume of clhanol.
Various secondary products are simultaneously
formed: 1.45-2.42 g of glycerol. 21-46 mg of
ethanal. approximately 3(1) mg of succinic acid
and 40-611 mg of acetic acid per liter. The
presence of all of these compounds indicates
the existence of a mechanism similar to ycasl-
bascd alcoholic fermentation. Yet In this case,
the glyce ropy ru vie fermentation portion win id be
greater, since the aveiugc glyccroUclhauol ratio x
100 r I8~2(M instead of ft:
During anaerobic metabolism, total berry acKI-
ity diminishes. Pcynaud and Guimbcivau (1962)
demonstrated in rigorous laboratory experiments
that tirtirk and citnc acid concentrations icmaincd
constant while malK acid coaccnlralKms dropped
sharply The degree of this decrease depends
on the variety: 32* for Petit \tidot. 42* for
Cabernet Franc. 1 5'*' for Grcnachc Gris and
57'i for Grenacbc Noir. As with clhanol produc-
tion, temperature affects malK acid degradation
(Figure 12.18). It regulates the speed aid limit of
the phenomenon.
Malic acid diminution is a major effect of
carbonic maceration. Ethanol is produced after
VV. 1218. Malic icid dcpoitaiion durliqi imc
mcUholttn. »cci>Mli*g (i> icafccuuic (Ffan/v i
I9S7)
a double decarboxylation. Yeasts use an identi-
cal mechanism (Figure 12.19). Two enzymes have
been confirmed as being involved in these reac-
tions— they are even considered as markets of an
anaerobic metabolism The specific activity of the
malic es/ynic reaches its maximnm between the
Mil and 4th day of .macrobiosis at 35 C. Dnring
the sanK period, the alcohol dehydrogenase is pro-
gressively inactivated. This is probably linked to
the accumnlalion of clhanol I Flan /y el <ii . 1987).
^
Fifc II 19. Mali* ue-l ik^miUiba by the pafc hem 1
Handbook of linokigy: The Microbiology of Wine and Vinifkalions
Tracer of mil, nui nun. mi. . and shikimK acid,
but not lactic acid, arc generally considered h>
be formed by this anacrobK nictaboli.Mii. while
Ihc ascocbic acid conicni decreases Ascorbic acid
concentrations decrease There arc also significant
changes in the nitrogen compounds, including
an increase in the amino acid coaicnl. probably
dissolved fiont Ihc solids in the must, as well as a
decrease in protein nitrogen.
The aiaciobK metabolism abio causes a break-
down of the cell walls, with hydrolysis of the
pectins leading to an increase in Ihc methanol
content, which may teach levels np to 80 nig/I.
corresponding to the hydrolysis of approximately
S00 nig pectin.
Finally, within 30 mimics, the development of
an anacrobK metabolism in the grapes leads io a
significant decrease in Ihc ATP and ADP molecules
responsible for energy transfers in biological
systems. After an initial decrease of approximately
2<*< at the time of passing into anacrobtosn.
the energy charge (BC = (ATP + ±ADP)/(ATP +
ADP + AMP)) stabilizes for 8 -"10 days before
decreasing again. In anacrohiosis. the regeneration
ability of energy- rich botdslATP. ADP) is limited
(Flanzyrtnr 1 .. 1987).
An important result of carbonic maceration dur-
ing red wincmaking is the characteristic aroma
produced The nature and origin of the molecules
involved in this aroma remain unknown. Accord-
ing io Flan/y el <rf 1 1987). the formation of aspar-
tic acid from malic acid, along with succinic
and shikimic acid, may be the source of aroma
precursors. These researchers also noticed differ-
ences in higher alcohol and fermentation ester
concentrations with respect to wines that did not
undergo carbonic maceration. The principal differ-
ence is the increase in various aromatic deriva-
tive concentrations: vinyl-bcnttrnc. phenyl- 2-cthy I
acctaie. ben /aldehyde. vinyl-+gaTacol. vinyl-+
phenol. cfhyM-gasicol. c thy M- phenol, eugcnol.
methyl and ethyl vanillalc Ethyl cinnamaK. in par-
ticular, was proposed as an indicator of carbonic
maceration wines.
Pcynand and Guimbcrlcau 11962) pointed out
that the simultaneous action of the intracellular
berry and yeast cell metabolisms were responsible
for the agreeable aroma produced in fcrmcnlors.
They conducted controlled laboratory experiments
to elaborate on these findings. After 8 days of
anacrohiosis at 25 C in the toul absence of
yeasts, whole grapes release weak aromas which
arc not always agreeable Reduction aromas are
even produced in a nitrogen atmosphere The
rescarehcrs concluded:
llihcic niibkichcnk-alinatlbfiniiinaafcuca-
tial MitH-untcs in imcMthkitb.. It doc* an jppcai
la be in ihc ri^hi dircdun Th»e alucivuiHiat
<ki Mil concur wih Ihc dcvckipntcv of i^cablc
jnifllu nrtcil duiiiu wlncniitin)! Milk iJibo«u:
aaccrcunn. The aroma iapmvcmcn may be due
in piaicutiito the actbanl 'ycaah.
The carbonic maceration aroma is probably
dne to the successive action of the anaerobic
metabolism of berry, yeast and perhaps bacteria,
but the mechanisms of these transformations
remain to be determined. In 1987. Ilan/y el ill.
again took np this hypothesis.
119.4 Crape Transformations by
Carbonic Maccrafion
In a fermentor undergoing carbonic maceration.
Ihc grape berry R transformed by anacrobK
metabolism reactions of its own cells These reac-
tions arc independent of any yeast involvement
and have been covered in the preceding section.
Tcsue degradation favors Ihc maceration phenom-
ena involved Phenolic compounds, anlhocyanins.
nitrogen compounds and other component! of the
solid parts of the berry arc diffused in the juice of
the pulp
The data in Table 12 18 express Ihc conse-
quences of these phenomena. A slight increase
in nitrogen and possibly mineral concentrations
is observed. There is also a systematic increase
in total polyphenol concentrations. The dissolu-
tion of anthocyanins also results in an increase
in color intensity This increase is considerable
for certain varieties, bat in general the juice
obtained is simply pink In this case, tempera-
ture also plays an essential role (Figure 12.20).
At elevated temperatures. pncuolK compound
Tabic 12.1ft MndilkaiKino
25C )• a CO, ur •* rotten il
tempo* a ■in of Cabcinci-fniac »ml Pel* Venlot onipe Juki
mmpheie)<IVviu«»<landGuimbeneau. 1902)
In jnacIDbiinb (8 «l:i. > .1
Com pone «
Cabemci In ik
Poll Venn*
Com ml CO) N.rogen CoMrol
COi Nitrogen
Reducing *ugaiMtf/l>
:in
Hhyl alcohol <g/l)
□Ivcerol (g/l)
023
Acaaldchvde fn^lt
12
Mel hvl alcohol (mtWD
Total a «n.j)c 11 (■u/L)
532
FtrnunpiMic index*
9
Cub. iniemiv'
020
Tim*
050
pH
325
Total wkliv ImEo/ll
96
A»h *Jkalint) <-Kj,ll
52
SHj-
8J
Sum of cut fan (mEi|1|
150
Taiurfa acid 1 ml q.l 1
»2
Malkac.ltmEo/lp
50
Ciric acKl<mEq/l)
25
Phosphoric acid (aEq/1)
2.1
Acetic acid<mEq/l)
ii„
Succinfa»cklIinEq<ll
SumofunbKoOiKq/ll
147
0.72
065
026
060
3JS
3 JO
Kid ii
pound <
bolb.nl
H. laflucmc nl icmpcmuit on phenolic coat-
f&Bfa* in juke pulp during amciuhic mcla-
Fhincjr eta*., 1987)
cancel
din In Is
Acta
Cayon
rations Increase over 8-10days and ihcn
1.
iding u Bouncix ( 197 1 . cited in Ribc'rcan-
rVfl/.. 1976). only 0.7 g of phenolic
compounds per liter arc found in juice which
has undergone caibonK maceration fioni grapes
originally containing a 4 g/l potential concentra-
tion Approximately 150 mg ofanlhocyanins puss
inw Ihc juice onl of Ihc 1650 nig conciiKd in a
kilogram of fresh grapes
Tin decreases during carbonic maceration It
is expressed as the degree of yellow coloration
with respect to the degree of red coloration (opti-
cal density at 420 nm divided by optica! density
at 520 mi) This ratio diminishes during carbonic
maceration. The anthocyanins diffuse more rapidly
in the juice than the colorless phenolic compounds
In general. Lin am and anthocyanin extraction Is
more limited in carbonic maceration than in Iradi-
tionalwincmaking. This can be an advantage or an
inconvenience, depending on the type of wine.
A ronia evolution should of course be taken into
accoanl when considering grape transformations
by carbonic maceration, bnt no experimental
results currently exist.
Handbook of Enology: The Mictobi«l«¥y of Wive anil Vindications
12.9.5 Microbiology of Carbonic
Maceration
Caiboik maceration cream funicular develop-
mental conditions for yeasts and bacteria. Tlcsc
conditions ate different In die two stages or the
process. They arc also different with respect k>
traditional wincmaking.
In the firsl stage, the ycasls originate from the
grape or are added as a yeasl stirrer. They develop
In juice produced by the progressive crashing
of a portion of the grapes ii a carbon dioxide
atmosphere (containing no oxygen) and a non- or
slightly sulliicd cnvironmcnl At the lime of run-
ning off and pressing, yeasl populations attain 10*
and sometimes 2 x 10* cells/ml This significant
yeast development is explained by tbe low cthanol
concentration and the presence of bloom con-
stituents (oleic and olcanolic acid). These unsat-
urated fatty acids, like sterols, activate the fermen-
til Mm and compensate for the absence of oxygen
lo a certain degree. This large and active popu-
lation ensures a rapid fermentation of sugar dur-
ing the second stage of this process The increase
In nitrogen, assimilable by ycas&. during anaero-
bic metabolism certainly favors fermentation Yet
If elevated temperatures (35'C) arc attained dur-
ing the carbonic maceration phase, all or part of
the yeast population may be destroyed and alco-
holic fermentation may become stuck, creating an
opportunity for bacterial growth. In this case, both
volatile acidity and inhibitors such as ornithine arc
produced These Inhibitors reinforce fermentation
difficulties and Increase the risks of stuck fermen-
tations (Flan/y el al.. 1987).
Carbonic maceration consequently facilitates the
development of lactic acid bacteria and the makv
Lulie fermentation process Tie risk of bacterial
spoilage is also greater, especially during diflicull
alcoholic fermentations
Many factors promote bacterial development
• lie absence of sulfiling or at least the Irregular
sulfiting of a heterogeneous medium:
• the presence of carbon dioxide, promoting their
growtn.
• the involvement of the latent phase population
in a slightly alcoholic cnvironmcnl.
• the presence of steroids and fatly acids from the
bloom.
During the second stage, malolactic fermenta-
tion occurs in favorable conditions die to the
increased pi I and the Improved nitrogen supply.
Bacteria may develop in the presence of resid-
ual fermentable sugars, with the consequent risk
of spoilage Alcoholic and malolactic fermenta-
tion should never overlap l Section 3X2). 1-rcshly-
pKkcd grapes must be sullitcd prior lo carbonic
maceration, even if it Is diflicull to distribute the
snlfur dioxide evenly among the berries. The vari-
ous phases of the mKrobiok^ical processes should
also be rigoroasly monitored.
119.6 Using Carbonic Maceration
Different systems can be developed to make use of
carbonic maceration. iRsucccssdepcnds on anaer-
obic metabolism Intensity, which itself depends
on fruit Inkgrity. degree ol ' .macrobiosis, possible
traces of oxygen, duration and temperature.
Harvesting, transport and vatting methods must
take account of the integrity of the grapes and
grape clusters. Flan^ el id. ( 1987) have described
varioas methods. Including placing picked grapes
in small containers lo avoid crushing and using a
system » till the fermenurs gently, after possibly
weighing the grapes, to limit the bursting of grapes.
Pumps should never be used *> transfer grapes
from the receiving area to a fermentor. Conveyor
systems arc preferred since they maintain tissue
Integrity better than worm-screw pumps.
Anacrohlosis is generally effected in a hermetic
fermentor. but grapes can also be wrapped in an
airtight plastic tarpaulin and ptuccd in a wooden
crate The crates filled with grapes at the vineyard
arc transported to the winery and stored.
The grapes are not dcslcmmcd before carbonic
maceration. Berry integrity may sometimes be
compromised by necessary mechanical operations
but with certain varieties. In ccrttln regions,
the presence of stems may introduce herbaceous
notes and a degree of bitterness during carbonic
Red Wiacmalang
391
maceration. Stem elimination should therefore be
considered ii -UK cases. Dcslcmmcrs without
rollers that do not crush ibe grapes should
be used Laboratory experiment have shown
thai the metabolism Is less inlensc for berries
thai have been detached from ihcir peduncle
than lor whole grape claslcis. Similarly, carrcnt
mechanical harvesting meihods do not pcrniil
carboiK maceration lo be cITcclcd in satisfactory
conditions. In the future, new equipment combined
with adapted vine growing methods will perhaps
permit a mechanical harvest be tier suited to the
needs of carbonic maceration.
Whatever the precautions taken white filling the
fc mica tax. some grapes arc inevitably crushed and
release juice. During the anaerobic phase, other
grapes arc progressively crushed, increasing jnicc
volume. In an experiment al 25 "C with the Cartg-
nan variety. 15*» of Ihc total frce-nn juice is
released in 24 horns. <*fi on the 5th day and 8<W
on the 7lh day of maceration. Variety, maturity and
fcrmciKX height are factors influencing the for-
mation of thct free- run jnicc. The homogcnifiition
pumping-ovcr operation, when effected, is also a
factor It B sometimes used for even distribution of
the sulfnr dioxide <3-8 g/hl). which is necessary
to avoid microbial spoilage The use of lyso/ymc
has been envisaged (Section 93.2) to prevent the
premature development of lactic bacteria
A fermentor undergoing carbonic maceration
there line contains the following:
I Whole grapes immersed in a carbon dioxide
atmosphere, poor in oxygen. They are the mosl
affected by aaacsobic metabolism. In addition,
they are located in an civiionntcnl with an
increasing cthanol conccnlnruon At a certain
partial pressure, cthanol diffascs into the berries
in anacrobiosis At pressing, the piessjuice has
a higher alcohol content than the jiKc from a
solely anaerobic metabolism.
2. Whole grapes immersed in must from crushed
grapes. They undergo a less inlensc anaerobic
metabolism than ( 1 1
3. Mast from certain crushed grapes undergo-
in-; ycasl-bascd alcoholic fermentation. Crushed
grapes macerate in this juice. Ihc fermenta-
tion of which occurs al the base of the fer-
mentor It mast be carefully monitored lo
avoid bacterial spoilage. Acetic acid bacteria
may develop when the fermentation develops
slowly. The addition of a yeast stirrer in full
acliviry helps to avoid this problem It also
gives protection from Ihc inopportaac devel-
opment of Lie tic acid bacKrEt in the case of
slowed ycasl acliviry. When the pH is exces-
sively high <pH 3S). tartaric acid may be
added to the juice at the boitoai of the fer-
mentor tup to ISO g/hl. uking into account the
KWil must volume anticipated at the end of the
anaerobic maceration phase). Snlliting is also
indispensable for inhibiting lactic acid bacteria
(3-8 g/hl). Hcmogcni/ation pump-overs must
be minimized when making these additions,
otherwise, free-run juKc volume is increased
During this fermentation phase, microbial activ-
ity must be monitored throagb the disappear-
ance of sugar and malic acid. Ihc increase in
volatile acidity and possibly the analysis of
il+MactK acid, whose presence indicates bac-
terial activity
When Ihc addition of sugar Ichaptali /alios! Is
judged necessary, it Is effected after derailing, at
the start of Ihc second fermentation stage.
Anacrobiosis Is obtained by tilling an empty
fermentor with carbon dioxide from an industrial
gas cylinder or a fermenting tank After Idling,
the carbon dkixKlc supply must be continued for
24-48 hours to compensate for possible losses
and dissolution in the grape. After this period,
fcrnicn latlon emissions compensate for losses The
extinguishing of a candle flame when placed in the
lank verifies anacrobiosB.
The temperature and duration of the anaero-
bic phase arc essential parameters of carbolic
maceration. The clevaiKu of the temperature is
less important with carbonic maceration than with
crushed grapes, which have more active fermen-
tations. In hot climates, this fact ■■■as used to the
wincmakcrs advantage, when controlling temper-
atures was more difficult than today. The anaer-
obic metabolism, however, must tike place at
392
I land book of Etiology: The Microbiology of Wine anil Vindications
a relatively high tmpcratuic (30 -35 C) for this
■Klbod lo be fully effective. Yet the Kmpcra-
lurc must mil exceed 35 C. above which this
mciubolisin Is affected Maceration Tor 6-8 days
.it 30 -32 C is recommended An insufficient tcin-
pcialuic can be compensated Tor by prokmging
■uccnilion time — for example. 10 days at 25 Tor
15 daysal 15 'C— bul ihc rcsuli is not necessarily
identical In sonic regions, excessively lew tem-
peratures (I5~20'C) restrict the use of carbonic
■laceration, as the reactions arc slowed down. Sys-
tems have been devised to warm the grape crop:
thb. operation Is always complex. Immersing the
grape crop in warm mtst or wine is laborious aid
several days arc needed to obtain a perfect homo-
geneity of heal A tie in pis al healing the grapes
directly on the conveyor belt, using i
technology, have net with limited su>
The moment of dev-atting should be chosen
according to the style of wine desired. This difficult
decision is based on experience but lakes in in
account Ihc evolution of density and Kmpcraiure.
color and tannic structure, aroma and juice flavor
along with the degree of grape degradation and
pnlp color Pimping the must over once or twice
before devatting enhances the musts aromatic
intensity and tannic structure.
During dev-atting. the grapes should be carefully
pressed using a horizontal moving-bead press or a
pneumatic press. These presses do not affect tissue
structure Due lo the presence of whole grapes,
pressing capacity must be considerable (one-third
to one-half higher than in traditional wincmalang).
Pressing is also slower. Grapes may be crushed just
befoie pressing to simplify this operation Since
the press wine is potentially organoleptically richer
than the free- ran Juice, the press-Tree- run ratio
should be as high as possible.
Al the time of devatting. the free run Juke has
a density between 1.000 and 1 010 and the press
juKe between I 020 and 1.050
Table 12.19 compares free run and piess jnicc
composition for traditional (crashed grapes) and
carbonic maceration wiucmaking In carbonic
maceration press wines, the alcohol content is
higher (caused by clhanol fixation) and the acidity
lower (due lo malic acKI degradation! These
wines ab*< have lower concentration of phenolic
compounds and other extracted components: their
dissolution is diminished
Due to their complementary composition, free
run wines and press wines should be Mended
immediately alter pressing, before the completion
of alcoholic and makilactic fermentation. Bacterial
con tun mat ion in the tree-run wine, leading to
picmaturc malolaclic fermentation and Ihc risk of
an increase in volatile acidity, is the only reason for
fermenting the fiec-run and press wine separately.
Microbiological analysis should be systematic at
this stage, followed bysnltiling and icsccding the
Tabic 12.10. Run-uffandpicu<
i-inc»iuh-»»ci
•■npann^indlii
■ ml liwuklnu » ilk caifooak
imitxation (Hani* rt <■*., 1987)
Cbafoncm
Wine -at;
canted
haj wing
BHfB
("iiKiim n
atfralioi
Ficc-mh
Prcu
Kiee-oin
PlCH.
AkoholK- MniMlht'. v,)|..i
Dcmfev M 15T
12.05
0.9949
10.90
0-9991
11.15
0.9906
I3O0
0.9920
Glycerol (g/l)
929
9.75
9.10
7.91
Dry c«l»cl <g/l)
TdalackUv (g/lH,i0 4 )
23£
3 JO
32.0
350
255
350
192
2 Si
pll
350
4JU
3.93
3.90
Tajloiruaenlmp/ll
154
425
144
123
Cofar iMcnshv*
388
912
510
is~
Tannic mancr'
1342
2550
1582
1440
--In III -.-•'■ .a1 l.l (.:. Jt 43» .1 ..
'Slim of ifiiuil lunilra al 3» JiU
»!m
Red Wincmaking
393
must with fresh yeast h> complete Ibc alcoholic
fermentation, if •cccssaiy.
During the second fermentation phase, the
complete transformation of sugar into alcohol is
generally vciy quick It Is carried out at 18-20'C
k> preserve aroma components. Aflcrwanls. the
favorable conditions permit the easy initiation of
malolaclic fcnncntiiion. Despite the cxislcncc of
two distinct phases, carbonic maceration requires
less time than traditional wincmaking. This method
Is therefore well adapted for wines that arc quickly
put oh the market.
12.9.7 Characlcrblics of Wines Made
by Carbonic Maceration
Table 12.20 (rlau/y eliil.. 1987) compares the
composition of traditionally made wines (crushed
trapes! and wines having undergone carbonic mac-
eration (effected at 25 and 35 C). The importance
of tcmperaiarc in anaerobic metabolism Is shown.
At .15 "C. this technique permits the same tannic
struct! re as traditional wincmakiig. In general,
density and dry extract, li'eil actdit>* and phenolic
compoand concentrations are lower with carbonic
maceration than with traditional wincmaking. This
wincmaking technique produces a lighter wine,
containing less substances from Ibc solid parts of
the grape The method has advantages when used
with rustic grape varieties — it avoids Ibc excessive
extraction of aggressive olfacltvc and gusGilory
element lacking finesse. In other cases, carbonic
maceration may result in insufficient stricture and
an impression of thinness, or results anywhere in
between.
In Table 12.20. volatile acidities were observed
to be relatively high— often greater than 03 g/l
in H.so, sometimes attaining 009 g/l (0.65 and
084 g/l in acetic acid). These numbers indicate
the inherent hac trial risk associated with this
wincmaking method.
The structural difference of a wine having
nndcigone carbonic maceration, with respect lo
a traditionally made wine, as pointed out by
laboratory analysts, is icflccKd in its organoleptic
characters. Carbonic maceration produces supple,
round, smooth and full wines For this reason,
they arc often used in Mends to improve wine
quality. However, this positive characteristic in
certain situations can be icgallvc in others: wines
Tabic 12.20. Aiuhtiml coapar»i>n of ibc .
(CM25is«l »'C(CM35)»nd«fi*c«imc m
Afteriheeiidol nulotutlt fcimcnuikm. 1983
mposik.ll of oil
iajpt) I I'lta/y •
:% hiving underline cirfxinlc Mice
thedpape* (C< liuiiiil'. m. curried «
-/.. I9S7)
CG
: VI23
Alcoholic MienphCr vol.) HA IIJ
A»h()c/1l M 2.7
A*h alkalini} (-Et|/i> 345 325
Ghcciol (p/l) 8.0 70
Total o«n)t.tM«!!/l) W6 146
Total ackliy IbVI HiSO.) 3.10 3.10
Votalik acidly (g/l H, SO.) 041 0.34
Tartaric acid (mKq/'i 24.9 24.0
Mat* acid (mlii/ll ° °
Fit 3.71 3.01
h-MWui imii.vh 35.3 32.8
Total SO, <«b/i) 38 57
Optical denaiy 520 x 10' (red) 394 393
Optical dnfty 420 -. 10' (yellow) 101 94
Optical denaiy 280 x 10' (tannin) 810 770
Inalpttvphcnokiplyj1li.nci.il 1373 1430
AnlhocyaniiiMg") 0509 oils
...III
3.00
3 JO
3.30
D31
043
034
0»
2S.7
222
193
19.9
3.74
3SS
3.71
380
348
423
34.8
384
»*
Handbook of linokigy: The Microbiology of Wine anil Vindications
gui become thinner and more fluid and. depending
on Ihc variety and maturity level. Ihc less abundant
tannins can also be more bitter, piobably iIk to the
presence of the stcms.
CarbonK maceration is certainly most interest-
ing from an aromatic viewpoint It produces wines
with a unique aroma. Sonic have accused this tech-
uh|uc of producing nnifom wines and of masking
the aromas of quality varieties iRibcrcau-Cayon
Hal., 1976). Other authors (Flaizy el ill.. 1987)
find that the aromas of certain varieties (Mus-
cat and Syrah arc intensified. This, technique has
also been observed to incicasc the aromatic iitcn-
slty of relatively neutral varietal wines (Aramon.
Carignani.
Changes in the concentrations of secondary
products of alcoholic fermentation have been
reported In panic alar, aromatic snbstances specific
*< this wincmaking method seem to be produced.
Vet the nature and origin of the corresponding
molecules arc not always clear, in spile of
the considerable research thai this technique has
incited The typical aroma seems to he acquired
daring the anaciobic metabolism phase, but the
yeasts seems to be involved in its expression
The description of Ihc specific aromas of car-
bonic maceration wines is confronted by the well-
known difficulties of tasting vocabulary Accord-
ing to experts, carbonic maceration wines have a
dominant fruillncss with notes of cherry, plum and
fruit pit. whereas traditionally made wines have a
dominant vlnosity with notes of wood, resin and
licorice. In addition, the various aromatic compo-
nent arc more harmoniously blended in carbonic
maceration wines.
CarboiK maceration Is besi applied in mak-
ing prvnar wines for early drinking Experts,
however, do not agree on the aging potential of
these wines For some, carbonic maceration wines
lose Ihcir specificity after two years of aging, bul
arc always belter than the equivalent traditionally
made wines. For others, these wines evolve poorly
after a year of aging: they lose Ihcir character-
Mic aromas and do not undergo the harmonious
gustatory development of Iradilioaal wines. When
evaluating the differences of opinion regarding
Ihis technique. Ihc variety should be considered
and carbonic maceration conditions should also be
taken into acconnL The temperature is particularly
important, since It determines Ihc intensity of the
anaerobic metabolism.
Carbonic maceration is essentially used for icd
wincmaking. It Is besi adapted to certain varieties,
such as Camay Reservations have always been
expressed about using this technique in icgions
known for their fine wines with aging potential,
dnc to concents that varietal character may be lost.
The technique isaiso used for rose' (Scctloi 14. 1. 1)
and fortified wines, and has been used experimen-
tally to produce while wines and base wines for
sparkling wines and spirits, but has not been further
developed Contaminated harvest. I more lhan I5*i
rot ten grapes) and mechanically harvested grapes
should not undergo carbonic maceration.
REFERENCES
AmeiineM.A-.8eiv H-tt '- Kunfcec R.E. tXighG.S.»*l
Singleton V.L. 1980 Tlir WnW") . of Viae MtAing
Avi Publishing Co.. Weapon. CT.
Amnni-hxilci K. 199.1 Locahvtfbn <k* jnlW-.icc
ci den Dik dam k uia. Bode dc kui c>la-
hllUc.Tbcsc DoctoM dc I'UnwcaU dc Bonlnuv
II. Mention Qiwlogic-Ampctaiogie.
Aupuriin M. 1986 Etude dc llntucncc dc ccttaim
t.Kicun uir k* compint\ phcMilipm ifci ranis cl iki
vin.Thwe Doctoral dc rUnitnU± Boidoui II.
Bkuiin J. aadPcynaud E. 2001. Comuiwur n maiil
rfu tin. 3™ Edikin. IXinod. Par*.
Bouki ).C. imI F*cudier J.L. 1998. In OEitoloffe. Fon-
dramii n imljff^in 1 e< imoloffijuei. Pbn/y C. edi-
■cur. Lavoe.icr. Tec cl Doc. Pirn.
Boukon R.B.. Singleton U.V.. Bnwn LP. and Kun-
kec R.E. 1995 Ptincipltt •mi Pratiices of Witenuik'
iag. Chapnuo & Hall Enobgy l.<bni> . New York.
Bourwii M. 1971. tile ptr J. Ritn*rt*u~Ga\on a «f\.
1976.
Cantxi* A. I9TI Lei lacicue. dc <lnwlui»n do, coa-
pmc* phcnoliquc* au cma <k b vimtkuinn These
Doctor* dc lemc cycle, L'nivcnkc dc Bonkaui II.
IXihcrnct M. 1971 Recherche* wr b Himinuc dc
H/« iWjfira a b hunne dc Boiryl". Dunsn. Thnc
Dociom dc leme cyfc, L'nncahc dc Boidcauv II.
Fern: L. 1922. die pit J. Riberetu-Guvm a or*.. 1976.
Pent L. 1958 Trtaic it'ibiotogie fo.wgMpi,m. INAO.
Peullbi M. 1997. fffinrtfn <£«<•/«#(». 82. 29.
Klia/v C. 1998. (fjiologie. FiuHiements iriani/ieues el
la^whiffqueM, Lavontet. Tech el Due. Pari*.
Fbnry M. 1935. CR. Actid. Affic. 21.935.
Fbary C. I "jii.-. M. and llciml P. 1987 U, ringSo*
i ,■':•■ 'no !-,-: ii'.Hj , ,.,,— ..■.',.],■. INRA. Plm.
Cbyoa U. 1905 Prepttuiim n rotwritruuf </« imx
PCch cdaeui. Bordeaux.
Clone* Y . Rfeeieau-G*y«in P. a*d Ri.eicmrGa.un 1.
1981 CR And. Agile.. 023
ncaick-Klinp T. 1992 In IV>«- LftVrobuifoji ( „rf /:,.,-
fRftr>i>/«£i (cd. Gil. Fled) Hait»ood Academic
PUblnhca.Chui. S- ixrbiul
lafcn-US*iicade S. 1970 Aw. Techno). Affic 19 12).
141-134.
Lifon-Eafuuicadc S. *ul Rfccmu-Cayon P. 1977 CR
Anti. Affic. SSI.
Lifun-Ealuuicadc S.. Cine E.. LoavBud-Funcl A. ml
Rfccnr-i-Cayon P. 1983 Com.. W»>w Uif. 17.55-71.
Fcvjoud E. 1981 Cuiii.aim/rr itf Timtil i.'u fin.
Dunul. p*r».
Pcynoud E. und Gulmbcitcau G. 1902 Aw. Fh\-ao).
!'<•>.. J(2). 101-107.
Pcyaaud E.. Laron-Lalbua.-*dc 5. and Guiabcacau G.
1900 Am. J. Eiwl. Vfiic. 4. 302.
PiahlC. ;=-! Nieben J.C. 1993 The r/oirfopmntf ■/
[.eunimniiK ueno* miittlti lit ciiHuirs 6» direct
nomldio*. Chi. Haiuca'i. lahoiaiun DancmiiV
A/1. Hanhala. Dcnatifc.
PnihlC. Untiul-Fuacl A..K»n.|S4idl..Morrai» E.
and toycux A. 1988 Qm U#w Kb. 22. 197-207.
Ri)cicau-G»v»ii I ml Fcviaud E. 1901 Tuiie iT<Eho-
fojiV.Val. II. Beongei. F»ib.
Rfccnrau-Cayon J.Peynaud E.andUfouicade S. 1951
bid. Affic. Afro.. 08. 141.
Rfrcicju-Gayim J.. Pcynaud E.. Rl>c<cau-Ca><>u P. and
Sudaud P. 1970 Siieiweset TrdmiqaenluMti. U»l 3:
Wnijiftiitni- TtaupmMtttoHM du l(/i. IXinod. Rim
Rfccnrau-Gayon J.. Peyaaud E.. Rfeciau-Gayno P. and
Sudnud P. 1977 Sriemvi el Teiimiqiies du Vin.
\fel.4: Clirifictiiim a SMtilisrtiaa- Miterielx n
bisiihtioti*. Duood- Pari*.
Rfrcnrau-Gayon P. 1953 CK A*Y*f. A^rir.. 39. 807.
Rfrenrau-Gayon P. 1973 Wris. 12. 144.
Rfccmu-Gayon P. 1977 CR Acid. Afficbi. 120.
Rfccorau-Gayon P.. Xudoud P.. Milbc J.C. tad ('«•
b» A. 1970 Com. \ipte \bi. 2. 133.
SfcjiriM J. . r-l :■..■!.:.. M. 1981 CR AntS. Auric. 07.
300.
So«e»T.C. 1979/ Sci. Food Affic. 30.623-033.
Sudcuid P. 1903 ljudt c«pc rime-ale de la < iniacaiba
en poujx These Doctcui-IroKaicui. Eacuhe de* Sci-
ences <fc Bonkaux.
Tuumni A.. Muna IP. and Doacehe B. 1994 J. 6a. Si.
Xlffieel U'i. 2.x ill. 19.
13
White Winemaking
13. 1 General notion?, aiul distinctive charac eristics of while winciiaking
132 While grape quality anil picking criteria
13 J Jiicc extraction
13.4 Pmtccting juice from oxidation
135 Clarilicatiot
13.6 Jiicc treatments and the advisability of hciKHiile treatments
13.7 Fcmicitilion operations
138 Making dry while wines in barrels
13.9 Controlling reduction odot defects daring while wine aging
13. 1 GENERAL NOTIONS
AND DISTINCTIVE
CHARACTERISTICS OF
WHITE WINEMAKING
13.1.1 The Esscntbl Role of
Pre- fermentation Operations
in Dry White Winemaking
Although red wines air obtained by ihe alcoholic
fcmicitalion of musts in Ihe presence of Ihe solid
parrs of ihe beny (skins and seeds), while wines
are exclusively produced by Ihe fermentauon of
C rape jn ice Thus, in ihe paid nc lion of while wines.
)nKc cxlraclion and varying degrees of clarification
always precede alcoholic fenucntation. It Is the
absence of skin contact ii the alcoholic phase, and
nol the color of the grape, thai distinguishes while
wine making from red wincntaking. While wines
can therefore be made from red grapes having
while jiicc. if the grapes arc pressed in conditions
■hat prevent grape skin ailhocyanins from coloring
Ihe niisl This is the case of IHiiiks tie imirs from
Champagne, made from Pinol grapes.
That Is not to say Ibat while winemaking docs
nol include any maceration If this term designates
'- .,'. ...■...;..■ ..
r. r*,. ..i -a. .-
398
I landbook of Etiology: The Microbiology of Wine anil Vinifications
solubilization ol solid components in Juice, a ccr-
tiin degree of maceration is inevitably associated
wiU white wincmaking. It occurs in tic abscace
of alcohol dun ag the pre- fermentation phase, al the
time of jnice extraction and clarification
Varietal aromas and amnia precursors an- lo-
cated ii the grape skin or in the underlying
cell layers in mosl quality cullivais I Volume 2.
Chapter 7). Yet these zones big also the richest in
grassy- smelling and biltcr- lasting substances, espe-
cially when the grapes arc nol completely ripe,
ate stricken wilh tut or arc fn>m a lemur less
favor.iWc for producing quality wines. The taste
of a dry white wine, made from a given grape,
therefore depends greatly on the conditions of vari-
ous pre- fermentation operations: harvest crushing,
pressing and clarification.
All wincmaking inclidcs a selective extraction
of grape components: while wincntaking docs nol
escape fn ■in this general principle Wincmaking
nol only consists of carrying out the alcoholic
fcimcnuiioa of mast or grapes bal also, and
especially, extracting the best part of the grape
berry while limiting the diffusion of substances in
the liquid phase capable of generating olfactory
antl gustatory flaws.
In red wincmaking. fractional extraction of
grapes occurs primarily during alcoholic fermen-
tation and maceration. The wincmakcr influences
the future taste of a red wine by adjusting vattiag
times iScclkMi 123). By adjusting various oper-
ations during vailing, the wincmakcr approaches
day by day. over a period of 2-3 weeks, the
desired tannin, color and aroma concentrations
for the wine. During vatting. time is the winc-
maker's ally.
In while wincmaking. conditions for the extrac-
tion of bctry components arc radically different,
since maceration phenomena occur before alco-
holic fermentation In this case, pre- fermentation
treatment conditions control the passage of com-
pounds responsible for the qualities and fktws of
grapes into must. The quality of a dry while wine,
made from given grapes, depends above all oa
grape and must manipulation daring pmdnction.
In other wonts, the art of making dry white wines
lies in knowing how to press the grapes and clarify
the musts in a manner that simultaneously extracts
and preserves potential grape quality. For certain
varieties (Sanvigaon. Muscat, etc.). a limited skin
contact (pcc-fcrmcntation skin maceration) before
pressing can be useful in facilitating the diffusion
of varietal aromasand Ibcir precursors in the juice.
The wincmakcr has only a limited amount of lime
to extract components from the gtapc skins before
the Jake begins to fcraicnl— generally a few hoars
to a few days maximum. In addition, the choices
that are made during the pre- fermentation phase
arc definitive: pressing time and program, jnice
selection, possible skin maceration, blending free
run and press jnice and degree of must clarificaiion.
Therefore, in dry white wincmaking. the impor-
tant choices are made before alcoholic fetmenta-
tion Afterwards, corrections and adjustments are
practically impossible When alcoholic fermenta-
tion is Initiated, the taste of the dry white wine
is already largely determined liven the decision
to tank or barrel ferment is made fairly early In
fact. Ixtrrcl- fermented wines should be barreled at
the initiation of fermentation, to avoid the wood
dominating the wine laUrlScctlon S8i. Tats deci-
sion must be made as catly as possible so that
barrel purchases can be planned property It is kx>
late id barrel wine after a tank fermentation, even
if wine quality woald have justified barrel aging.
JaKc clarificaiion is another example of defini-
tive p re fc rare n tat ion decisions: it Is impossible to
mitigate its consequences subsequently, during fer-
mentation In fact, no satisfactory methods exist
H> simulate lagging fcraicntitions of ovcrclarifkd
juKe or to eliminate vegetal and reduced odors that
appear daring Ibc fermentation of poorly clarified
Botrytized sweet wincntaking constitutes an
extreme case of the fundamental decisions Involved
concerning fractional berry component extraction.
The most important decisions made by the wine-
maker essentially concern picking grapes at the
ideal noWc rot stage (Section 14.2.2). Noble rot
Is nol only an overripening by water loss, like
raisining. bat also and especially an intense enzy-
matic skin maceration (tilted lu a ftifnrii-spccitic
metabolism. The decisive part of botryti/cd sweet
wincmaking occurs in Ibc grape on the viae.
White Wine making
399
In conclusion, each lypc of wincmaking cob-
tains a key phase during which ihc decisions of
the wincmakcr have a determining and almost
irreparable effect on wine laMe: vailing for red
wines; pre- fermentation operations for dry while
wines, and noble rot development and picking con-
ditions Tor boiryli/ed sweet wines.
13.1.2 White Wine Diversity
and Current Styles
White wines arc generally thought K> present
a greater divcisily of styles than red wines
(Ribcrcau-Gayoi el tit . 1976). In fact, apart from
still and sparkling wines, white wines can be dry or
con tun a varying amount of residual sugars (from
several grams to several do/en or. sometimes,
even more than 100 grants per liter). These
differences can occur in wines made from the same
varieties and parcels — the grapes being picked
at different maturity levcK. This is Ihc case of
great German. Austrian and Alsacian Rieslings and
Gcwurftramincrs. Hungarian Fu minis. Bordeaux
Scmillotsand Sauvignons. Loire Valley Chen ins.
etc The fixed acidity ofdry and sweet while wines
can also vary greatly (from 3 to 6g/l expressed
as H.S0 4 ). Moreover, dry wines ntay undergo
malolaclic fermentation, universally used for red
wines Intense or discrete, predominantly marked
by ihc variety or oily by secondary products of
alcoholic fermentation, while wines also seem to
have more diverse aromas than red wiics. This
relatively rich typology characterizing white wines
may be divided into two general categories which
alsoc
I . Premium wines improve during bottle aging by
developing a bouqnet.
2 Prmteur wines, incapable of aging, arc to he
drunk young.
In addition, barrel matured while wines arc
partially or loeilly made in new barrels In other
wines, the organoleptic character supplied by the
oak is not song hi— these wines arc made in neutral
vessels (tanks or ascd barrels). Finally, like certain
red wines, some white wines are distinguishable
by their oxidative charactcrrajc (sherry and yellow
wines). Yet most are made in the virtual absence
of oxygen and under the protection of antioxidants
snch as sulfur dioxide and ascorbic acid to preserve
their fruity aroma
The diversity of while wine types and wincmak-
ing methods has strongly diminished during the
last 20 years, die u Ihc trend towards a workl mar-
ket, a standard iratkan ol consumer tastes aid a gen-
eral trend of producers to inutile a few universally
appreciated models. The growing influence of the
wine critic on the market has certainly amplified
and accelerated this convergence of white wines
towaidsa few widely recognized types. Four cate-
gories currently distinguish international dry while
wines: neutral. Chardonnay. Sanvignon and aro-
matic white wines
)al Neutral White Wines
Neutral while wines do not possess a particular
varietal aroma. They only contain young wine fer-
mentation aromas — essentially due » ethyl esters
of fatty' acids and accutcs of higher alcohols pro-
duced by yeasts (Section 2 31. when the fermenta-
tion of clarified juice occurs at relatively low tem-
peratures (16-18 C). These wines are appreciated
especially for their lh)rsl-<| Benching character, due
to their refreshing acidity possibly reinforced by
the presence of carbon dioxide (0.6-1 g/l). They
should be low in alcohol and without bitterness
Their fleeting aroma rarely Lets for more than a
year of storage: these white wines arc generally
bottled a few months after Ihc completion of fer-
mentation and should he drunk within the year that
follows Ihc harvest A particular varietal aroma is
nol sought nor is an expression afierroir expected
in Ihesc while wine beverages*. They arc generally
made from high-yielding, slightly or non-aromatic
varieties snch as L'gni Blanc (Italian Trcbbtano).
Maccabcn (Spanish Viura). Aircn (also of Span-
ish origin and having Ihc highest planted vari-
etal surface area in the world). While Grcnachc.
Claircitc. cK. Unfortunately, neutral white wines
arc sometimes produced with noble varieties, die
to excessively high crop yields and unfavorable
soil and climate conditions This is often the case
of Scmillon at yields greater than 60 bl/ki. or
iiH-
I I and book of Etiology: The Microbiology of Wine anil VnifiBtkws
Sanvignon grown in hot climates regardless of
yields In lire 1970s, rhese simple and inexpensive
while wines sold well, especially when promoted
by a strong brand name. Today, the demand for
Ihcm has dn>pped. as toe marker oricntalcs itself
■■wants more expressive white wines, particularly
in Anglo-Saxon countries. Oxidised wines possi-
bly containing several gianis of sugar and non-
oxidised wines containing several grams of sagar
have all but disappeared.
(b) Otardonrcns
Chanlonnay is the principal current international
while wine sGrndard. While Burgundy wines sup-
plied the original model (Meursanlt. Chassagnc-
Monlrachcl. CbaMcv car. I The lop cstalcs from
this region are among Ihc besi dry while wines
in Ihc world. Their wines ate powerful, firm, aio-
malically inlcnse and sweet . although Ihey do
■ol contain icsKlaal sagar The great while Bur-
gundies arc distinguished by their aging poten-
tial During the aging process, they develop a
remarkable redaction bouqact. In its /one of ori-
gin, the Chardonnay variety produces grapes rich
in both sugar and acid, often reaching 13** poten-
tial alcohol for an acidity between 6 and 7 g/l
(expressed as sulfuric acid) and remarkably low
pH (3. 1 -3 J). The traditional Burgundy wincmak-
ing method, with barrel fermentation and on- lees
aging, has profoundly influenced current white
winentaking methods. Today, cnological research
has shed light on and justified these Burgundy-
origin empirical practices, put u use workl-widc
and nol just for Chardonnay
During ihc last 20 years. Chardonnay was
hugely planted in European Mediterranean cli-
mates and New Work! vineyards. Along with
Cabernet Sanvignon for red wines, il is ccrerinly
the variety best adapted lo climatic conditions
warmer than its original Cormier. All Chardonnay
producers try k> attain the Burgundy archetype,
like Cabernet Sanvignon wines strive to attain the
tap- ranked growth model of the Mcdoc. Excel-
lent C hard on nays arc found in many vilicullural
regions throng horn Ihc work!, bul the diversity of
expression of this variety in different Burgundy
temnn or climates still
for the wine buff.
K' I San unions
Inspired by the wines of Central France (Satccne
and Pouilly-EuuK). Sauvignons constitute another
important world standard for dry while wines.
Their ofien inlcnse and complex typical aioma
is easily recognized. Ccrtun volatile substances
responsible for Ibis aioma as well as their precur-
sors in Ihc grape have recently been identified i Vol-
ume 2. Chapter 7). The Sanvignon aroma is mote
sensitive to climatic conditions during maturation
than the Chardonnay aroma II is therefore less
constant and stable and more difficult lo reproduce.
The aromatic expression of Sanvignon is often
disappointing in Mediterranean climates It has
consequently been less universally successful than
Chardonnay Due to its cool climate. New Zealand
wilhoul a doubl produces one of Ihc most aro-
matic Sauvignons in the New World On average.
Sanvignon wines have a lesser aging potential than
Chardonnay wines, except in very particular situ-
ations Sauvigaoa also originated in the Bordeaux
region and Is nearly always blended with Semil-
lon in this area. The Sauvignoi contributes the
fraitincss. the lirmness and (he acidity, while the
Scmilktn gives the wine body, richness and bou-
quet during aging. These two varieties are particu-
larly complementary. During recent years. Sauvi-
gnon wine making methods have undergone many
changes— including a return to barrel fermentation
of musts originating in the best icrroirs as well as
on-lccs aging of new wines, whatever Ihc fermen-
tation aKihod(banelorcink>.CunenlCbardonnay
and Sauvignon wincmaking methods arc very sim-
ilar, bnl ntalolaclic fermentation is rarely practiced
on Sanvignon wines (Section 13.7.6).
I'd I Aromatic White Wines-
Various aromatic white wines compose Ihc fourth
group Sometimes, these wines are tamoa* and
made from premium varieties. Their geographical
territory has remained limited n> their original
regions An exhaustive list of these wines is nol
White Wine making
401
included in ilus icxi. -m a few examples will be
given.
Wiihii ills group, lie dry unite, premium qual-
ity German and ALsacian wines arc wonb mention-
ing. These wine styles arc also made in Austria and
continental Europe Tie ikisi notable varieties arc
Riesling. PinotCrls and Gcwur/tramincr. Laic har-
vesting of Ihcsc varieties produces premum sweel
wines capable of considerable aging. They have a
chaiactcrislic aionia rcminisccni lul least in part)
of thcit giapc or jnice aroma. These floral or Mus-
cat varieties arc distinguishable from simple savor
varieties sich as Sanvignon. Cbardonnay. Chenin.
etc The juice of simple savor varieties Is lot very
fragrant, bat their wiies have a characteristic vari-
etal aroma essentially derived from odorless pre-
cursors located in the grape The role of volatile
terpene alcohofe aid certain norisoprcnoids in the
aroma of Muscat varieties has been largely stud-
ied and proven (\<>I»k 2. Chapter 7). The specific
aroma of Ibesc difTctcnl varieties, however, n for
from being totally elucidated
Several regioial varieties abo exist which, for
diverse reasons, have until now only produced
typical wines in relatively limited /ones Some
of them have never been plaited oulsidc their
legion of origii. while others lose their character in
warmer climates, French varieties Include Chenin
Bfcinc i Save nicies. Loire Valley). Viognier (Coi-
drici) and Peril and Cms Manscng tiurancon).
Albarino is in the north of Spain aid the remark-
able aid rare Petite Arvinc is in the Swiss Valais
13.2 WHITE GRAPE QUALITY
AND PICKING CRITERIA
Varietal aroma finesse, complexity and intensity
arctic primary qaalilics sought after in a dry while
wine. Ik personality is due to varietal expression
or. mote precisely, its particufctr aromatic profile on
a givei lerwii: Fcnricntitron amma components
arc present in all wines aid ate not very stable over
time These esters aid higher alcohols produced
by yeasts arc nol sufficient to give a white wine an
aromatic specificity, but they were the first to be
measured by gas phase chromatography because
of their relatively high conccitrations in wines.
Consequently, in the past, the importance of their
contribution to the aromatic quality of dry while
wine was exaggerated II has been widely accepted
that aromatic quality is mainly die *< the primary
aroma— the aroma originating in the grape — even
though the volatile compounds responsible are far
from being identified and the production mecha-
nisms from grape lo wine remain unknown. The
handicap of rienir.il varieties Is thus explained: no
wincmaking method can compensate. For all that,
the varietal aroma Is not the only character of a dry
white wine. The balance of acidity and softness,
sensations of volume, .stricture and pcrsisiencc
and the impression of density and concentration
also play an important tote in quality appreciation
Healthy ripe grapes must be used to obtain a wine
wiln all of these characteristics. The grape disease
stale and malirity level, in particular aromatic, are
the essential harvest selection criteria for making
quality dry white wines. Harvest time and methods,
i mechanical or manual) influence these two essen-
tial patametcis and are therefore very important.
13.2.1 Dior Stale
While grape varieties arc susceptible to gray
rot die w Boirytis cinereu development on the
giapc (Section 10.6). In a given region, tic mote
forward white varieties arc more sibject to this
disease than red varieties. Obtaining healthy grapes
with Sauvignon. Sentillon and Miscadelle grapes
Is niKh morc dilficilt than with Mcrtot and
Cabernets Muscats in Mediterranean climates.
Chardonnay in Champagne and Chenin in the
Loire Valley are also affected.
Prom early contamination of the grape cluster
t latent since bloom). Boirytis can develop explo-
sively near harvest time. Feared by wincgiowcrs.
this pathogen is triggered by severe rains near
reiiiisim and during maturation. The fungus con-
taminates bolh green and burst berries, degrading
i In skin — i In site ' i an hi. c- and aroma prcciisors
Even a rektlively small percentage of boiryti/cd
gtapes in the crop always scrioisly compromises
the aromatic quality of dry white wines. Gray
rot on white grapes results In a decrease in
varietal aroma, a greater instability of fermentation
-::
Handbook of rinology: The Microbiology of Wine anil Vinific.uions
i and Ihc appearance of olfactory flaws
These consequences of gray 101 on Ihc aroma of
while wines arc much more serious than oxidasic
casse. This casse is a direct manifestation of the
taccasc activity on wine color, especially with
red and rose wines (Section 12.62). bnl it can
be observed in certain while wines— in particular,
bottled sparkling wines — even several years after
bottle fermentation.
Although empirically witnessed with all aro-
matic varieties, the harmful effects of gray rot on
the primary grape aroma has only been quantihed
with the muscat variety by measuring monotctpene
alcohol concentrations in musts (Boidron. 1978).
When Boiryiis contaminates 20** or more of a
grape crop, the total lerpcnlc alcohol concentra-
tion of IronOgnan Muscat or Alexandria Muscat
drops by nearly 509 with respect to healthy grape
mast concentrations (approximately 15-3 mg/l).
The most flagrant lerpenc alcohols (linalol. gcrau-
iol and ncroli are the most affected These alcohols
arc partially transformed into less fragrant com-
pounds, such as linakil oxides. a-fcipcnc alcohol
and other compounds (Rapp era/.. 1986). them-
selves original component! of healthy juice. This
rapid degradation of icrpcncs by Biitryih cintwa
can be observed in the laboratory in a fungus cul-
ture on a medium supplemented with monotctpene
alcohols
Cray rot is also observed to affect the specific
aromas of other varieties. A relatively small per-
centage of gray rot (less than IC* i diminishes the
Sanvignon varietal aroma in wine This aroma Is
dae ai lcas( in part to very fragrant voLiiile thiols,
existing in irate amounts (a few nanograms or a
few down ng/l) in wines (Volume 2. Chapter 7).
These aromas are essentially found in the grape
in the form of odorless precursors linked id cys-
tine (Tominaga etal.. 1996). Botryln einerea
may directly degrade the free and bound aro-
mas of Sanvignon. bat this has not been clearly
demonstrated This type of degradation would only
explain an aroma loss corresponding io the per-
centage of botryti/cd grapes Yet the reaction of
fragrant thiols with quinoncs formed by the oxida-
tion of grape phenolic compounds has been clearly
proven to exist BtHryla lactase activity in a mnsl
containing phenolic compounds inevitably leads
to the formation of quinoncs. The quinoncs trap
Sanvignon varietal aroma as it is formed during
alcoholic fcrmenGtlion. When Sanvignon mast is
insaffkicHtly snlritcd during the pre- fermentation
phase, ii is oxidized The resulting combination of
thiols and quinoncs produces wine with a slight or
non-existent varietal aroma I Set Iron 13.4.1).
Paradoxically, noble rot docs not destroy the
specific aroma of white varieties used to make
grcai bolryti/cd sweet wines (Section 14.2.1).
In the Sautcrncs region, the lemon and orange
fragrances of Scmillon and Sauvignoa arc even
enhanced, as is the mineral character of Riesling
or the lychce aroma of Govurztramincr in the
Alsatian or German noble rot wines. The bouquet
of dry wines made from healthy grapes and sweet
wines made from bolryti/cd grapes of the same
variety and from the same leinar has even been
observed to converge during bottle aging In the
ideal noble rot case. Ihc intense skin maceration
of the ripe grape under the action of fungal
enzymes promotes the diffusion of free and bound
varietal aromas in the must These aromas are
concentrated without being degraded. This process
is different from raisining. in which the grapes
are concentrated by Ihc sun which bums the grape
skin. Most of the varietal specific aromas arc lost
and a character peculiar to raisins is acquired,
varying little from one while variety to another.
Theoretically, a small proportion of noble roiled
grapes coaM be added to a grape crop intended
for dry while wincmakiig. bat in practice this is
difficult At Ihc lime of the healthy white grape
harvest, however. n>ost of Ihc rol-infcclcd grapes
on the vine correspond io early Bmrylis silcs
developed on the unripe grape and thus gray rot
Gray nat also greatly diminishes the intensity of
fermentation aromas of dry while wines. Among
Ihc cxoccllaktr enzymes liberated by Batiytis in
Ihc infected grape, esterases exist whose activity
persists in juice (Dubounlicu eiitt., 1983). They
are capable of catalyzing the rapid hydrolysis
of esters produced by yeasts during alcoholic
fermentation Figure 13.1 shows the hydrolysis
kinetics of these different fragrant compounds in
a dilute alcohol medium in the presence of a
V. Il A- 'A ilk'Iii.ikili-
403
Fig l-li. Kaanaik' h : ..li. .:.-.■
cihanol) of ..1i:i,-.y.-i euen. b
ciiraci in.- Aotnr.
(pH 3 J al IK"C llfi
»y >• ctocclluuir fun cm
(Dubounlieu rt .tf .. IU8J)
Bmrytis enzymatic extract. The occurrence of gray
tot is more dcinmctiial Id Mumil variety wines,
con tuning essentially fermentation aromas.
Finally, gray m< scnonsly affects ihc aromatic
distinctness of dry white wines V.iiictil ;
arc masked while dusty, dirty and nioldy ;
appear. It also prumoles Ihc development or mncld.
camphorated and waxy odors, appearing later
during maturation and especially in Ihc buttle This
type or olfactory flaw is comparable Id a premature
oxidative aging of while wines (\olumc 2. Sec*
uon8.2J). Gray rol B not solely responsible
and while wines made from hcallhy grapes can
also contain Ihc flaw The responsible compounds
and Iheir rormalion mechanisms irmain k> be
discovered.
The level of Bmrylis cinereu contamination ofa
grape, in the form of gray rot. therefore constitutes
a determining crileriot for evaluating grape qual-
ity, whether red or while. But white grape crops,
and consequently dry while wine quality, arc more
affcclcd at lower levels of gray rol con tarn ina-
Ikw than red grapes The visual examination of
the pcrccncigc of bolrytizcd berries, despite ie-
insufficiencies, was Ihe only method available lo
winegrowers A dozen or so ycais affcr the pk>
necring research of Dnbcrnct (1974) on Bmryiis
cinereu laccasc. new methods of analyzing this
enzymatic activity in juice (Section 10.66) were
developed. This new way of quantifying Btmy-
lis development on the grape appeared prompting
Two methods were proposed: the first, a polaro-
graphic method, measured Ihc oxygen oaisamp-
Uon in a mnsl sample with a Clark clcclrodc
(SaJgncs eiul.. 1984): the second, a more sensi-
tive cototimctrK method, used syringalda/inc as
a specific reactive that produces a pink quinonc
in the presence of laccasc IDubourdicu elal.,
1984: Grassin and Dubonidicn. 1986). RcsulR arc
expressed in laccasc nniti (Section 10.6.6)
Juice from healthy grapes is evidently devoid
of laccasc Coming from infcclcd grapes, it can
contain from one lo several dozen units per
milliliter, depending on Ihc fungal devctopment
stage, the variety and the climatic conditions influ-
encing Ihc concentration contained in the grapes
Before Ihc appearance of Bmniis conidiophores.
Ihc infected berries contain liltle laccasc activ-
ity 1 1 -2 uiti/ml). Activity considerably increases
with sporulalion (15-20 units/ml) and conliuncs
to gmw. due u concentration, during the shrivel-
ing of Ihc grape (Tabic 1.1.1 ) Universal etiological
tolerance thresholds are always difficult u estab-
lish for a grape defect they depend on the level
el quality or perfection desired fora wine. Ideally,
a while grape crop shoald not contain any botry-
tized berries, the laccasc activity should be zero.
Tabic II L Develop
I' "...■■. heny .hi.. Uct
DubounUeii. I9KA)
or Hau\th i
rti.iv ofj.fc
Ihc
Development of flair vim
I j i live i-t ft it'.
uiuii/al-
Hc-lhv oapc
Full mi wthout cnnkliiphoics
1-2
Appearance of coakliophom
li 2il
Shriveled -aiicd papc*
20-70
~-i
Handbook of Etiology: The Microbiology of Wine anil Vniftatkws
«c al least less than 1 unit/nl The oxidasic cassc
threshold fur red wines is greater Hum S uniis'ml
and i he sensitivity limil of ihc laccasc measure
by Ihc colorintclric method Is 0.5 units/ml. li ihe
event ol .a gray roialuck. the grapes mnst be man-
sally soned in ihe vincyaid. this is the sole means
of maintaining ihc qnaliry of Ihc hcallhy portion
of ihc harvest (Section I32J).
Although less widespread than gray ml. sour
iol (Section 1063) can seriously affcci Ihc dB-
casc stile of grape crops in ln_.ili.rd areas, when
Ihc ■utii rath* occurs in a warm and humid cli-
mate. In ihc Bordeaux region, white grapes, in
particular Sauvignon. arc more sensitive lo this dis-
ease than led grapes, h has not been extensively
studied and R poorly known, despite ic-. serious-
■ess. The grapes tike on a brick-red color within
a few days, while telling sonic of the Juice flow
oil. and they simultaneously give off a strong
acetic acid odor The microbial agents responsi-
ble for this acclic fermentation aic a combina-
tion of aerobic yeasts < Hamaatapam uoanmi I and
acetic bacteria. Fruit Hies arc known k> be the
contamination vector (Hisiacb elal.. 1982: Gucr-
roni aid Marchclli. 1667). but the exact causes
of ihc berry contamination by the microorgan-
isms which habitually make up the microfauna of
the grape surface have not been elucidated. The
development of sour rot is encouraged (like gray
rot) by excessive swelling of the berries following
heavy precipitations during maturation. The pres-
sure of surrounding grapes can often deuchccrciin
grapes from the pedicel. Contamination can occur,
beginning at this rupture rone. Microscopic epi-
dermal tissues permitting juice flow, invisible lo
the naked eye. may afc*> be a cause The evolution
of the grape crop towards gray rot orsour rot fan
these situations depends on environmental condi-
tions. When excessive temperatures (higher than
30'C) block the development of Boirxlis caata,
sonr rot quickly appeals and is capable of destroy-
ing the entire harvest in a few days. While the
development of gray rot in humid climates ceases
with Ihc return of hot and dry conditions, sour
rot continues id growth inexorably— whatever the
mclcoro logical conditions. Young, vigorous vines
with a superficial rool slruclnrc planted in well
drained soils are the mosl sensitive *< sonr rot.
This phenomenon is aggravated by bin! and insect
damage in vines kicated in urban areas and rones
well lit at night.
Sour rot obvkxnsly damages dry white wine
quality more than gray rot. Mnsb> made from
partially sonr grapes can contiin more than I g
of acetic acid and several grams of gluconic
acid per liter (Section 1065). They have very
high sulfur dioxide combination rates caused by
kc tonic substmccs formed by the acclic bacteria
metabolism. Their propensity for premature fcr-
mentations makes natural settling particularly dif-
ficult lo effect. Finally, to combat Ihc sprcading of
sour rot. Ihc harvest must often be suited before
complete maturity and Ihc grapes must be rigor-
ously sorted in Ihe vineyard
The presence in Ihe harvest of even a small pro-
portion of grapes infected by powdery or downy
mildew leads to the appearance of characteristic
olfactory flaws, having an earthy and moldy smell.
These odors can adversely affcci Ihc aroma of
dry white wine Fortunately, these types of grape
spoilage have become rare.
13.12 Maturity and Setting
the Harvest Date
The need for picking ripe grapes to make good
wine is well understood. Yet optimum grape crop
maturity (whether red or white) Lsdifficnltio define
and there Is no universal notion of grape maturity
It depends on Ihc latitude of the vincyaid. the
climate, the vintage. Ihe variety, and the parcel as
well as the type of wine desired.
Must sugar concentration and acidity do nol
solely define the maturity of grapes destined lo
produce dry aromatic wines. Aroma and aroma
precursor concentrations arc also determining fac-
tors. No systematic rcfctlionship. however, exists
between optimum grape aroma concentrations and
maximum grape sugar concentrations— no more
than between the latter and optimum grape acidity
levels for a given type of wine. The characteris-
tics of Chardonnay maturity are not Ihc same in
Mcursault and Champagne or on a ranked growth
temrir and a generic appellation. It is therefore
White WiiiiiiKikiu-
405
impossible to establish a general rule lor this sub-
ject The notion of aromatic maturity Lsoflcn used
by certain cnologisis and wincmakcrs. Ibis lan-
guage can be misleading Optimum maturity can
only cottcspond to a level of g rape maturity thai
produces the bcsl wine from a grape crop of a
given parcel. Furthermore, the optimal aroma com-
position of a grape Is iol easy lo define. In fact.
Ike grape, like all fruits, progressively loses is veg-
etal and herbaceous aromas during maturation, lo
acquire fruity aromas which are more or less stable
lovvards the end of maturation
The formation of these diffcrcnl aromas in wine
Is relatively complex. Some exist in a free slate
in the grape, others arc formed from precursors
located in the must, during the prc-fcrmcitalion
phase under the action ofgrapc enzymes, ordurlng
alcoholic fermentation through yeast metabolism.
The grape has a potential for both undesirable
herbaceous flavors and sougbl-aficr fruity aro-
mas. These rvvo potcntiabt evolve in the opposite
diieclion during maturation Theoretically, such
changes should be measurable According lo the
theoretical representation in Figure 13.2. Ibegrapc
has an optimum composition in the 5 week foUow-
m. but It is not yet possible lo moaiior
TIM<*>crli)
Kip; 111 Thc«icikal«.cbcauiifiliccvi>luibaofSiiu<
faom (iKipn tfcinnp I he 7 uc<Ia IoIIuuIb^ itraiic
I: cvolrt»n of *up«f uiKcUnibi (I = mtaianim to
ccmra»n In Fancc). II: cvokaion of ukUy (ofiiaui
between 5 and A g/l H,SO.I. Ill: cvoluion of vepei
flnvoiUrbii0ryuniiHF= minimum percept ion I Iiki
old). IV: evolution of fruh character potential (aibira
unit.)
analytically the aromatic evolution of the principal
varieties — with the exception of Muscat, whose
free tcrpene alcohol concentration gives an indi-
cation of the intensity of the characteristic floral
aroma. Until etiology makes farther progress in this
area, standard maturity assessments, half-bloom
and lialf-i'iTi«s(«i dales and giapc-tasling. which
helps to evaluate the aromatic maturity of the har-
vest, must be used H> determine the harvest dale.
Standard mainrity assessment, from rSita'stm
until harvest, follows Inc evolution of three princi-
pal parameters: berry weight, sugar concentration
and total acidity It Is also useful to measure the
malic acid concentration and the pH. but these
analyses arc not often carried out by wincmak-
crs A maximum sugar concentration without loss
of berry weight indicates the completion of mat-
uration. Ovcrripcnlng. which begins when berry
weight diminishes, is generally dcpKled by an
additional Increase in sugar and possibly acid con-
centrations Ovcrripcning is rarely sought for white
gtapes used for the production of dry white wines,
due lo the accompanying aroma losses
A minimum concentration of must sugar has
been empirically determined for each variety,
within a particular region, for producing dry while
wines of satisfactory quality For example, in the
Bordeaux region. Sanvlgnon and Semillon must
have al least I 1 *) g and 176 g of sugar per liter,
respectively. Below these limits, regardless of
wincmaking methods, the wines obtained have a
vcgclal aroma They lack finesse and rarely express
the pctsotality of the lemur Similarly. Ihc opti-
mum acidity of ripe white giapc musts is specific
to both the location of the vineyard and the variety
used In Bordeaux. Ihc optimum acidity al the time
of the harvest is between 5 and 6 g/l (expressed as
HjSQi) lor Sauvignon and 4-5 g/l for Scmilkw
These value Inn its correspond to average acidi-
ties and sugar concentrations of samples taken
over several ycais al the time of ideal maturity
(Table 1.1.2)
When grapes have reached their minimum sugar
concentration . harvest is possible but several other
conditions must also be satisfied: grapc-tasling
indicates Ihc disappearance of herbaceous atomas
whereas fruit)* aromas, characteristic of Ihc variety.
Tuhfc 111 Average S«
gntulh IBoftkautl
Handbook of Enofcgy: The Microbiology of Wine anil Vin ideations
Acidly <g/l H>SO.)
Acidity Ig/I II1SO.1)
;■"■!
1991
1992
1993
1994
1995
1996
arc present. appn>\ 1 m:iic ly -Mi days have passed
since half- rerai urn (pins or minus It'! ): and ihc
acRlity is within in Ihc optimum mngc for Ihc givei
varKly and vineyard. In general. Ihc slower ihc
rale or decrease in acidity* during maturation. Ihc
kngcr ihe harvesl can be delayed without fcaruf
Rising fniuy varietal aromas In ihc best lemurs
for making aromatic dty while wines capable of
considerable aging. Ihc grape remains fruity and
sufficiently acidic In the final stages of maturation.
These lerrnirs have slow and complete maturation
conditions. Convciscly. excessively hoi climates,
early harvest, and excessive water stress in the
sammcrarc unfavorable lo the aromatic evolution
of while grapes.
Table 13J gives an example of $,tuvignon
maluralion on Iwo ditfeicnl Bordeaux soils: a
sandy-gravcly soil (C) aid a sandy-clay soil on
compact limestone IC). Soil C fillers well and has
a low water reserve. Tic walcr supply is limited.
Tubfc 111 Mmuj
. of 1993 Boufcauv SamipKin u
oil (G) ind u»h cUy .
«k a .eou-.y>il<C)<-ieq
KM. I996|
Ctar-HicriMk
SB
■ P fc data
Aug. II
Aug. 19
Aug. 25
Sep. 1 Sep. 9
Je|
16
Id
-"1
Bern* •
•evh tg(
Wriinai pei
««agc
Sugar*
I rat kin lg/1)
Total ■
catty
lmeq/1)
Malic i
•calfi
&eq/l>
P«
li(C)
Ben] ■
.e^tatg)
Venation tci
««age
Sugar*
I rat kin (g/ll
Tuala
cidiy
imeq/l)
Malic;
ICll 1 1
.eq/l)
P«
White Wine making
«n
making ihc vine more forward Iban in Mill C.
which has fewer hydric constraints The grapes
on soil C may be picked up « 2 weeks later
than ihiw on soil G and arc sligblly overripe
Soil C grapes undcigo a slower maturation Al
Ihc saiK maturity level in Ihc two soils, soil C
grapes arc sligblly niorc acidic with less malic acid
and a k»wcr pH. Over the years, soil C grapes arc
ohscrved in remain fruitier during Ihc maluralion
process than soil C grapes The harvest dale can
consequently be set « correspond with practically
Ihc maximum sagar concentration desired On the
gravely- sandy soil C. Ihc characteristic Sauvignon
anna can be almost tocdly kw in the coarse of
a week Early b;irvesling on this type of soil is
necessary. iol only due lo the forwardness of the
ftvvwr bat also due especially » the instability
of Ihc varietal aroma. In a given vineyard, an
understanding of Ihc forwardness and hcbavkir
of parcels greatly influences Ihc reasoning behind
determining harvest dales.
13.13 Harvest
White grape harvesting forqaality wine production
has long been known lo be more difficult and
rcquirc more precautions than red grape harvesting.
More sensitive lo oxidation, easily masked by
olfactory flaws, while wines have a more fragile
aroma than red wines. The aroma can be partially
kisi or altered as early as the harvest, if certain
rules arc not folk>wcd Harvest conditions musl
be sach thai Ihc grapes picked arc healthy and
Iheirenological maturity (sagar. acidity and aroma
concentrations) is as uniform as possible. Leaves,
petioles, dirl and assorted debris should be avoided
in the harvest Prom harvest to their arrival al the
winery, the grapes must be as intact as possible lo
limit mast oxidation and stem maceration.
The grapes should be harvested al a temperature
below 20T. Ii warm climates, harvesting may
have lo occur at night or in Ihc early morning: bul
moistarc on the grape clusters should be avoided,
as it can be a significant cause of dilution.
The choice of harvesting method depends on
grape maturity and disease stilus on ok hand and
ic constraints on the other .While grapes
can be harvested manually or mechanically, all at
once or in several stages, with or without sorting
in the vineyard or on sorting tlblcs at the winery.
In tcmreratc-climalc vineyards sensitive logray
rot. multiple selective manual harvesting optimizes
dry white wine quality Only healthy grapes
reaching the desired maturity level arc picked
Infected grapes are eliminated in the vineyard
This is ihc most effective sorting method Spoiled
grapes and grape clusters are left at ihc vine, unripe
grapes arc picked al a later dale. Leaf removal
and cluster thinning, carried oul during the year,
combine k> avoid clasicr crowding and promote
sun exposure. In Hits manner, ripe grape clusters
are morc easily identified by the grape- picker. Well
planned pruning. Ihc early elimination of base-
bud shoots and laterals, leaf removal near t&w'soii
and grape-cluster thinning should all be carried
oul with the objectives of promoting a healthy
sanitary stile and homogenous grape maturity
These effort, nol only improve wine quality bul
also facilitate harvesting.
Multiple selective harvesting has long been
rccogni/cd lo increase grape quality. Chaplal
I 1801). restating cstaMrdicd principles, wrote:
Only healthy and ripe papa, »hould be picked;
all Infected grapes should be disc-anted wftfa caic
and unripe papc* should he left on ihc vine.
The harvest Is carried uut two i» ihiec times in
place* where wine quality -■> a pical concern.
In peocal. the fiei cuvee » the bcu. Some
countries neveflhetes* tarvcsi all papes at the
santc lime. The ctaraticriMic* of the pood and
ladaiceipicssedaiihesametinte.Amuchlouer
ifi.ili, wine ■• i Km produced compared u fed the
pnlcWBl of ibe piapc%. if nunc precaution were
uLcn during Ihc haocu.
In certain years, grapes may have a homogenous
maturity and perfect sanitary sale. In these situa-
tions, all of the grapes can be harvested at Ihc same-
time — multiple harvesting is nol necessary
Due to its lower cost, lis speed and in simplic-
ity, mechanical harvesting has been increasingly
adopted over Ihc lasl 20 years. Ik effect on dry
while wine quality can be negligible in optimum
sanitary and maturity conditions but mechanical
i;*
Handbook of linokigy: The Microbiology of Wine anil Vinifkalions
harvesting of :i heterogeneous crop always sacri-
fices wine quality. In this caw. il is of economic
and cnological intcicsl to haw ihc infected grapes
removed by a picking (cam before harvesting ihc
healthy portion of Ihc crop.
Mechanically harvested white grapes must be
protected against oxklalion Saltiting. however,
mast be avoided since it promotes the extraction
of phenolic compounds. The additkin of dry ice to
the crop is a preferable alternative. Some countries
asc ascorbic acid, but this antioxidant Is only
authorized for treating wines in the EC
Whether manually or mechanically harvested,
the grapes shoald be transponcd rapidly to the
winery in containers that minimize berry crushing.
13.3 JUICE EXTRACTION
13.3.1 General Principles
In dry white wincmaking. pre- fermentation oper-
ations (grape and JuKc handling and Ircalmcuct)
are deciding factors in final product quality
(Section 13.1.1). Their role B multiple. They must
extract and clarify juice in a relatively limited
amount time while minimizing juice kiss. In addi-
tion. Ihc diffusKin of certain grape skin subscinccs
in the jukc. in particular fruity aromas and aioma
prccursois. must be pn>motcd during these oper-
ations. The dissolution of hcrbaccons-odor and
bitter- usling compoinds. associated with the solid
pare of the berry, mast simultaneously be limited
The formation of substances capable of decreasing
the stability of extracted fruit aromas must also
be avoided Oxidized oroxldi/abtc phenolic com-
pounds in particular are able » trap certain aromas
(Section 13.2.1).
Before describing the different techniques used
and their consequences for juice and wine com-
position, juice extraction principles should be
discussed
The fermentation of juice containing too many
saspended solids (rcsilling from JuKc cxlrac-
lion) dors not produce quality dry while wine.
In fact, high concentrations of suspended solids
in juice are known to have detrimental effects
on wine quality (Section 135). The first crite-
rion of a juice extraction method, therefore, is
its ability to produce clear juice with a turbidity
as near as possible to desired levels (200 NW).
All wincmaking ts a scries of elementary oper-
ations and each one must be conceived with
Ihc idea of facilitating the others that follow.
The lower the concentration of suspended solids
in the jukc after draining or pressing, the eas-
ier it is to accomplish juice clarification: con-
versely, clarification becomes impossible after a
poorly adapted pressing that prodnccs excessive
saspended solids. When designing winery equip-
ment, thts criterion is not always sufficiently taken
into account. The production of saspended solids
during jaicc extraction has other disadvantages:
it indicates that the grape has undergone severe
mechanical treatment and conscqaently a greater
amount of herbaceous character sabslanccs are dif-
fused in the juice.
Proper juice extraction shoald also limit oxi-
dation phenomena. Ihc dissolution of phenolic
compounds from the skins, seeds and stalks, and
pH increases linked lo potassium extraction from
Ihc solid parts of the grape. Reselling oxidation
phenomena and juice browning can be evaluated
by measuring the absorbancc of tillered juice at
420 nm The dissolution of phenolic compounds
is measured by the phenolic compound index (the
optical densily of the JuKc at 280 nm is subtracted
from the optical density at the same wavelength
of the same juice percolated on PVPP). The pH
increase during pressing merits being followed
more systematically in practice. The evolution of
jukc electrical conductivity during pressing can
also provide interesting information on pressing
kinetics.
The objectives described above are better
attained when Ihc following conditions arc satis-
fied:
• low pressing pressure:
• limited mechanical action capable of Iriluraliig
grape skins:
• slow and progressive pressure increases.
• high volume of jukc extracted at low pressure:
White Wine making
409
• j ii ice extraction at a tciipcialuic lower than
20 c Ci
• limiitcd crumbling aid press-cake breaking dur-
ing preying;
• inininioni ait con tact— rapidly protected from
alt exposure and sallilcd
The transformation or grapes into juice can he
obtained by different methods Jnice extraction can
he immediate ot preceded by a skin maceration
phase. It can be continuous ot in batches, with
ot wilhoit crushing and desk-mining Con Unions
and Immediate jiKc extraction processes (very
widespread until recently in high-volume winer-
ies, despite i& disastrous consequences on juice
quality) air fortunately being abandoned: they will
Iherefoic be covered only briefly . Immediate whole
ot crushed grape banrh pressing and skin maccta-
i- 1" will be described in more deciil.
13.3.2 Immediate Continuous
Extraction
In this process (Figure 13 31. the grapes, crushed
by rollers beforehand, fall by gravity into a contin-
uous inclined dejuicer containing a helical sciew
and arc transferred into Ike continuous press placed
Fie <** CokImioc
below. Continuous dc juicers are capable of treat-
ing large amount of grape crops (several hun-
dred kilograms per minute), liberating a high
proportion of free run (70* ) They have the dis-
advanugc of producing jnice with a high concen-
tration of suspended solids and elevated turbidity
( 1000- 10000 NIT'). The pctccnttgc of Ices aftct
natural settling b. between 30 and 5(fi. Clarifying
Ibis volume of suspended soJKLs is problematic
II icqiircs cosily kugc-scalc filtralion or ccuin'i-
galion eqiipmcni which has insufficient treatment
rales. Moreover, the high speed of continuous
dejuicets limits the diffusion of arena compo-
nents from Ihc skins ln*> the jnice Sometimes
even at the same tntbidily aftct clarification, juice
having undergone con tin ions dcjnking has more
difflcult fc rate n unions than juice from Ixitch press-
ing— mote apt to extract compounds indispensable
u> ycasP.
Con (in nous presses extract the remaining 30**
of joKc contained in the skins alter continuous
dejuieing The skins arc rushed inlo a cylinder by a
large helical screw against a restriction to compact
Ihc skins and form a plug. Due to Ihc tearing
and grinding of the grapes caused by Ihc screw.
Ihc jnice obtained with this equipment, regardless
of iK performance, is billet, vegetal, colored and
high in tannins and has an elevated pH (Pcynaud.
1971; Manict and Mcklingcr. 19761. The wiics
obtained could never make up a quality blend
Uippellittiim). Speed is Ihc only advauttgc of this
press, which is capable of throughputs of up to
100 metric tons/hour The use of this extraction
method is rate today, die u Ihc devempment of
high-capacity pneumatic presses that arc capable
of high throughputs while maintaining inequality
of l\i Kb pressing.
13.3.3 Immediate Batch Extraction
without Crushing
Also called whole cluster pressing, this extraction
process Is based on pressing methods used in
Champagne In Ibis famous srurkling-winc region.
Ihc objective is u obtain while jnice even from
ted grapes. The grape skin must not be tritnrakd
during handling or pressing.
410
Handbook of linoUigy: The Microbiology °f Wite anil Vinificalions
Intact grapes arc placed into the press To
maintain skin it.suc inkgrity. they arc nol ousted,
pimped or destemmed. The juice is extracted
in batches: ibc tilling, pressing and emptying
operations are carried out successively and make
■p a cycle. In small installations irc-ating premium
qaality grapes, the small containers (wooden nhs
or cralcs) used m transport the whole grapes are
also ascd Id till the presses. Ttey arc easily
maneuvered on pallets by means of a forklift
li>r larger- scale production, if trailers are used
to transport grapes from tte vineyard lo the
winery, they mast be capable or emptying their
contests into the press without the need for a
Blast pump, which inevitably crushes the grapes.
Various techniques arc effective. Trailers can damp
by gravity into a scrcw-driven hopper fecditg
a conveyor bell reading to Inc press. Hydraulic-
lift ii. iik in capable of elevating themselves to the
level of the press may also be used. The grapes
arc then transferred into the press wilh a helical
screw. A helical screw-bused system does not
resalt in a signilicant amoaat of bars! berries aid
does not adversely affect wine qaality as kmg as:
ID tte transfer distmcc does not exceed 4-5 m:
(ii) the height of grapes above the screw is kept
>t a minimum (a few do /en centimeters): (iii) the
screw diameter is sufficient (3O-40cm): liv) its
rotation speed is slow. Belt-driven hoppers have
recently appeared, containing a conveyor belt they
transfer whole grapes in ideal conditions, but this
system can be difficult to clean
Three principal types of batch presses arc
ascd: vertical presses, moving head presses aad
pneumatic presses These same presses arc used
forrcdwincmaking (Section 126.4. Figure 12.11).
Press operating conditions have a greater influence
on the qualify of fresh grapes than fermented skins,
which contain only approximately I5'« of the tool
wine prudaccd.
Vertical screw presses arc lac oldest, since their
operating principle was invented by the Greeks.
They arc the arctetypal press (Hic'rct. 1986) At
the beginning of the 20th century, vertical screw
presses were progressively replaced hydraulic
presses thai made use of hydraulic prcssurc to
compress the berries In vertical presses with a
sis- ■ t- 1 l- basket. Ihc hydraulic jack lifts the Ixtskcl
and compresses Ihc berries from top- k> bottom in
tte direction of tte fixed prcssurc plate In fixed
basket presses t traditional Champagne presses), the
top to bottom compression is produced by a mobile
pressare plaK equipped with a hydraulic jack that
lowers ilsclf.
The quality of jaKc extracted by vertical
hydraulic presses is indisputable. slice Ihc pressure
is exerted without tritaraling tte grapes. The juice
has a low concentration of suspended solids due
to the filtration rcsalting from tte cake thickness.
This type of press requires elevated pressures, from
4 to 5 bars daring tte first pressing to 14 bars for
tte last. Tte extended pressing time and tte perco-
lation of juice across Ihc skins increases the con-
ccniratkin of fragrant compounds from the skins in
tte mast The primary disadvantages of these verti-
cal hydraalK presses arc their slow throughputand
tte labor-intensive operation of breaking the press
cake. In most instillations, they have been replaced
by rotating horizon td presses, either moving-head
or pneumatic, permitting the cake lo be broken ap
mechanically.
In threaded-axle moving-head presses, depend-
ing on Ihc direction of basket rotation. Ihc plates
(heads) approach from each end (compression
phase) or separate from each other (decompres-
sion phase). The separation of the heads provokes
tte brcak-ap of Ihc cake. The press is tilled and
emptied through central openings in tte basket.
These presses generally contain internal hoops
connected by stainless steel chains fixed lo the
beads. This set- up effectively breaks up the cake
hni also sharply increases the formation of sus-
pended solids For this reason, models designed for
tte production of champagne and sparkling wine
arc withoat hoops and chains In Ibc most popular
horizontal head press (Voslin). two basket roth
lion speeds make rapid prcssurc increases and cake
break-up possible. Rolating-axlc hoci/ontil presses
arc preferable to fixed-axle presses Rotating the
axle in the opposile direction of the bosket dis-
places the heads more rapidly and limits the num-
ber of basket rotations necessary between pressing
cycles. Nkwing-bcad presses generally have live
to six pressure levels (ap to 9 bars). Pressing can
White Wine making
411
be controlled manually or automatically — ihc pro-
gram is nxxliLiicd according lo ihc nature "I Ihc
grape. The pressing qKility obtained with ihls rypc
of press depends a k>i on ihc choice of pressing
cycle. Increasing Ihc pressure loo quickly and
excessive, too rapid and ill-limed cake break-up
lead lo vegetal and oxidised juice wilh suspended
solids Slow inaia.il pressing, while monitoring
throughput and Juice inrbidily. obtuns ihc best
resale*.
Figure 13.4givcsan example ofa pressing cycle
win a horizontal moving-head press (Vaslin. 22
V'l'i in manual mode asiag whole, healthy and
ripe Saavignon. All pressings, as well as the firsl
Ihicccakc brtuk-ups.aic excelled at a slow basket
KiH II*' Wkik-ckmcr picuiap cycle In manual
■ode with a nKivinp-hcad picu i\ Jslio 22 VT). y,
in (ii = pmuiic level: tl, in fl< = prcuuit Hep;
C = « ni ml> I i Db' . it ly inf "' uli hrt.il-up
rotation speed. Bach lime aflcr retracting Ihc heads,
mnsi cxlraciion rs noraially icsumed ai a picssuie
lower than before bieaking up Ihc cake. Tabic 13.4
slates Ihc varkias pressing limes and juice volamc
and inrbidily for this diagram. The drained juice
and ihc juice from ihc tirst pressing are Ihc mosl
turbid As soon as Ihc pomace cake is formed, ii
acts as a filler aid 50*4 of the juice is extracted
without bieaking up Ihc cake The juice from the
firsl two pressings « n, and n : > constitutes the
free ran juice. The press juice is composed of the
last three picssings and represents approximately
15'* of ihc total cxtracicd volume. Toial pressing
Uik exceeds 3 hours, bul Ihc overall turbidity of
Ihc pressings (about 500 NTU) is satisfactory with
respect to the 200 NTU preferred for a juice before
fermentation. Consequently, the percentage of lees
obtained through natural settling is generally less
■ban 10*.
Correctly carried out to obtain quality jaKc.
pressing wilh a hon/ontd nK>ving-hcad press is
neccssaiily slow. Additionally, dac Id iKchauical
i i-simiii::- imposed hy Ibc baskcl. presses larger
lhan 60 hi cannot be manufactured: thas Ihcir use
for quality white wines and small instillations is
to limited.
In pneumatic presses. Ihc pressure cxcilcd lo
exiracl the juice is applied u the grape clusters
by an internal membrane which is inflated by an
on- board air compressor. The maximum pressure
attained by* a pneumatic press is 2 bais. Differ-
ent models exist perforated baskcl or closed tank,
cqaipped wilh drains, with an axial bladder or side-
mounted membrane and filled axially or through
doors They can function manually or aaiomali-
cally with more or less sophisticated programs.
Tabic 114.
■trapes mi
Evokan
of Btisl initial!. <kiriof>
ad picu (Win 22 VT)
hok-vhrucr g
rc«ii»p of Sauv^mn
Prcuiag
I
mcdnlnl Volume (M
ftikcfi
Tuibklay (NTL'I
-.:
Handbook of Fnology: The Microbiology o( Wine anil Vindications
Closed tank presses are preferable to perforated
brisket presses, since the grapes con be more easily
protected with a blanket of carbon dioxide during
filling. These prcsscscan also be used lor skin mac-
eration in the correct conditions (Section 13.33).
Additionally, the tank press has a greater mechan-
ical resistance than the perforated basket piess for
the same metal thickness. Juke collection by the
drains in tank presses also limits juice oxidation. Ii
buskct presses, the juke flows out In a thin layer,
increasing oxidation risks Membrane tank presses
arc currently the most popalar. espccErlly for high-
capacity presses The membrane is located on the
half of the Link opposite the drains. The largest
membrane presses cirrcnlly have a 350 hi capac-
ity. Filling, pressing, crumbling and emptying is
depicted in Figure 135 During the pressing phase,
the unk Is immobile with the drains racing the bot-
tom. During crumbling, the membrane is deflated
and the basket rotates.
Axial tilling is only an option when the press is
filled with crashed grapes (Section 13.3.4). This
nKlhod leads to an increase in suspended solid
concentrations, obtained after pressing
As with horizontal moving-head presses, the
pressing quality of pneumatic presses depends on
the chosen pressing nKlhod and cycle. The gen-
eral rales are the same The maximum volume
ruin? Prcuii; (nwiiB i>t*u.j»B
Fift liS Operation »f I ck»cd-Uak mcnibr..iK picu
Fift 13." Suada-d picM-im; program of u Buchci
pncuamlt picu. Tl. T2. T3. dual km of unmixed
picnuic; TJ. bo picuuic period; TS. unilu
pn-.uuK period; TO. «mimi«i pic.Miic period. R I . R2.
R3. annuel uf mm in oi during caimhling in dlftcKH
pha*ci ot ihc cycle
of juice must be extracted at the lowest possible
pressure and crumbling must be limited Cram-
Ming generates less suspended solids than in
movlng-hcad presses but the oxidation promoted
Is not negligible. Figure 136 Is a representa-
tion of the standard pressing program used by
Bnchcr presses. Time and pressure parameters arc
adjustable. Table 133 gives an example of a Sauvl-
gnon pressing with a Bnchcr 22 hi press, which is
interesting to compare with Table 13.4 (moving-
bead press! The total pressing time is half thai of
a same capacity moving-head press. Juice turbid-
ity Is significantly lower, especially at the start of
pressing, even though the largest volume of juice
Is extracted at this time. Turbidity varies relatively
little during pneumatk pressing. Compared with a
nK>vIng-hcad press, a pneumatic press can liber-
ate a significantly clearer juice more rapidly using
much k>wcr pressures Suspended scdimcntdcposil
Is often less than 5'f
The most recent pneumatk* presses have fully
programmable systems, permitting the operator to
m luihiday durii
ronling to in juni
• hofc-t-kBicr pnrwing of Siii
i» p-opain (figure 133)
Proiuic
Time (min)
Volume 1 hi)
Juiced
Tuih
idiy<NT*U)
Uw (0 .2 bun.)
55
13.2
BB
4W
lncnri»ingfM>m().2i
2 r«ir»
2~
ID
hi
350
Mivia-jm<2 hie.)
8
02
2
;i.i
Total
■;n
15
li-r
4f>3
White Wine making
41.!
carry out several dozen sequences wiihii a press-
ing cycle Within each sequence, lie pressure,
duration and uumbcrof tink rotations diringcrum-
Ni«i! arc ilcli licit. In this manner, tic pressure can
be Increased by adjustable increments. The juice
produced has a low inrbidily and somciinics docs
not require clarification The picssing programs
that limit the number of crumbling* and thus juice
oxidation are by far the best. By measuring juice
flow i. ik' the system detciniincs the pressure level
at which the picss must operate and the amount
of lime that this pressure should be maintained.
The peess incic men tally increases the pressure and
determines the time and ink'nsity of crumbling.
The most impoHant characlcnstic of pneumatic
pressing is the kiw increase in the concentration
of phenolic compounds in juice during Ihc pressing
cycle (Maurerand Mcidiugcr. 19761 The juices of
the final pressings, or at least a greater percentage
of final ptess juices, may be bkrnded inki the
finished product
In conclusion, different batch pressing systems
have been successively used in the making ol qual-
ity while wines. Clear juice extraction and slow
krtal pressing limes were two inherent character-
istics of vertical presses. With this kind of press,
even if the wincmakcr had wanted to press mote
quickly and less carefully, this was impossible.
With a moving-head piess. clear juice extraction
is dependent on pressing slowly and conectly.
Yet these presses have often been incorrectly used
to work more quickly, producing juices of infe-
rior quality with suspended solids The creation
of pneumatic presses radically changed the con-
straint on while grape pressing Clear juices arc
obtained with much reduced pressing times aid a
k»wcr proportion of inferior quality press juice. The
1 ■ ■ t. .I picssing lime, however, must be sufficiently
long to permit the diffusion of charactcrMic aro-
mas of the grape into the jiKc
13.3.4 Advisability of Crushing and
Dcstcmming with Immediate
Extraction
Crushing consists of bicaking the grape skin for
immediate liberation of pilp and pan of the
jnicc. Grooved rollers with adjustable spacing
have long been used for crushing Turning in
opposite directions, they seize the grape clusters
and crush the berries. The machinery shoikl be
adjusted so that the stalk and seeds arc maintained
intact during this operation. The crasher should
be placed above the press to permit gravity
filling. This set- up eliminaKs the need for a must
pump, which always generates suspended solids
This principle is not always respected. In hlgb-
volnmc wineries, pneumatic presses ate frequently
filled axially— using a must pump. Draining is
facilitated by the periodic rotition of the basket. In
Ibis case, the presses arc ascd as dynamic drainers
before pressing These operations always increase
the formation of suspended solids.
Crushing before pressing has the advantage of
permitting a more significant draining of the grape
crop during the filling of the press. Press capac-
ity can be increased by 30-5G*i Additionally,
crushed grapes are pressed slightly more quickly
than whole-clusters. On the negative side, the juice
obtained is at least two times more turbid than alter
whole-cluster picssing Ices volume after natural
settling is approximately 2ifi if a moving-bead
press is used, and slightly lower with a pneumatic
press. The suspended solids are liberated In the
drained jiicc before Ihc press cake is capable of
playing Its role as a filter. Consequently, when
a closed tank press is filled with crushed grapes,
instead of draining immediately, the drains should
he closed daring the first half of filling to limit the
formation of suspended solids.
The mechanical action cxctlcd on the skins by
crushing seems k> promoie the diffusion of aroma
components inu the jnicc. Nevertheless, by rapidly
liberating the Julie, crushing curtails skin contact
The two phenomena work against each other. The
role of crushing on primary aromas Seated in the
skins is uncertain
Crushing Is generally thought u increase herba-
ceous flavors! he xanol. i7J-3-hcxcnol and iriiru-1-
hcxcnoll in juice and wine, especially In the case of
Insnnicicnigrapcmaluiiry (Table 13 6) Even after
adjusting jnicc to satiable Inrbidily levels before
fc mica tat ion. the wines obtained have considerably
Handbook of Etiology: The Microbiology of Wine anil Vinificinons
<mg/l> of C,
(kcvanol- unci
nti,) i* Scallkm wtaa
Slate of pnfo,
H»rvc*i A"
HincM B*
t linked
Not c naked
2.1
1 J
IS
10
I!.'j'. i'l\ A ail □ at m)\ii.1i-1 b} 10 ill. v
higher C 6 alcohol conccniralions with early har-
vested aid c ru.shcd grapes.
Dcstcmming while grapes intended for imme-
diate pressing also presence certain disadvantages.
The stalks act as a drain during picssiig. Remov-
ing ili cm increases draining lime and the number
of c rumblings required To facilitate the pressing
of mechanically harvested giapcs. ccrtiin ntan-
■faclurcrs (Bucbcr) have eqnipped their pneu-
■.■■..Ik picsscs with complementary drainage sys-
tems. The presence of Milks during pressing also
limits the concentration in )iKc of thcrmolabilc
proteins which cansc pn>lein cassc in while wines.
Wines arc thetcforc stabilized with lower ben-
mniif concentrations when produced fn>m juice
extracted from non-dcslcmmcd grapes I Volume 2.
Section 6.6.2).
If press capacity permits, crushing and dcslcm-
■ling should be avoided and the grapes should
be hand- harvested and prcsscd imnKdialcly. These
operations arc only necessary when the grape
undergoes skin maceration beforc pressing.
13.3.5 Skin Maceration
Experience and cautiousness have led to the
creation of general while wiucmaking principles
recommending as little maceration as possible
with the solid parti of the grape cluster. The
diffusion of substances ftum these solids ink)
the juke leads to rations Daws in the wine:
vegetal aromas of unripe grapes, astringency and
bitterness of phenolic compounds from seeds,
skins and steins and mokly. earthy and fungal
odors from spoiled grapes. It should therefore be
avoided. With grapes of heterogeneous maturity
k-icK and disease stains, immediate and rapid juice
extraction followed by rigorous press selection is
indispensable. Draining looms, nscd in the r<5<K
for stocking crashed grapes before pressing, were
abandoned for Ihls reason (Ribc'reau-Gayon el til .
1976). They provoke oxidation and an uncontrolled
maceration in the presence of stalks.
Yet with certain varieties, when soil and climatic
conditions combine to produce perfectly ripe and
healthy grapes, skin maceration can be sought for
the better extraction of grape skin components that
participate in the .noma, body and aging potential
of dry while wines. In this case, the positive
elements tatgcly oatweigh the negative elements
linked b> insufficient maturity or a high slate of
disease.
Slow pressing contributes u the extraction of
aromatic elements from the grape skin In fact,
press juice is the result of a certain degree of
maceration. In certain cases, adding it to wine is
desirable, in other cases, it should be avoided.
The higher the sugar concentration and aromatic
Intensity and the lower the pH. the more adding
press jnice improves wine quality. In vineyards in
the Bordeaux region, incorporation of Sauvignon
or Scmillon press juice before fermentation is
systematic when the grapes come from old vines
and the best parcels. It is avoided with jnice
from yoang vines, insaflkicnily ripe grapes and
excessively high yielding vines Section ( 13.22).
Skin maceration consists of voluntarily permit-
ting acontact phase between the skins and the jnice
in controlled conditions. An adapted rank is tilled
with moderately crushed, deskmmed grapes. Sev-
eral horns later, the drained jnice is collected and
the drained pomace is pressed
Results reported, as well as winemakcrs opin-
ions on skin maceration and the quality of wine
obtained by this method, are sometimes contra-
dictory. This r> not surprising, since the nature of
the grape (variety, dC-casc stilus and maturity) and
maceration condilions( temperature, rank and grape
handling) greatly influence its effect. Accotding
Id certain authors (Ough. 1969: Ongb and Bctg.
1971: Singleton el at., 1975). skin contact lasting
for more than 12 hours results in coarse, phe-
nolic wines of inferior quality. Others (Arnold
and Noble. 1979) lind that the skin maceration
of Chaidonnay significantly improves aroma qual-
ity and wine structure without increasing bitterness
White Wine making
41 S
:iml aslringcncy The besi icsbIk. in ibis particular
case, have been obtained wiih relatively toig niac-
oration (16 bonis) Sboncr maceration b;is been
iceoninicndcd Tor Austria! varieties (Hanshoffcr.
1978).
Skin maceration grew increasingly popular in
France during Ibc mid 1980s (DnbourdKu eial..
1986: Ollivicr. 1987). This operation pa«dnccs
satisfactory resnlis wiih while Bordeaux varieties
(Sauvlgaon. Seniillon aid Mnscadcllc) as well as
with MBsca&.ChanloiaayandGrovniaBScng — as
long as it is earned out wiih Ibc proper material
on hcallhy grapes negative response to biecasc
aciivity lest) wiih homogeneous maturity.
The grapes, completely destemmed. are trans-
fcrred to the maceration tank with a must pump,
the lank having been rilled beforehand with a trycr
of carbon dioxide lo avoVA oxidation. Sulliting is
avoided, to limit the extraction or phenolic com-
pounds. Different installations arc possible.
The first solution consist-, of carrying oni the
skin maceration in a pneumatic press, if it is air-
light When maceration is complete, tic JnKc is
drained and the skins arc pressed. This system has
etiological advantages and Is simple The grapes
arc only transferred once, this eliminating oxida-
tion The primary disadvantage is the immobiliza-
tion of the press
Skin maceration is generally carried out in a
lank equipped with a system pcrmilling the drained
jnKc ( 7C* i to be removed and the drained skins lo
be transferred to the press by gravity. The volume
of this lank must be triple that of lie press
Skin maceration in a membrane tank (Elilc-
Pcia) is a process situated between maceration
in a pneumatic press and lank maceration. At
Ike end of maceration, the jnicc is collected lirsi
by natural draining and then by infilling the
tank membrane, incrementally increasing pressure
10.1-025-0.4 bar). Niicry per ecu of the juice
can be collected by this method. The drained skins
arc transferred by gravity into the press with the
help of a screw conveyer The juices obtained
by Ibis method do iol contain many suspended
solids (200-300 NTU> and arc particularly well
protected from oxidation
Grape lempcrainre mnsl be maintained below
I5'C during maceration Circulating cold fluid
through the jacket or cooling-coil to refrigerate
tbc i.r-.k directly is lot possible without agitating
the grapes, but this operation Is nol recommended
because it pioniotes the formation of suspended
solids and the cxtraclkHi of phenolic compounds
The crushed grapes may also be cooled wiih a tube
heat exchanger. This process requires considerable
cooling capacity and draws tic grapes through
small-diameter piping with many bends. Increased
production of suspended solids may result Another
method consist of incorporating liquid carbon
dioxide into Ihc grape crop during Hlling al the
outlet of tbc must pump The grapes arc cooled
without a supplemental mechanical treatment In
addition, the oxygen dissolved in Ihc musl during
crushing Is eliminated by the flaw of COj. The
grapes arc also transferred lo the lank under an
men atmosphere It requires 0.8 kg of CO; to cool
120 kg of destemmed grapes by l*C.
Maceration times vary from 12 to 20 hours,
depending on the winery- At controlled temper-
atures 1 10- 15 C) and in ihc absence of oxygen.
Ibis time period seems lo permit a suitable cxtrac-
tioa of aromatic compounds from the skins without
Ibc risk of significant dissolution of phenolic com-
pounds.
Pressing macerated grapes docs not pose any
particular problems Due lo Ihc destruction of the
pec He structure by grape en /vines, lie grapes can
he pressed al low pressures with only one lo
two crumblings necessary. The first pressing is
immediately reincorporated with Ihc free run jnicc.
The final pressings arc left separate The decision
lo incorporate tbem with Ihc frcc nil and olhcr
pressings Is made alter clarification.
Skin maceration result in a decrease in musl
acidity and an increase in pH (Table 13.7). These
changes arc linked lo Ihc liberation of potassium
fa>m Ihc skins and the resulting partial salification
of tartaric ackl The acidity can decrease by as
much as I - 1 5 g/l I expressed as H;SOj ). bul the
degree of these changes depends on the variety
and the lemur. Acidity and pH often vary less in
Chardonnay than ii other varieties such as While
Crcnacbc(Chcynicr«7rt/.. 1989).
Handbook or Etiology: The Microbiology of Wive anil Vlnifkaiions
Table 1J.7.
(l98Skuvc
lofllKlkX of friE-icr-tnuli.il ihc
si.i(I)i*HiunlKu ." </.. 19B0)
alkm on total aim Jcutily bcfiin
: ckiri6calnD
Variety
ConimP
Pfc-lcimcnutioa man
<«■,»»
li-:il acidity pH
(g/1 H)10.)
Tunlicklkv pH
(aVI H,SO.)
Dual too
(a bouM
3.03
3. IS
3-13
405
400
4. 75
530
3.3S
3.3S
333
3.30
Table I3.K Influcatc of *■ IS-hnur pR-tciaicniuion i
mu.i phenol* compounds (IJubounlKU rl .rf . I0K0)
Table IJ.'l. Influence of pic-fcinicniatiii
Co.|ou*U I liul-iiinlKii .! <*.. I9S0-I
Variety
In
ednlc
pfcuiap
Civ-fc™
1 .aCCnitk.ll
OD 2S0
iJl.l
,.■1, ...ii
i poind
OD280
Poet
mlk compound
iadcx
indct
Suvfci
..-. 1
0.7
3.3
73
3.3
Souv^a,
■■3
OJ
3.3
8.1
4.7
• „,.,.-,,
■■4
50
3.3
SB
i.»
Willi respect to whole grape picssiig . skin Mac-
eration also punt Acs ai increase in optic density
al 280 in anil the pbciolic compound lndc\
(Table 13.8) bit the diffcrciccs observed ii wines
arc less marked tTablc 13.9* and tic optic density
al 280 nn rcntaiis well under 10— the upper limit
generally accepted for while wiies.
Maceration increases the amiio acid conccitra-
tion In juice, resulting in an imptoved fermen-
tation speed, which Is often observed in prac-
tice. Macerated grapes also produce jiicc aid
wine thai is richer in ncnlral polysaccharides
(Tabic 13.10) and proteins than picsscd whole
clusters Wines made from macerated grapes
Table 13.10. Influence at pic-fern
on (Will polysaccharide coaccnttw
tTAibounUcufT.rf.. I9K0)
pK-icimcnUiki
White WiiiiiiKikiu-
41"
require higher hcikniK' concentrations to be sta-
bilized (Volume 2. Sec lion 662).
Skin maceration makes Ibc most of the aronialic
potential of Ibc grapes and in general it signifi-
cantly enhances varietal aroma without increasing
bcrroccoas flavors. In Muscat wines, these sensory
differences can be analytically interpreted by rvca-
suring lav and bound Icrpcac alcohols Baumcs
el al. < 19891 observed increases of 576-742 p.g/1
in free Icrpcncsand 689-1010 u.g/1 in bound ter-
renes. Measuring 4- mcrcapu>4- methyl pcnttn-2-
one in Sauvignon wines also indKakrs Ibc obvious
iolc of maceration in the varietal aroma of this
variety in wines. The 10 ng/l concentration in the
control wine almost doubles to 18 ng/l in wine
made from macerated grapes.
13.3.6 Cryosclccfion and
Su p ra ct I rac t ion
Chauvct elal (1986) initially developed these
techniques to improve the qualify of juice intended
for sweet wincmaking (Section 14.2.4b). bat they
arc also of interest for dry white wincmaking. The
process consist of cooling whole grape clusters
in small crates for 20 hours or so in a walk-in
freezer at a temperature of —2 to —VC Two phe-
nomena— cryosclcclion and sapracxtrac lion — arc
at Ihc origin of these changes in juice composition
observed with respect b> traditional pressing
Cryosclcetion corresponds with pressing grapes
at low temperature. Only the sweetest grapes
remain unfrozen and release their juke A quality
jaicc is obtained, the volume increasingly limited
as the Km pen lure rs lowered After (hawing, a sec-
ond pressing rcleascsa lower quality juice, coming
from grapes with a lower sugar concentration.
Supracx traction corresponds to pressing whole
grapes alter they have been (hawed The freezing
and thawing of the skins and lower epidermal lay-
ers results in modifications in tissue uluastructurc.
In certain aspect, it produces an cffccl compara-
ble to skin maceration Notably, aromas and aronta
precursors arc released more easily from the grape
Extraction of skin phenolic compounds is. how-
ever, lower than with skin maceration and even
immediate whole grape pressing (Ollivicr. 1987).
Sugar ex traction from the skins Is also increased by
0..i ■( ></« potential alcohol during sn pracx traction
Despite its slowness and elevated cost, supracx-
traction promotes the aromatic expression of cer-
tain noble white varieties.
13.4 PROTECTING JUICE
FROM OXIDATION
13.4.1 Current Techniques
Oxygen is often said to be the enemy of while
wines. In fact, except for rancio wines, whose fla-
vor results from intense oxidation during produc-
tion, while wines arc protected from oxygen (or at
least from oxidative phenomena) during (he wine-
making process and maturation. These precautions
arc cikcn to prolcci ibc fruity aromas of young
wine aid to avoid browning. They also promote
(be later development of a reduction bonquct in
premium wines during bottling aging.
The oxidation of substances in while wine can
occur at any time daring wincmaking While the
need to protect white wine from oxidation alter
fermentation Is generally accepted, protecting must
from oxidation Is not unanimously considered
necessary
Most wiucmakcrs prefer limiting air contact
■Mill crushed grapes and white juice as much
as possible. An adapted sulfar dioxide addition
to jaicc blocks the enzymatic oxidation of phe-
nolic compounds. This philosophy is based on
empirical observation: juices of many grape vari-
eties must conserve a green color during the prc-
fcraicatation phase to be transformed into fruity
white wines Oxidation phenomena must conse-
quently be avoided as much as possible.
Other otologists believe, on the contrary, (hat
musts loo well protected from oxygen give rise to
wines that are much more sensitive to oxidation
Parlbcmiore. pressing experiment carried onl in
an air-free environment have shown that these
wines brown quicker in conttct with air than wines
made from traditionally pressed grapes (Marlinicrc
and Sapts. 1967). In addition, these wines arc mote
difficult to stabilize with sulfur dioxide
-■X
Handbook of linokigy: The Microbiology of Wine and Vindications
Mailer- Spain ( 1977) was the liiM to contest Ihc
■ccd Id sulfite while juice befotc alcoholic fcr-
mentation. His research clearly showed ihai adding
pare oxygen lo non-sulfiicd juice before clarifi-
cation improves Ihc stability of white wine color
wilhonl producing oxidation- type flaws. This pn>
cess. called bypcruxidalioa or hypcroxygenation.
consists of oxidizing juice polyphenols lo precip-
itate Ihem during clarifkration and eliminate them
daring alcoholic fermentation
Mast oxldalion results in a varying degree of
color stabilization of white wines, depending on
variety (Schneider. 1989: Chcynicr ttal.. 1989.
1990: Monlonnct el nl . 1990). Hypciuxygcnatiot
has aLso been ascd successfully on an experimen-
tal basis to discolor and improve the quality »'
second pressing Pinot Noir and Mcnnicr jnicc in
Champagne (B Link and Vatidc. 1989) The impact
of Ibis krehnkjac on Ihc aiomalic quality of the
wine lanes according H> the variety and Ihc tast-
ing panel. The effect on aroma is sometimes judged
favorable omcnlral for Alsatian and German vari-
eties. Chardonnay and Chassclas (Fabre. 1988.
Miller- Spilth. 1988: Chcynicr el ill.. 1989). Nit
hyprroxygc nation, or simply not protecting musts
Irom oxidation, considerably affects Ihc aroma of
Sanvignon B tine (Dnbourdicu and Lavignc. 1990).
The 4-mcthyl-4-mciraplopcntan-2-onc concentra-
tion decreases when Ihc must is less well protected
from oxidation (Figure 13.7). The mechanisms of
this phenomenon will he discussed in the nexi
Section ( 13.42). Jnicc oxidation also decreases the
aromatic intensity of other varieties sac h as Scmil-
ktn and Pclil and Gros Manscig. whose aromatic
similarity to Sanvignon is dae to ihc pariKipa-
tion of sulfur-containing compounds i Volume 2.
Chapter 7). The best Chardonnay wines also seem
lo be made by limiting juice oxidation Boultoa
eiiil. (1995) shared Ibis opinion, believing that
juice hy peroxidation harms Ihc varietal atona of
13.4.2 Mccbu
»f Juice Oxidation
Oxygen consumption in juice Is essentially
dae to the enzymatic oxidation of phenolic
compounds Two oxidases (Section 116.2) arc
involved (Duhcrncl and Rihdrcau-Cayon. 1973.
I
I'ilt IJ.7. InftUcnc
cent rat ion in Swv
gnc. IP90)
1974): tyrosinase in healthy grapes: and ticcasc
ftoai BtnryHs cineiva. which only exists ii juice
ftoni boiiyti/cd grapes. Laccase activity can be
spccilically measured to evaluate grape health
analytically (Sections 10.6.6 aid 13.2.1).
The substrates of tyrosinase arc almosl exclu-
sively cinnanK acids and their esters with tar-
taric acid (cafEtric and conmarK acids). It trans-
forms caflaric acid into quiaoncs (Section 11.62.
Figure 11. I2).Thcsc oxidation reactions arc cxire-
mcly quick. The oxygen consumption speed
in jnicc. when fiist put into contact with air.
can exceed 2 mg/l/min whereas it is aroand
I -2 mg/l/day in wine A certain degree of oxida-
tion in jnicc inevitably resale during while wine-
making befotc protection by salfnr dioxide. The
decrease in Ihc speed of oxygen consamptron dur-
ing successive oxygen saturations is caused much
more by the depletion of the substrate, caftaric
acid, than by the inhibitivc effect of the oxida-
tion products formed Adding caftaric acid re-
establishes the initial consumption rale ( Moutouncl
enil.. 1990) bat laccase is capable of catalyzing the
White Wine making
411
Cob«b»«l miM ( — # — > ,
uund -■' <*.. iWi.i
■din of j bcilhy
»f o*c «Kh IMS
i (—0—)- <MoU-
oxidation of a kirgc variety of substances. It nol
only acts rapidly toil als i continues ncr a much
longer time period (Figure I."' ■:- :.
The i|uiDoK formed from caftaric acid has
several |»T»sihle destinations:
I. iH.ui combine with quinonc imps in juice — in
particular, glutathione (Section 1162. Figure
1113). a highly reductive Iripcpudc with a free
sulfhydryl group, found in concentrations up
lo III) mg/kg in certain varieties. Tie prod-
uct formed is S-glntathionyl-2-caftiric acid,
initially called the Grape Reaction Product
(GRPHCncynicrff «/. 1986) Th P. oxidation-
reduction reaction regenerates the o-diphcnol
function Tyrosinase has no action on this glu-
ttlnionc derivative, but it can be oxidized by
lactase. The caftiric acid quinonc can also com-
bine with other juke-reducing agents, such as
ascorbic acid The coupled oxNIation regen-
ctules cafbiric acid As long as glutathione
(GSH) and ascorbK acid concentrations are
elevated, consumption of must oxygen docs
not result in quinonc accumulation or juice
browning
2. The caftaric acid quinonc can also cuter into
coupled oxidations with llavonoiils as well as
GRP. Fktvonoid and GRP quinoncs arc formed
These then combine with glutathione to form
di-S-gluuthionyl caftaric orGRP2.
3. The caftaric acid quinonc is capable of con-
densing with i-diphenoh. first with caftaric
acid The color and Insolubility of the products
formed increases with their degree of condensa-
tion Flavoaol qninoncs also enter into conden-
sation reactions, resulting in strongly colored
and later insoluble prodnce..
The oxygen consumption speed of while juice
and the nature of the products formed therefore
depends on initial concentrations of caftiric acid,
glutathione, ascorbic acid and fiavonoRls in juke
Variety and. nun! likely, grape maturation con-
ditions influence the proportion of caftaric acid
and glutathione found in juke The differences
in reaction of two juices to the presence of oxy-
gen arc Illustrated in Figure 1.19 (Rigaud etal.,
I990| Colombard juKc is rich in glutathione and
ascorbic acid, and let oxKUition frees few caftark
acid quinoncs. In an Initial phase, as caftaric acid
Is formed, il Is reduced by ascorbic acid When
ascorbK acid is depleted, caftaric acid combines
wild glutathione to form GRP. which accumu-
lates Juke color changes from green to beige
There is no browning and few reactions coupled
with the flavonoNls. when quinoncs arc not avail-
able. Ugn) Blanc juke contains relatively little
glutathione and no ascorbic acid: il behaves dif-
ferently Oxygen consumption is more rapid and
a large quantity of quinoncs arc formed, result-
ing In a pronounced browning of the juke with
a continued increase in the orange nuance The
cafciric acid quinonc enters Into reactions coupled
with flavouoids and GRP. whose concentration
decreases.
The oxidation phenomena linked lo the prop-
erties of tyrosinase and laccasc are rapid and arc
present as early as crushing and pressing Unsul-
lied jukes exposed lo air consume a variable
quantity of oxygen according to their caftaric acid
and flavonotd concentrations
Handbook of Etiology: The Microbiology of Wine and VnifkaboBS
Fiii I."' Oiktaikin of (a) Conmbaid and (hi Itpjii
• tartaric acid: ■ GRP: *, m(i» acfct: * moiMticj
The effects of jik'c hypcroxygenation on ihc
strbility of while wine color are variable, due to
(be existence of several icaclional mcchanp>ms.
If color stabilization hy oxidation is sought,
other jir'c components must not be adversely
aliened — especially aroma. The difficulty of
obtaining fruity Sauvlgnoa wines from oxidized
or brown-oraigc iliiicd Jakes remained ■■ex-
plained for a king Unte. bur il is tow known
thai various thiols, playing a role in ihc Sauvi-
gnon arunta. are very sensitive to oxidation. They
Knd io produce drsuliiir bonds in Ihc presence
of oxygen and. mote importantly, combine very
rapidly with quinoncs Snlliting prolccis these aio-
mas by blocking quinonc formation This treatment
R effective, even if implemented on partially oxi-
dized jnicc. since it reduces Inc quinoncs (Darrict.
1993).
13.43 Techniques for Protecting Juice
from Oxidation
The wincmakcr can implement various comple-
mentary tcchnK|ucs to limit juke oxidation:
• snliiting— antioxidant and antioxidasK activity:
* adding ascorbtc acid— - 'antioxidant effect:
• cooling grapes and musts u slow oxidation
reactions.
• healing musts at 60'C for several minutes to
destroy oxidases:
• handling grapes in the absence of air u limit the
dissolution of oxygen:
• clurification to climinalc a portion of the tyrast-
nasc activity associaled with solids and to limit
the oxidasic activity of juice.
Sultiting is the fiiM. most simple and effective
method of protecting jiice from oxidation. To
destroy tyrosinase. SO mg/l of sulfur dioxide
per liter must be added Ri the juice. If the
giupes arc healthy, this addition definitively blocks
enzymatic oxidation mechanisms Sultiting must
be carried out with higher concentrations to
inactivate tyiusinase in highly colored press jukes,
containing quinoncs.
The entire snlfur dioxide addition should be
made at the same lime It should be fully homog-
enized into the juice. Snlfiting at concentrations
below 50 mg/l sbonld be avoided, since this only
delays oxidation phenomena and jnicc brown-
ing, in lime, all of Inc oxygen contained in the
jnkc rs consumed The worst method consist, of
White WiiiiiiKikiu-
progressively adding small quantities or SO?. The
total amount of oxygen consumed in these condi-
tions by 11 juice in contact with air Is greater than
in an unsulfited juice. The final color of the two
jukes, in Ktiis of oxidation, is practically equal.
Sulfiting grapes promotes the extraction of skin
phenolic compounds Protecting with ascorbic acid
( 10 gJkl) dors not have this disadvantage. t>m this
powerful reducing agent is not anfioxktasic Like
low sulfur dioxide concentrations, it only limits
browning by reducing quinoncs but docs not limit
oxygen consumption. Grapes must be in limited
contact with oxygen when using ascorbic ackl For
example, they should be handled in the presence of
dry ice when filling maceration tanks or pneumatic
presses Jukes not protected by sulfur dioxide
should not be allowed to stagnate in contact with
air in press pans during pressing. Their large
surface area promotes rapid oxidation.
Cooling grapes and juices is extremely effective
in slowing juke oxidation, and il should be
used systematically. Oxygen consumption is three
limes faster at 30 C than at I2C (Dubcrnet and
Ritercau-Gayon. 1974). Cooling with liquid CO;
while rilling tanks and presses associates handling
the harvest in an inert atmosphere as soon as juice
appears with the effects of cooling.
Cryoex traction or supracxtraclion (pressing
whole grapes at temperatures below O'C) consider-
ably limits oxidation. This technique enhances the
fruity character of dry white wines, compared with
pressing at ambient Krmperaturcs. Not only arc aro-
mas and aroma piccursors freed by the freeing
and thawing of the skins, but oxidative phenomena
arc also limited during pressing
Clarification (Section 13 5) limiKoxidasic activ-
ity but docs not prevent juKc from browning.
A sufficient soluble tyrosinase activity remains
in fresh juice unprotected from oxygen, allow-
ing rapid browning. Clarification R a means
of eliminating oxidation products— in particu-
lar, condensed llavouokls formed during coupled
oxidations.
Healing juke theoretically destroys oxidases but
it must occurquickly after extraction. The healing
process must also he rapid It is rarely used in
practice.
13.5 CLARIFICATION
13.5.1 Formation and Composition
of Suspended Solids and l,ccs
Freshly extracted grape juke is more or less tur-
bid. It contains suspended solids of diverse ori-
gin: earth, skin and stem fragments, cellular debris
from grape pulp, insoluble residues from vine-
yard treatment products, ck Macromolcculcs in
solution or in the course of precipitating are also
involved in juice turbidity'. Among them, grape
peclic substances play an essential role (Volume 2.
Section 3.6). With rotted grapes, juke turbidity is
also caused by the presence of polysaccharides,
especially (l~3:l-o)-jl-i>glucanc. produced by
Botrytis cinerea in the berry. A few milligrams of
Ibis substances is enough to provoke serious clar-
ification difficulties (Volume 2. Sections 3 7 and
1 13.2). These glueidie maciomolcculcs influence
juke turbidity through the Tyndal effect (Vol-
ume 2. Section 9.12). Acting as protective col-
loids t\blumc2. Section 9.4). they also hinder
clarification by limiting or blocking particle floc-
cufcifion and sedimentation phenomena as well as
clogging filter surfaces Natural grape pectinascs
tor those added by the winenutker) acting on the
colloidal structure of the jukc facilitate natural set-
tling After several hours, the juice separates into
two phases: a nunc or less opalescent clear juice
and a deposit varying in Ihkkucss. The latter con-
tains different colored successive strata: greenish
brown in the lower portion of the deposit and
green to light beige in the upper portion. Souk
w inc makers distinguish between the heavy deposit
that forms first during natural settling and the
light deposit that accumulates more slowly Clar-
ification consist, of separating (by racking, for
example) the clear juice from the Ices before alco-
holic fermentafion.
The quantify of Ices formed during juice extrac-
tion and the speed of sedimentation speed depend
on variety, grape disease status, maturity and espe-
cially wine making methods (crushing, draining,
pressing, etc.) (Section 13 J).
In normal conditions, juke lurbtdity generally
decreases during grape maturation (Hadjlnicolaou.
422
Handbook of Etiology: The Microbiology °f Wiie anil Vinifications
1981). This evolution results from Ihc hydroly-
sis or pcciic substances in Ihc beny by pcciic
enzymes of Ihc grape (cndopolygalaciuronasc and
pcclln esterase). In «lty weather conditions, ihc
grape remains pnlpy and ihc Juice Is mote diffi-
cull 10 extract and clarify, due lo a lack or pcciic
activiry. Towards ihc end or maturity, the solu-
ble acid polysaccharide (pectin) conceiiraiion in
Juke generally evolves In parallel wiln jnicc Inr-
bhliiy This is a good potential indicator of clanfi-
calion (Robertson. 1979; Dubourdkn el til.. 1981:
Olllvicr. 1987) When the pcclli concentration In
juke continu;dly towers during uialuralkn. the
Juke b. generally easy to clarify In the opposite
case, clarification is more difficult and exogenous
pec Ik cn/ymes must he used
High levels of mi In the harvest increase juke
turbidity and make clarifkallon difficult, due to
the protective colloidal effect of glncan prodnccd
by BtXrytis. A low concentration of mi (less thai
5'£ 1 tends to facilitate Juke clarification, due k>
a pcctlnasc activity in conciminalcd grapes that Is
■early 100 times higher than in healthy grapes.
Jikc extraction methods have a prime influence
oa the formation of suspended solids. Slow batch
pressing while minimiflig crumbling obtains the
clearest juices (Section 13 3 J).
The exact physical structure and chemical com-
position of Ices remain unknown. They an: made
■p of varying sized particles of less than 2 mm.
They arc generally observed fc> contain essen-
tially insoluble polysaccharKlcs (cellulose, hemi-
cellulosc. peclk matter) and relatively few nitro-
gen compounds, essentially insoluble proteins not
■lili/ablc by ycasis (Table 13.11). They aLso con-
tain mineral salts and a significant amount of
lipids— most likely from cellular membranes This
lipidic fraction contains a sllghlly higher propor-
tion of unsaturated than saturated fatty acids The
principal fatly acids arc linolcic acid (CI82),
palmitic acid (CI6:0) and oleic actd <CI8:I)
(Table 13.12). They citcr inlo the composiiion of
membrane phospholipids of grape cells baiasmall
proportion aLso exists In a free stale, adsorbed to
lees particles (Lavigic. 1996). These panicles an:
definitely utiliaiblc by yeasts
Tub!.- Ull Ixcicompnilbni
die a <t.. 1994)
%)(Akun-
Compose*
■■
ToiaI ■cm a 1 pulyucthii rides
Aiha
Acid foh'MKchiridci
Tut ill
71.9
20
55
52
7.8
93
Tulle 1X12 TnUl folly acid
lb* of kc% (': ) (Akiaodic a
corapmi-
<*.. 1994)
1 ti m |-nCM 3S
Laurie* jcidCI2fl
Pllmkic acid C 16:0
ftriaiokk «idCI0:l
Sieark acid C IH;D
Oleic- at id CIS: 1
Ltnokk»eklCIB:2
8J
25 Jl
55
222
250
13.5.2 Influence of Clarification
on Dry While Wine
Composition
Wine makers have long observed an improvement
ii dry white wine quality resulting from proper
)nkc clarification. Clarifying juKc improves wine
quality more dramatically, when compared with
the unc landed juice, when there is a high con-
centration of suspended solids in (he Initial JuKe.
Wines made from juices containing too many sus-
pended solids have heavy, green aromas and bitter
tasks. They are also morc colored, richer in phe-
nolic compounds and their color is less stable to
oxidation. At the end of fermentation, they often
contain reduction odors, more or less difficult lo
eliminate by aeration and racking. Inversely, the
fruity character of the variety is more distinct and
stable in wines made from clear juice. Some of
these empirical observations based on Gistlng have
been interpreted by analysis
Since Ihc 1960s, clarification has been known
to Improve the fermentation aromas of dry while
wines (Trowcll and Guymon. 1963; Bcrtrand.
1968; Ribcrcan-Gayot rial.. 1975). Wines made
from clarified juices have lower concentrations
White Wine making
423
of heavy-odor higher alcohols, aid higher cob-
ccntratons of ethyl esters of fatly acid* and
bigber-akohol acetates, which have more pleasant
Cfcirificalion also Hints the concentration of
C, alcohols in wines (Table 13.13) (Dubounlicn
rtof., I9B0). Before femicitilion. juices csscn-
iLilly contain C b aldehydes (hcxanal. aj-3-hexcnal
and .7i,iiiO*he\enall fomicd by en /ymalic oxida-
lioi of linolcnic and linoleic acid during pressing.
The ilc tiilcd ntcchanisms of these reactions ate
described in Section 1 1 6.2. These compounds are
not very soluble in juice and mosl likely remain
paflially associafcd with the must deposit Dur-
ing alcoholic fermentation, they are systematically
icdnccd him the corresponding akohoLs by the
yeast and pass into solution in Ihc wine. The elimi-
nation of musl Ices therefore helps to lower vegetal
an>mas in dry while wines The influence of clar-
ilication increases when pressing and handling of
grapes become more brutal and maturity decreases
Initial researeb demonstrating Ihc etiological
value of clarification generally reported the
perecnlagc of clear JuKe obtained by different
clarification methods but rarely specified the
turbidity of the clarified juices. Yet relatively small
turbidity variations haw been shown to have a
determinant influence on alcoholic fermentation
kinetics and wine composition.
More recent research has focused on the
influence of the degree of clarilicalion on the
production of off-odor sulfur-conlaining com-
pounds during alcoholK fermentation and the
more or less stable reduction ofT-odors that result
(Lavigne-Cmcgc. 1996: Lavigne and Dubourdieu.
1997) (Volume 2. Section 13.62). Some heavy
sulfur-conlaining compounds ptoduccd by yeasts
increase jnkc turbidity (Table 13.14). Yel. con-
sidering the perception threshold and olfactory
descriptor of Ihcsc various compoundslVoluBK 2.
Section 13.62). only methionol lmcthylthio-3-
propunol-1). with a dKigiceahlc odor of cooked
cabbage. Is significantly involved in the off-odor
observed when jnicc Intbkliry exceeds 250 NTU
Methionol Is stable in wine and it cannot be elim-
inated by racking and aeration This troublesome
Tuhfc- 1X13. InlUcact
i.( .-.
ml fhiriicsiun <■
» Ci akohoh com
-cniaibm
(hciiool-f hcictmb) 1
(Dubounlku ei .i-
1980)
Mum iranrm
\:u,i
tuibidny (NIT)
C, alcohols i* «
,,.-.,,,■.1,
Non-.tiilhcd mini
400
20
Clarified nun
200
in
lea
ND»
2.1
tihcicd lew
8
09
Tabic 13.11.
(Uvipae-Ca
cge. 1990)
1 tuibkliy
if heavy uiltur-conulnino cumpouiub I
\ (|ig/l)
aUNtuca
2-Mcnapa-clkinul
Met hyl-2-icicihydM>-thiophc none
2-Nfcihylhiu-ci'hanol
fthyl mcihyih»>-3-fiopa*..itc
Mcitavkhn-3-rmriiot-] acetate
Mcife\hhu->pmp*Kil-l (meihD
Mei»yhhk>-4-buu»ol-2
Dlmcihyl-uilfovklc
Bcn/Whiimie
>Meihvibbfinp«onic acid
Handbook of Etiology: The Microbiology of Wine anil Vmiikaliuaa
Pit; I J. I». unuc.
lUvigne. 1990)
of lit.' In vofalilc uilfclr-eollUining! Cumfouatb by yei
consequence of insufficient clarilnration Is defini-
tive The degree of juice turbidity must therefore
be adjusted wild prcc isWn lo maintain Ibc aromatic
finesse of dry while wine.
Increased mcthionol fonuatooa in insultrc lenity
clarified jniccs cannot be interpreted as a methion-
ine cnrichikcnt of juice by Ices. In fact, the tecs do
■of contain soluble amino acids and the acid pn>
leasc is incapable of freeing them by hydiolyzing
proteins in thcjuKc. Furthermore, even prolonged
contact between the juice and Ices docs not result
in an inenMsed concentration of amino acids.
Some experiments demonstralc Ibc role of the
lipid ic fraction of the Ices in the excessive produc-
tion of mcthionol during alcoholic fermentation.
This fraction most likely promotes Ibc incorpora-
tion of methionine in yeasts which is transformed
inu mcthionol according to the Bhrlkh rcaciion
(Sec lion 2 4.li The lipidic fraction of the Ices has
also been shown lo be involved in limiting acetic
ackl production by ycaso. The practical conse-
quences of these phenomena arc obvious, if juice
turbidity is loo low. an insufficient concentration
of long-chain unsaturated tatty acids risks induc-
ing excessive production of ace Ik ackl by yeasb.
If the turbidity is too high (greater than 250 NTU>.
an excess of these same fairy' acids promotes exces-
sive mclhionol formation
Jnicc lurbhlily also influences the production
of volatile sulfur-cotiaining compounds by yeasts
(Volume 2. Section 8.621: H ? S. methanethiol.
diiKthyl disiillide. carbon disullidc. Wine made
from juice fc mien led at 5I0NTU has a pro-
nounced reduction off- odor (Figure 13.10). In the
same wine fcmienicd at 270 NTU. dimethyl disul-
fide Is present in conceniralious above Ibc sensory
threshold but decs not produce a redaction charac-
terized flaw However, when the methanethiol con-
centration exceeds its sensory threshold (OJ u.g/1).
which is the case with a less clarifcd must, the
quality of wine aioma is immcdiaicly lowered.
Methanethiol Ihns pkrys a major role in reduction
flaws in dry while wines following insuffkxnt clar-
ification. Snlfnrand certain pcslicidcrcsidKsin the
Ices explain the effect of juice turbidity on the for-
mation of light sulfur-containing compounds.
At equal turbidity levels, sulliting also influences
the production of heavy and light volatile sulfur-
coutuinitg compounds by yeasts. Mcthionol and
hydrogen sulfide, for example, increase greatly
with the sulfur dioxide concentration ised This
concentration masl not exceed 5 g/bl. added in
totality as soon as the jnicc is received. The
tree sulfur dioxide concentration should not be
adjusted before alcoholic fermentation, or during
or after clarifKation. since this practice does not
provide greater oxidation protection for the jnicc
and systematically promotes the pioduciion of
sulfur-coaiaining compounds by yeasts
The role of elirilication in fmity varietal aro-
mas is not well known and wiacmakcrs* observa-
tions in this respect are sometimes con trad ictny.
White Wine making
425
Insuflic iciitly clarified juices, especially ihi«sc con-
taining insoluble products of phenolic compound
oxidation can produce wines wilh decreased vari-
etal aromas On Ihc contrary, ovcrctarilicalioa (less
than 50 NTT) also decreases the froity aroma
of dry while wines. This phenomenon has been
observed with Muscat. Chardonnay. Sauvignon.
Semi lion. Mansengs. clc. It is exacerbated by dif-
ficult fcrnicitalion conditions, excessively slaw
fermentations with increased volatile acidify pro-
d iic (ion. The varietal aionia or wines made from
excessively clarilicd Juices is sometimes masked
by an artificial, banana, amylic or soapy aroma,
linked lo the presence of a significant quantity of
esters.
Il isdiflicult 10 recommend an optimum Inrbidily
that is valid for all varieties. A range between
ICO aid 250 MTU ■ generally used, since it Is
a suitable compromise between a good alcoholic
fermentation and aromatic finesse.
13.5.3 Effect of Clarification on
Fermentation Kinetics
Slow and stuck fcrmenttlions of dry while wines
are well known consequences of clarification
This phenomenon, varying in intensity according
lo jnKc composition and clarification methods,
has Incited much research and been interpreted
differently in the past
Clarification depletes mnsf microflora. Inoculat-
ing juke with yeasis alter clarification has k>ng
been known u basvn the initiation of fermenta-
tion but does nol noticeably change lis duration
or the quantity of residua! sugar present upon its
completion (Rlbcrcau-Gayon and Ribcrcan-CTayon.
1954). Clarification Is nol simply an unycasting
of Ihc j nice.
A vahcry of physical actions also contribuic to
the stimulating effect of alcoholic fermentation by
suspended solids By providing nnckation sites
for gas bubbles, suspended solid particles have
been suggested k> promote the elimination of COi
from the fcrmcnlation medium— this limiting its
Inhlbltivc clfect on ycasls. This effect is very
limited at lank pressures found in dry whit:
wincmaking. Suspended solids arc also though!
lo promote ycasl multiplication by serving ;
support. Asa matter of fact, the addition of vt
supports such as infusorial earth (Schandcrl. 1959).
bcmomic (Groat and Oagh. 1978) and ccllaktsc
(Lame el til. 1985) improve the fermentation
speeds of severely clarilicd musts bat. a! eqnal
Inrbidily. docs not have the same effect as fresh
suspended solids
Suspended solKLs also supply yeasts wilh nutri-
tional elements and adsorb certain metabolic
Inhibitors. In fact, these two cficcfc arc related
and significant The lipid fraction of saspended
solids provides the principal nutritional supply
I Section 1.15. 1)— in particular, long chain unsat-
urated tally acids (Cih) that lie yeast can incorpo-
rate into its own membrane phospholipids Sugar
and amino acid transport systems across Ihc yeast
membrane arc conscqacntly improved Due lo their
hydrophobic lipid content, suspended solids arc
capable of adsorbing loxK inhibilivc fatty acids
freed in the juice during akoholic fermentation
(C s . C|„. C|>). The combination of these two
c(Tccts( lipid ic nutrition and toxic rally acid adsorp-
tion) produces a survival factor effect for ycasK
(Section 35.2) lOllivicr el «u\. 1987; Alexandre
rial.. 1994).
In Table 13.15 and Figure 13. II. seven loKcor-
responding lo different clarification levels were
constituted from fresh Muscadclk juice. Lowering
juice turbidity prolongs alcoholic fermentation In
the case of the most clarified juice (lot C). this can
lead to a stuck fermentation Supplementing lol C
wilh cither colloids or soluble maciomolcculcs re-
establishes fermentation conditions similar U kit F.
the least clarified. In the final stage of fermentation .
the colloids or soluble macromokcuks act as sur-
vival factors (Section 35.2). maintaining a higher
viable population (Figure 13.11). The adsorption
of Cg and C ti , Tally acids by saspended solids is
easily demonstrated in a hydroalcohoDc medium
model in the laboratory (Table 13.161 In the same
conditions, they have a fixating capacity similar
to a 05 g/l commercial preparation of yeast hulls
IScclion 3.6.2) (Lafon-Lafourtadc el al., 1979).
Slow akoholic fermentations observed in extre-
mely clarified juices arc always accompanied
by Increased acetic and conccn nations in wines
Handbook of Enotogy: The Microbiology of Witc anil Vindications
Tuhlc 1.1- IS.
1*1 coaaplcln
nluc*ccofihcciilli>kbl
nofakoholic fcnncMaik
o*p<uli»n nl .linlicil Muwidclk ■■
»<Olii.icrrt«'.. 1087)
IUH inditlticnl ..m(la»n.
nihe kapih
Comfo^ol
TitiiiacnV
CS CC CE
CN CN + HF
CN + SM
Tu*>iday I.NTU)
Piutei* <mg/l)
Ti«jl folvtio tunic < ■p/ll
leoph of akobolk fcracntaikin (day*)
:■.»■■- ii b . . - 1 1 ■-■..,•..! xi ihc end of k 1 Mr. -i . i ii ■
Tabic 13 IA. Influence ofibe amount of hare ton
OUfallV ItilldMliplua Imp/l)allci24 hiiun of i.
(OlIhktrfHf.. 1987)
Fift I J. II. laButoic of hiibi lurhila. on \c.M pop-
ular io* duriag akoholk- fen»enialkin: CS. cold Ki-
lled mow: CC. (oik clarificrtba: CN • SM. ccn-
niliwtd aiV • Mkfck iiuimiiiok.-ulc: CE. cold
MHlia^ - (n. r, nun ktlirili.-j* ion iCN.it ■IriliiptdmiW;
CN -t HF. cenrinmed «nuu - collokbl ha*
(Seclkn 2.3.4). The degree of ihis cfTcct varies
depending on Ihc yeast strain used (Tabic 13.17).
Il is intensified by hiyh sniiar concentrations
13.5.4 Clarification Methods
The mosl simple and effective jilcc clarification
ncthod is nalniul settling — the natural settling of
Tnfcfc 1317. Influence of muM luibkliiy on
acidity In wine* ia'1 H,SO.) foi two Sit vbt»
Turbidiy
VLI
fXJS
500
0.10
a. u
250
0.12
0.14
100
n 20
0JD
SO
n m
OiS
saspended solids followed by a careful racking,
f-rcc run and ihc lirsi pressing on Ihc one
hand and subsequent pressings on Ihc other are
collected separately in proportionally wide tanks,
preferentially by gravity, and are Ihcn sulfilcd.
Soot after pressing, an initial cLinlkation should
be carried oul to separate Ihc gross Ices already
fomed. The snpcrnatanl is pumped from Ihc top
of Ihc tank. The hose is progressively lowered
into the tank while Ihc surface of the liquid (well
illuminated by a hand-held lamp! is observed. The
operation is slopped as soon as the hose ncais the
Ices.
White Wi tic making
427
The Ices from the last pressings of juice are often
blown Even after filtration. Ihc resulting juice
should km be blended with Ihc lice run: it should
he fermented separately. The gross tecs from the
first pressings arc effectively clarified by filtration,
which shouU be carried oul as soon as passibtc
since this deposit Is very fcrnicntiblc. The fillrukr
cun be blended with jnice which has already under-
gone an initial racking.
The jnice should be cooled lo5-IO'C before the
second sedimentation « slow ihc initiation of alco-
holic fermentation and limit oxidation. Its dura-
tion varies, depending on the jnKe. With certain
pressing nKlhods. the second racking is sometimes
not necessary because Ihc juice Is already sufli-
cicnlly clear.
Precisely adjusting clarification levels requires
Ihc use of a ncpaclomcicr. This device should
be standard equipment in every winery thai pro-
duces dry while wiic. A direct nephelometric
measurement is much more rapid, convenient and
accurate than determining Ihc percentage of par-
ticles in conK centrifuge lubes, as recommended
in some works. Figure 13.12 gives an example
of corresponding values between turbidity and
solid percentages. The optimum turbidity range
of 100-250 NTU corresponds lo OJ-05'* of
Kid U12. K\»mpk ol"« he eomrfaibit between luibi*
iv cxpnucel in NTU i*d penrenugc »l solid piakln
particles The ncph dome trie n
more precise than the solid percentages In addi-
tion, the nephelometric reading is direct, whereas
Ihc solid pcrccnctge measurement require at least
5 minutes of ccntrifugation.
Samples arc taken from the middle of the decant-
ing tank to monitor jnice turbidity evolution dur-
ing clarification. When optimum jnice turbidity is
attained, a second racking is carried ont as indi-
cated above. Once this operation is accomplished,
the turbidity of the clear racked juice mnsi be veri-
fied. If it is loo high due tocrror.additionalsctding
is necessary If. on the contrary, il is too low. fine
lees must be added to the clear juke
To obtain the highest quality juice from linal
pressings, suitable for blending with juice fiom
lirsl pressings, commercial peclinascs should be
nsed to maximi/c clarification. Very low turbid-
ity, from 10 to 15 NTU. shoukl be obtained The
light Ices of these press jukes arc highly col-
ored by phenolic compounds and should be elim-
inated. Like heavy Ices from press juice, they arc
nol worth filtering. Optimum press juices turbidity
1 100-200 NTU) is obtained by adding the appro-
priate quantity of fine Ices from the natural settling
of Ihc corresponding free run.
When juice clarification is loo stow, due lo
insufficient activity of natural grape pectinasc.
settling can be accelerated by using commercial
peclinascs from Atpeigillut m'ger. These prepa-
rations should be pure and not contain cinna-
mate esterase activity (Volume 2. Section 8.43).
to limit vinyl-phenol production by ycasls. Like
grape peclinascs. commercial peclinascs have sev-
eral activities-- notably a pectin esterase activity
thai dcmcthylivs peclic chains and an cndopoly-
galacturonasc activity that hydroly/csosidk bonds
between galacturonK residues The use of exoge-
nous peclinascs can result In excessive juice clarifi-
cation. Juke Inrbidily must often be adjusted after
settling by adding fine Ices Juice clarity mnsi be
fastidiously adjusted lo assure a complete fermen-
tation and allow Ihc expression of aromalk quality
of dry while wincs.
Aficr settling. Ihc jukes from Ihc last press-
ings must be evaluated lo determine if they can
be blended with first pressing juke There arc no
428
Handbook of Etiology: The Microbiology of Wive anil Vindications
general raks to guide Ibis decision lasting, eval-
uation of visual cokirtlhc kasi oxidized possible),
pkenolic compound index, sugar concentration and
especially pH aic taken into account For example,
for a Bordeaux Sauvignou. press jnkes with a pH
greater or equal lo 35 arc iol blended, bat ihe
addition of quality press ji ice to free ran intensi-
ties the varietal aroma of certain Sauvignou wines.
Prcssjuiccs should be blended before alcoholic fer-
mentation.
Other .juice clarification methods exist •sing
■tore or less expensive equipment ecntrifnga-
tion. filtration, tangential mkroliltralion and car-
bon dioxide or nitrogen flotation For varions rea-
sons, these techniques do not produce as high
qnalify wine as natural settling. Centrifugal ion
always causes a ccroin amount of oxidation
and nitration systems generally produce wines
lacking aromatic intensity These Kchniqacs are
even less justified today, since pneumatic presses
cxttuct relatively clear juke requiring minimal
settling Replacing conlinnous extraction systems
with pneumatic presses in large instillations has
made all of the mechanical clarification systems
practically obsolete.
13.5.5 Clarification Methods for Lees
The volume of Ices obtiined after natural settling
represents a sizable proportion of the harvest
Two types of filters are used for Ices filtration:
dialomaccoas earth rotiry vacuum fillers and plate-
and- frame tillers (using 1-15 kg of pcrliie/bl of
lees tillered) These two methods extract ckar
Juke <lcss than 20 NTU). without clogging, at
a rule of 1—2 hl.'h.'nr. Their recuperation rale is
■car Wi when the lees to be Mlicred con tun
Hf* solKls There rs practically no Jnkc loss
(Dubonidkn eiol. 1580: Serrano «v«f . I9S9).
The Juke obtained can be bkndcd with ckar juice
from natural settling without a quality difference
dclcctabk by lasting or analyse.
Plafc-and- frame tillers are easier to use than
rotary vacuum filters — especially for small winer-
ies They also have the advantage of not exposing
the juke lo air.
13.6 JUICE TREATMENTS AND
THE ADVISABILITY OF
BENTONITE TREATMENTS
Sugar concentration and acldiry adjustment are
described in Sections II 4 aid 115. They should
be earned out afkr clarification.
In the past. bcnuniK treatments were also
recommended to eliminate proteins in the juice —
responsibk for instability of juice clariry (Mili-
savljcvk. 1963: Ribctcan-Gayon Hal., 19761
(\bluntc2. Sections 55 and 561. The nsc of
ben unite in juke before fcraicntation offers long-
known advantages but afco presence ccruin more
recently discovered inconveniences
Treating jnkc with bcntoniic is recommended
for wines vvhkh are lo be clarifkd shortly after the
completion of alcoholic fcmicntalion. Additional
handling of the wine is avoided in this manner at
lime when the wine Is thought lo be more fragile.
However, prok in set bill ty less carried oulon jnkc
are imprecise and therefore not practical The same
ten unite concentration is generally used on juices
from a given winery. In spite of this treatment,
while wines are sometimes unslabk at bottling and
require an additional bcnlonilc treatment.
If while wines are lo undergo barrel or cmk
on Ices aging I Sections 13.8 aid 13.9). beiuiilc
treatments are not recommended for two tea-
I. Maintaining wine for several months on Ices
containing bcnlonilc. with weekly stirring, has
been observed to damage oiganolcptk quality
2 On-lecs aging naturally stabilizes while wines
with respect lo protein precipitation ■; Vol-
ume 2. Section 56.4) The mechanisms of this
phenomenon have long occurred unnoticed.
Yeast autolysis progressively releases different
ntannoprokins in wine with a strong stabilizing
power wilh respect to the prokins responsible
for prokk casse. On- Ices maturation of While
wine diminishes the ben unite concentration
necessary for stabilization by a factor of 2 to 4.
After barrel or tink maturation, on-lecs aged
whik wines are treated with bentonik at relatively
White Wi tic making
429
low con ccn (rations, determined by precise and
reliable ]'i- iii.- j- stability tcsk
13.7 FERMENTATION
OPERATIONS
13.7.1 Filling
Fcnucnlors ait tilled wiih clarilicd Juice. Approx-
imately Hfi of Ihc tank volume is kli emply lo
.!'.■ i.l ihc overflowing of foam iSeclion 3.25) pro-
dnccd during ihc tumultuous phase of alcoholic
fcimcntalion.
Different clarified jniccs musl oflcu be asscm-
Med when filling a high-capacity fermenting tank.
This operation retinites several ctemenciry precau-
tions Befoie blending the clarified jukes from
different tanks, fine Ices which nave sctlkd after
racking mast be reincorporated into the juice In
addition, juice that has not initialed fomentation
should not be Mended with fermenting juice, since
the yciists fermenting one juice produce HjS in
the presence of free SO; from the other juKe The
initiation of fcratcn tat ion must occur aflcr the con-
stitution of the blend.
13.7.2 Yeast Inoculation
Within the last 20 years or so. the nsc of active dry
yeusi t ADYl in wincmaking has increased consid-
erably. It has replaced the traditional practice of
yeast starlets in many wineries. In this formerly
widcNprc.nl method, a juice is strongly sulfilcd
1 10 g/hl) to eliminate spoilage yeasts and pronto*;
the growth of wine ycass It Is then inocutaled into
newly filled fcmicniorsat a concentration of 2-5'i
: it'le i several dajs o! spontaneous fermentation.
The kinetics of spontincoas dry white wine fer-
mentation arc fairly haphazard The speed and
dcgfcc of completion of fcimcntalion vary, depend-
ing on Ihc indigenous strain present. In addi-
tion, clarification un- inoculates' juice b> a cer-
tain degree A slow fcimcntalion due to a tow
yeast population can result Sometimes quality wild
microflora can produce spontaneous fermentations
with excellent result. Selection of wine ycasistrains
began by isolating strains with successful sponra-
ncons fc rate n la lions.
Today, approximately 30 active dry yeast strains
belonging *> Soccluotmyees ceirvisiiie aic used in
while wincmaking. They have been sckrckd based
on more or less empirical criteria of their cnotog-
ical aptitudes in different wincmaking regions of
the work!
The active dry yeast strain chosen for white
wincmaking has significant consequences on fer-
menution kinetics and Ihc devekrpment of varietal
aromas. A difficult fcimcntalion always gives rise
todnll wines kicking aromatic definition and inten-
sity The most important quality of a yeast strain
intended for dry while wincmaking is the ability
to ferment completely a juice with a lurbidlty of
between 100 and 200 NTT" containing up to 220 g
of sugar per liter, without excessive production of
volatile acidity. This ability to ferment clarified
juKe is not widespread among wild ycasls. and
wild yeast inoculum of spontaneous fermentations
sometimes do not contain such strains.
Some Strains like 7IB. produce high cslcr con-
centrations — in particular, higher alcohol acetates
contributing the fermentation aromas of dry while
wines. Their use is only recommended for neutral
grape varieties— Incy mask Ihc varietal aroma of
noble varieties
Other strains, like VLI. were selected for
their low vinyl-phenol production. Tncsc com-
pounds possess rather unpleasant pharmaceuti-
cal anrmas. Above a certain concentration, they
dull the aroma of dry while wines (Volume 2.
Sections 8 4.2 and 84.3) These strains have low
ciuuamaK decarboxylase activity. During alco-
holic fcimcntalion. this enzyme catalyzes the par-
tial transformation of ^-countaric and fcrulic acid
found in juKe into viny 1-4- phenol and vinyl-4-
gaiacal. Since Ibis enzyme is inhibited by phe-
nolic compounds, only white wines can con-
lain quantities of vinyl-phenols likely to affect
their aroma. The use of strains with low cinna-
mate decarboxylase activity is recommended—
particularly for white jukes containing high com-
ccnlialKinsof hydroxycinnaniK acid.
The role of yeasts in the varietal aroma of
wines is poorly understood. With the exception
of the terpenic aromas of Muscat varieties and
the Sauvignon antma. little research has been
4in
Handbook of Etiology: The Microbiology of WJic anil Vindications
dedicated In ihc aromatic characteristics or other
varieties. Ycasis have teen shown to free oily
small amounts of free IcrpcK alcohols from
Krpcnic glycosides present in juice. The yeast
strain used for fermenting MiscaljiKc. thcrcforv.
does not greatly inllncKC the Icrpcic alcohol
composition in wine However, many wincmakcrs
have observed the particular aptitude of ccrttin
Sacch ceirvisuie strains (ECS. 2056. VL3) to
intensify the varietal aioma of cciiain grape
varieties (Sauvigion. Ccwtlr/tramincr. Maiscngs)
(\tolumc2.Chapicr7).
As a result, the wincmakcr must understand
•ol only Ihc fermentative behavior of Ihc yeasl
strain used but also it. effect on Ihc specificity
of the wiac made. The composition of Ihc grape:
aroma precursors is responsible for the aromatic
intensity of the wine. The role of the yeast is
to transform this grape aroma pofcnual in*" five
aromas. Ipwfaclo. Ihc aromatic character revealed
varies according to the vintage and lerroir. A good
yeast strain permit* the expression of Ihc finesse
and complexity of the aromatic character of the
grape, bul it docs not detract ftom this character
by revealing 11. rws masking it with excessive
fermentation aromas or caricaturiig it by revealing
only some of Ihc particular nuances If yeasl strains
ate chosen judiciously, the nse of selected ycasis
will not lead to a standardization of drv white wine
While j ii ice should be inocnfciled with active
dry yeast al a concentration of 10- 15 g/hl or 10*
cells/ml of juice, immediately after clarification.
The cells are reactivated beforehand for 20 mil
in a water and must mixture ( I : I ) at 4o r If the
mist was cLinlicd al low temperatures ( 10- I2*C),
it is not necessary to wait for the must tcmpcralire
to rise before inoculating, since early inoculation
guarantees the implantation of ihc starter
The talk or Ixirrcl contents should be homoge-
nized at the lime of Ihc inocilalion. Ii this manner,
the suspended solids arc well blcidcd during yeasl
growth In high-capacity tanks without an agiulor.
UK blending operation Is difficult and the starter
should be pimped into the kres at Ihc bottom of the
tank, as opposed to over the possibly ovcrclarificd
mist at the top of the tank.
13.7.3 Addition of An
and Juice Aeration
The general mechanisms that link ihc nitrogen
and oxygen needs of ycasis during the alcoholic
fermentation process i Sections 2.4.2. 3.42 aid
352). explain the advisability of ihc addition
of ammonium salts and the necessity of aeration
during dry while wincmaking.
Assimilable nitrogen concenlralKWh (ammonium
cation and amino :ic ids except for proline I in while
jiiec from cxxd-climalc vineyards (northern and
Atlantic) are generally siffkicit to assure normal
yeasl multiplication but. even in these climates
favorable to while varieties, an insufficient nitro-
gen supply to the vine or excessive summer dry-
ness can sometimes result in juices deficient in
assimilable nilrogci. ViiKultnr.il conditions favor-
ing this situation are varied but always foresee-
able: superficial root systems of young viics: win-
ter root asphyxiation In poorly drained soils, light
soils with an insufficient water reserve: and grmid
cover strongly limiliig water aid nitrogen sup-
plies. The wincmakcr should pay close attention to
these potential problems, since nitmgen-deficicnl
white juices almost always produce heavy white
wines with lilllc fnit and aging potential
White juices with concentrations of less than
25 mg of the ammonium cation or 160 mg of
assimilable nitrogen per liter should be supple-
mented with amiKHHim sulfate Assimilable nitro-
gen concentrations in suspected niliogcn-dcliiicnl
while juices should be systematically analyzed
using the formol index (Acmy. 1996). a simple
method easily performed al the winery with a pH
meter Some while in lists can contain less than
40 mg of assimilable nitrogen per liter In these
extreme cases, supplementing Ihc must with 30 g
of amiKHiiim sulfate per hcctolilcrObc maximum
allowable dose permitted in the BBC) is nol suf-
ficient to re-establish a suitable nitrogen supply
Id yeasts. Chronic nitrogen deficiencies shoakl be
corrected at Ihc vineyard by appropriate vilicullural
practices.
Ammoniacal nitrogen Is added either all al once
at the lime of Iioculalioi or in two additions, the
second occurring al the same time as the aeration
White Winemaking
431
on I be second or (bird day of alcoholic fermen-
tation The second method sometimes permits a
more rapid fcrmcutilioa.
In the pusl. «Ik to fears of aroma loss through
oxidation, while JdKcs were not aerated during
alcobolK fcriKnlalion in big h-capaciry tanks.
Opinions unlay arc less categorical. The varietal
aroma of while wines is affected by oxidation
during pressing and draining (Section 13.42) but
an aeration in the first half of fermentation has
no effect on Ibe fruit annua of aromatic varieties.
At Ibis stage, the considerable reducing power of
yeasts wry effectively pcotccts the aromas from
oxidation. If the addition of oxygen sometimes
diminishes fcrmcilalion aromas (esters and fatty
acids) of dry white wines, it is caused by the
resulting stimulation of alcoholic fcriKnlalion.
The risk of slow orstuck fcrntcn titioas. associaicd
with strictly anacrobK conditions, is more serious
than a minimum loss of iransKnl fermentation
The juke should be aerated during pumping-
over operations — maintaining suittblc air and liq-
uid contact Oxygen gas can also be diicclly
injected into fermenting juKc by an aerating
device. This oxygen addition (2-4mg/l) should
occur in the fiisl days of alcoholic fermentation
during the yeast exponential giowth phase. Oxy-
gen permits the synthesis of sr rols— essential cell
membrane components and yeast survival factors
during the stationary phase. Aeration becomes acc-
essary for the completion of alcoholic fcmicitalion
when juice turbidity is low and sugar coaccnlra-
tious are high. Adjusting juice clarity, measur-
ing and correcting (when necessary) assimilabk
nitrogen concentrations aid aerating al the right
moment along with yeast inoculation arc the prin-
cipal factors governing successful fermentations in
dry white winemaking.
13.7.4 Temperature Control
In ir.Hliinn.il while winemaking. alcoholic fermen-
tation ■■..is carried out in small containers (barrels
or small Inns) located in cool cellars wilb temper-
atures between 12 and IOC In these conditions,
the fermentation temperature remained close to Ihc
cellar temperature from throughout Ihc fermenta-
tion. It rarely exceeded 22-25 'C during the most
active phase of fermentation These barrel and
lun fermentation conditions still c\ist in historical
quality white wine legions (Burgundy. Saulcrnes.
Graves. Loire Valley. Alsace, etc.) In the last
15 years, many wineries, in a desire to improve
while wine quality, haw reverted back to (he age-
old technique of barrel fermenting Temperature
control problems .uc much less of an issue in these
small containers.
The temperature of high-capacity fermenting
tanks must be con trolled lo avoid excess! w temper-
atures during fermentation Today, most wineries
haw tanks equipped with temperature control sys-
tems. A cooling system maintains water at a low
temperature (4-6'C) in an insulated tank A second
system distributes the cool water through exchang-
ers, placed inside (he fermenting lank or set up
as a jacket on the outside. These cooling systems
were developed fairly recently. In Ihc 1950s, ihc
migration from small to huge fcrmcnlors occurred
without taking inlo account Ihc heat exchange con-
sequences of these changes.
As in red winemaking. excessively high temper-
atures (above ,10'C) can be the cause of stuck fcr-
mencilKHis. but this problem rarely occurs today.
slice wincmakcrs have means of cooling juice
before aid during fermentation They are also
aware of the iced to avoid cxccsslw temperatures
lo limit aroma loss. Fermentation temperatures
above '<> c diminish the amount of esters pro-
duced by yeasts and increase higher alcohol pn>
duclion i Bcrtrand. I%8). The effect of temperature
on varied! wine aroma is much less clear. At wry
high temperatures (28-30 T). Ihc rapid release of
carbon dioxide entrains certain snbslaiccs. causing
aroma loss, but lower- temperature fermentations at
18'C or at 2i~24'C do ioi necessarily produce
wines with a significant varietal aroma difference
Fermentations at 18 C or lower arc therefore nol
a means of enhancing the fruit character of aro-
matic varieties. Pre- fermentation operations and
selection of yeast strains haw a much greater influ-
ence The temperature kinetics of high-capacity
fcrmcnlors should simply be modeled after tem-
perature evolution In barrel fermentations, with a
432
lUintlUok of Enology: The Microbiology of Wine anil Vindications
i temperature of aronnd 22-23 C at mid
fermen tilK* — progressively decreasing tothcccl-
tar temperature by the end of fermentation
Untimely temperature drops should be avoided
al all stages of fermentation in both turrch aid
Ginks. For example, a tink temperature should nol
be lowered from 23-16'C over a few hours. 1o
avoid suhscqucni Icnipcratare coiirol. The ther-
mic shocks thai j casts undergo in these coudi-
ilons promote stow aid even stuck fennea tit ions
(Section 3.7.1).
13.7.5 Completion of Alcoholic
Fermentation
The daraiioB of dry while wine fermentation
depends on several parameters: juice extraction
conditions: sugar aad assimilable nitrogen concen-
trations: turbidity: yeasl strain, aeration: and fer-
mentation temperature The wincmakcr can adjnsl
and control all of them. A stow or stuck fermen-
tit ion rs most often the result of carelessness and
always affects wine quality The alcoholic fermen-
tit ion of a white wine should not exceed 12 days.
Longer fermenutions should not be sought after
except in Ihc case of exceptionally high sigar con-
centrations.
Juice density is measured dally to monitor
alcoholic fermentation kinetics When the density
drops to approximately 0994-0993. sngar con-
centrations arc then measured daily to verify the
completion of fermentation Fermentation is con-
sidered complete when less than 2 g of reducing
sugars per liter remain The fermentors are then
carefully topped off. Snbscqncnt operations depend
on whether malolaclK fermentation is carried out.
If in. ili 'I. kin fermentation is not desired, the
wine temperature is lowered to around 12'C.Thc
tees arc stirred daily by agicaoou or pumping,
avoiding oxygen dissolution. This operation makes
use of lie reducing power of yeast tees to
protect wine from oxidation. The formation of
reduction odors in the Ices Is simultincously
avoided (Section 13.9).
After 1-2 weeks, the wine B sultilcd al
4-5 g hi Until recently, on-tces aging In high-
capacity tmks was not considered possible, due
to the appearance of reduction odors The Ices
were rapidly eliminated by racking shortly after
ss lining. Today, by raking ccrtiin precautions,
white wines can be on-lccs aged even in tanks
(Section 13.9).
13.7.6 Malolactic Fermentation
Malolaclic fcrmcncition is always sought with rcd
wines bit is practiced less oflcn for white wines.
Its use depends on Ihc variety and wine region.
Chardonnay in Burgundy and Cbassclas In
Switzerland are two classic examples of on-lccs
aged while wines which systematically undergo
malolactic fermentation after alcoholic fermenta-
tion The primary objective of this transformation
Is to dcacidify Ihc wine This is especially true of
premium quality white Burgundies. Before mam-
lactic fermentation, their tatal acidity can be as
high as 7 g/1 expressed as H ? S0 4 with a corre-
sponding low pH Marotaclic fermentation also
Increases Ihc biological stability of the wine. For
example, to avoid an accidental malolaclic fermen-
tation in the bottle, champagnes undergo malo-
lactic fermentation after alcoholic fermentation. In
controlled conditions. Malolaclic fcrmencillon also
con tri bn les u Ihc an>matic complexity of Chardon-
nay wines. A Chardonnay wine Ibat has not under-
gone malolaclic fermentation cannot be considered
a great Chardonnay Malolaclic fermentation docs
nol lessen Ihc varic til aroma of chardonnay: on the
contrary. It develops and stihili/cs certain aromatic
and rexlural nuances, making the wine more com-
plete Unfortunately, nol fully understanding the
varietal aroma of ehaidonnay . Clology Slill cannot
provide an explanation of these phenomena al the
molecular level.
Today, many Chardonnay wines made through-
out Ihc worU according k> the Burgundy model
undergo malolaclic fermentation more for the aro-
matic consequences lhan for dcac id duration and
stabilization In these same cases, the juices are
often acidified to be capable of matohictic fcr-
mcncition These practices may shock a European
wincmakcr but are employed lo produce a certain
type of wine.
For most other varieties, such as Sanvignon.
Scmllun. Cbcnin and all Atsacian. German and
White WiiiiiiKikiu-
433
Austrian varieties, malolactic fermentation notice-
ably lowers ihc Irmly character of white wine-..
Other Methods should be used u lower ucidily
when this operation Is necessary, but the n>lc of
lactic acid bacteria on Ihc annua of wines made
from these varieties should be studied, al least b>
justify the tradition of avoKling malolaclic Icniicn-
tat ion with these varieties.
When malolaclic Icrmcn union is desired, the
wines arc lopped off and maintained on Ices after
alcoholic fermentation, withoit si Kiting, at a tem-
perature between 16 aid IS C The containers
must be filly lopped off and the lees stirred weekly
Id avoid oxidation. With proper wmemaking meth-
ods, in particular moderate si liking, malolactic
fermentation spontaneously initiaks after a blent
phase of variable length that can be shortened with
Ihcnscof commercially prepared malolactic inocu-
lim (Section 13.731 Some wineries even keep
non-sul filed wines, having completed ntalolaclic
fcmicitation. at low Kmperalures from one year
to another hi nseasa malolactic sutler cultnrc. In
regions where malolactic fermentation is system-
atically practiced. iK initiation docs tot pose any
particular problems, since the entire instillation
(notably the barrels) contains an abundant bacterial
inoculum and malolactic fermentation is difficult
to avoid If wine begins to oxidize while wait-
ing for the initiation of malolaclic fermentation, it
should be lightly sulfiKd (2 g/nl). Malolaclic lei-
mentation Is not compromised and the aiuuiaiK
character of the wine R preserved. Once the malic
acid Is degraded. Ihc wines arc sulfilcd al 4-5 g/hl
and maintained on lees until bottling.
13.8 MAKING DRV WHITE WINES
IN BARRELS
13.8.1 Principles
Dry while wines capable of substantial aging
are traditionally fermented and matured in small
con tuners This practice was widespread at the
beginning of ihc century In France, it continued
in certain prestigious Burgundy appellations. Al
the beginning of the 1980s, barrel fermenting
and aging of white wines smged in popularity.
affecting nearly all wine regions in the work!
However, the use of barrels is not suitable for
all wines: also, implementing: a barrel program is
difficult and very costly
The yeasts play an essential role in the original-
ity of the traditional Butgundy method of barrel-
aging while wine Contrary ID red wiic. which is
barreled after the two fermentations, while juice is
barrel fermented and Ihcn aged on Ices in Ihc same
barrel for several months without racking. Daring
Ibis aging process there arc interactions between
the yeasts, the wood and the wine. Unknown Id
cnotogy for a king time, these different phenom-
ena arc better understood today. They encompass
several aspects: Ihc rote of cxoccllular and pari-
etal ycasl colloids: oxidation-reduction phenom-
ena linked to ihc presence of Ices: Ihc nature
and transformation by ycasK of volatile substances
yielded by Ihc wood to the wine, and barrel fer-
menting and aging techniques
13.8.2 The Role of Exocdlubr
and Parietal Ycasl Colloids
The yeast cell wall is composed of glucKIK col-
loids— essentially 0-glucans and mannoprotcins
Ik detailed molecular stricture is now well under-
stood (Section 1.2.2).
The macromolccular components of Ihc yeast
cell wall, particularly Ihc mannopmicins. are par-
tially released during alcoholic fermentation and
especially during on- lees aging In the laboratory
on a model medium, coitact lime, temperature
and agitation of Ihc ycasl btomass promote the
release of these substances! Volume 2. Section 3.7)
(Lktnbcrcs Hal., 1987). All of these conditions
occur in traditional on- Ices barrel aging A wine
barrel fermented and aged on totil Ices with
weekly stirring ( baton n age t has a higher glucklK
ycasl colloid concentration than a wine fermented
and aged on line Ices in a tank for Ihc same lime
period (Figure 1.1. 13) The difference in couccb-
tralion can attain 150-200 ng/l.
The release of mannoprolcins is the result of
an enzymatic analysts of the Ices. f)-Clncanascs
present in Ihc yeast cell wall (Section 1.2.2)
maintain a residual activity several months after
i tandbook of linology: The Microbiology of Wine and Vinificalions
VjL'il. |17lV)(l»lllB>
Fifi 1 3. IX Evolution uf (be total polysaccharide c
i~f nniihin la ofcic wine during link afiaf «■ line I
( l."rhr.Tl,|'i:-J"-l^llcM 1
cell death. Tbcy hydroly/c ihc parietal glaums —
anchor poiats of ihc mannoproKins released into
Ihc wilt
The direct organoleptic iiflucncc or polysac-
charides on ihc body or fullness of on-lces aged
while wines has never been clearly established, bnl
Ihc polysaccharides released during on-lccs aging
( from Ihc yeast cell wall, for example) arc capable
of combining with phenolic compounds in white
wines tChatoinci el til . 1992) The total polyphe-
nol index and Ihc yellow color thus steadily dimin-
ish in ihc course of on- tecs barrel aging Moreover,
after several months of aging, wines thai are barrel
aged on total Ices air less yellow lhan Ihe same
wine aged on tine Ices in a tank (Figure 13.14).
The lees limit Ihc cllagic tannin concentration,
originating from oak in particular. Tannins given
off by ihe wood are lixed on Ihc yeast cell walls
and ihc polysaccharides ( mannoprotcins) released
by ihe lees. A wine conserved on Ices therefore
has a lower overall tannin concentration as well as
a much lower proportion of free i. reactive) tannins
(Figure 13.15).
In addition, on-lces aging lowers while wine
sensitivity lo oxidative pinking. This problem,
characterised by a color evolution towards a
grayish- pink (Simpson. 1977). occurs when wine
is slightly oxidised during stabilization or bottling.
A? O H |KT Mnl I h ■*»
l-'iji I J. 14- Evobaii>a<iflto?clk>wcok>r(OD J2D)i>fa
ttiactanl .!,■.-...■■ line ,.■:■. I* .,..-,[ .-.-.-:.. in r. ' h...".vl,
on loul Ice* t : iK'taiimw « (V . 1992)
Young while wines, in particular Sauvignon. whose
masts were carefully protected from oxidation,
arc especially sensitive to this color change. The
compounds involved in these phenomena arc not
known Contrary to anthocyanidins. they arc nol
discolored by varying the pll and sulfiting. but the
pink color disappears upon exposure lo light liven
if the pink color of these wines generally disap-
pear aterscvcral moninsof bottle maturation, this
problem can lead to commercial law suits The sen-
sitivity of white wine to this ovulation can be eval-
uated by measuring the difference in absorbaice of
the wine al 5(10 nm. 24 hours after adding hydro-
gen peroxide 1 1 i-.uk- 1.1. 161 This value multiplied
by 100 is the sensitivity index. If it is greater
than 5. there is a definite risk of pinking Wine
sensitivity to pinking remains fairly constant in
the course of aging racked wine on fine Ices, in
barrel or in tank, but diminishes rapidly on total
Ices (Tabic 13.18). The yeast Ices probably adsorb
Ihc precursor molecules responsible for pinking,
bnl neither casein lining nor PVPP treatment is
capable of significantly decreasing wine sensi-
tivity to pinking. The addition of ascorbic acid
( 10 g/flli at bottling is the only effective preventive
treatment.
White Wine making
w.
* »r clbpk tannin* of a barrckd wine aped «n ko, or :■ ibu ke% <Ck»tonr
Tabic 13.18. Evoknioaoflbc pinking icnati
aged on line kc% ami on total Ice* iLavqtac.
Hj index in the an
unpublished tcui
i)
Aped November lunuarv
March
April
Onincko, IT 13
On total lee* 17 4
8
8
i^vkinUMIiiii)
Kifc 13.10. Pinking %cn**ivhy determination of a whie
wine 24 boun ..Hen he ,idiln»>n of hydrogen penxkk
($iapv>n. 1977). Dashed line = interpolation of the
adsorption curve wit bout pinking, of OD 400. 410. 420.
MO. 025. 050. A OD M0 =diHercnee between the
cakulated and mcuMinrd vatic at OD M0. PS) (pinking
iMHvky iadci) = 4 OD M0 x 100
The release of mannoprolcins during on- Ices
aging also increases tiitirn and piotcli stability in
while wines (Volume 2. Sections 1.7.7, 5.6.3 and
5.6.4).
I .VS.. % Oxidation-Reduction
Phenomena Linked to the
Presence of Lees
Maintaining while wines on tola! Ices in tanks
aflcr su I filing is diflicnll withoit Liking cer-
tain prccantKfns (Section 13.9). Drctgiecablc sul-
ftirons odors rapidly appear, nuking racking nec-
essary. Wilb proper must clarification and Minting
(Section 135.2). barrel aging permits piolongcd
coiiact wilh lotil lees withoit the development
of reduction odor flaws Inversely, when a dry
while wine is separated fn>m its kres and stored
in new haricls. II ntonr or less rapidly loses its
frnit characicr and develops oxidative odors These
.. waxy and camphorated odors intensify
436
Handbook of Etiology: The Microbiology of Wine anil Vnifkatkws
(tiring bottle -mint;. The lees arc thus iadispens-
able to the proper evolution or dry while wine in
barrets They act as a reducing agent, in a manner
similar to tannins in the maturation of red wine
While wines haw a higher oxidation -reduction
potential in barrels than in unks (Dubourdicu.
1992). Inside the barrel. Ihis potential dimin-
Rhcs from the wine surface towards the Ices
(Ftgnrc 13.17). Over lime, the barrclssccn to lose
sotK of their oxidative properties. Woodclfcigilan-
■ins. released in lesser qnan tides as the barrel ages,
contribaic lo its oxidizing power. A reduction ten-
dency consequently occurs more often in used bar-
rcbt than in new barret. Stirring homogenizes the
wine oxidation-reduction potential (Figure 13.18).
Lees reduction is blocked, as well as surface wine
oxidation. The stilting of on- lees wines is as indis-
pensable in new barrets as in used barrels, bat for
different reasons. Wine in new wood is protected
from oxidation and wine reduction is avoided in
used wood by this operation.
During aging, the lees release certain highly
reductive substances into the wine, which limit
wood-induced oxidative phenomena. These same
compounds appear lo slow premature aging of
bottled white wines. The nature and formation
mechanisms of these compounds arc described in
the text section
DqaiMcmi
Pit; I J. IT. l*nuc(HCiifihc^)n|jmah^oiiihci>VRbikin-ic<lu^Mn|alc«nlof «hitc»iK(Dubauidiai. 1992)
numn
1X1*11 1>
Fifi I J. IK UDucncc of Mimnp anthc ovMfainn- ntUKtbn puctful of land-aped «fc« «i«e (tXih«iu«lku. 1992)
White Wiiiii.Kikiii-
41 "
13.8.4 Nature of Volatile Substances
released by Wood and their
Transformation by Yeasts
Among ibe maiy volatile substances rckascd
by wood in wine. voLuik phenols, p- methyl- y-
octalac tones and phenol aldehydes arc the principal
compoinds responsible for the wood aroma of
barrel-aged wines Volatile phenols, in parlKular
cugcnol. give wine smoky and spicy aromas. Cis-
and fiviru-»clhyl-y-oc talac Dues arc responsible
for the coconut aroma Volatile phenols, essentially
vanillin, produce vanilla notes ) manic aldehydes
have grilled aromas bnl Ihcir perception threshold
Is much higher than concentrations found in wine.
Their olfactory Impact Is Ihns negligible.
Barrel- fermented wines overall have less wood
aroma than the same wines barreled after alco-
holic fermentation (Cbatoiict tfal., 1992). This
phenomenon is essentially linked to ibe reduc-
tion of vanillin by ycasls ink) vanillic alcohol,
which R almost odorless Furank aldehydes are
also rcdiccd inlo alcohols
On-kcs aging after alcoholic fermentation also
Influences the wood aroma of white wine In terms
ot aging, the wood character is less pronoanccd
and better Incorporated If Ibe wine is maintained
on total Ices (Cbalonncl el nf . 19921 Yeasts arc
capabk of fixing and continue to iransform ccrtiin
volatile compounds as they arc released from
lie wood.
Laic barreling (after alcobolk fcrmcnuijou)
and prematurely eliminating Ices will produce
white wines with excessive wood character. These
methods have unfortunately been practiced in Ibe
past in certain regions, where wincmakcrs have
applied red wine aging techniques lo white wine.
13.8.5 White Wine Harrd-A^ing
Techniques
Due lo its high cos! and ibe care required, the use
ot new barrels is only economically feasible for
the production of relatively expensive, premium
quality wines produced in limited quantities Barrel
aging should be applied u dry whlfc wines capable
of aging and slowly developing a bottle-aged
bouquet These wines arc Ibe most sought after
by connoisseurs and letch the best prices. After
several years of aging, the wood character of
these great while wines R perfectly incorporated
into the overall bouquet. Their aging poknlial
is improved, bnl not conferred, by the Judicious
use of barrels. Tic use of new harrcls is not
necessarily suiubk for dry white wines intended
lo be drunk young and made from fruity varieties
Fruit character is less intense, as the wood masks
aromatic expression To satisfy the current (but
perhaps temporary) demand for oaked wines in
certain markets, it can be tempting to barrel
ferment and age ordinary wines In this case, the
battel is simply a means of compensating for an
aromaiii.il ly deficient wine. The widespread use of
these praclKes may actually lower the consumer
appeal of oaked wines over time
The most popular bunch in France for white
wine making are made from linc-graii oak from
foresK in central France, notably Allicr In Ihesc
mature forested areas, sessile oak tQucrvus ses-
iilis) essentially constitutes the oak population.
Fine-grain oak contains much higher concentra-
tions of odorous compounds, in particular (•-
methyl- I'-ovtalac tones, than course-grain oak. The
latter — largely pedunculate oak IQutnw peihm-
culma) — comes from isolakd trees or brushwood
under full-grown sessile oaks. The coarse-grain
i Limousin i is less odorous, but contains much
higher tannin concentrations While wines made
with l.imonsin oak therefore have a nunc pro-
nounced yellow color and tannic character. The
technique is rarely nscd for wines, particularly
whiles, but spirits arc generally aged in coarse-
grain wood.
Toasting, carried oul daring barrel production,
considerably influences the arouialK impact of ihc
wood in wine. Barrcfe arc misted to between
medium and high so that the very fragrant line-
grain wood docs noi dominate the fragile aroma
of while wines (Table 13.191.
Intermediate- grain wood from Burgundy may
also be used for white wincmaking This wood is
nol very tannic, it is kss fragrant than line grain
and is best adapted lo medium Rusting
Fine-grain oak from central and northern liuropc.
in particular Russia. also produces acccpublc barrel
Handbook of linokigy: The Microbiology of Wiie anil Vindications
iaU*dio4M)no iMciu.it>* 0(1 he color nod wood chaiactcf of
I* |aM.'H
■ (■*'!)
M«lr.M. ?> in, l .i<i>«.li
ViRW-«ncolI..g.U
rmyi-MirtKni a. j/n
I*$ mM (tijL'l)
Prnail + (Mttwi ciig/Q
|M II— >l||.y.»
l...i^ll„., [|
viKUftidKiKt.^ii
Vauui 1 (in.'H
S)!l»jlWri,Jr IM.I)
T.iiUlkliyil- pt"»k <«mfi
»nil. Il has a similar composition b> thai of oak
growa In central Prance In identical wlacmakiag
conditions, while wines made in line-grain Russian
Ixirrch and French barrels ate very similar in taste
(Cbatonactr-Jn/.. 1997).
American white oak [Quercus nihil) b very
frog ran l It is rarely used for premium white
'.'jinui.Lkiiii. . as excessive concentrations of fi-
■Klnyl-i-iit.il .11. n '!,- arc apt to be released, uully
■tasking Ike wine's character. American iuk is
reconimcidcd for rapidly oaking ordinary white
Barrel preparation for white wlncmaking is rel-
atively simple. New barrels, delivered nnsullilcd.
arc simply rinsed with cold water and drained for
a few minutes before use Used barrels, stored
empty aid regularly silfurcd. are apt *> release
SO? into the juice during Idling Asa result, abnor-
mally high levels of HjS are formed during alco-
holic fermentation aid are capable of generating
strong redaction odois ILavignc. 1996). ThR pbc-
■ Ls particularly pronounced if fcrnrentiag
Jiicc rs barreled Used barrels must consequently
be filled with wan 4B hours before asc. to elimi-
nate the SO? likely to be rekrascd in the fermenting
The barrels arc placed in a cool cellar (I6'C)
and are Idled cither before or at the scut of
alcoholic fermentation A Hfi hcadspacc should
be left, to avoid foam overflow during maximum
fermentation intensity The fine lees and/or the
yeasts should be carefully put into suspension
to homogenize the juice before barreling After
barreling, the tank dregs (Ices and deposit) should
be scrupulously distributed in each barrel of the
lot At the start of fermentation, barreling repfciccs
aeration during the yeast multiplication phase. If
the lunch are filled with juice before fermentation,
aeration 1 by introducing air or oxygen) Ls necessary
when fermentation is Initiakd. If these different
precautions are not taken, fermentation is irregular
from one barrel to another in the same lot. In
barrel as in tmk. difficult fcrmcntaliois arc often
the result of human error or negligence.
V. llA- 'A ilk'Iii.ikili-
439
As sooi us fermentation Is nearly complete, the
Ixirrcls arc toppal olT with juice from the same
kit Sluggish fermentations can often be reactivate!
by topping off Ihc hint* I will a wine lol thai
has recently completed a successful fermentation
This technique is equivalent h> using a starter
i Uf.i i composed of a populaiton in the stationary
phase— icsistinlio inhibition factors. At the end of
alcoholic fermentation. Ihc battels arc stined daily
unlil saltiling (Section 13.7.6). Wines indcrgoing
malolaclic fermentation ate not sullilcd unlil iLs
com pic to*
During barrel maturation, stirring and Kipping
off should occur weekly, with free SO? concentra-
tions maintaiied around 30 mg/l.
13.9 CONTROLLING REDUCTION
ODOR DEFECTS DURING
WHITE WINE AGING
13.9.1 Evolution of Volatile Sulfur
Compounds in Drv White Wine
During Barrel or Tank Aging
Barrel- fcnrici led and aged dry while wines arc
most often main tuned on yeast Ices dnring the
entire aging process. In this type of wincmaking .
if Ihc olfacloiy redaction aionias do nol appear
during alcoholic fcroicuuiKM. they rarely oceir
later. Stirring wine frequently k» pit the lees in
suspension and limited oxidalHn across harrcl
Stives ilhibit the formation of off-odor siilhir-
coittining compounds in the wine.
During Ihc rurrcl aging of wine. volatrrc wine
thiols. H;S aid mcthancthiol — lormally preseni at
Ihc end of fermentation —decrease progressively
(Figure 13.19). This phenomenon occiis moir
rapidly in kw Ixirrcls. prohibly due to greater
oxygen dissolution and Ihc oxidi/ing effect of
new wood tannins (Lavrgic. 19%).
Despite the relative ease of hintl aging dry
while wine on lotal lees, the wincmakcr must still
payckiscallcntion K> Ihc wincmaking factors (clar-
ification and sulfiting) thai influence the produc-
tion of sulfir- con tailing compounds by yeasts In
fact, even if a wine is in a new or used turret.
racking and the delinitrve separation of the foul-
smelling lees from Ihc wine must be carried on.
if a rcduclioa Daw exists al Ihc end of alcoholic
fern ten tat ion. The quality of txirrcl aging is greatly
compromised, particularly in new turn-Is. in the
absence of Ices. Ihc dry while wiie is not piolccicd
from oxidation
Fift 1 J- !•». KvohliiiD of light uilruceoatatniag compounch In barreled »bHc wis l» ihout Bitwi) ua tolil fc«
(Uvipoe. IP90)
4-n
Handbook of Etiology: The Microbiology of Wine anil Vmifliatlons
Controlling reduction atona defects In dry white
wines during aging in high-capacity Links H more
difficull The presence of ices inevitably leads K>
the development of reduction odors within the first
nonth of aging, whatever the redaction stilcof the
wine after ulcohollc fcnncntilioa (Figure 13.20).
In 11 km cases, tank-aged dty while wines air
systematically racked aid separated from their
tecs. In these conditions, if the gross tecs are
eliminated early enough, before reduction annua
defeeb occur, the wine can be aged on fine Ices
without risk Early racking helps to stabilize light
sal fur-con tuning compound concentrations This
opinion was clearly suited in the last Boidcanx
etiological treaty (Ribcrcau-Cayon el of.. 1976). It
was founded simply on observation, without any
supporting analytical data
The pn&lpil iLiniicr uf Uuiing white wine*
on ycui Ices )■ hiyh-ia pacify taiil* R Ihc
development of fcydmoen sulfide iml •cm pun
odor.. But even If the presence of ycjau n hoi
jccumpmkd by these tlanclcilMli olf-odorMol
tutcs.thciiapklclimiauiKinicuiksiafrcshcrirKl
■ure aioaark win which baiet comervc Iheii
■olive chmcieiwks-
Acrativcly racking wine in the tank without
scparaliig the lees Is not siflicicnt to avoid the
Fig I J. 2I>. Evolution »f lilt hi uiltui-contaiaunr compounds In white wuc on (out kev in link lUvioac. I99A)
Fift 13.21. EvolubnoflkyhlMilw^oatainiaigcompouncfcii
iaUak n.l.r.i . da f. lcc%(Uv^K.I99(l)
White Wine making
I'ifi 1122. i:vt>hiH>n of 11,\ nli.1 Brlkim
l^cMl.v.ip-. 1995)
development of disagreeable odors. Immediately
aflcr nicking. H?S and methane thiol concentrations
diminish, bui within a month, the lime necessary
for the Ices to settle on the bollom or the tank, the
defect reappears (f-igurc 1321). The compacting
of the Ices under the pressure exerted at Ihc bollom
of high- capacity tanks seems n> promote reduction
phenomena in white wines aflcr sultiling ( Lavignc.
19%)
The ability of yeasK m generate foul-smelling
sulfur compounds in these conditions progressively
dimincihcs during aging and totally disappears
after a few weeks. The loss of the Ices sulfilofc-
ductisc activily. catalyzing the reduction of SO;
into M.S. explains Ibis development.
Lees
13.9.2 Aging Dry While Win.
High-Capacity Tank o
As king as Ihc sullitorcduclasc activily r
yeasts, dry while wines fermented in high-capacity
Links cannot be stored on their Ices without the
risk of developing icdnction off-odois. However,
if the Ices arc Kmporarily scparaled from the
wine until Ihc reductive activily slops, they can
be reincorporated arte rwards— there is no longer
a risk of generating sulfur-containing compounds
(Lavigne. 1995).
In practice, wine is racked several days after
snlfiting. The Ices arc stored separately in bar-
rels This initial step of the aging method slabili/cs
sulfur- containing compound concentrations in the
wine on line Ices, and al the same lime avoids
reduction odors from the gross lccs Simultane-
ously. H?S conccn nations progressively diminish
in the barreled Ices. Only one day aflcr scpam-
lioa from ihc wine, these lccs no longer contain
methancthiol. Aflcr appioximatcly one n>onih. the
lees arc reincorporated inlo the wine. At IhLs stage,
nol only do the lees no longer generate sulfur-
contiining compounds bnl their addition also pro-
vokes an appreciable decrease in Ihc concentration
of methancthiol in Ihc wine (Figure 13.22).
The use of fresh lccs is an authorized etiological
practice, used u correct the color of prematurely
oxidized while wine The ability of Ices lo adsorb
certain volatile wine thiols has been discovered
more recently (Lavigne and Dubounllcu. 1996)
Yeasts, taken al the end of fermentation and
added to a model solution containing methancthiol
and cthanclhiol. are capable of adsorbing these
volatile thiols. They are fixated by the yeast cell
wall man no proteins During aeration, a distill *r
bond is formed between the cysicinc of the cell
wall mannopiotcins and the thiols from the wine.
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Dubetnct M. (1974) Reeheiches sui b tyrosinase dc
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Applkalbm icchiuik>tik|ucs. These Doctoral 3emc
Cyck. I'nivcakc dc Bontcau. II.
Duhnurdku D. (1992) U boi.\ el la ou.diie ilei i»ii
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lavigne V.(l990)«n ; Fr. Ottwl.. 155. 3b- 39.
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Uvninc-Cnicpc V. ( 1996) Reehcichcs sue ks composes
volaiib soulres fi>rmcs pat b kvuic au cuuis dc b
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Doctoral. Unhcrsie dc Bonkaux II.
Lbubcics R.M. Dubourdku D. and Vilkiar J.C.< 1987)
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ManinkreP.andSapis J.C. (1967) <-»«. \tgocMn.2.
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MauretR.and MekUnpct F. (1976) Poi. HUidvb 31
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Mououna M..RigaudJ..Souuuci I.M.andChcyakt V.
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MulkfSpath. H( 1988) OofeciiflK. Man 21-25. 15- 19.
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Rcchcrehcs dc Itaivcrske dc Bonkauv II.
OllrvkrC. Moacsiieel Tb.. I.anre Ft. anil IXnSoui-
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OjghC.S.<l969)Ara. / Enol. Miic. 20(2). 93- 100.
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22(3). 194-198.
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Pcyaaud E. (1971) Camuasunce ei irmxdl du tin.
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R*W(VJti*(i.i;.i)ii I aid RtKicau-Guyua P. (1954)
.Vfioi* (iwijp™ tpcrniiumij ite\ biduiliies Agfiiislet
el ATimaiHam. Mailiil.
Rfecmu-Gayon P.. Lafua-Lafamadc S. j ml Itcn-
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iind Sudraud P. ( 1970) Triiu iTQenologie Srimcrs
el Mutinies du Ma. Vol. 3. pp. 303-364. DumhI.
Pom.
RnEiudJ..CheyiikfV-.S«u«»iei JAI.aad Mouounei M.
(1990) Jto: FT. Omot. 124.27-31.
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(IWUIAwlf. O/V. 57 (03K). 309-311.
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Mtv/in. Bipro timer. Siuupart.
SchoeidceV. (19891 /Jfc tUww(Mfwi«. 1. 15-20.
Seipeai I). (1990) AdapMlna du Snjvi^ao* el du
SCmillon 1 Id divcftilc urobfii|uC CI ajtncpcdolo-
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dc rUnivcnK Vicloi Sepakn Bonkaui 2.
Scirani. \1 . Pael/nldM. j«I Dubaunlku D. (1989)
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ToauuagaT.. Muncufl. und Dubouidicu D. (1996)
In Mine Symposium hienutional d'OetwIu/ic.
pp. 44-49. Tceb & Doc Uvobicr. Park.
14
Other Winemaking Methods
14.1 Host wines
142 Botrylizcd sweet wines (Saulci»cs and Tokay)
14J Champagne and sparkling wines
144 Ponificd wines
145 Flor wines
445
449
458
47G
14.1 ROSE WINES
14.1.1 Definition
In many countries, especially wiihii the EC. laws
have been established lo dcline wine Ycl red.
while and rose" wines arc never spccilkrallydclincd.
all hough a classification system would be pur-
IKularly useful for rose wines In tin. al least
ii Fiance, ccttiin treatments (potassium fcrro-
cyanidc I. authorized Tor while and rose wines, air
prohibited for red wines. In addition, each of these
different kinds of wines may be subject lo specific
regulations.
Various characteristics five rose wines their
charm These fruity wines have a light struc-
ture aid are served chilled: they can accompany
an entire meal. Although some have obciiued a
good reputation, they are generally uol premium
wines. Several winegrowing areas (c.g Cotes dc
Provence) have acquired a reputation for produc-
ing fresh, fruity rose" wines. They arc certainly not
easy lo make and arc nol always given all the nec-
essary attention (Castino. 1988). nor arc the best
grapes usually set aside for making rosl wine In
some cases, making a rose may be the best way of
attenuating certain defects in red wine grapes, eg
insufficient ripeness, rot. or off- flavors. Rose wine
may also be a by-product of drawing juice onl of
vat. lo enhance the concentration of the remain-
ing red wine In Fiance, rose wines are usually
dry. while in other countries, notibly the United
Stiles, many off-dry or sweet loses may contain
10-20 g/1 residual sugar
Due lo the diversity of grape crops and wine-
making techniques used, il Is practically impossible
r r*>- rftnaWAir. ,
? »-!.-■ •> • :«■ r«s
44f>
Handbook of linokigy: The Microbiology °f Wiie anil Vinifkrations
M establish a tcchnokigKal definition of rose
wine-.. Further complicating ihc situation, mix-
ing red ant while grapes Is authorized in ccruin
eases, rin i blending red and while wines is prohib-
ited, wilh very Tew exceptions Color is therefore
the only criterion for defining rose witcs — falling
somewhere between the color of red and white
wines. Characteristic value ranges for the differ-
ent color parameters should be cs&iblishcd for the
variots kinds of rose wines (sec Tabic 14.1).
To a certain extent tkc current trend is towards
lighter our wines.
Rose - wines have sonic similarities to red wines,
they arc often made from red grape varieties
and contain a small qiantity of an (hoc van ins and
turn ins. They arc also refreshing, like many white
wines, and for this reason white wincmaking
techniques arc also used in their production.
There is a large range of rose wines in terms
of cotor intensity and attempt, have been made
u characterize them by analytical parameters
(Garcia- Jarcs el ill.. I993a.bl Clarets, at one end
of ihc spectrin), arc light red wines having sonic
body: they require a short skin maceration and
arc softened by malolactic fermentation. After
standard rose wines, the other end of Ihc spectrum
comprises lightly colored wines i California Mash
wines), refreshing and light like white wines.
Stained while wines' resemble white wines but are
made from red grapes or have been in accidental
contact with red wines. The expression Nunc <k
Mane was created to distinguish while wines made
from white grape varieties
Although appearing slightly yellow in color,
while wines made from unmaccraled red grapes
can contain a small amount of anlbocyanins. in a
colorless state dnc to the SO?. A pink coloration
TuMc 14.1. C«m|
•cdwincMBIoui*
idriuin of tire cnmpinki
and Peynuud. 20011
on o( «»e »ml
Color
ntf»iy
/cm
Tout
pill) f bc£»l
imgil)
Ib»c
Youip red u ine
Red wine who
0.7-2.1
22-5.1
8-18
in n
>40
20-50
90-250
>350
after the ;nldiiion of concentrated HCI indicates
their existence. This reaction is very sensitive
and clearly differentiates stained' wines from
Mane ik Mane wines, which do not change color.
Slight contamination in white wines can occur
even in correctly washed and maintained wooden
containers that haw contained red wine
Many red grape varieties arc suitable for making
rostf wines. The Cotes dc Provence vineyards spe-
cialized in rose wine production have developed a
blend of grape varieties that provide wcll-bakinccd
color, banquet, and body (Flanzy. 1966). Cinsanlt.
Syrah. and Mourvcdrc add aromatic depth . finesse,
and elegance to the basic vane lies. Carignan and
Grcnache.
Among the variety of methods for making rose
wines, immediate pressing and drawing off arc the
moslcommon Carbonic maceration (Section 12.9)
is not very widely used, but il produces inter-
esting, complex aromas in full-bodied rose wines
(Adrc el at.. 1980). However, it frequently results
in wines that arc too deep in color forackLssK rose,
even if the anaerobic phase is short and Ihc temper-
ature Is controlled (35 : C for 36 hours or 25 ; C for
48 hoars), so they must be blended with lighter-
cororcd wines
Il R important to monitor grape ripeness Grapes
intended for line, crisp rose wines shonld not
te overripe (potential alcohol not exceeding \1'A
by volume and relatively high acidity), while
those intended for fuller- bodied, softer roses need
slightly higher potential alcohol levels and kmcr
acidity Only healthy grapes should be used, as
wine quality is likely to suffer if over IS'* of the
raw material is aliened by rot
1 1 is best %> harvest Ihc grapes when the tem-
perature is cool, al nigbl or in the early morn-
ing, to preserve their rruiliness. Most grapes for
rose wines are handpKked. but a properly adjusted
harvesting machine also gives good results.
14.1.2 Importance of Color in
Characterizing the Various
Types of Row Wines
Characterizing rose wines by analytical parameters
has long been sought. Table 142 indicates that
Other Wincmaking Methods
Tabic 14.2 Pfccn
of id.. 1976 >
Dlie com
pour,*. »«
l, ">"'"
ind color of diflcrcni type*
ii f Fir nek rn
scwinc
I [RIM
"~ ay ° m
Wins
Total
Anihocvjni*.
T»«nim
Color
Tin
1*
T»«nin/
phenolic
indev
(■0/1)
l-p'll
itfcmiy*
imhocvani.
Aajou
0.10-2.00
050-
I.Sn
Bcurn
7-14
14-74
ISO -430
0.76- 1.18
4J- 10.4
Honk- Jin rose
7 11
35-41
440-850
069-167
I0B-2I.I
Bunkaiix child
10-14
1 is- too
720-800
105-150
S.3-6J
Cikc> de Prove ih:
c (direct
prcuing)
7-11
14 W
80-320
0.38 1.19
DJO
: .98
5.6- 158
Cotes de Prove ik
cll'rec o
in)
7- IS
II 02
63-270
031-1.70
058-
1.62
2.1-78
Midi Idircci piew
«g)
10-14
13-35
180-320
063-1.19
ii SO
1.17
SD-IS8
-OJ>4Sl + OI>ia
•oinaxoDsaic
K. I iiiiii
mlllMViIUI
UttUlM)
autbocyanin ionic utralions arc between 7 and
50 ntg/1 for rose wines obtained from directly
pressed fresh grapes For rose wines obtained by
drawing off alter a short maceration, the maximum
concentration is lilt mg'l
The rannin/anlhocyauin ratio permits the two
wincmaking methods (dircci picssing and drawing
oil" i to be differentiated This ratio diminishes as
■he maceration lime incrcascsand it is higher when
the grapes arc directly pressed
Andre el at. ( 1970. 197 1 ) strongly insisted that
color is important when evaluating rose' wines.
Rose wine color has a very large range of intensity
and lint When the color is intense, it verges on
bright red: rcciptocaUy. Ike clearest wines have
a yellow nuance The color intensity expresses a
more or less full-bodied structure.
The same researchers discussed the resale of
a tasting thai demonstrated the inltucncc of cotor
on the evaluation of rose wines Six rose wines
were submitted h> the evaluation of a tasting group.
The wines were classed according to their average
score and were ranked according to three different
criteria:
• a classification taking only color into anoint.
• a classilicalKm resulting from a traditional
lasting (cotor. odor, lasici:
• a dassifkation elicited without Liking colorinlo
account (dark glasses).
The first iwo lastings produced similar result
but the blind tasting led lo very' diflcrcni results
With standard rose? wine, without specific charac-
ter, cotor was the essential element of its evalua-
tion, as toag us Ihc wine had no Daws
Rose wine color is directly influenced by grape
variety. It depends on the unibocyanin conccntru-
lion in the skins and their dissolution speed. All
else Icing equal. Carignan produces mote colored
wines than Crcnache. and Cinsuall produces the
lightest wines. The tinl of the color also depends
on variety. A prevailing yellow color is the result
of a higher extraction of tannins with respcel lo
anthocyanins. The color of rose wines made from
Crcnache rs predominantly yellow, but Carignan
produces dark pink wines, with purple nuances.
141.3 Rose Wincmaking
by Direct Pressing
This widely used method consists of using while
wincmaking techniques on red grapes, directly
pressing fresh grapes A certain degree of mac-
eration Is necessary to obtain cotor. It is done
directly in the press cage, while the crushed grapes
are being drained. I"nrs method therefore docs not
require as quick an extraction as while wincmaking
methods.
Pressing methods have an important effect on
wine quality. Increasing the pressure, of course,
increases total phenolic compound extraction. In
addition, aflcr each lime that the press cake is bro-
ken up. extracted tannin concentrations increase
™*
Handbook of Etiology: The Microbiology of Wine anil Viniflciiions
■khc quickly Ihun anth<vyanin concentrations, aid
ibc yellow Dm Increases. The different press juice
should I here fore he selected in the course of cxttuc-
lion and he discerningly blended wilh ihe Tree run
juice. The press Julie float the last pressing cycle
■my be eliminated because, besides its vegetal
tistc. ii supplies more tannins than anlltocyanius.
Immediately aftcrcxiraction. the Juice should be
protected from oxidation by sulliting (5-8 g/nl>.
In Ibeory. clarification seems less iinportint in
rose wlucmaking thai in while wincmaklng. but
this practice refines wine aroma and diminishes
the iron concentration. Must can be treated with
ben (unite Authocyanln fixation results in a slip hi
color decrease but it is brighter and less sensitive
lu oxidation It Is not advisable i» use ben unite
wilh pectolylic enzymes
ExUcbk clarilkatioi of the must is tot required.
As in the treatment of while must (Scclioi 133).
turbidity levels below 50 NTU may lead k>
diflkrulty in fermentation, while levels above 250
NTU may tesall in herbaceous off-odors Low
doses (03-2 1'i'bl) of prclolylK enzymes may
facilititcscilliag
Fining with casein, gelatin, or bcatoaltc may
be helpful ii clarifying the must (Flanzy. 1998).
especially when the grapes me botryti/ed. tricing
care to use small enough doses not to affect the
flavor of the finished wine. Settling residues may
be clarified in the same way as those from white
mast are clarified (Scctioi 1333).
Commercial yeasts should be selected for their
fermentation capacity aad performance in reveal-
ing aiomas. Tcmpeiulure should be maintiincd at
approximately 20'C. The poor fermen lability of
certain musts may lead to problems in completing
fermentation, alleviated by adding uitiogcu and.
especially, oxygen.
In the past, malolactic fermentation was not
customary— the freshness ;ind fruitincss of these
wines was considered indispensable. Today, this
second fermentation is used to make these wines
fuller. It is often difficult to carry ont and requires
a more mode Rile sulfitiag of the grape crop.
Rose" wines should be kept at relatively km*
temperatures to preserve their aromas, with an
adequate dose of free sulfur dioxide (20 mg/l).
Immediately after sulfiling. wines are slightly
discolored and appear more yellow, but in Ihe k>ng
term the color is mote stable, with a more affirmed
pure red nuance. This type of wine is generally
Intended to be enjoyed young so It is important to
avoid loss of color, especially as wine that has been
heavily sulfated to prevent malolactic fermentation
may lake a king lime to recover. Color may be
stabilized by adding uaala extracted from grape
seeds (10 g/hl)
14.1.4 Making Rose Wines by Skin
Contact or Drawing Off
Dccpcr-cokHCd. fuller-bodied rose" wines ate pro-
duced by leaving the skins and seeds In contact
wilh the juice for a short time, to extract more
anthocyanlnsaad tannins However. excessive skin
con tm may result in loo mach coktr. accompanied
by marked aslringcncy and bitterness.
The jalec may be kept In contact wilh the
giapc solids in the press for short periods of
time (2-20 hours): this technique is known aspre-
fermentation skin contact. This ptocess may also
take place ina vat fora longer period ( 10-36 hours),
then sob ic of the juice is drawn oil and fermented
as a rose wine. Skin contact is primarily aimed
at making rose" wine, while drawing off is mainly
Intended to produce a mote concentrated red wine
froai the remaining must In the vat. wilh the rose"
wine made froai the diuwn-ofT juice as a by-
product In the Cotcsdc Provence vineyards, which
specialize in rose wine production. 40« are made
uslag skin contuct. I ' ' < by drawing off juice, and
the remaining 50f by pressing the red grapes
immediately iMasson. 2001 ). Pre- fermentation skin
con tic t is known toenhaace sof Inessaad fruit, while
reducing acidity.
In both cases, (he crushed, stemmed, sulfitcd
grapes are transferred cither directly loa pneumatic
press with the drains closed, or to a vat. After a
variable period oa the skins (2 -36 houts). Ihe juice
is separated from the solids, cither by piessiag or
by drawing off all or furt of the liquid from the vat.
The mast is then fermented in ihe same way .is juice
froai grapes that have been pressed immediately.
In certain cases, only part of the juKc( 10-20* >
may be drawn off from Ihe vat. Once a ccroin
Olhcr Wincmaking Methods
Tabic I4.X ComputnanofdiHcR
■■. :Si.lr.i.i,- ,1 1908)
WiKMfcMg
method
Tata
1 phenol,,
index
Adhocjnis
(■g/ll
Tianiu
(■gA)
Color int ciuiv"
Ti.nin/
Mjctalkm lot 12
Without SO,
SO) - ID g/hl
>—
11
Id
7
26
100
100
320
M0
OJl
0.--2
133
14 J
I2J
7.0
vulnmc of juice has been drawn. Ii is refilled with
crushed grapes This technique is noi only to pro-
duce a rose" wine bnl at*) to enhance the pheno-
lic conlcil and cotor of ihc remaining red wine,
by increasing ibe solid/liquid raiio )■ Inc vat in
some cases. Ihc main purpose of drawing off Is lo
inprove ihc quality or lie icd wine.
Con tact II me. temperature and sulfi ling are factors
thai influence phenolic compoand dlssolniou and
color in lose" wines (Casiino. 1988). Sulfur dtox-
hIc is known to have a certain dissolvcnl power
(Section 8.7 J). It is not manifested during iradi-
tioial red wincmaking. die to the preponderant
effects of other factors (duration, icmpcmlare and
pumping- over). Yet when maceration Is limited. Ihc
effect of sulfiiing is obvious. Table 14.3 shows the
inipacl of the wincmaking technique on Ihc color
intensity and phenolic compound concentrations of
lose" wines Sulliting promotes an thocyan in dissolu-
tion and color enhancement. It is not easy to control
the conditions thai will prodnce the required color
and phenolic structure, as they depend on the spe-
cific characteristics of the wine.
The saccess of sach rose wincmaking Is based
above all on the use of healthy and perfectly mature
quality grape varieties. Malolactic fermentation isa
general practice and becomes all Ihc more necessary
as Ihc maceration phenomenon increases A low
acidity soflcns the tastes of the tannins.
14.2 BOTRYTIZED SWEET WINES
(SAUTERNES AND TOKAY)
14.11 Introduction
Due to their lack of turn ins. while wines lolcralc a
large divcisily of straclurc such as varying acidilics
and ihe presence of carbon dioxide (sparkling
wines) or sugar (sweet wines). Most sweet wines
are while wines. Only a few special red wines, or
fortilicd red wines, sach as port, contain sagar. bul
Ihey receive additional alcohol.
Sweel wines correspond to an incomplete fer-
mcnttlton. leaving a certain proportion of grape
sugar thai has not been transformed into alco-
hol. Wines are arbitrarily Judged to be semi-dry.
sweet and syrupy sweel Uii/uomix) according lo
their sugar concentration: up lo 20 g/l. 36 g/1 and
above 36 g/l. respectively The sugar concentra-
tion is sometimes expressed In potential alcohol:
for example. 12 + 2 signifies a wine containing
]2'i volume alcohol and 36 g of sugar per liter
(2'i volume of potential alcohol).
Semi-dry and sweet wincmaking are fairly sim-
ilar to dry wincmaking. but Ihc grapes must have
a sufficient sugar concentration and the fermen-
tation mast be slopped before completion, either
naturally or by a physical or chemical process.
Syrupy sweet wine making Is different The
required high sugar concentration cannot be
attained during maturation Certain processes must
concentrate ihcjalcc and certain wincmaking steps
are unique lo these wines
Drying, freeing and noble tot are used lo
eonccnlraie juKc. Due the Importance of noble rot.
it will be discussed In more detail later in this
sec boa.
Grapes can be dried naturally by ihc sun. when
left on Ihe vine, or by artificial heating (Sec-
tion 1 1 2) This ovcrripcnlng pmcess can be used
to make different types of wine. The drying of the
g rapes results in a varying degree of concentration,
but the cn/yni.ilic systems of the Hull permit a
grcafcr concentration of sagars than acids. Grapes
-SI.
Humllkxkk of Etiology: The Microbiology of Wive anil Vinirications
can also be dried for np to several months ■■ a
clowd mom which may or may not be healed This
method results in masts containing up to 400 g of
sagar per liter, capable of producing syrupy sweet
wines. The fermentation of these Juices is difficult
and the price of these wines Is high, due to volume
loss and production cash.
Freezing grapes on the vine produces ice wine
(Eiswcin). well known in Alsace and Germany.
The grapes ate left on the vine until the winter
frosts. The temperatures, -ft lo -7"C. lead to the
partial freezing of the least ripe grapes By pressing
the giupes at low temperatures, only the juice from
the ripest grapes, containing the n>ost sugar, is
extracted. Cryocxtraclion (Section 142.4c) seeks
to reprodacc Ihisnaiaral process jrtitictilly Mak-
ing such wines is to subject lo weather condiliois
and not always possible every year. The method is
difficult and expensive, and should only be used to
make premium wines.
14.2.2 Noble Rot
The biology of Botryla cinerta and it. develop-
ment in the form of noble or vulgar rot have been
described in (Section 106). This overripening pro-
cess, noble rot. permits the production of great
boiryii/cd sweet wines. These exceptional wines
can oily be made in specilic conditions Their pro-
duction is therefore limited.
The Saulcmes-Barsac region Pi certainly one of
the most highly esteemed areas for noble rot sweet
wines but other regions exist in France (Loupiac.
Saintc-Cmix da Mont. Monba/Jllac. Anjon). in
Germany (Moselle) and in Hungary (Tokay).
Noble rot presupposes fangus development on
perfectly ripe grapes. Scmillon and Sauvignon
grapes must attain 12-134 vol. potential alcohol
and have a pH of less than 3.2 before any fungal
development. At this time, the berries are golden
with slightly brown thick skins. This result can
only be attuned on certain terroirs. with low crop
yields (40-45 hl/ha). before berry concentration
by noble rot. Mycelial filament* penetrate through
microlissures and decompose the grape skin. This
decompositio! is the result of an intense enzy-
matic maceration of the grape skin. The grape then
attains the pmtrii plein (fnll rot) stigc and has a
brownish color. The berry docs not buist: il main-
tains Ik shape but the skin no longer mainuins
the role of a protective barrier from the external
environment. The berry acts like a sponge and is
concentrated as the walcr evaporates The grapes
are harvested when they attain the roil stage. The
concentration in the berry leads to an increase in
internal osmotic pressure that causes the death of
the fangus. The second phase of Boiryiis einerea
development mnsl therefore occur soon after the
full rot phase, before sabscqacnt Boiryiis develop-
ment can result in gray rot. The distinction between
noble rot and gray ml is not always obviovs
Late-season weather and harvesting conditions
are essential lor noble rot development. Maximum
grape concentration shoald not always be sought,
because gtay rot may develop when conditions are
not ideal. Attempts have been made to harvest at
the full rot stage and then concentrate the grapes
by artificial means
Alternating hnmRI and sunny periods are essen-
tia) for reaching the perfect state of maturation.
Gtay tot occurs when Bmryiit tinereti dcvckips in
extremely hamid conditions. The development of
noble rot retinites a particular climate, ideally with
morning fogs to assure fungal growth, followed
by warm aflcmoou sunshine to concentrate the
giupes. for a relatively long period of 2-4 weeks.
In Bordeaux vineyards, these meteorological con-
ditions correspond with the establishment of a
high pressure ridge extending the anticyclone from
the A /ores to the north-east. Noble rot can also
develop rapidly in the Girondc region after a short
period of rain, caused by oceanic depressions, fol-
lowed by a sunny and dry spell (low humidity.
<M ) with winds from the north lo north-east This
type of weather is generally associated with the
presence of an anticyclone in north-eastern Europe.
Noble rot develops progressively on different
giapc clustcis and even on different grapes on
the same grape cluster. The grapes mnsl therefore
only be picked when they attain ihcir optimum
maturation state. Selective harvesting ensures that
grape* pickets only remove Ihc nobtc-ioitcd grape
clusters or grape cluster fractions during each
picking. Climatic conditions dictttc the number of
selective pickings each year — up lo three or four.
Olhcr Wincniiiking Methods
451
Harvesting can continue oniil novcmbcr in Ihc nortl
hemisphere Dk lo ihc evolution or grape crop
quality fmnmnc day to anothcracconling loi lunatic
conditions ;iid ihc evolution or rot. juice should be
viniticd separately according Id harvest dale
Vincgrowing lor ihc purpose of making botry-
ll/cd sweet wines requires more mclicuktus caie
than for making dry while wines. This is partic-
ularly ihc case In oceanic climalcs. favoring Ihc
carl >' implantiuon of Rtrtrylis on sensitive vari-
eties In ihc Saulernes region. Scmillon and Sauvi-
gnon require shorter cane pruning and Ihc rigorous
control of vegetative growth, in particular early
delcating (before terms/in) in ihc grape /ones
Variable proportions of bunch ml can coexist
will noble rot. Tic involvemcnlof accllcacid bac-
teria results in Ihc production of volatile acidity.
Giay pji can also font odors and usies rcscnt-
Ming mushrooms, niokl. iodine and phenol. In
addition, considerable amounts of carbonyl- based
compounds nay be prodnced. which bind sulfur
dioxide and make these wines difficult « sdbili/c.
They arc produced by aceiic haclcria in Ihc
GlucontibtK'ter genus present on the grapes. 5-
> Aolnii lose is one of ihc main snbstinces rcspon-
sibtc for ihrs phenomenon (Sections 8.4J and
8.46).
14.13 The Com posit km of Musts
Made from Crapes Affected
by Noble Rot (Section 10.6.3)
and the Resulting Wines
Noble iol can rcdnce crop volute by up lo
50** Low crop yields of 15-25 nl/ha affect grape
quality.
The fungus consumes a large quantity of grape
sugar Id assure growth According u Ribfrcaa-
Cayon^<rf(l976>:
V.i'. marc urgir and i*kc Ihc »n.c votimc
n obtained im« bra I by papes uih icspt.i lo
r> 'I ■■ ■:- 1 : " led -"'ii ;■'"■■ for (be ■-.. - 1- i . .v.i.--. Named
conceal at Ion und the ivuihinp ccaaidcrahlc crop
. iclt Imci die nsjMmiMi- lurlhc&C richer and
better if ill i;- ■■ i ■■.-..
This lung us also consumes a lonsidcraNy highci
proportion of ackl than sugar. This pheni
is beneficial lo wine quality, since the acidity
increases much nunc slowly than the sugar con-
centration. It Is not a simple concentration by water
evaporation, but rather a biological dcacidifkatioi
Botrytis cinerea has the rare property of degrading
tartaric acid: its concentration decreases more than
the malic acid concentration and the pH conse-
quently increases by 0.2 unit.
Table 10.11 takes the modifications caused by
noble rot into account The lower sugar and acidity
concentration due In weight and volume loss in
the 1000 berrtcs corresponds with a significant
increase in the sugar concentration and a slight
decrease in acidity of Ihc juKc.
Boiryiis cinerea forms glycerol and gluconic
acid from sugar These two compounds play an
important role in characterizing rot quality Glyc-
erol is produced at the start of Bmrylis devel-
opment, lis concentrations increase as Ihc rot
becomes more noble. Gluconic acid is formed
much later and corresponds with a poor rot evolu-
tion Wines made from healthy grapes should con-
lain less than 05 g of gluconic acid per liter, noble
rot wines between I and 5 g and gray rot wines
more than 5 g The higher the glycerol/gluconic
acid ratio, the better the rot quality is
The ocvcVapntcn t of Botiytis cinerea also result
in Ihc production of two polysaccharides (Dnbour-
dieu. 1982) One. with a complex structure, has
antifungal properties and inhibit! alcoholic fermen-
lation. The other isa^-glncan with colloid protector
properties and it impedes ctanficalion of new wine
It is prodnced inside a viscous gel. located between
the skin and the pulp. Ib> level of diffusion in the
juice Is related lo grape handling and treatment con-
ditions All brutal mechanical operations, such as
crushing, pumping and pressing, diffuse glncai in
the juice and make the wine more dill icnlt loe huify
Boiryiis cinerea development corresponds will
en/ymaiK changes in the grape. Lactase, an oxida-
tion en /j inc. replaces the grape s tyrosinase and is
much more active on phenolic compounds than the
latter. Noblc-rottcd musts arc probably relatively
well protected from oxidation, since most pheno-
lic snbstraKsof Ihc grape arc already oxKli/ed by
tic trnte of harvest. Specific varietal aromas seem
to be fairly well protected from oxidation during
4H
Handbook of linokigy: The Microbiology of Wine anil Vwific.uions
■oblc rot dcvclopncnl (Srxrion 132.1). In addi-
tion. Botrxtis synthesizes a nuntber of enzymes
(ccllulasc. polygalacturonase), permitting its pene-
tration into the grape An esterase thai hydn>ly/cs
fcrmcnttlioicslcis has been isolated. thus account-
ing for the differences in atonia between wines
made from grapes affected by noble rol and from
other white wines
The molecule soukin also participates in the
aroma (roaslcd. crystallized fruit, honey) of noble
rot wines ( Section 10.6.4).
Another cbaracleristic of wines iii.n.k- in ■in
grapes affected by noble rol is their relatively high
volatile acidily level. lis origin can be accKlental.
die to the presence of lactic acid bacteria in
JuKc and especially anew acid baclcria on grapes.
Yeasts also form sonic volatile acidily. due fc>
the corresponding fermentation difficulties of these
jukes (Section 2.3.4).
Operations likely lo reduce the production of
volatile acidily include (Section 14.23) sapplc-
■Knling the must with anintoniacal niltogcn. which
skonld be adjusted to 190 mg/l at Ike start of fer-
mentation, combined with seeding with a suitable
yeast and aeration. The aim is K> increase the cell
population to a maximum, as this minimizes the
forn ration of volatile acidity For this reason. Euro-
pcan Union legislation has specdied hither limits
for volatile acidity in botryli/cd wines Lafou-
Lafoureadc anil Ribcreau-Gayon (1977) sought K>
specify the origin of Ibis volatile acidity Two lac-
tic acid isomers and ethyl acetate were analyzed
in wines. Lactic acid bacteria produce laigc quan-
tities of Ihc former and acclK acid baclcria the
killer The authors concluded the following:
• If d (— atactic concentrations are more lhan
200 itg/l. lactic disease nay exist.
• II i (+)-laclk conccnlratWns are more thai
200 mg/l. malolaclk fcniicitalion is respon-
sible.
> If ethyl ace tile concentrations are more
160 mg/l. acclic ackl baclcria are involved.
Hu-
ff these three paramcieis aic less lhan the
indicated values. Ike yeasts are entirely responsible
for the volatile acidity Volatile acidities between
0.9 and U g/1 n H;S0 4 <I.I-I6g/I in acetic
acid) of many Sanlerncs wines were found lo be
produced exclusively by yeasts, wilk 10 baclcrial
involve men l
14.2.4 Noble Rot Juice Extraction
la i Pressing; flrapcs
The general rules of while winemaking should
be followed when transporting noble-rotted grape
crops The depth of grape crops, in particular,
should be kept loa minimum during transport, lo
avokl spontaneous crushing.
Upon their arrival al the winery. Ike grapes
skonld be manipulated with care. Rough handling
provokes Ihc excessive formation of suspended
solids and vegetal Lists II would also make the
wines more difficult u clarify, because of the
diffusion of glucai (torn Boinl/s cinereti m the
juke Winemaking kckniqucs should make use
of gtavily. as much as possible. Pumps on self-
emptying gondolas are often too bin til
Noble-totted grapes are generally crushed and
macerated in Ihc liberated jnice. This operation
helps lo extracl ihc sugar. The grapes are nol
destemmed lo facilitate Ike citculalion of jnice
in tfce pressed skins, but manual dcslcmming is
sometimes practiced alter ihc liist pressing with
Ike stems In Ikis manner. Ihc tmnic and vegetal
substances of Ihc slcms are nol conferred lo the
must during subsequent pressings at high pressure.
The extraction of noble-totted jukrc is difficult a
high pressing pressure must be used and Ihc press
cake musl be broken up between pressings
Due to their high viscosity', these grape crops
cannot be drained before pressing They are
directly Iraisfcrted lo the press cage by gravity
or a conveyor belt Pressing is ccruinly the most
difficult and essentia! operation in botryliTCd sweel
winemaking. and incorrect operation of the presses
can sacrifice quality These operations in particular
musl be carried out slowly and delicately
Continuous presses should nol be used. They
are too brutal even when equipped with a large
diameter screw turning very slowly They shred
Ike gtupc ctop and pioducc juices wilk a k>l of
suspended solids, and glucan-rick wines thai are
difficult to clarify.
Olhcr Wincmaking Methods
453
The older vertical cage hydraulic presses arc
effective and prodncc lew suspended solids in
Ike juice. The press cake is beoken up manu-
ally with these presses and Ihc pressing cycles
arc slow Moving- head presses arc therefore pre-
fcrrcd because of ihcir ease of operation and salis-
ractory yield. After two K> three pressing cycles.
Ihc last cycle can be effected by a hydraulic
press. whKh permit* higher pressing pressures
Pneumatic presses, used for other types of grape
crops, arc not well adapted for pressing noblc-
rotled grapes. Their pressing pressure is insuffi-
cient Although manufacturers nave created special
models with up to 3 bars of pressure, these pneu-
matic presses do not properly extract the juices
with the highest sugar concentrations, above 21 or
2i'i volume of potential alcohol.
Selecting fhe best juice from Ihc various pressing
cycles is a difficult problem With healthy grapes,
the drained juice and the juice from first pressing
cycles arc the richest in sugar the last pressing
cycles are vinilied separately. With noblc-roiicd
grapes. Ihc juice with the highest sugar concentra-
tion and the highest iron and tannin concentrations
is Ihc most difficult to extract: it is released in the
last pressing cycles The addition of this j nice to
the blend can therefore improve wine quality but
should be done prudently
(b) Cold P rasing (Cn»cMracli<in>
Cryocx true lion permits the selection of the most
sugar-rich and therefore ripest grapes. Cbauvcl
ei ul. (1986) first proposed this technique
Cryocx traction is based on a law of physics
Raoults's tuw siatcs that the freezing point of
a solution lowers as the solute concentration
increases. When the Kmpcraturc of a white grape
crop Is lowered to O'C. only Ihc grapes containing
the Icasl amount of sugar arc frozen. By pressing at
Ibis temperature, a selected juice is obtained which
represents only pan of the total juke volume. The
potential alcohol content of this juice, however, is
higher. By further lowering the Icnipcralnrc of the
grape crop and the pressing. Ihc number of frozen
berries is increased. Asa result, the selected must
volume is further diminished and Ihc potential
alcohol content increased Table 14.4. which gives
Ibe results of an experiment, specifics the increase
in potential alcohol content for grape crops pressed
al different temperatures The selected must vol-
ume represents between 60 and KIM of the total
volume, depending on the temperature
The selection of Ihc most sugar- rich and thus
ripest grapes by cryocx Irac lion is Ihc primary
cause of improved wine quality l-rce/Jng may also
concentrate grapes diluted by rainy harvest sea-
sons. A phenomenon called snpracxtraction is also
involved Freezing and thawing grapes results in
tissue destine lion and a better sugar extraction
In practice, the grape crop p. collected in small
containers and fro/en in a walk-in freezer at vary-
ing Icnipcralurcs as low as — I6'C. The grapes arc
ihcn pressed in a horizontal moving- head press or
in a pneumatic press The freezing point of the
richest grapes is suflicicnlly low thai their juice
may be extracted without difficulty. Table 14.5
Tabic 14.4.
andrcktul
tilled of ptruiap ii
muM* Horn noble n
rapcaiurc (cryocx
H £opo> (Sauicmc
imclnnlonihc vohiMcai
• XChauvet eiiJ.. 19801
idlktihol potential I'
«vol.)ofichxtnl
Coal ml
Planing
Selected
HUB
Residual
■■l
Vbhiac tution
ptllCHDl
alcohol
t'i vol.)
rmfccuuK
(°c>
Vofcim.
(hi)
Potential
alcohol
<3 vol.)
VBhlHM
(hi)
Potent ial
alcohol
<-. ™t.)
hlcMlcd aini
10.7
-5
110
I9J
10
II J
80
100
-6
815
190
215
I2J
79
100
-85
745
2D.7
33
98
09
10.2
-10
OS
::.-;
17
13 1
04
100
II
07
23.2
33
10.7
07
170
IS
Ml
234
40
IM.
fMI
I landbook of Enotogy: The Microbiology of Wine anil Vnifkatioas
Tabic U.S. Improvcacui
1985 by lalnp ciyociiiaciJ
i>f Sjmemo muu tfjalily in
anlChauvci rt.d.. 19801
VIocy.nl Gape
icapeauiw ui
picui* (C)
pctcrtinl alcohol I'i vol)
CoMnil Selected
(lives the results obtained (or live Sanlcracs vine-
yards in 1985. Instilling a cryocxiiuction system Is
expensive bui energy costi arc reasonable In any
ease, this technique can only be applied (or limited
grape crop volumes.
The improvement in mast quality is not only
limited to an increase in pnleniial alcohol content.
These musts have the same characteristics as their
kite- harvested counterparts: they arc more dlflicull
u ferment: the risk of volatile acidity production
is greater; and the sulfar dioxide binding rate
p. higher However, these cryocxtraclion selected
wines are clearly preferred over the control wines
(Cnaavct eitrl.. 1986) due to their increased
concentration, (inesse and distinctiveness
This technique Is applicable to all types of white
grape crops but is especially interesting for loblc-
rotlcd grape crops Excessive hnmRliry during
ovcrripcniag can prevent the giapes from obtaining
the desired concentration levcK Prolonging the
concentration phase would only increase grape
degradation and lower crop qnaliry. but. by picking
early, cryocxtraclion can be used to eliminate
excess water and select the grapes capable of
producing quality botryli/cd sweet wines. The
quantify Is diminished but qnaliry Is maintained.
This method can complement mannal sorting
during the harvest, but cannot replace it
(c) Sulfiting Jiicc
SuKiting has several dices during white wlnc-
niaking:
I It protects against oxria lion by inhibiting bic-
casc. prodnccd by Bmryiis cinerea. Due K> the
considerable oxidation of grape phenolic sub-
strates during overripening. however, the oxi-
dation risks arc less significant than one would
think.
2. By blacking fermentation for several hours, it
permits a coarse clarification by sedimentation.
i It Is also capable of inhibiting the development
of acetic bacteria, often present In huge quan-
tities on holryti/cd grapes, as well as lactic
bacteria, responsible for ntalolaclK fermenta-
tion. Finally. sulOtlng restrict, the proliferation
of some spoilage microorganisms, particularly
apicnkiled yeast, that waste" sugar and focm
unwanted by-products in the wine.
4. ll destroys antifungal substances and thus
facilitates alcoholic fermentation
Sulfiling juice intended for bolrytixd sweet
wine production has often been criticized. This
operation leads to increased concentrations of
bound salfur dioxide, which remains definitively In
the wine. Subsequent SO, additions mast therefore
be limited to remain within legal total SO; limit,
thus com prom ding the microbiological stibilizu-
tion of wine In practice, this inconvenience of
snlliting Is attenuated by the fact that only 40-6fXI
of the SO. added in juice Is found in the hound
form In wine. The rest is oxidized into SO).
To conclude, a light snlfiling at 3-5 g/hl.
for example, is generally recommended at Ibis
stage.
(d) Jiicc Clarification, licnlonile Trcalrncnl
and Jiicc Corrections
Ckirilicalion r. an indispensable operation in while
wincmaking. but its advantages are more disputed
for botryiiocd sweet wines. It has been blamed
for making wines thinner. This criticism seems
excessive — In most cases, clarification improves
aromatic finesse and gastilory qualities However,
the natural settling of bolryti/cd musts Is difficult
to effect: the suspended solids and the highly dense
jukc have similar spec Hie weighs Mas! viscosity
is also high due to the high sugar concentration
and the presence ofglucKIK colloids produced by
Olhcr Wine making Methods
455
Bairytis chierea. In practice. ii.niu.il settling Tot
18-24 boars results ii partial clarification, per-
milling large particles to be eliminated The ;iddi-
tion or pccmlyric cn/ynics Id noble-rotted juice
presents in nLirilk.it Ion advantages. Pcctinascsarc
secreted by Bawyiis cinerea in ihc grape ami arc
found in abundant quantities in Ihc JuKc. The aver-
age pectinase activity ratio bcrvvcci hcallhy grapes
and noble fallen grapes is estimated to be 1:10.
Natural setlling for 3-4 days at O'C permits a
more effective clarification. This kind of clarifi-
cation shoukl only be nsed when there arc doubls
about rot quality.
As with dry while wine making . ovciclaritication
can lead k> large fcniicnlation probkrms and
iicrcascd acetic acid production. Must turbidily
should aol be as kiw as in dry while wincmalang
(100-200 NTV); 500-600 NTU orcvci a slightly
higher turbidity is perfectly acceptable. Moreover,
botryiiecd sweet wines arc not subject to the same
probkrms related in insnfticicnl clarification as dry
while wines—the development of reduction odors
and vcgclal taslcs.oiKlability.elc.
Benlonile has been widely used as a setlling
aid for juices in large fcrntcniDis during whik:
winciiaking bui is no longer used Id make rurrcl-
fcrnicnlcd wines thai arc aged on Ices The effec-
tiveness of this treatment in the making of noble-
rotlcd sweet wines has long been qnestioncd. The
high colloid concentration in these wines appears
in diminish the adsorbing power of bentonile with
respeel to piokins. Moreover, adding ben tonne
before fermentation as opposed to after racking
does not facilitate sedimentation. The prcsence of
ben unite can also make racking, following the
completion of alcoholic fermentation, more diffi-
cult. For these diffcrcnt reasons, nines are Healed
with he n ion He a few months before bottling, after
a piotcK insCibility laboratory lest.
Depending on the legislation, various adjust-
ment such as Ihc addition of sagaror modification
of acidity can be made Id juice beforc the initia-
tion of fermentation. These adjustments should he
limited lo avoid discquilibruliug Ihc wine.
The addition of ammoniacal nitrogen often pro-
motes the fermentation of these musls. depleted by
Ihc development of Bairytis cinerea. An addition
of 10- 15 g of ammonium sulfate per hectoliter.
25-40 mg of ammonium ion (NFU) pc> liter, is
gene ml ly rceom mended.
The addition of 50 mg of thiamine per hectoliter
is often beneficial. II is nol only a growth factor
but can also limit the combination rale of sulfur
dioxide during wine storage by promoting the
decarboxylation of kcioaic acids (pyruvic acid
and a-kcloglutank acid) ( Lafon- Lafourcadc el oT.,
1967).
When dry while wines and noble-rotted wines
arc made in the same winery, adding a small
quantity of lees from hcallhy grape must, after
clarification, can considerably improve Ihc fcr-
mcnclbility of noble-totted musts and diminish
the production of volatile acidity (Duboardica.
ii ii published results! Depending on the amount
available of the hcallhy grape mast sediment sus-
pension. 2-4 liters of sediment should be added
to each barrel II u 2'lu resulting in a turbid-
ily increase of 200-400 NTU. The scdimcnl used
should be sulfikrd. unfcrmcnlcd and conserved at
a k>w temperature.
14.2.5 Fermentation Process
(a) Fermentation Diffkulllcs
Noble- rolled masK arc known to be difficult to fer-
ment. The high sugar concentration Is the principal
limiting facur bat nutritive deficiencies provoked
by the growth of Bairytis cinerea arc also respon-
sible Among possible deficiencies in Ihc mast,
nitrogen is a key factor Masncnfef ai. 1 1999) ana-
ly red 32 musts intended lor dry while wincmaking
and reported a mean available nitrogen content
of 182 nig/I. measured by fomiol titration. The
same analysis of 20 samples of botiyiiycd must
intended for sweet while wine production yielded
a mean value of 84 mg/l only (Bcly el til.. 2003)
Polysaccharidic antifungal substtnecs affecting the
fermentation process arc also involved. For an
identical sugar concentration, juice is more diffi-
cult k> ferment when grapes are deeply attacked
by Batryiis cinerea.
It is advisable k> adjust the ammonium sulfate
content to 190 mg/l at Ihc beginning of fermen-
tation to facilitate the process and minimize the
456
Handbook of Etiology: The Microbiology of Wine anil Vindications
production of volatile acidify Later addition of
nitrogen supplement, is likely to have a negative
impact.
Inoculation of Ibc juice is slroigly recom-
mended The chosen yeasl strati should be highly
cthaiol tolerant and should produce link volatile
acidity in diflkull fermentation conditions The
dry ycasR should not tic introduced directly inlo
the jiKc to starl the fermentation for this type of
wincmaking. A yeast starter should be prepared in
diluted must. supplciKnted with NHi* and yeasl
hulls. Ihen seeded with dried active yeasl al a dose
of 25 g/il of Ihc tola! volume lo be inoculated.
The strrtcr is added to ihc must on Ihc second
day of fermentation, al a rale of 2't of Ihe taul
volume. This increases the maximum >east popula-
tion, which controls fcrnicnlalion rate and volatile
acidity production (Section 2 3.4). In one experi-
ment, adding yeast in this way reduced Ihe linal
volatile acidity conlcnl by 2"<
Noble- rotted wines were tradilionally fermented
in small wixxlcn barrels This method creates
favorable conditions for this kind of wincmak-
ing The jnice from each day of harvesting can
be scparaKd according lo quality Temperatures
ate also better controlled, since the fermentation
occurs al near ambient temperatures, bui ihe cel-
lar may need lo be healed if juice temperatures
arc too low or if Ihc ambient temperature drops
too bw. Barrel fermentation also permits a contiu-
nous uiicroac ration, promoting yeasl activity and
a complclc fermentation. BarrcHcmicnlcd musis
wilh high sugar concentrations produce more alco-
hol than the same mast in a tuige I'crmcnior.
because the fermentation stops earlier in a tuik.
Furthermore, the presence of carbon dioxide pro-
tcts fermenting must fn>m oxidation, and pheno-
lic compounds, the main oxidation snbsiraKs. ;irc
destroyed in the grapes by Brtryn'i cimrea as il
develops.
Tank- ft mailed musts have an even greater need
for aeration, since this fermentation occurs in
stricter anaerobic conditions In sweet wincmak-
ing. oxygen should be introduced during the sta-
tionary phase of Ihc yeast population growth cycle,
rather than during the growth phase, as is the case
when making other types of wine (Section 3.7.2).
Lafcr aeration has been shown to prevent c
increases in volatile acidity, which mainly ocenrs
in the early siagcs of fermentation. Temperature
control Is also indispensable in these conditions to
ensure that the fermentation temperature remains
within reasonable limit* (20-24'C).
Rapid and vigoroas fermentations result in
less aromatic wines and should be avoided, bnl
exaggeratedly slow fermentations arc not a factor
for quality.
fb) Stopping; fermentation {Mutage\
Sugar concentration and alcohol content determine
Ihc gustatory equilibrium of this kind of wine The
sweetness of sugar must mask Ihc burning char-
acteristic of alcohol. Reciprocally. Ihc latter mnsl
balance Ihc heaviness of a high sugar concentra-
tion For this type of wine, the alcohol content and
potential alcohol strength should approximately
satisfy the following rckitionship: 13 + 3. 14 + 4.
15 + 5 This equilibrium is generally obtained by
blending several wine hitches, some containing
moic sugar, others nunc alcohol.
Fermentation rarely sups spontaneously at the
exact ak oho I 'sugar ratio desired In sonic cases, il
can go too far: in other cases, il becomes exces-
sively slow and the increase in volatile acidity is
more signilkani than Ihc decrease in Ihc sugar
concentration. Al this point, the fcrnicnlalion mnsl
be stopped. This operation generally consisted of
adding sulfur dioxide lo Ihc wine
Sulliirous gas was traditionally added directly lo
warm wine, since Ihc yeasts are more sensitive to
SO; at higher temperatures More recently it has
been supposed il is better, before adding SO), lo
wait the wines were allowed lo cool and a portion
of the yeasts was eliminated by racking. For a
given antiseptic concentration, the lower ihc initial
population. Ihc smaller is the residual population.
The wine should be protected from air while
racking, if possible by an inert gas atmosphere, to
avoid the oxidation of cthaool and Ibc formation
of traces of ethanal. which combines sta>ngly
wilh SO;.
In any case, a massive concentration of sulfur
dioxide (20-30 g/M) should be added, lo block
all yeasl activity instantaneously aid avoid even
Other Wineinaking Methods
457
United cthanal production by the yeast*. Scver.il
days after the addition, the Tree SO? concentration
should he verified. A value of 60 mg/l is suitable
for storage. Adjustment can be effected at this
Date if necessary.
14.16 Aging and Stabilization
The organolcplical quality of botrytized sweet
wines improves considerably after several months
of barrels maturation and several years of bottle
aging The boaqucl lakes on liacssc and com-
plexity — reminiscent of coafect fruit aid toasted
almonds. The wine becomes harmonious on the
palate. The sweetness is perfectly bulaaccd by
the alcohol and a note of acRliry gives a refresh-
ing finish. The wines arc not heavy and syrupy,
in spite of their high sugar concentration These
transformations remain poorly understood even
today They occur in conditions, particularly oxida-
tion -redaction conditions, reminiscent of red wine
maturation and aging.
Premium botryti/cd while wines arc barrel-aged
for 12-18 months, sometimes even 2 years or
more The bangs arc maintained ipright. As a
tcsalt. the barrels must be topped off once per
week during this maturation process. The bar-
rels are hermetically closed much earlier than for
icd wines ( 1 -2 weeks after atutagei. Due to the
risk of re fermentation, all wine must be handled
with the utmost cleanliness during the tapping off
operation.
The first racking is generally effected at the
beginning of December, after the first cold spell.
The objective is to separate the cexusest Ices and
conserve the finest. Racking* arc then carried out
every 3 months. At each racking, the barrels are
carefully rinsed with cold water and then sterilized
with hot water at 80'C or with steam After being
drained, they are sulfilcd before being refilled.
Microbiological stabilization is difficult with
these wines, Re fermentations arc always possi-
ble, despite their low fcrmcntability linked k> the
involvement of nobkr rot First ol all. the SO? com-
bination rate can be high (Section 8.4). It R not
always possible »> obtain the necessary concen-
tration of free SO? concentration, while remaining
within the total S0> concentration limits imposed
by legislation. The free SO. concentration should
he maintained at approximately 60 ntg/l. This diffi-
culty rarely occurs in the presence of noble rot and
is generally linked to at least a partial involvement
of i! raj rot. Dnc to their antiseptic properties, sor-
bic acid and fatty acids(C h and C|,)can be used as
chemical adjavauK to sulfur dioxide (Sections 9 2
and 9.3) daring the saaragc of these wines.
Various physical processes can complement the
effect of SO: and help to stabilize these wines
(Section 9.4). Wine conservation at low temper-
atures carouud C) hinders but docs not defini-
tively inhibit yeast development, bat beating wine
at50-55 u C forscveral minalcscan totally destroy
the yeast population (Section 44) Heat- sicnli zed
wines must be stared in sterile conditions to
prevent subsequent contamination Sterile stor-
age, however, poses practical problems and is
not possible in wooden casks (Volume 2. Section
12.2J).
The difficulty of storing high sagar concentra-
tion wines, capable of refcrmenting. has incited
the dcvckipatcnt of other techniques. A dry wine
and a sweetening reserve* can be prepared sep-
arately The sweetening icscrvc is a partially fer-
mented JaKe containing 2-25'i vol. alcohol and
150-200 g of sagar per liter. Only the second
wine fraction is difficult h> store, but its volume
is limited ( 15-2CW of the total volume). The wine
can therefore be saltilcd at a high concentration
( 100 mg/l) and stored at a low temperature. The
two wines arc blended jasl before bottling in a
sterile environment. Wine annua conservation is
maximized with this method but the kchiiqac is
only applicable to wines containing a maximum
of30g of sagar per liter. Hoirjiizcd sweet wines,
however, cannot be made by this method
In additon. the presence of glucan makes botry-
!!■■'.■■- sweet wines difficult to clarify by filtering or
fining. This substance has colloid protector char-
acteristics (Volume 2. Section 115.2). Yet when
correctly barrel ntalared. a bcnionile treatment is
sufficient to line these wines and is sometimes not
even necessary In this case, the majority of clar-
ification problems arc dnc to poor rot quality and
poorly adapted working conditions.
Handbook of linokigy: The Microbiology of Wine anil Vinifk-alKvns
14.2.7 Tokay Wbic
Tokay is a famous botryti/cd sweet wine predated
In Hangary. Several types of wine exist aider
Ihe same name The most renowned rs Ihc Tokay
A/su. vvbKb is a perfectly balanced, perfumed
and delicate sweet wine. As wild Sanlcrncs wines.
BiXrylii cineira transforms Ihc g rape to font noble
ml. bul in different conditions
These wines arc prepared with A/su grapes
which arc concentrated on Ihc vine, by both drying
and aohlc rot. The grapes arc ground I mechanically
today) to obtaii a type of paslc. A bigb-qaality
■cw wine in the final stage of fermentation,
concentrated in alcohol, acidity aid extract, is Ihci
poured over Ibis paste. The wine and paslc aic
tben itacciuted for 24-36 hours, permitting the
sagar aid different aromatic elements of Ibe Am
grapes to be diffused in Ihc wine. This mixture is
then pressed to separate Ihc pomace and the wiie.
The wine R tben aged ii a cask. The amount of
paslc added to ihc wine corresponds to the different
types of Tokay Atsi. The wine is classified by
the number of back-baskect ipMiimyotf of paste
(20-25 kg) per 136-1 iter cask. The following types
of Tokay can Ibis be distinguished
• Three back- baskets per cask Tokay A/su this
couuins at Icasl 60 g of natural sugar per lilcr
and it must be aged for at least 3 years in barrel
and bolllc.
• Hoar back- baskets per cask Tokay A /si: ibis
contains at Icasl 90 g of natural sugar per lilcr
and is aged for at least 4 years.
• l-ivc back- baskets per cask Tokay Am Ibis
couuins at Icasl 120 g of natural sagar per lilcr
and is aged for at least 5 years.
• Six back-baskets per cask Tokay A/so. Ibis
couuins at Icasl 150 g of natural sagar per lilcr
and is aged for at least 6 years.
The quality of Aati et&aciii (essence of A«u|
Is even higher. It is produced )■ certain specific
vineyards with vine-dried and loNc- roiled grapes,
ideal for creating Ibis wine. The corresponding
■list hasa high sagar concentration and is difficult
to ferment. The wine contains about 250 g of
sagar per litr and lis alcohol conient is between 6
and %'i vol.
Tokay wines arc improved by air-exposed aging
ii cool cellars. These cellars arc galleries dag
into calcareous rock which maintain Ihc wine al
a constant 10'C temperatures Dae to their high
sagar and alcohol concentration, these wines arc
not very sensitive u microbial spoilage and can
be conserved in partially tilled casks
14.3 CHAMPAGNE AND
SPARKLING WINES
14.3.1 Introduction
There arc many methods for making sparkling
wines . bul this term refers exclusively to wines
that have undergone alcoholic fermenuttou in a
closed vessel. Artificial carbonation by saturation
with carbon dioxide gas (CO;) docs nol produce
Ihc same qaality bead, and wines made efferves-
cent by this method arc known as artificially car-
bonated wines".
Champagne Is the most prestigious sparkling
wine. It Is produced according to Appellmitm
itOri&ine Cmarolee regulations, in a delimited
area, using strictly defined grape varieties and
wine nuking techniques.
Sparkling wine is generally made in a two-slage
process. The first consists of making a base wine
wilb particular characteristics. The wine Is blended
and cold- stabilised, then fcrmcnlcd for Ihc second
lime In Ihc case of Champagne, this second fer-
mentation (called prise dc mousse'*) tikes place
in the bottle that will ultimately be delivered to
Ihc consumer, once Ihc yeast deposit has been dis-
gorged The use of ihc expression ineiftotk clttaii-
penoise for sparkling wines produced, with the
same method, outside the delimited Champagne
appellation is now prohibited and has generally
been reputed by "mtftbotle tmttiliimnelle" . The
advantages of second fermentation in bottle have
also been combined with clarification by filtration,
achieved by emptying the wine into a val aflcr its
second fermentation, then tillering and treating it.
if necessary, prior lo bottling in the final bottles
tor shipment i Section 14.33).
Olhcr Wine making Methods
459
In Ibc case of splitting wines made by ibe
Charmaf ot cave close" method . the second
fcmicitallon tikes place in airtight vals (Sec-
lion 14.35). TV wine Is then filtered, bottling
IK|Kur is added, and ihc blend is bolllcd under
Hi mil . tcady for delivery'.
In ihc past, sonic wines became sparkling spon-
taneously, when ihcy were bottled with some resKI-
ualsagar before fcrmcntilK>n was completed. They
were unreliable lo make, as Ihc second fermenta-
tion was unconirollcd and could produce insafti-
cicni pressure, or. on Ihc contrary, result in excess
pressure lhat caused the bottle Id explode Several
variations on this technique air still used today,
but are now mach better controlled Fermentation
is stopped by refrigerating the wine once it has
(cached a specific residual sagar level Once it has
been bottled, it is allowed to warm up naturally
and fermentation is completed This technique is
relatively simple to asc as the chilled must after
stopped fermentation nay be siofcd as long as
icqniicd. then bollkd at an appropriate time.
In the later l~ lh century, problems in control-
ling the second fermentation led the Champagne
wineg towers to devise their present system of sep-
arating the complete fermentation of a dry white
base wine from Ihc second fcrmcatabon in bot-
tle, with the addition of a controlled amount of
sugar corresponding exactly lo the carbon dioxide
pressure required.
14.3.2 FcrMcnting Base Wines
tat Principles
The aim Pi lo make a base wine with a moderate
alcohol content, generally a maximum of ll'i vol .
as more cthanol will be formed during the second
fermentation in bottle and the total overall alcohol
conicnl is to remain bctow 13* vol The base
wine shoakl also have a certain level of fresh
acidity, lo ensure the right balance in Ihc finished
ptodact. The grapes are thus harvested at an
earlier stage of ripening than in olhcr Appellation
tfOri&ine CoianHee vineyards. Consequently, the
grapesmnst be pressed very carefully lo avoid skin
contact aid the resulting bitterness and herbaceous
character. In the case of Champagne, this is
especially vital when pressing Ihc bkick Pilot Noir
and PinolMcunicr grapes that arc blended with the
while Chardonnay Picking and pressing conditions
must, therefore, be carefully conliolled
Handpicking is followed by sorting lo eliminate
any defective, damaged, or rotten grapes, as a
relatively low percentage of spoiled grapes have
a negative impact on quality The whole grapes
arc transported in recipients containing 45-50 kg.
with boles to drain off any juice, thus avoiding
skin contacland accidental fermentation, as well as
maintaining ihc grapes in acrobk conditions These
containers are carefally washed after each use.
lb) Pressing: and Extracting the Must
The grapes must be pressed soon after picking,
without crushing, to avoid contact between skin
and juice, and Ihc various fractions of must arc
kept separately Each pressing operation takes
approximately four hours. Two types of presses
arc used. The hydraulic presses traditionally used
in Champagne arc round or square, with a large
surface area to ensure lhat the grape layer is no
more than 80-9) cm thick. Pressure is kept low to
avoid crushing the skins Horizontal presses have
also been used for a number of ycais now. but
only platen presses wiihoal chains or. preferably,
pnea malic presses (Valadc and Bkinck. 1989). The
different fractions of Ihc must reflect Ihc aacven
ripeness of the grapes and the varying composition
of the vac nolar sap. The etnfe corresponds to the
juice from Ihc middle pan of lie grape flesh, which
is both the sweetest and the most acRllc. The outer
part of the flesh is sweet, but less acidic, due lo
the salification of the organic acids in the vicinity
of the skin. The flesh closest lo the seeds has the
highest acidity aid Ihc least sugar.
Pressing methods arc standardized and the juice
is col Ice ted in small vats, known as heltms". Tra-
ditional Champagne presses used lo hokl 4000 kg
grapes Two or three pressings in quick succession
and loosening Ihc pomace after each one (Valadc
and Pcmot. 1994) produced 2050 I of top-qnalily
must (enough to till lei of the 205 I barrels used
in Champagne) This was known as the cmvV
The next two pressings produced 410 I (2 barrels)
of premieres tunic'* and a thiid gave a further
460
Handbook of linology: The Microbiology of Wive anil Vlnificaiions
barrel of dcuxicmc taillc' . The tls.il pressing in a
standard hydraulic press produced 200-300 I of
press wine ( rcbcchc'l. inlcndcd for disllllalioa
raihcrlhan for making Champagne In presses with
a large surface ;irea. (he edges arc subjected lo less
pressure than the cenkr. so ihe poniace is brought
from the edges towards the center between each
pressing These presses may be operated automat-
ically (Valade and Perm*. 1994).
The must Is scparakd in a similar way In the
case of horizontal presses, which arc. however,
caster to use.
A 1993 regulation specifics thai 4000 kg grapes
must produce 2550 hi of clarified must, allowing
for 2-4* sediment. Only the cimfe (2050 hll
is used to make prestigious Champagnes, while
the taillc (5 hi) produces fruitier, faster-maturing
wines that arc generally included in non-vintage
brut blends
Analysis of the different pressings, as they conic
out of the press, shows significant variations In
composition (Tabic 14.6). As pressing continues.
util acidity decreases, as do both the tartaric and
nallcacld levels Mineral cot tent and pH increase,
as do the phenolic content and color intensity,
while the sugar level remains relatively constant
The aromatic intensity and lincs.se of wines made
from successive pressings also diminishes, but wc
do not have the means k> analyze these changes.
It Is essential to take great care harvesting,
clarifying, and separating the must to
.* the quality* of the finished Champagne
(Moncomblc etnt.. 1991) Not only do Ihesc
precautions make it possible to produce practically
colorless must with very link* sediment from Mack
grapes, but they arc also vital to preserve the
llnessc and quality of the end product.
(c) Musi Clarification and Fermentation
For the reasons outlined above, the must should
be perfectly clarified prior to fermentation (Pcrnot.
1999). Generally, the must is clarified immediately
after pressing at the pressing room in the vtncyaitls.
Some Champagne producers clarify the must again
on arrival in the fermentation cellar. .Sulfite is
added (5 -8 g/hl) and the sediment is left lo settle
down naturally In some years, depending on the
condition of the harvested grapes, pcctlnascs may
be added u facilitate Ikiccnlation and settling.
Champagne musts contain large concentrations
of nitrogen compounds, especially proteins, which
contribulc to (he quality of the finished Cham-
pagne, especially, persistence of the bead How-
ever, proteins arc also involved in Instability prob-
lems, leading to turbidity. Sonic producers add
tannins 15 g/bl) lo flocculate any unstable proteins.
Bcnlonilc may also be used for this purpose, at
doses not exceeding 30-50 g/hl.
Traditionally, the initial alcoholic fermentation
t bouillagc'*) took place in oak barrels, in cel-
lars where (he temperature was never higher than
15-20'C. Some producers still barrel- ferment their
wines to enhance aromatic complexity and fla-
vor. However, most base wines arc now fcrmcukd
in coated steel or. mainly, stainless-steel vats, as
they arc easy to maintain at temperatures below
20'C. The aim is for the fcrnicn tation to continue
Tunic 14.0. Pkyii
lufCkim-iiipfft
t (VjUIc and Mauri. 1189)
DciHty
Sugar <g/l)
Total acidly (p/l H r SO.)
Ta-unc uiid (g/fi
icid (g/l)
-II
n <«^l>
Total •■■open <mgN/l) 575
Avaitablc nitrogen (•pN/1) 202
Olhcr Wine making Methods
461
smoothly and uninterruptedly UMlil ihc residual
mincing sugar content Is below 2 p.l Although ibc
nmsl is usually chaplali/cd. Ills R 1101 always nec-
essary as ibc aim is b> achieve an alcohol coiicnl
no higher than 10-11% vol. Completing fermen-
tilion Is not usually a problem, especially as the
mnsl R systematically seeded wilh scleclcd yeasts.
'.'■ liiilu.iKltil.- iKlhods In olher sparkling winc-
pnxlucing aicas aic generally lused on those used
li Champagne, bnl may be simplified to icdncc
costs. Although other grape varieties do not have
the same qualities as the Chardoanay. Pinoi Noir.
and Pinol Mcuntcr used in Champagne, ihcy make
suitable base wines, provided ihcy arc picked wilh
a sufficiently high acidity level, eg Chcnin Blanc
In Ibc Loiie Valley. L'gni Blanc in Bordeaux.
Mau/ac in Limoux. Maccabco in Catalonia. cK.
<d> Malolacllc Kcrmcntation
Champagne R an clegail. fruity wine that requires
a certain level of acidity (around 6 g/l H ? SO,i
The drop in acidity that occurs during the second
fermentation must be taken into account in making
Ihc base wine (Section 14.3 J).
Accoidiig to E. Pcynand (cited by Ribbreau-
Cayon el ill.. 1976). when he analyzed Cham-
pagnes in the 1950s, he found that malolacuc
fermentation was uncommon and there was a con-
siderable difference in flavor between wines with
and without malic acid. The same author added
thai there had been many changes since thai time
(increase In pH. decrease in salfiling. etc.). result-
ing in a grcakr vulnerability to bacterial activity
and increasingly frequent uncontrolled malolaclK
fermentations (hat were unknown in Ibc past
There Is still some discission concerning the
beneficial effect of malolacllc fermentation on
Champagne aromas. If it Is properly controlled.
It improves the quality of acidic wines, especially
Chardonnay.asthc bacterial activity enhances (heir
aromas (Section 1.1 7.6). In olher cases, it may
result In wines lacking freshness thai age too
rapidly and may even necessitate Ihc addition of
tannic acid lo raise the acidity level (Volume 2.
Section I.4J).
To ensure mKrobtologKal stability and avoid the
serious consequences of mahilactic fermentation
during second fermentation (prise dc mousse")
or bottle aging (conservation sur lallcs). malK
acid must be eliminated from Ibc base wine pilot
lo bottling. This solution has been found most
effective and Is the most widely used, although
other methods arc under investigation
The factor with the greatest impact on malolac-
tic fermentation Is the snlfur dioxide content, as
it is Inhibited by even small doses of free SO;, li
is also affected by Ihc combined SO? content and
becomes very difficult, or even impossible, at total
S0 2 levels above 80-100 mg/1. Careful observa-
tion (RibCreau-Gayon el al., 1976) showed a corre-
lation between Ibc total So, contcnland malolaclK
fermentation in Champagne during the second fer-
mentation in bottle. Current miration technology
makes it possible to sterile- botllc the busc wine and
inoculate it with pure yeast. Ibus avoiding uiak>
laclK fermentation in bottle without excessive use
of SO.. Lyso/yme may also assM In stabilizing
the base wine (Ccrbaud el al., 1997; Pllattc el al.,
2000).
However, it Is not always easy lo start nalobc be
fermentation at the right time in wines with very
high acidity (Section 13.7 6) and it R essential lo
adjust Ihc SO. content and temperature for that
purpose. It is also possible to inoculate a properly
prepared starter, but this is a ralber laborious oper-
ation. Seeding with reactivated bacterial biomass.
initially developed for red wines (Section 12.75).
has considerably improved malolactic fermentation
conditions in Champagne base wines (Laurent and
Valadc. 1993) and suitable products arc now com-
mercially available.
14.3.3 Second Fermentation in Bottle:
The Champagne Method
(a) Preparing and Bottling a Curie
The new base wines are clarified, racked, til-
lered, and fined in the usual way. Base wines may
be fined using Kinglass (15-2.5 g/hll or gelatin
(4-7 g/hl). with or without tannins (2-4 g/hl)
(Marebal end.. 1993) Tannin may be required to
deal with Ihc Instability resulting from these wines
relatively high protein content, while it is essential
to maintain a sufficient level of protein to give
462
I landbook or Etiology: The Microbiology of Wine anil Vindications
a high- quality head Moderate doses of bentonitc
nay also assist in prolcin subili onion.
Aaivee is prepared by blending wines of differ-
cnl origins (vineyards classified 100*. 86*. etc.).
qualities, aid. possibly, vintages. This is indispens-
able in maintain the quality and character identi-
fied with the piulKcr froai year b year and Is
still mainly determined by tasting. Tnerc Is a sin-
gle Appellttfitm it Origine Cawdlee for all Cham-
pagnes, while the other two appellations in the
region. CdKaux Cnampcnois and Rose" dc Riccys.
arc only applicable to still wines The hierarchy
among Champagnes is mainly dependent on the
selection of base wines used to blend the cumY.
Once the citre'e has been blended, the base wine
ci cold-subili/rd lo prevent uitralc precipitation.
In some cases, it may be lined Jnst before or after
cold subiliailiou.
Preparation for boilling may also include nitra-
tion Barrel- fc mien led wines arc adequately clari-
fied by simple settling of the Ices, but wines fer-
mented in vat always rcqnlrc filtration. cspccEilly
immediately after cold stabilization.
During bottling, the llragc liqueur (syrup con-
tuning 500 g/1 saccharose) is added for the second
fcrmcnuilon. calculated lo produce the carbon diox-
ide rcqnircd for a pressure of 5-6 bursal I0-I2"C.
Theoretically. 20 g/l saccharose must be fermented
u produce 5 bars pressure. Table 14.7 indicates the
quantities of sugar required *■ produce the desired
pressure in Ibe bottle aflcr fermentauon
An active yeast sutler, consisting of selected
strains of Succluamiyees cererisine . is added at the
same time K> ensure that the second fcrmcuuuon
( prise dc mousse ) will be successfully completed
Tunic 14.7. Sugar concur of the dope liqueur nevoid-
mytolbf ;icv.-.:n rcqullttl i V.bdc .ml I .mil . 3 « 1 .
I'.v.m].;- ni|.,li'l -.u'. ■ hi.mi Sugar
AilOC At 2(I"C iy.il fmeicacKn'll
20 O
212
220
2'- 1
2i II
25a
25J)
2ia
In the bottle. This starter may consist of fermenting
must but is better prepared with active dried yeast.
Dried yeast develop well in must that does not
contain alcohol, and may be inoculated directly,
particularly In while wincmalang (Section 13 7.2).
However, in a medium containing alcohol, eg.
when required u restart a stuck fermentation
(Section 3.8.3). they mist be reactivated prior lo
use. so that they arc in a suiublc physiologi-
cal condition Laurent and Valade (1994) recom-
mended an effective method for preparing dried
yeasts for use In second fermentauon in bottle.
It is advisable to seed the bottle with an ini-
tial population of 1.5 :< It/' cells/ml. Bckiw that
amount, fermentation is slower and some sugar
may remain unfcrmcnlcd. white above thai level
(eg. 2.10* cells/mil. fermentauon is faster but
some yeast strains may produce yeasty off-odors.
The tirage liqueur and ycasl starter can be
added In vat prior to bottling, or to each bottle
individually during the bottling process, eg. via
two measuring pumps
Other substances may also be added at the lime
of boilling (c.g. 3 g/hl bcnlonilc or 0.2-0.7 g/hl
alginate) to facilitate elimination of the yeast sedi-
ment when Ibe bottles arc disgorged
(b) Second Alcoholic Fermentation and A&tig
on (he l-ces*
The bottles arc closed with crown stoppers made
airtight by plastic seals They arc stored in a hor-
i/oinal position in box pallets or sucks, inter-
spersed with Litlis of wood to steady the layers.
It R imporunt that the bottles be placed hori-
/ouully. firstly u ensure that they remain air-
tight during fermentation, and. secondly, to provide
a maximum iuKrfacc for exchanges between the
wine and its Ices. Fermentation ukes one month,
or sometimes longer, at the constant tempera-
ture of 11-12 C In underground cellars in Kpcr-
nay and Reims This slow. even, low- temperature
fermentation is another quality factor In pro-
ducing fine Champagne, especially the finesse
and persistence of the bead when the bottle Is
opened. Carbon dioxide pressure increases grad-
ually (Valade. 1999). inhibiting yeast growth and
slowing the f craicn tation rale, especially at low
Olhcr Witcmaking Methods
463
pH and high alcohol levels. Attempts have been
made in enhance Ihc fermentation rale by adding
nutrients, wilh inconsisKnl results li is more effee-
live K» increase ihc initial yeasl inoculation, as well
as to adapi Ihc yeasl strain and Manor preparation
conditions in Ihc lypc or base wine.
The wine is silll noi proper Champagne . even
when Ihc second fcnncntitMo in hollk Is cut-
plcted. i.e. all Ihc sugar has been fermented The
wine spends a kmg pcnxxl aging on ils yeasl scd-
iiKnl. whkh gradually decreases in volume and
becomes more compact. The bottle must remain
ii a horizontal position to provide a maximum
winc-scdimcnl contact surface. The ycasls release
subsumccs iSeclioa 14.3.4) into the wine, initially
by excrcton. then by diffusion from Ihc dead yeast
cclrs These arc mainly amino acids, eilher synthe-
sized by Ihc yeast or previously absorbed from the
wine. Autolysis involving cell wall enzymes has
also been ohscrved. All these complex phenomena
play a significail rote ii Champagne qualify The
improvement it quality during this stage is corrc-
Litcd u the composition of Ihc base wine, which
explains why other sparkling wines benefit less
from aging on the lees. Non- vintage Champagne
Is aged on the tecs for a minimum of 15 months,
while the minimum for vintage Champagne is
3 years, but they may stty on their Ices for up
to 8 ycais. or even longer for some top ciiwto.
As long as the sparkling wine remains in
ton tic t wilh Ihc yeasl sediment under anaerobic
conditions, the tees act as a redox buffer and
ihc wine Is perfectly prescived. Champagne from
hollies several decades old tasted in Champagne
cellars were found to be it perfect condition, as
Ihcy had never been disgorged. Once Ihe bolites
have been disgotged. not only docs the Champagne
slop improving, but there is also a risk of defects
developing due to redox phenomena.
The main risk during storage. especially if bolites
arc exposed to light. Is the development of off-odors
in the wine. These reduction odots are due to the
formalion of thiol groups by photodcgiadatKin of
sulfur amino acids naturally present in Champagne.
The reaction, photosensitized by rihollavin I vitamin
B2). pioduccs me thane thiol and dimcthyldisullklc.
which arc responsible for sunlight ftivof' i gout
dc lumierc > (Volume 2. Section S 65) (Maujean
and Scguin. 198.1). Maujean eial ( 1978) showed
that this drop in redox potential due to lighl
exposure only occurred in disgorged Champagne
The formalion of thiol groups and ihc resulting
sunlight flavor also depend on Ihc reduction
conditions of the wine prior to light exposure This
defect may be prevented by using glass bolites
wilh low transmission values at wavclcuglhs bctow
450 mn Adding ascorbic acid, together wilh so.,
just before Ihc bottles are finally corked (when the
dosage is added) is an effective preventive a
tc> Riddling; and Removing the Yeast Sediment
The next sbigc consists of gathering the yeasl sed-
iment on Ihe inside of the cap. This is tradition-
ally done by riddling the bottles on special tucks,
which hold the bottles neck down, at a variable
angle. Riddling consists of turning the hollies with
a slightly jerky movement, bringing them gradu-
ally to a vertical position, completely upside-down .
over a period of a month or mote.
This operation tikes a variable amount of
time, generally from three weeks lo one month,
depending on the type of wine and its colloidal
structure, as well as Ihe type of yeast and iis
capacity to form clumps. Riddling is an awkward
stage in the Champagne production chain, due lo
ihc space required for Ihc riddling racks, the labor-
intensive protest, and the fact lhat the bolites
arc immobilized for a relatively long period of
time. A great deal of work has been done lo
simplify this operation. The first approach consists
of adding various substances lo the wine in vat.
prior to bottling, intended lo facilicik: settling of
Ihe yeasl sediment. While the results have not been
negligible, this technique has not made any great
improvement in the process
More significant progress has been made by
reproducing the iutennilieiii movements of riddling
on the scale of a box pallet (several hundred
hollies.! Bach box pallet is instilled on a movable
base, which is tilted manually lo change Ihc angle
of the bottles, gradually bringing them into the
vertical . neck- down position. This system may be
mechanized and programmed (gyropallctl lo riddle
Ihe bottles much more efficiently, completing the
464
Handbook of Enology: The Microbiology of Wine anil Vindications
cycle in one week instead of Ihc one month required
for manual riddling. This system is now widely
■sal. in spile of lie high iniiial investment requited.
Another approach to simplifying riddling con-
sols of using ycasl enclosed in liny calcium algi-
nate beads for Ihc second fermentation in bottle
(Dutcurtrc <■!«//.. 1990: Valadcaul Rinvillc. 1991).
The scdinKnl settles on the cap almost immedi-
ately when the bottle is timed upside down aid
riddling is no longer necessary. Of course, this
assumes that fermentation and aging on the Ices
continue normally with the enclosed yeasts. The
second condition is that yeast cell growth docs
not birsl the beads, producing a powdery deposit
that is difficult to eliminate This problem is now
avoided by using a double coat of alginate on
the beads. Several million bottles have now been
processed with enclosed yeast, and work is contin-
uing to monitor the aging and development of the
Champagne. Once this technique has been demon-
strated not u affect quality, it will be possible to
envisage Ik nse in large-scale production
Id) Dl^Drgingand final Corking
Once the sediment has settled on the cap. the
wine Is disgorged. In the past. Ibis operation was
done manually by removing the cap quickly while
racing the bottle slightly so that the few milliliters
of wine containing the sediment would be expelled
without emptying Ihc bottle or losing too much
carbon dioxide pressure.
Nowadays, the bottle necks arc almost always
frozen prior to disgorging, in an auumalcd system
that also adils the dosage lk|ucur. corks Ihc bolllcs.
and Ills the wire closure. The bolllcs are held
upside down and the necks plunged into a low-
krmpcraturc sail solution that freezes about 2 cm of
wine above the cap. trapping the sediment. When
the bolllcs arc turned upright, the cap R removed
and Ihc frozen plug is expelled.
The botllc is then topped up with dosage liquor
( liqueur d expedition'*), a syrup made of reserved
wine containing approximately 600 g/l of sugai.
used to adjust the final sugar level of the Cham-
pagne. Brut* Champagne generally has 10— 15 g/l
(1-1.5% dosage), while Demi-sec has JO g/l
t-Vi dosage).
The dosage liqueur can be acidified with citric
acid, if necessary It also contains Ihc quantity of
sulfur dioxide required to eliminate any dissolved
oxygen, and may be supplemented with ascorbic
acid (50 mg/l). This offsets the sudden oxidative
effect of disgorging: the redox potential may
increase by ISO mV. or even more, depending on
the redox buffer capacity of the wine.
According to E. Pcynaud (cited by Ribc"rcau-
Gayon el til.. 1976). Dosage Is not simply a matter
of sweetening the wine, bnl of improving it The
quality of the dosage liqueur, the way it is aged.
Ihc types "' WIK used, the quality of the sugar,
and the preparation formula all pfcty a major role
in the quality of the finished product ." The dosage
iKjacnr con tributes to the overall flavor balance.
14.3.4 Composition of Champagne
Wines
(a) Analysis of Champagne Wines
The analysis results in Tabic 148 show the effect
of bottle fermentation on Ihc wine's composition.
The alcohol content increases by I .!'* vol during
the second fermentation and may drop by a few
tenths during preparation for shipping, depending
on the composition of the dosage liqueur. If the
base wine is not properly cokl-siabili/al. total
acidity may decrease slightly during the second
fcrmcntibon due to Ihc precipitation of potassium
bydrogcnoGulralc and by Ihc breakdown of small
amounts of residual malic acid under the action of
Ihc yeasts. Otherwise, there is little variation in the
acid content unless a small amount of citric acid
is added in the dosage liqueur. This decrease in
overall acidity results in an increase in pH
One of the most significant characteristics of
Champagne must and wine ts their high nitro-
gen content, especially in the form of ammo acids
tDcsponcs ei of., 2000) (Table 149). which facil-
i titcs the initial and secondary fermentations. The
amino acid content of Champagne is twice or thrice
as high as thai of Bordeaux wine (Rihcrcau-Cayon
el nl.. 1976). The same authors gave the followiig
analysis rcsulLs forChampagac and Bordeaux: 462
and 184 mg/l of toctl nitrogen. 1 1.2 and 6.3 mg/l
niacal nitrogen, and 2 16 and 100 mg/l of
I'.ictii nitrogen, respectively.
Other Wine making Methods
Tabic U.K. Co. parson of
( 1993 viatapc. mean anal**.
Mm *muv ji 20°C (pAInT)
Akobolai 20C(*i vol.)
Supo (g/l>
P H
Toial acidly (i»l HflO-O
\oIitilc acidity (p/l H.M.'I
FB=c SO, (mg/1)
Total SO, fauVI)
Taita(icackl(£/I)
Mill. ... .1 i ,■ I ■
IWsMUlMmg/l)
Calcium <«k/I|
Coppcr(mg/1)
In»(H0A)
Sodium img/l)
Tout nil iugenlmtjN/1)
'.: niM.-.i,]L.l DtlftlpCII ■; :u-'l !
OD 530 nm
ODWI am
CokiriMcnsHy
Shade
Conductivity (mSfcmlat 2D"C
S:iluiMH>n IcapCfMUIC ,n 20 "C
111c nd Ji ibe
line til bmillap
11.038
0X187
D.123
2.59
Iflcrsccond Icimcntjlbn
and addilKnol dixtapc
toibiut tpjalily
DD28
1 1 KM
0.1 JJ
3S9
The m til nitrogen content of Champagne vanes
trow 150-600 mg/l (Manjcan eittl.. 1990) and
thai or ihc mnsi is considerably higher. Chanlon-
ii. iy and Plnol Noir/Meunicr grape varieties have a
high nitrogen content, and Champagne is the wine-
growing region where it reaches Ihc highest levels.
Table 14.9 compares the mean amino acid con-
lent of base wines made from diffcicni Champagne
grape varieties.
According to the liicralnic. Champagne must
contains 25- 100 ntg/l of proteins On BSAcquiva-
lent). while the level Is considerably tower in base
wine: 14-32 mg/l (Tusscan and Van Lacr. 1993).
In harvested grapes. 75'i of ucil nitrogen is in
amino acid form, while it accounts for 95'» in new
base wine (Tnsseau el ill.. 1989).
Several proteins have molecular masses between
20 and 30 Kda. while one with a molecular
mass of 62 Kda is piobably combined with sug-
ars (glycoproteins). They have isoelectric points
between 25 and 65 (Brissonnct and Manjcan.
1993).
Besides proteins and polypeptides from Ihc
must, tne sparkling properties of Champagne
also involve carbohydrate colloids (polysaccha-
rides and glycoproteins) (Marchal etui.. 1996:
Bcrthkr el id . 1999) released from Ihc yeast cell
wait, during aging on the IccslFcuiikitef <i/.. 1988;
Tusscau and Van Lacr. 1993). ThR yeast autolysis
is certainly accompanied by more radical transfor-
mations, '"fie amino acid content of some sparkling
wines has been reported lo increase depending on
Ibe contact time, and stirring ibe yeasi back into
suspcnsK<n has been recommended to enhance this
Boidron el ill 1 1969) compared Ihc lolalilc fer-
mentation compounds involved in Champagne aro-
mas with those found in other sparkling wines
Champagnes characteristically have lower conccn-
Irations of methanol, higher alcohols, propanol.
466
Handbook of Etiology: The Microbiology of Wive and Vinifkaiions
Tubb 14.0. Average unlno .......I tnwei
wiaca made fntm diftcicat Oampugnc jir,
(Auavcdonuiltcnie miannd detected wia
<Kcuik% la mg/l)(Dc-.»ojicsf--<rf-. 2000)
AllBHghK
NJ
Ghfta malic Kid
38
Gfcjjminr
07
Pmllac
77?
Glycine
27
A koine
III
Cinillinc
38
Valine
32
CyWcine
24
Mcihbninc
7
koku-inc
4
Ixuciac
14
Tyr^inc
a.
p-thaiic
ii
Pfcenvl-jlaninc
14
f-N-buryiir Mil
67
11 hi*, limine
4
Oraihinc
7
Ljhk
8
Htuidioc
s
Arprninc
20
Tatil
I3M
PmlincM amine
3885
elbyl butyailc. and isoamyl acclulc. which have
a negative died on aroma Tkls Is probably due
to ihc wincmaking conditions (c.g. Icmpcialuic).
Olbcr more positive, aromatic compounds such as
clhyl capralc and cihyl lactate i related to malcv
taciic fe mien til ion) ate moic abnndani in Cham-
pagne.
In ihc pasl. il was relatively common lo find
huge residual ycasl populations in Champagne
bottles Ycasl connK between 16 and 48 x
10* cells/ml have been luid in Champagnes oi
ihc markci (RibCrcau-Cayon eltil.. 1976). Cnr-
icil riddling and disgorging techniques, purlieu-
Lirly sedimentation additives and tine-iuncd rid-
dling programs, have made substantial progress in
eliminating residual yeasts.
lb) Kffcncsccncc in ChantpapjK Wines*
The excess carbon dioxide pressure lespousiblc
for effervescence is an essential characlcnslic of
Champagne
When Champagne Is poured into a glass ihc
foam, which rs an important quality factor, appears
even before the liquid II is well known thai
while listing a poor initial visual impression has
a negative impact on the overall assessment, and
tits is certainly the case with the bead of a
sparkling wine (Robillaid. 2002). A good quality
bead consists of tiny bubbles that remain separate
and spherical in shape Large bubbles produce an
unattractive, grayish bead thai usually disappears
very rapidly.
Effervescence also reveals the wine s aromas,
as the bubbles contain odoriferous compounds in
addition to caiboa dioxide (Maujcan. 1996).
Il is. therefore, important *< consider Ihc cri-
Icria for the formation and stability of bead in
sparkling wines. The following analysis Is based on
a 1997 review by A. Maujcan (Laboratory of Enol-
ogy. Reims University) and B. Robillaid (Moct cl
Chandon Research Laboratory), as well as sev-
eral other publications (Maujcan. 1989. Robilfcud.
1993: Ligcr-Bclairand Jcandcl. 2002).
The bubbles in spirting wine aic due to carbon
dioxide, formed during Ihc second fermentation
and dissolved in the wine. A bubble of CO? must
push the surrounding molecules apart before il can
emerge A great deal of energy is icquiicd lo form
a liquid.'i'O; iuKifacc. but Ihis is minimized by
n ik leal ion phenomena.
Bubbles may be formed directly from dissolved
gas (induced homogeneous nuclcalion). When
Champagne rs shaken up. eg. during shipment,
parent bubbles produce smaller bubbles, some of
which are stabilized by contact with proteins and
fkial on the surface. The drop In pressure when the
bottle is opened causes them to explode, producing
other smaller bubbles, which explode in turn, and
so on This chain reaction is responsible for a
violent gush of wine, which ntay leave the bottle
half-empty (Maujcan. 1996).
B ubblcs ate more usually formed by adsorption
of ihc gas on a solid particle (induced heteroge-
neous nuclealion). It has been demonstrated that
Olhcr Wine making Methods
467
i radius of 0.25 »n is required for the
bubble s internal pressure to be sufficiently kiw in
relation to thai of the wine, in enable the bubble to
grow and rise through the liquid Plasties have a
higher surface energy than glass, creating a greater
affinity forCO;. so that bubbles coming offaplas-
IK surface will be larger than those released in a
glass VCSSCl
Several factors arc involved in effervescence
kinetics* following degassing (Casey. 19B7: l.iticr-
Bclair. 2002 and 2003: LBjcr-Bclair el ul.. 2000).
The lirsl factor is the physical ■.unit- of the
sol hi sirfacc (particles in suspension or vessel
wall), particolarly the number and radins of
microcavitics on which the bubbles are formed,
detaching themselves once they Have reached a
certain diameter. This produces a bead, or line of
bubbles, which always rise from the same spot Of
course, the niicrocavitics must be hydrophobic or
they would be filled with wine
Other factors, such as viscosiry and chemical
composition, are inherent to the liquid It is quite
probable that some carbon dioxide molecules are
immobilized by binding with other substances.
Electrostatic interactions ntuy also lead lo the
adsorption of CO; on the surface of macro-
molccnlcs. as shown by the significant changes in
effervescence kinetics when proteins or polysac-
charides were added *> synthetic wines (Manjean
etal., 1988).
The instability of the bead is defined by three
parameters:
1 . Swelling babbles: The gas from small bubbles
B absorbed into Lugcr bubbles, etc. This results
in a course irregular foam with an unattractive
appearance
2. Draining: This refers to the liquid that drains out
of the foam over time. It leads lo a reduction
in from volume and a distortion in the shape of
the bubbles. The foam gradually dries out leg
as on the head of a beer gfciss).
3. Coalescence: A break in the film between two
smaller bubbles produces a larger one. resulting
in a coarse foam that disappears quickly
Several experimental processes have been pro-
posed for measuring the spontaneous or forced
degassing kinetics in sparkling wines (Maujcan
rial.. 1988). as well as for assessing the persis-
tence of fount (Manjean end.. 1990: Robillard
eial.. 1993).
The Mosalux apparatus (Maujcan e i ill.. 1990) Is
used to determine three characteristics of sparkling
wine bubbles:
1 . Fuamabilily or maximum foam depth expresses
the liquid s capacity to contain gasoncc it sum
effervescing visible in the foam formed when it
is poured inio a glass.
2. Foam depth describes the conscinl depth of the
foam when the liquid is bubbling in the glass
and corresponds u the bead of foam.
1. Foam stability measures the time required
for the foam to disappear once the liquid
slops effervescing. This parameter is oil)' of
theoretical interest in laboraiory work.
Measurements showed that frunutbility and
foam stability are mutually independent— wines
may produce a lot of foam but it is not neces-
sarily very slabtc. A close correlation has been
observed between foamability and prolcin content
A decrease in prolcin content of a few mg/1 can
lead to a M'fi drop in foamabiliry (Malvy ■■■' irf.
1994). However. Maujcan el til ( 1990) did not find
any simple correlation between prolcin content and
foam stability.
Prolcin solubility affects its impact on foam-
ing in sparkling wine Hydrophobic proteins may
also be adsorbed at the gas- liquid interface, on the
bubble skin' .stabilizing it by decreasing surface
tension. Proteins with lower molecular weights are
more rapidly adsorbed at the interface. Proteins
that have an effect on effervescence have isoelec-
tric poinLs in the vicinity of wine pH (25-3.9).
This charactcrislic does not promote solubility, but
makes the protein more hydrophobic Thus, pro-
teins alien foamability by changing the surface
tension when ihcy are adsorbed at the liquid-gas
interface of the bubbles. Glycoproteins have an
even greater impact on foaming, as lie bydropbilic
468
Handbook of Etiology: The Microbiology of Wine anil Vinificaiions
osklK fraction increases ihc viscosity of Ihc liquid
tilm between ihe bubbles and reduces Ihc draining
of Ihc Ik|*kl phase. Although yeast mannoprotcins
arc less hydrophobic thai plant glycoproteins. Ihcy
arc present in kugc <|tan lilies in Champagne wines
and apparently coutribiic to their stihilitv (Fcuiifcil
eittl.. 1988).
. < •_ 1 1 1 1 depth decreases during aging on the Ices,
bat is largely compensated by the improvement in
stability.
It is well known lhal Ihc various stages in the
wine making process have an impact on loom qual-
ity. Robilfcinl ef «f (1993) examined Ihc impact of
filtering base wines. This operation removes solid
ot colloidal particles that provide a base for bub-
ble formation (nuclcatioa). considerably reducing
the intensity of effervescence and thus. Ihc foam
stihilitv of the corresponding sparkling wine. The
smaller Ihc pores of Ihc filter medium. Ihc more
marked the impact on foam stability-.
Treatment with plant charcoal or bentonite also
causes a considerable decrease in foamabiliry.
rcLiicd to the reduction in protein content. On the
contraiy. fining with gelatin, combined with silica
gel or tannin, improves foaming qualities.
14.3.5 Other Second Fermentation
Processes
(c) Transfer Method
The aim of this method Is to benefit from the
advantages of second fermentation in small but-
tles and aging on the ycasl Ices, while avoiding
the problems associated with riddling and dis-
gorging. Once Ihe second fermentation and aging
arc completed. Ihc wine Is filtered and transferred
to another botlle. This process is not permitted
for Champagne, allhongb inert Is a tolerance for
qnartcr-boltlcs which arc filled after filtration, fol-
lowing second fermentation in full-six hollies.
This process is still occasionally used to prepare
hall- hoitles. but lis use is due to be prohibited in
the near fntute.
After second fermentation and aging. Ihc bot-
tles arc simply taken to Ihc racking area They ate
emptied automatically into a metal vat. under car-
bon dioxide pressure equivalent to thai created in
Ihc bottles by fermentation, to prevent degassing
The wine in Ihc vat is refrigerated to -5'C
by circulating liquid coolant through a suitable
beat-exchanger. This makes Ihe CO; more soluble.
Dosage liqueur is also added in Ihc vat and the
wine is left to resl lor a few days. It is Ihcn plalc-
tillered to remove all the yeasts and bottled. As the
wine is kept allow temperatures under pressurized
carbon dioxide, it retains all Ihc dissolved CO;.
Tbissystcm has a number of advantages. II clim-
inales the labor cos& of riddling and disgorging,
as well as Ihc lintc Ihc wine is immobilized on
lie riddling racks. Dosage liqueur is much more
evenly distributed ll is also possible to blend sev-
eral batches of wine after Ihc second fcmcnci-
lion to obtain Ihc desired qnality. Cold- stabilization
prcvcnti tartrate precipitation and fillralKan ensures
lhal Ihe yeasis are completely eliminated, leaving
Ihc wine perfectly clear.
If these operations are properly conducted, they
give satisfactory resale However, wines made by
the Champagne method were always preferred in
comparative tastings, probably due to the fact thai
small amounts of oxygen were dissolved in Ihc
wine during transfer operations, however carefully
Ihcy were controlled It has also been demoastrafcd
that exchanges occnr between Ihe carbon dioxide
molccalcs resulting from the second fermentation
and the industrial gas used to protect the wine
during Ihe transfer process. Finally, filtration may
modify the wine's foaming qualities
<d) The Charmat (Cove Close! Method
Second fermentation in bottle is technically de-
manding and is. therefore, only justified for high-
quality products made from fine base wines thai
are likely to benefit from aging on Ihc yeast Ices.
As long aging Is nol economically viable for
cheaper producer, a simpler, less expensive process
(Ihc Charmat method) has been developed to
produce sparkling wine from lower quality grapes.
Figure 14.1 shows a simplified diagram of a sys-
tem for second fermentation in a scaled vat. The
various base wines are Mended and transferred u>
Ihc second- fermentation vat (C) and yeast starter
(valAI as well as syrup (vatBl is added Id
provide Ihc quantity of sugar required for the
second fermentation and the dosage of the finished
Olhcr Wincmaking Methods
Ki& 14.1. A buik-mcibotl
equipped will a in Mint) s\Mcm . C. ic
lut B.i4><in>incirK bunting: G. nrln^ci
for *fjrklin£ vviac: A. \cjn( i
iintl tcracai.-ii»n lank: D. icfriprat b
lion gnnip; H . fiber. I. hauler
■laikin tank: B. *upji
ilc K. bait ling link: F. i
pialKl. The fcraicitalion vat(C) is equipped
with healing and cooling salens to maintain a
temperature of 20-25 'C. When ptcssaic in ihc vat
reacbcsS bars, fermentation is stopped by reducing
Ihc temperature ami snlfiting slightly. The wine is
then transferred in a refrigerated val (D) and kepi
at — 5'C far several days for cold stabilization.
The wine Is filleted and Ihen transferred to
another val (Hi. connected K> the bottling line. The
entire operation is carried oit under pressurized
carboi dioxide (P). to prevent degassing.
These laigc-votumc processes certainly cannol
achieve the santc quality as bolllc fcr»cnlalion.
This is partly due lo Ihc fact that Ihc wine is
not aged in conlacl with Ihc tecs, as a sufficient
level of interaction can only be achieved in a
small container The quality of the grapes used and
the speed of the process afco have an impact In
Ihc Charmal method. Ihc ycasl is oflcn eliminated
after only a few days' fermentation K> reduce
costs. In view of the olhcr factors involved, il
Is by no means obvious thai aging on the Ices
would improve quality. Systems have, however,
been developed for maintaining Ihc wine in vat on
iK tecs, stirring them inlo suspension lo accelerate
exchanges. The success of this opciation depends
on the quality of the base wine and keeping
fermentation temperatures knv to stow down the
reactions.
The Charmat iKlhod may give better results
■ban bottle- fermentation in bol climalcs. as it pre-
serves the base wine's freshness and fruil. Finally.
Ihc Charmal method Is most appropriate for pro-
ducing sparkling wines from aromatic varieties
such as Muscat, as aging on the Ices attenuates the
Muscat character, without significantly improving
quality.
te> Asll Kpurnanlc
This Is probably the most famous sparkling Mas-
cat. Unfortunately, when the mast is fermented lo
prodacc a completely dry wine, it ktses all the
distinctive grape aromas and has an unpleasant,
bitter lastc. Long experience has led lo the devel-
opment of a km- temperature fermentation process
that Is interrupted every lime it starfe speeding
np. The must Is clarified, lined, and centrifuged
as many times as necessary until the yeast and
available nitrogen nave run out Analysts results
show clearly that the tolal nitrogen, particularly
<I70
Handbook of linology: The Microbiology of Wiic and Vindications
available nitrogen, decreases every Uik Ihc fer-
menting must Is filtered, piobubly as il was fixed
c* ihc yeast. The residing wine is relatively stable,
die in nitrogen deficiency, wiih 5-7'i alcohol by
volume and 80- 120 g/l sugar This wine nscd lo be
put into bottles for second fermentation, but il was
irregular and uncontrollable. Second fcrmcntiuoi
low lakes place In a scaled val (Chamiat paxess).
using a blcid or wines from different vincyaids.
clarified by fining with gelatin/tannin and nitra-
tion. Fcnnenuikw starts at 1S-20 C and is then
slowed down by reducing Ihc temperature. When
the pressure reaches 5 Kirs, the wine is chilled lo
or aid clarified again. Tie Knipcraturc is Ihcn
reduced lo -4'C for 10- 15 days lo slabiliw the
wine. Followiag further filtration orccnlrifugation.
boiiling lakes place in an environment pressurised
will CO; to prevcit degassing. Some producers
■sc sterile rillmtioi and others pastcurioc the wine
» prevent il from fcrniciling again in Nmlc The
finished Asli Spumanlc contains 6-9*i alcohol
by volume and 60- 100 g/l sugar. A number of
other sparkling wines ate produced using similar
methods.
14.4 FORTIFIED WINES
14.4.1 Introduction
Fortified wiacs are characterised by their high con-
centrations of alcohol and sugar They arc derived
from the partial fcrmcitalion of fresh grapes or
grape Juice. The addition of alcohol prematurely
sups Ihc fermentation This fortificalKM can be
c Heeled in one slcp or in several.
These wines were evidently created in Ihc pasl
in response to technical problems encountered
in warm regions. Sugar-rich grapes and elevated
temperatures resulted in explosive fermentations,
easily leading lo stuck fcrmcneitiois The partially
fermented wine was unstable, especially since
salfiting was far from mastered at the time. Lactic
acid bacteria subsequently developed, causing
tic tic disease and Ihc production of volatile acidity
(Section 381. The addition of alcohol during
fcrmcntilKm wasasimplc means of stabilizing the
wine and produced an alcoholic and sweet pnxlucl
wilb an agreeable laslc. As bile as the 1960s, these
wines represented a significant part of CalifornEin
and Australian production.
Today, other means can be used to produce
standard types of vviics in these climatic condi-
tiois Grapes are harvcslcd at sugar concentrations
compatible with complete fermentations, even in
relatively hot climates. Fcrmcnti lions are better
controlled through sulfiting. aeration and temper-
ature control. They are also complete Malotac-
Ik fcriKnlation can now occur without haclcrial
spoilage
Due hi greater demand, traditional dry red and
while wines have replaced fortified wines at many
wineries. Today, only the most famous fortified
wines remain. Specific natural factors aid wcll-
adaplcd technology permit these wines to develop
their fine aromas and rich flavors French vim <kni\
ihitioels and port wines are certainly among the
most prestigious foilified wines, but other fortified
wines from Greece. Icily aid olhcr Mediterranean
countries also exist
The Office International de la Vignc Cl du Vin
lOlV) defiles fortified wines as special wines
having a taw! alcohol content (both potential and
actual) above 1 75'* vol. and an alcohol conicnl
between 15 and 22* vol.* Two types of fortified
wines ex St:
I . Spirituous wines receive only brandy or recti-
fied food-quality alcohol during fcrmcntition.
2. Syrupy sweet wine
must or aiisiellf
alcohol
cai receive concentrated
addition lo brandy or
In both cases, the natural alcohol potential of the
grape juice in list be at least 12% vol At Icasl 4'i
vol of the alcohol in the final product must come
from fermentation
Storage conditions, up lo botlliig. vary depend-
ing on the type of fortified wine. Due lo their high
alcohol coilcnl. these wines are very resistant to
oxidative phenomena Some actually develop Ihcir
desired characteristic through a certain degree of
oxidation. These wines undergo a Hue barrel aging.
Olhcr finer and more delicate fortified wines arc
protected from air and arc bottle aging
Olhcr Wine making Methods
4"!
14.4.2 French Fortificd Wines (Km
Doux S'atureli)
fa) DcfinltMin
These famous wncs I VDN) air found in a
doyen iippelliitiiitis across three legions in the
sonlh of France (Brngiraid rt«i\, 1991). Banyub..
RiM-s.iiu.-s. \!au A' and various Muscat igipellmiimt
are among the best known. These wines, fall under
lie OIV definition of fortified wines bul French
legislation tixcs the two lypes of fortified wines
mentioned in Section 14.4.1 differently With the
spirituous wines (VDN). only the added alcohol
is taxed. The other fortified wines, are taxed
on Inc total alcohol, including the alcohol from
fermentation of the must.
Production conditions are more constraining in
France than as specified by the OIV. Not only
is the area covered by each appellation clearly
defined, but the grape varieties are also specified.
Non-Muscat varieties are Grenache. Macabcn.and
Malvoisic. while only Muscat of Alexandria and
Muscat a Pctits Grains are permitted in fortified
Muscat wines.
Crop yield limits are set at 40 hl/ha. with only
30 hl'ha allowed to be used for making VDN.
Crape juice must contain at least 252 g of sagar per
liter (approximately 145'* vol. potential alcohol).
The proportion of alcohol added at the time of
fortification must comprise between 5 and Ufi of
the must volimc. The must is forlilied when the
fermentation has already transformed a little more
than half of the natural sugar The final prod act
must contain between 15 and W'f vol. alcohol
content and at least 2 1 5'* vol total alcohol Total
alcohol includes the alcohol and potential alcohol,
which corresponds with the quantity of alcohol that
the resklual sugar conki produce by fermentation:
Residual sagar content (g/l)/ I6.S3
= potential alcohol ('* vol.)
The minimum resKlnal sugar content varies from
59 to 115 g/l. depending on the appellation.
The initial must concentration and the percent-
age of added alcohol are verified by the follow-
ing relationship P/o. where P = residual sugar
weight (g/l). and a = polarinclric deviation which
depends on the proportion of glucose and fructose,
itself related to the quantity of sugar fermented
In crape .juke. Hie glucose 'fructose (G/F) ratio is
equal K> I Clucosc diminishes more rapidly than
fructose during fermentation A French fortified
wine must have a P/o of between -2.00 and
-3.00. Fraud P. suspected bckiw -3.5. A fortified
wine artificially made from dry wine, alcohol and
saccharose or concentrated mnsl (G/F = It would
have a P/o of -5.23.
Table 14.10 provides supplemental information
concerning the chemical composition of French
fortified wines.
tb) Vindication
Several lypes of French fortified wines (VDN)
exist. The white VDN are made from white or
gray Grenache or Macabcu grapes. They do not
generally undergo a maceration, but arc occasion-
ally lightly macerated They are light, fruity non-
ox id i/cd wines made lo be drunk young.
Red VDN are macerated. The juke and pomace
are generally separated after several days of
vatting. Fortification most often occurs on the
separated juke but in certain cases the alcohol
is added to the pomace and the maceration is
continued for 10-15 days. Richly colored forlilied
wines with high concentrations of dry extract arc
obtained by this alcoholic maceration. These wines
are capable of being aged for a long time.
After separating the must. Muscat wines are
made similarly to white wines. However, macer-
ation increases aromatic extraction, making these
wines therefore requires a lot of care lo respect the
finesse of the aromas.
Grape maturity is regularly assessed to deter-
mine the harvest date accotding to the variety
During overripening. the maturity mnsl be care-
fully followed because sugar concentrations may
increase sharply lo attain 250-270 g/l (lS-It»
vol potential alcohol). The acidity also dimin-
ishes considerably The full aromalK potential of
muscat wines is obtained at a sagar concentra-
tion ofamaid 225 g/1. BiHryl is cinerea negatively
affects fortified wine quality, especially in the
I I and book or Etiology: The Microbiology of Wine and Vinifiiations
Table U.U>. AiulylkaUlnQCI.
emtio ol Fie
nch fortified
o iocs (Vl)\) (Biuginnl
rt.rf.. 11*91)
Minimum
Average v ahiei
KUiimim
nlu
value*
Aknb.il tome* (r. vol.)
US
15 1
lo 17
18.9
Total ak-ahol f5 ml.)
215
215 1
lo 225
230
Dcmk) .« arc
1.010
LOIS
lo 1D30
1035
Suptrtp/I)
45
70i
la 125
ISO
PA. <IS-C)
I.S
-20 1
lo -25
-35
Taut di> cU»ct <tj/l)
•■:.
HOi
lo 140
170
Reduced <lr> euiact (g/l)
18
20 1
to 25
J
Aibx* !n/l)
1.4
121
to IS
35
Ashes alkalinity (p/l K,CO,|
IJ
\2\
to 22
3.1
Taal«klily <gJlH,SO.,)
in
J JO
to 13
SO
VoUnUe JK.kU) (p/l ll.M.i.l
D.I5
DJO
loDOO
k>>.-.:
pH <20'C)
2.90
300 1
lo 380
4.20
IkuH IFoU ■ \*k\Y-
wUc VDN
IS
25 1
lo 4C
ss
KdVDN
:::
n
to '■•■■
70
Akkbydw 1 -pli
25
no
to 120
i.v.
llwhcritolwbi-p.lt
SO
70i
m90
ISO
Glycerol <gfl>
.1.(1
60 1
lo 100
I'd
Buiyleoc glycol lg/1)
D.30
:. 50
la 020
1 .20
Uctic acid (g/l)
IU9
::■ R)
loOJ
(03
free SO, (mg/l)
Oi
to 15
20
Total SO, <«g/l>
lw»
100
to ISO
k>v.:
case of Muscat Botryfi/rd grapes should not be
macerated
The first steps or wine making wiih maceration
consist of modcralcly t rushing and dcstcmMing
(be grapes The grapes arc then transferred to
the lank and sulfllcd at 5-10 g/hl. The fermen-
tation tcnpcralnir is scl al approximately Jo C" U
favor maceration. Maceration units vary from 2 to
8 days, if the fortification occurs after nasi sepa-
ration. In this case, the fermentation speed should
be reduced beforehand Wines arc macerated (or 8
lo 15 days when continuing the maceration after
lortilkalioa
When there is no maceration, the grapes aic
drained and pressed to extract the juice, accord-
ing to traditional while wiucmaking methods
(Section IJ J) Immediately after extraction, the
juice is stibi)i/cd by su Kiting at 5—10 g/hl and
preferably refrigeration The must Is then clarified
by natural settling and racking or ccnlrifngation.
Yeas i Marie r may be ascd and fermentation lent-
pcraluics arc kept relatively low. 20-25'C fand
even 18 C for Muscat). K avoid loss of aroma.
lei Fortification (Mul-agri
The addition of alcohol to fermenting must slops
yeast activity, increases the dissolution of phenolic
compounds during maceration and provokes the
precipitation of insoluble sabstanccs. A near-
neutral wine brandy is used The addition of non-
wine spirits is not permitted The alcohol addition
may be done in several steps lo slow and spread
out the fermentation phenomena.
The moment of fortification is chosen according
to the density, which decreases during fermen-
tation. The density must not drop below a cer-
tain established limit, called the fortification point.
Choosing the correct fortification point is essen-
tial to wine qnalily. The wine must have a sugar
concentration corresponding K> the type of product
desired and conforming to legislation
Fortification rabies arc used lo achieve the exact
alcohol content required, using cither wine spirits
at a minimum of 96.0* alcohol by volume or a
blend of spirits and mast The addition of wine
spirits is c lice led with cither 'Xf.i vol .alcohol
Olhcr Wincmaking Methods
473
or with varied Mends or alcohol and must The
second form of addition arose from the need lo
have a Bix official picseni for lax purposes when
using alcohol In the past, wines were slabili/cd
with high SO. doses to slop the fermentation,
when waiting for iht authorization lo use alcohol.
Nowadays, ihc wine spirits air denatured by
mixing with must that has just stifled fermenting.
in Ihc presence "I a government inspector
ll Is recommended to slop rcniicntition lo/uiii&e)
tcfoic fortification by rcfrigcrafing the musl
or eliminating the yeasts by ccntrifugaliot or
filtration.
Sulfifing destined to neutralize the ctnanal
formed aid to block oxidations definitively stabi-
lizes the wine. A ffee SO- concentration between 8
and 10 ing/| should be maintained Approximately
10 g of SO> per hcctoliKr should be added, con-
sidering the high pH (35-4.0). sugar concentration
and alcohol content of these wines.
id > ' 'onsen iit i- 111 and Aging
Due lo their diversity, many storage and aging
methods exist for these wines. All arc generally
aged for a year in tank, nndergoing repeated rack*
ings lo assarc clarilicalion. Different methods spc-
cilic locach type of wine air used aflcr this period.
Muscat wines are stoicd in tanks until bottling.
Precautions air taken lo avoid oxidation and to
protect aiomas: I5-I7T temperature, sufficient
humidity, use of inert gas. etc.
After a selection based on listing, many led
VDN. having undergone maceration, arc placed in
6 hi casks exposed to the sin. Oxidation phenom-
ena cause these wines lo take on an amher tint and
characteristic aromas The wines are often fined
and cold selbili/cd before being placed in casks.
Carrying out these operations at ihc time of bot-
tling may thin the wine. A simple filtration at this
lime is preferable. A once- traditional method lo
obtain the same oxidative transformations consists
of leaving slightly emptied glass carboys outside,
exposed to natural climatic variations, but it is now
lately used
The finest and most delicate white and red VDN
can be matured in 225 I oak barrels in cellars
at moderate tcmpcralaics (15- I7'C) without any
particular oxidative phenomena, according to tra-
ditional tine wincmakiig methods The wine Is
matured for approximately 30 months and bottled
after fining with gelatin. Reduction phenomena
aflcr the wine is bottled are responsible for the
actual aging process.
Rancio wines ate made traditionally and locally
The production of these wines is not codified The
method consists of maintaining a 6 hi banc) par-
tially filled Each year, wine is removed from the
banc) to be bottled and replaced with newer wine
VDN are subject to Ihc same clarification and
stabilization problems as other wines. Iron casse.
proKic casse. tartrate deposits and colored matter
can cloud Ihc wines. Standard preventive measures
can help to avoid these piobkms. Oxidasic casse
is another accident linked to giapc rot.
The high alcohol eoiicnl of these wines gives
Iheniaccrciin level of microbial stability, bnl acci-
dents arc still possible due to their high sugar
concentration and elevated pi I. Some yeasts toler-
ate 16-17'.* vol cthanol and arc capable of caus-
ing ic fermentations. In addition, these yeast, resist
elevated concentrations of free sulfur dioxide Par-
ticular strains of Lactabaciiha lillgmilii have been
identified in certain French VDN. They aic apt lo
develop, provoking lactic disease, responsible for
abundant deposits and gustatory flaws.
Stindard operations lower the risks of devia-
tion — hygiene, fining, filtration, sensible bsc of
sulfur dioxide, etc —but pasteurisation rs the only
treatment Ihat completely eliminates germs and
stabilizes wine. The correct use of this method
docs nol cause organoleptic modifications, even
with Muscat Slcrilc filtration can also be used
14.4.3 Port Wines
<ai Prodacllon Conditions*
Poti wines conic from the steeply' sloping Douro
region In Portugal (Ribc'rcan-Gayon etal., 1976:
Barren. 1991). The schistous soil, the jagged relief,
the high temperature variations between seasons,
low rainfall and intense sunlight characterise the
Douro. These conditions lead to highly aromatic
pigmcukd grapes with high concentration of sugar
and phenolic compounds The temnrs
-"4
Handbook of Enology: The Microbiology of Wit* anil Vwifications
in a decreasing scale front AoF according lo soil
nalurc. grape variety, vine age. altitude, exposition.
cK There is a great diversity of cultivated varieties
in this region (15 red and 6 white). The grapes
arc picked very ripe bnl arc nol vine dried. They
arc sorted very carefully fc» eliminate bad grape
clusters and spoiled grapes. The must, with ;i
minimum of I \'i vol. potential alcohol by volume,
bnl which usually contains 12-14** vol.. is sainted
(9- 10 g/ll) and may be acidified, if necessary.
A relatively slow partial fcrmcuuiioa Is sought.
Extraction of skin component incurs during a
concurrent maceration. The wine was traditionally
fcrmcuKd in ia&viei. SO cm-high granite vats con-
tuning 2.5-1 10 hi. an ideal shape for ensuring
that all the grapes would be perfectly crashed The
grapes were Uoddcn tot several hours each day
until the thiol day of maceration The fermenta-
tion occuncd simultaneously The pomace cap was
immciscd by mechanical means at regular inter-
vals. When the desired density was attained, the
la&tve was opened and the wine llowed into the
casks. B randy was added tu the wine in casks Id
slop the fermentation and raise the alcohol content
lo 18-19** vol.
Today, most wines ate made in modem wineries
and the crushing and maceration operations are
mechanized. The open or closed tanks arc equipped
with automatic pumping-over and mechanical
mixing systems. The manual work has all bnl
disappeared. These perfectly controlled technical
modifications have improved and regularized the
quality of port wine while increasing profitability.
Upon arrival at the winery, the grape crop
is dcslcmmcd and carefully crashed lo facilitate
maceration. The must is sulliKd bat generally
not inoculated with ycasfc. to avoid explosive
fcrmcuuiKws The temperature is maintained at
aroand 30'C during fcraicntition.
After reaching 4-5'* vol alcohol, the fermenting
mast drained from the tank is clarified, possibly by
a rotating filler, before being fortified. Correctly
choosing the fortification point is essential to the
quality of port wine and to obtaining the level of
sweetness desired. The quality of the port also
depends oa the quality of the brandy used for
fortilication All brandies used arc submitted lo
analytical and tisic krsls; they contain 11-1VA
vol alcohol. Pneumatic and mechanical horizontal
presses arc currently replacing traditional vertical
presses, since the former ate easier to asc. Pressing
is moderate During fortification, a fraction of the
tannin- and color-rich press wine is added to the
free run wine.
<b) Maturation and Characteristics'
of Port Wines
Eigurc 14.2 summarizes the maturation and aging
process of the different types of port wines During
the winter following the harvest, after the Hist
racking, the wines are classed according to taste.
The best batches, during an exceptional year, may
be reserved k> be dectued as vintage port, but most
wines are blended.
The blends axe aged in 5-6 hi oak barrels
i/v/v.'.v i for several years in oxRLativc conditions
that maintain an elevated oxidation-reduction
potential Metal ions, in particular copprrand iron,
play an essential role in polyphenol oxidation.
Even in the bottle, these wi
oxidation - red ucuoa potential The it
its ferric state, as if all of the reducing compo-
nent had been destroyed by oxygen. Their pro-
longed oxidation and intense cstcrillcatioa give
these wines a rich and complex bouquet
During agiag. the tannins bccoBK softer as
they polymerize or combine with anlhocyanins.
while coloring matter precipitates and the color
changes The less oxidized ruby ports maintiln
the I ru illness and robustness of yoang wines They
have a more or less dark red color. The older, more
oxidized tawny" pom ate goMcn red or golden.
White ports undergo a certain level of macera-
tion and arc aged in the same oxidative conditions
as blends. With certain exceptions, the wines ate
nol oxidized, lo maintain their fruity aroma and
pale nil.it.
Superior quality products 1 10-ycai-okl. 30-year-
okl ports, etc.) also undergo oxidative aging.
At the lime of bottling, these oxidized wines arc
stable In the presence of air. They improve very
little during bottle aging.
Vintage" portsarc the best quality wines. Afler
a brief aeration lo stabilize the color, they arc
Olhcr Wincmaking Methods
o
_
0t,.,.
-" <^
i—
l»
o
.
i c
"—■
^
1
1
1
(,__
J._
I - "
1'ifi 14.1 How tluiirj
ttirlfce pn»ti>l»n"l
HHyklofpoO nine (Bam*. 1991)
aged in full kinds, like many great fed wines
Vintage pons ate boltkd alter 2-3 years' barrcl-
aging. while Laic Bollkd Vintage pons*' arc
boltkd aflcr 4-6 years (Figure 14.2). They then
continue to Improve considerably In bottle A
rcdnction bouquet develops: it is linked k> the
low oxidation -reduction potential lhat maintains
the iron ion in the ferrous stale. Vintage pods
have a considerable aging potential and can be
aged for 20 years or more in the absence of air.
due h> their high polyphenol concentration These
wines arc very robust when young and. after years
of aging, maintain a high extract concentration, a
characteristic fruilincss and relatively high color
intensity, with rcd-mauvc tones dominating. Once
bottled, these wines arc sensitive to oxygen: when
the bottle is opened, the wine rapidly loses its
qualities.
The year can still be mentioned on the bottle
of non-vintage, quality port wines. These vintage
character wines arc called colhcila ports".
14.5 FLOR WINES
14.5. 1 Definition
The Office International dc la Vigne el dn Vin
defines '(tor wines* as:
wbnK
ClfOl .
lubaficdio .i hdlofiK'al aplBp period in contact
with ill. by I he <lc>ek>pac«l of lotycaals (liln-
loraHit£ vCUMM .illcr akoholk fcr*C nation of
■ he nun.' It ninth-, red ifced akolnl of aprkufctml
»piriu can be bieraddcd to I he wine. In thb ca*e.
■ he akohol comcm of the banned pnnlm muM
he cqualto orpreatcrtlun IV- vol.
Sherry in cnglish and gcrmau— Jcrc/ in Span-
ish— Xcrcs in french is the best-known Dor wine
In orYuvuo- type sherry, the aging process is essen-
tially physicochcniKal and biological development
is limited. The iHonao method will. however, be
described in this section Jura yellow wines arc
another well-known example of fktr wines.
14.5.2 Sherry Wines
i.i > Prodicllon Conditions*
The sherry production /one is situated in the south
of Spain, near the city of Xcrcs dc la Froikra. The
production of this prestigious wine was described
by Casas Lucas ( 1967). Goswell ( 1968). Gonzalez
Cordon 11990) and Jells (1992). This section is
based on the work of E. Pcyiaud (in Ribcrcau-
Cayon el ill.. 1976). updakd by J.F. Casas Lucas
in 1994
The Palomino cullivar constitutes nearly 95*i of
the grupc production for this wine The remaining
476
Handbook of linology: The Microbiology of Wine anil Vitrifications
5't consists of the Pcdm XInicncz variety. The
viae is cultivated on different leninrs. creating a
production hierarchy. The mists contain 12-14*.*
vol poicniial alcohol and an acidity of 2-3 g/l
expressed as H ; S0 4 (or 3-43 g/l In lartarK acid).
The grapes arc carefully picked and placed in
IS kg cases. In Ihc past. Ihc grapes were tradition-
ally exposed Id Ihc sun for a day on a mud Door
UtfnujttrY This practice, known as sotea. results in
a !"'• loss in grape weight, an increase Ii sngar
and tartaric acid concentrations and a decrease in
•talk' acRI concentration. Although favorable Id
qaa!hy. Ihc saleo practice hat all but disappeared.
Pedro Xinicnc/ grapes may slill undergo this prac-
tice, attuning a high sugar concentration within
15 days l Sec lion II 2.2). These grapes arc used k»
preparer a sweel wine (cream sherry) which is used
In variable proportions to sweeten dry wines.
The sherry wincmaking method is based on
while winentaking principles without maceration.
The jnicc extraction conditions are consequently of
prime importance moderate crushing, no contact
with metal, stow and light pressing. Juke selection
after pressing. The clarification and refrigeration
of these juices lend lo be generalized
Plastering (adding calcium snlfate to must) Is a
traditional practice in this region This operation
permit Ihc suspended solids to sclUc more rapidly
and Ihc wines obtained ate more limpid and
their color more brilliant There is a decrease in
pH. a diminution of ash alkalinity (due to the
precipitation of acRLs in the form of silts) and
an increase In totil acidity and buffering power.
Approximately 2 g of CaSO,(2 H.O) arc added
per liler. towering the pH by 0.2 units. The wine
may also be acidified with tartaric acid ( 15 g/l
Sulfur dioxide Is used during wincmaking and
storage to disinfect Ihc barrels, bnl concentrations
mast be limited so as not to binder the development
of ftor yeast Taking hygienic measures avoids
undesirable microbial con Emu nation
In the past. Ihc fermentation occurred in 5 16 I
oak bench ttotis tie extraction) lilted wilh
450-467 I of jnicc. Today, relatively low-capacity
sEilnless-sKcl containers arc increasingly used, to
Until excessive tcmpcralaic
lb) Biological Aging Principles
for Klor Yeasts
The wines, still on their Ices, are ustcd during the
months following the complclkin of fermentation
The best wines, considcicd Ihc mosiapt foraging.
arc racked, fortified to I5-I55'i vol. and stored
in a container filled to 5/6ths of in capacity
The alcoholic content of these wines prevents
microbial spoilage, bnl Dor yeasts spontaneously
develop on the surface of Ihc wine. After a certain
degree of development, another tasting result! in
a new classification, determining the appropriate
type of aging {eriwot) for each Ixia: biological
or oxidative
During biological aging, the Bar develops, some-
limes during several years. Certain yeasts arc
capable of developing on the surface of 15- K>*«
vol alcohol content wine in contact with air. This
Hint is produced by ycasev specific to the region,
coming from either grapes or previously nsed bar-
rels. These ycasB develop in acrobtoscv by oxi-
dizing clbanol. They belong to the Sueelktrtmyres
gcuns Over the years, taxonomy has included
these yeasts in Ihc S cerevisitie specks or has
Identified them as Sacchtaiviiyces ovifonnis. Sac-
tharmtyees htmmus. etc. These are therefore not
ordinary mycodemal yeasts, responsible for vari-
ous wine diseases dnc u poor storage methods
Martinez el til (1997) Identified the following
yeast strains in Sherry flar:
7-¥t Saceharomyres ceivrisitx Ifiicus
l-¥t Sacchammwes eerevhhte moimiliensis
S'i Sticchtavatwes cererisitie cheresiensis
Oi'A Sacthtrvmxcfs cerevistOe nmxii
i'i strains nol typkal of Sherry fitr {PtehUi.
Htmseneht. and Ctmtlitki)
The different strains develop and form jif at dif-
fcrcal rates, as well as having different mclabolk
effects. e.g. motthdtensh produces the highest con-
centrations of cthanal.
The yeast film is called fior and the biological
aging process has been known as criaaza tie fbr
for a long time at Xcrcs. The more or less rapid
and inKnse Itor formation and its aspect and cotor
(white, cream, golden, burnt) depend on many
Olhcr Wincmaking Methods
4""
factors, especially Ibc nature of ibe yeast ami the
ebcmical composition of the » ik. Tbc flor rarely
(onus above 165'* voljkoholand ii is impossible
above ITi vol. The presence of a little sugar is
favorable: ibc presence of phenolic compounds is
unfavorable and darkens Ihc wine color. Sulfur
dioxide, nitrogen compounds and other suhscinces
are also involved. All of these factors exert an
influence that is reflected in fa tire wlac amma and
quality.
During this type of aging technique. Ihc wine
docs not remain permanently )■ the same con-
tainer: ii is periodically transferred to different
bolus. These transfers air fractional, following the
solera system. Bona arc piled in rows. The barrels
ibaku) in each horizontal row (escolal arc full of
wine from the same crianm (that is. same degree
of aging). During aging, the wine is moved around,
blended, and redistributed to obtain the most uni-
form wine possible at the line of bottling This
system also permits new wine to be added regu-
larly, which helps Ki maintain the flor.
Figure 14.3 summarizes the solera system. This
example contains three escolas: 720 I arc taken
from the six bona of the lowest tow. or solera,
for bottling. The solera barrels arc filled with
720 1 coming from the live boias of the preceding
eriaclem. Finally. 720 I from the four bolus of the
highest tow Mil the last five botas. These foarbolus
are filled with new wine.
The boia tie asienia intended for shciry aging
has a volume of 600 I The wine volume is 5*ths.
or 500 I.
In practice. Ihc solera systems arc much
larger— ihcy usually contain several hundred
1'ijt U.J. Solca ijHtan, ihou-iip ihc pan ill dawiu.
off and (cduKtbuibuof wine loin ibc km bum rot
of hunch alia older crioirni Uiioc.Thh operation «>
bcpin by rcmoviiio 120 I lio« haiicU un ibc kmci
level (nrioleot lor bolt linit
Ixirrcls. Transfers arc made in groups of 12 lo IK
botas.
Wine is transferred three lo four times per year
in the solera system. The transfer volume depends
on the type and age of the wine desired. The ratio
of total syslcm volume to annual volume removed
determines average vvinc age.
(c) Wine Transforrnalions Daring
Biological Aging
The biochemical transformations provoked by the
criaicti & jlor have been studied. As oxygen is
consumed by the flor. its proportion decreases
in the barrel- head space and is replaced by car-
bon dioxide The wine transfers, however, aer-
ate Ihc wine. The oxidation-reduclion potential
of the wine (25O-300 mV) indicates a moderately
reduced stile. The flor acts as an isolating layer,
protecting the wine from excessive oxidation In
this manner. Ihc wine ages normally Ifino type),
acquiring a pale yellow color.
Volatile acidity diminishes to It) g/l hi H ? SO,
10.12 g/l in acetic acid). F.tuanal formation is
an essential characteristic of the crian<p de Jkir.
slowing during aging to produce a lotal of
220-380 mgfl. Doc lo its chemical reactivity,
ethanal is a precursor of many chemical sub-
stances thai contribute to the bouquet of sherry
wine (diethyl acclal. 50*60 mg/l). Sotolon i Sec-
tion 10.6.3) isacharaclcnsiKclcmcnlor the aroata
of fino wines.
The glycerol concentration attains 7-9 g/l im-
mediately after alcoholic fermentation During the
lirsi aianzti phases, ii is signihcanily depleted
After three years, its concentration can fall lo
several tenths of gram per liter.
1 attic acid is also formed, reaching 22 niBq/1.
This production cannot be explained by alcoholic
(7 mEq/l) and malolaclK (6 mEq/l) fermentation
alone. MalolaclK fermentation Is nevertheless
complete and contributes « wine quality
Free amino acid concentrations diminish during
flor aging, but the evolution of each amino acid
varies, depending on the situation After 7 years
lit* aging, the concentration of proline, the most
abundant amino acid in //»*-lypc sherries, repre-
senting Itfi of Ihc initial amount of total amino
478
Handbook or Etiology: The Microbiology of Wine anil Vindications
acids had decreased to only 31* (Botclla el irf ..
1990).
The crianza <** flor in shcrry-regkin winer-
ies produces fino wines. Manziiialln P> produced
according lo Ihc same principle in (he Saalocar dc
Baininicda icy ion This style of wine is aged foral
leas! 3 years Al the end of ihis long aging process,
the crianza <le (far disappears The aging pnxess
cai. however, be continued chemically {olimtat
wines) for 6 ycais or more These product* have
the following names according to their age: jino.
tmoiiiilltuto. iBtminillikbi viejo and ontoniilkufo
miy tie jo.
(d ) Oxidathc Aging of Ohirosn Wines
A nor lilm develops on nearly all sherry wines.
This Dor develops several months after the com-
pletion of alcoholic fermentation An initial forth
ncaiion at 15-153% vol .alcohol is practiced oa
wines ihm siiKd to biological aging. After a few
months of being aged under Ihc film-like growth,
the wines arc tasted and classified, confirming
those that arc destined to be aged biologically
and deciding which wines must undergo oxida-
tive aging. The Litter arc selected according to
film growth conditions If Ihc filmlike growth is
tot established in suitable conditions, the growth
Is completer)' slopped by an additional fortification
*i 175-18* vol.
From this moment on. the wines receiving the
additional fortification will age in Ihc absence of a
yeast film, without nor yeast activity. Only pnysic-
ochcmKal phenomena occur. During the aging pro-
cess. some of Ihc substances responsible for the
fruity character in the wine arc oxidr/cd by oxygen.
The barrel wood also plays a rule in the oxidation
process Us Kxlnre actsasa type of tissue of scmr-
pcriKablc membranes. The icaclions involved in
these phenomena arc slow and poorly under-
stood Basic wood substances arc extracted and
oxidized.
Oloroso wines can be aged according to the
solem system or in a more static manner with-
oat blending. This static method produces vintage
wines UuuukitY Oloroso wines arc generally lie her
in color and more robust than Jino wines. fc/vri
olonaa wines correspond loa lower class of wine
than otoaw.
Before bottling, the different types of sherry
are clarified by fining with albumin or powdered
blood. They are also sometimes stabilized by a
bcntonltc addition
There is a risk of bacterial spoilage during both
aging pa<ccsscs|bK)logical and chemical) For this
reason, each banc! is regularly lasted during the
process. At the slightest quality doubt, the wine is
transferred to sherry vinegar production.
14.5.3 Yellow Wines from Jura
Although they have their own character tChevcn-
ncmcnl einl . 2001). there arc a certain number
of parallels with Sherry. Like sherry, they undergo
an aging pnxess with nor development, bnt no
alcohol is added lo them
These wines are made from Sav-agnin grape. The
base wine containsapproxlmatcly 12'* volalcohol.
The wine is placed in small barrels, the barrels are
topped off and scaled They are aged in a cellar
for 6 years, without topping off. producing a head
space in the barrels. A filmlike growth progres-
sively forms on Ihc surface of the wine This flor
is composed of aerobic film- forming yeasts which
develop by respiration and cause various transfor-
mations — in particular, the oxidation of cthanol
Into clhanal The yeast most often encountered in
yellow wines belongs to the S. cererisiue genus.
The inoculation is spontaneous with these wines,
causing the film growth to be irregular and some-
times resulting In spoilage. The risks of Increased
volatile acidity arc greater than in sherry wines and
increase as the wine alcohol content decreases
To improve production conditions, film growth
can be accelerated by inoculation with a lilm-
producing yeast culture and by leaving bead space
when filling Ihc txincl. instead of waiting for It to
occur spontaneously by evaporation. Maintaining
a kiw temperature (12- I3"C) also limits bacterial
spoilage
In practical terms, tinjaune is aged for over six
years, and Is subjected to alternating cold winter
(5-10^C) and hot summer (25-30 : C) lempcra-
lures These variations cause the development and
Other WiaciniiLiap Methods
479
cli in inat Ion of a serin of par blooms over ibe
years, resulting in Ihc coexistence of live ycasl
in the sari ace <ki' anil dead yeast cells thai arc
deposited in Ihc Ices and autolyycd. The yeasrs
have an intense metabolic activity al 25 'C bit arc
much less active al IOTtCbarpci.ticrpi«/..2002).
The yellow wines from Jura arc characterized by
■heir high ethanal conccitration (600-700 nig/I),
■heir deep color aid their particular oiganolcptk
characteristics.
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Index
Kkl 241.249
■ 243
AccuUlehvde 63. 07
AcaaUehvde dehydrogenase, see ALDH
Acetic acid
accuniutitkin of. by ye*M 04-7
faraiibi of. by ycul 64-7
see aim Bictcru. acetic ucid
AYfofcuw »pp . 183- 92. 2D2
Acaola 08. 189
At .1 nx 292
Acklificaiioii 107 S. 343
Aekliy 107. 217. 307- 10. 370. 374-5
vohriik 00.91. 108-10. 112. 188. 190.345.300.
371,431
IS I
..„
123
Additive iccfcakfici 313-5
AdcnnuK diphosphate, see ADP
Adenosine triphosphatase, see .«'!'»«
Adenosine triphosphate, see ATP
ADM 188
ADP 53-4.42
Acaib* 102-4.341-4.450
maloUclk ilnciilm iod 375
momentary /permanent 103
see itto f\imping over
Aeiobic •cspiatbn 140
Aembnsk, 103. 104. 140. 190
Agi*g
acctk- ackl bacteria during 191
barrel 433-5.470
reduction odots duritg 439-41
bolilr 470
Hot you 475-9
lbiti6edv.ioes 472-3
o*-kes 428-9.432.434-5.402-3
oiicbiivc. olorom wioci 478
Ahmeur Bou Ahmeur 203
Alanine 71.72
Abariao 401
Alcohol dehydrogenase. nr<- ADM
Alcohol, potential 80-1.201
Alcoholic (cracvatkin 57-8
Alcobob. higher 74-0
A 11)11 188
AhilnaicbalU 4M
Alkipies 190
Atmijtr 470
Anuviume 150. 174. 370
AMMKkk
auimibtion ■c.hj<in-> 72-3.86
mabolan of 73-4
cbssiacaik>* 71
composition hy giapc variety 277
dcaminaibnof 74
in grape 240.253-4
permease, general, see GAP
u stianibns 85-7
-,»kMi pathways 70- 1
transamination 73
Aimuii
assimiblion mcchanisatt 72-3
in grape 240.254
iib.n must Mimuunt 80-7. II I. 430- 1.455
Anaboltsm 53
Anaembk respiration 140
Anaenibk.sB. 104. 140. 380-8. 391
Ant bests 243
Antbncyamns 240. 25b. 205. 208. 323. 328. 345.
347-8.351.388.440-7
350
Anlhanilfc acid 257
Antliacicrah. 211
AMhkHks 224
Antifungals 210-1
AntkukUm* 194.237
Atfnxkbuin 194
i n>- »>»si^>(> ..i
Amiscptki 111 -J. 197-8. 210- I. 213. 224
API into 125-8
Apptlhtiaii 458.462
Aobiaoic 202
A anon 277
Aqjinine 71.72. 1SS-6. 254. 277
Aqjon. Muniyc uxlci 237
Akmi
aceiaic* a ml 70
buUciy 371
caibonk aaccralka 388. 394
coapkxiyof 371-2
lieeiag of 323-4
liuiy 405.425
prapc variety comliucnu and 278
hitrbcralcohob and 70
maccaikn and 345
ovkfatkn 420
■a in dilution 76
Ulltlllniand 210
>cpeial 279. 340. 357. 384. 105
A Banks
hoirytB modification uf 289
evolution 250-9
in pope pulp 240
in pnpc skin 247
maturation imln^ for 202
mo.no .in? 261
tcapccuurc ami 205
■■. hre
429
.V rr'l ,-<.,'( a ■.." ■ .' I] .."„ ( 4
Awi 10 -9
AwoaSk acid 224. 234-7. 25 1 . 408. 4 19
aerated iim-%. ontaaokplk protection of 236-7
con mat k ovktatkin
pa>tcci»a jjfjiml 235
pn>tcci»n jjtjiipI 235-0
modcolactkin 234-5
properties of 234-5
A^paak acid 254
Asperptlut nigtv 94. 291. 291.127
teper/ftliit ».pp. 322
AMhma 196
Am) SpumaMc 469
ATP 53-4. 58. 59.62. 70. 71. 99
ATPaw 8.72
Autora.lkiuaphv 13 1
249
458
cicm.icclK
id 183-92.336.451
Sow ilii cinrf
n and 292
ratbonk an
ralnaaad 391
rnaractcriMk*
183-4
phjitobptc
.-builU'ation
il 185-6
184
cilobpy 183-4
development, in grape nun 189
evolution of. la ™ ine making 190-2
identification 184-5
notation 184-5
ktcatnn 41
Bclih.lr.mi 186-9
vuliliny cllc.l on 211
la ° iacnukiai 190-2
Baclcta. Cn«- negative 184
BaclCfB. (ina-ptnaivc 1 16-7
idem Ificat kin tcMs loi 120
Bactcro. bete m fermentative 124. 120. 143-4
Bacteria, ho mole rm em alive 124. 120. 141-2
Bacteria, tactic acid 1 15-30. 345. 370- I. 373-5.
378.452
acclk bacteria iulcaclka* 178
anahiB.. pbcnotypk 120-9
cell component* 115-22
cell nail 116-7
cylo plain. I2I
mxkui 121-2
pbnmk membrane 117-20
raaiBrtiT 124-5
PCK 120. 132-5
eiiochnimc.bckof 124
development in wine 161-80
DNA 121. 126
DNAanahis. 129-35
ckctaipboicik 129
genomk 129
hybridiraik-n 130-2
RFLP 129
elktool effect 166
fuapal interaction*. 178
gnmih adaplalkin in wine 167-8
gara-th. physkochcmkal facton 103-8
idemificatkin 125-36
by fatly aekl coapo%ikin 135-0
phcaoiypk analyst* 125
by protein eompovition 136
U iahftaon 99. 108
iahfcitorv of 370
iacr.pc.itv mteatlioM 177-8
location of 41
■talk ackl laaafoiaattoa by 172
mclah.ilB.rn 139-59
oigank ackU. principal wine 144-51
*ugar 140-4
akronou. evolution of 108-74
multiplkaiion 122
■union, ia wiac 101-3
encgyt.iu.ee* 161-2
my pen effect* on 167
pH effect 161
p be no Ik compound aciioa on 107
population ph»c* 168-70
death phase 109
decline phase 169
groulh phase 169
stationary phase 169
protein*, shock 168
RNA luhih 1*1
sorbkackland 12S
specks, oohakta of 171-2
sullhing and 169-70
sulfa, dioxide effect 165
mechunRmnfactkin 165
taxonomy 122-5
mokcular 122-3
pbeaotypk 122-3
Icapcnturc cllc.i 166-7
vbhlc but noa-cuki»abk 170- 1
wine coapt»Hnn. evohabn of 172-4
wine coapo*K»n\nalfey and 157-9. 168-74
yeast iacraclbns 174-7
Bacteria, mablactk fcracutatbn 108 9
Bactemlcokink* 170
Bacicrbcino, 177-8
Bade rb phages 178-80
Balling, degiee 261
Barrel, sulphur 218-9
Barreling down, direct 362
fk'iotumgr 1 433
llau-c.dcortc 261
Baycovia. in* Mhyl pv rocarhonatc
BerrmmUexe 299
BcnnnHc 428-9. 448. 457. 460
Benvoic acid 246
Bcigey's Manual 185
Bcny. see Grape beny
It mi in 63.90
Baterness 151.226
Black leaf 283
BoMCWt styk 344
Bofn de .menla 477
MQlfc
60
BotrttUrintrta 283-94
acetkaekl bacterid and 189
cutkkthkknexxand 284
detect bn of 292-4
cnviroamcntalcnndiiioas for 283
enzyme synthesis by 317
grape tarn lily and 292
urapeseoxitrvkyto 284-5
as iahfctor 99
ketones and 202
bcatbn of 41
overripening by 299
oxidaskcasseaud 213
phenolic coapouad oiktaiion will .120
yeast activator frum 94
«r iV«i Gray rot; Nobk mi; Vulgar rot
Book skkness 236
Hrtilinp . hoi 231
Btanax Helblbcrmk Pmdutt 264
Srrt/r/iontuv. spp. 43.83
Brevkin 178
Brix. degree 261
Bubbles 466
Bud fcailDv 281
Budding 15
Bunch rot 451
Buach Mem necmsis 283
Buiancdkil 68. 150. 189
Cabernet Ftaac 387
Cabernet Sauvkiaon
At 356
acidly 307
ammaikj, 257. 259. 282
bunch ucn necrosis in 283
climate and 277
clonal ickctkin
shaded 263
shatter 243
soil aad 280
nageac
279
282
> image 271
Cah-k acid 245
CaBarkackl 418-9
Cakiim 253.308.309
Cr/rdidrtspp. 19. 2D. 41. 43
acid m* and 292
contamination by 83
kkntilicatba 24
karyotype analysis 37
sorbkackland 225
Canopy management 281-2
Cap. pomace 335-6.343
Cap punching 349-50
avda Pbcagc
Carbon dnxkk 336.390
asphyxialbnhy 334
conccntntrom 238-9
ax iahfetot 98
storage under 237-9
Carbon supply 84-5
Cardinal 263
Carignan 243.277.391.446
Carotenokb. 259
Casckin 178
Cue
Iron 234-6.315.473
oxklask 211.213.215.291.332.340.342.360.
376.404.473
Castor and Polkix 279
Caubolam 53
: 8
Celhibsc 186
(ci.iifcijui.Ki 428
ChikoK •>>'■> Iki»« 255
Cbapoliaibo 85. 310. 313-5. 308.391
Cfcaicoal 97.111
Chink, no.,. 400.425
ebrul «kwlc
279
ma bin.
ihiiiicr
*32- 3
213
dii DiMcmbi 414-5
ClaMcba 277.432
Chcmollhuln.pli. 53
Chcmoonnnotropk. 53
: lr. ITI.tli,i|l , 139
Ckeni» 243. 257. 277. 399. 401
CM in 3.5. II
: bro
Chmmatogi
14
rsiphy. paper 368. 372. 373
iB*ic Jc.j<b...vU>c 324. 427-8
■k K-kl 240. 255. 320
bacterial •ciaboli*- 149-51. 172
Aoirifri rromvi and 289
in pope pulp 240
in pope ikin 245
Ciric »ckl cycle 58.03
riMfoqMtrwnt ipp. 291. 294
Cbiieltc 399
Cbrct 440
Cbrificaibn 104-0. 322. 331. 397
holrytircdvcct wine* 449
ibj«pjpiK method 458
EMOHK 06
method* ot 420-8
mint 105
in wile wiKMktno 422-5
Clea«i»p.c(op 305-0
Cluoaie 240.260.272-7.282.355.357
277. 281
:. bm
idootn
279
CfoarMum 123
CocMYac A (CoAI. acetyl 74. 70
Cabatxnl 419
Cobr
extract bnfcttrbiliflllnll 323.418
liiicsM. 332-3. 348-9. 352. 388. 447
«-i-m'i.. Winc«wkio|t, ml
Cobrimctry 293.403
Concord 257. 278
Cnyti M4gc 280
Cooling 415
Copper, pbyloloik 279
CoAi«p 404
Crw/urr 243
Couaaik ucid 289
Co*ym»e 54
Ciabtiec effeet 02
i "' i . .- j . : . . ..'i- ii " 476
CnmbliMi 409.412
C'tuthinp 331.353-5.413-4
CiyocMncife* 30O.42I.4SO.4S3-4
Ciyosckdioa 417
Cuiiium 347
Cii"* 459-00.408-9
Cyioducibo 22
Cytoiinim 243
Cyiouil 1 1
DeackUficaibn 308-10
I>.-iui mitt WTO ipp. 19
Dccaaok i.rd 227-8
Dctnlbtbn. vine 283
Degree-day*. pm= inn 204
Detteia ipp. 43
DCBl) . niM 80-1
DCUHIKBCH Of 261.345
Dcodonalna 226
IteMcmmlng 331-4.390.414
Dcvaitiirg 363. 391
Dexiran 156
Dacciyl 08. ISO
Dialihcimoaeier 81
Dkiirhonvh 203-4
Dkihyklkiihorute <DKDC1 228
Dipbcoskki 318
Dimcthybllyl pyrophosphate, kv I) MAM'
DuncibyldicaifooiiMc (DMDCI 228-9
I) iinci hi- Uii J iho rule
dccompoinbaof 229
DimctbylglyccrkMid 68
1 -I-..- iiging 464
Dnaiuaibn 188
DiuoHi«i power 215
DMAPP 257
DNA 14-5.35-6.38. 121
DNA replkatb*
KBi-comcnrntivc mcchannm 122
Douge 404
Draining 305.342.413
P n«.iiurc 300-2
Dav. log-off acthod 448-9
Dried Activated Vc«M (DAY) 94
Eflciva
.ceoce 460-8
Enrlkh
leactbn 74.424
Hi dm c in
i 203
EitMtin
300.450
Ekcimi
ihomn. puKe-lkld 30-7, 130
Erubdetr-Meyerholl pathway 55. 124. 141.
Emboli* a 248
Empcro
i 263
Eudopbsmk iclkuhim 12-3
Eneigy.fiee 53
non-utllircd «s heal 99
Enobse 55
KniKM)oi*li<un)ft fuih-vjv 288
Kift»c». posi-hurvesi 300.310-22
commercial * incmaking 322-4, 335
hydrohsis 310-8
oiidwbn 318-22
pecialyik 3IO.322-4.J84
Eigosierol 7. 10. 13.91-3
Esdiaiduti coli 121
EnvUa til
Estco 7b
MIumI 09.218
sulnw db«idc blading 194. 200- 1 . 210
EUbboI
iicid on and 292
j mi 1. 1 acid i ram port li.- icd I-. 72
anaerobic mmbolb.ni iind 380-8
batferbl membrane stiuctunr modified by 120. 108
bacterial .»iliii..ii of. acetk Mid 185-0. 188. 190
tok.. iMcnsiy .iihI 353
dctcctba. isotapk 313
u ictmcm.il tan iahfctor 9S-0
iallucacc on vcul cell -cmbraac 10
lactk acid bjcie.i.1 icnskirity id 100
IlluaoHokaatc 180
Ethyl acetate 292
Ethyl caibamatc 88. 280
Kthyl esteis
.nomas and 70
Ethyl pyiocaibonate 224. 228
EU kpistatbn 194-7.235. 300. 307. 314-5
Eiin>(-r . HiviJllljUliaiJli. mno IB 272
Eva pool ion. vacuum 310. 350
EUaciioa 300. 335. 452-5
EAD.irr Fta> inc-adcnlnc dinuckotidc
Fall)* acids 91. 100
bacterial 118-9. 135-0
u fcimcmatba lahbion 97. 107
kes 422. 424
membrane phospholipid ?
saturated short-chain 227-8
Eermentatbu activator* 89-95
adding yeast suncr 94-5
■uagi 94
Fermentation, akoholk
ambk« conditbnv effect on 339-41
ba.thcan.tr. 53-70
chcmkatcqualba lor 99
compkltan 432
coat railing of 339-45
malkackldcgtadainnaad 09
monitoring 344-5
icgubtlin 02-3
lemperaiuie ciTetts on 99- 102. 339-41
Fermentatbn. banel 431.450
Ecimenlaliun by-products
u fermentation iahfaior 90-8
Fermentation, champagne method 237.458
Fcrmcntaiba. glycciopyarvK 55. 58. 02- 3. 00
Fermentatbn kinetics 83-4
cbnfccaiba effects 104-0. 425-0
monitoring 80- 1
"Vi«->liWs 102-4
pnysicocbcakal ration affecting 99- 100
temperature effects 99-102
Fermentatbn. auk. lactk 108-9. 102. 177. 230.
344-5.401
acidity during 374-5
aciaitanand 375
conditions Kifilicd tor 374-0
hMorvof 307-70
ia.ttub.ion for 370-80.432
before akoholk fcimcntaitan 377-8
with tommcnjil leuft»io\ttw mtf.'i parpaiatb
379-80
with non-p polifcaiiag bacteria 378-9
with S.*-diirom\xw cmxiiiae 429
moniorimi 372-4
■a ied vvmcnrakiag 307-80
suUHing-ind 375-0
temperature effect om 100-7. 375
ia while wincmaking 432-3
hoe tamformaibniBcr 370-2
Ferment*! bus
aecekraibnof 91
compkibnof 345
cannol systems 81-2
con lolling 80-3
automated 81-2
inhibition of 95-9
mabbctk 230
monitoring 80-3
nohk .oticd musis 455-0
restarting 110- 2
speed 83.331
spontaneous 41-8
Muck 80.84.88.98. 100-12.330.431
actbnincaseof 109-12
bade rb phages and 174
causes 100-8.378.390
consecnjcaces 108-9
. I i,i' i ii.- .■ ii 300
lactk dbease and 173
sullitiug a ad 215
tcmpcmlure effects .-.-. 339-41
Fcrmcmoiji
aeottan kveb in 335
closed 330.341
comliuctbn of 330-7
& tcimi&iued )
continuous 381-2
ifcvjtnnff. iUomaii 16]
cquippi* 317-9
fillip 429
maieroKfor 136-7.303
■ i". ri Hi ... iir.'-i.i f $35
nullum cylindrical 351-S
(«<*» Tanks
fcnilHy.uiil 279-80
Fiimion 428
Fuedacklny 151
(/•ah detente 354
Fbvine-adeniuc dinuckolidc (FAD) 58. 0!
Fnvonokls 120.419
Fbvonoh 240
Fkmtba 428
Foam 400-8
anli-fixiBiiig LijKn^ 81
avoiding IbrimiiHin of 82- 3. 468
fomaibn. fee-lots influencing 81
FOEB cnlkctkm 178
Fag 301
Folk Bbachc 243
Fomol indci 87
Foaincmbn 471
Fnsu 282
Fruciolimnoskbsc
Fnxtase 55-7. 162. 187. 202. 246. 250- 1.471
Fmi Id 404
: ...!.-.. i.k -. 210. 224
its.lu.1 95
li"((MJ" 284
Fuaneol 279
liiraim 199
Gabciumnk Mid 255
GAP 9. 10.72
Gas inject »■ 349-50
Gases, incn sionge 237-8
Gay- 1 ussj i I
GDH 70. 254
Genetic ft n^c if lining 38
Gc*>mc renewal ibcon- 18. 48
Geanylpyiopnospliaie (GPP) 257
Gewunaraminer 265.277.399.401
hunch sicm nccnab in 283
clonal sckcibn in 279
1Mb:-: ,.
402
GUcicUim 241
Gbcanases 4.6. 11.321
Glucane. 4
GhKiM 150. 157.117.131.434.451
P- 3.4.4SI
Gn*. oncogene* is 253
GbconkaenJ 187.288.451
Ghuainbtrtet s Pr 183-92.292
Glucose 55. 102.471
bacientildcgtudaibnof 180-8
calabolk icpicssba by 62
in grape 250- I
sulphurdbxideand 202
iranspoii 10- 1
Tllili 70.72
dekydn(pemse. in GDH
Gimtminc 70.72.253
Glutiuiinc synthetase (GS) 70
GUathion '320.419
Glycerakfchyde 3-phosphaie (G1P) 55
Glycerokinase 153
Glycen.1
aceumubikinof 62-3
baetenuldegnidmbnof 151-3. 188
/gbconkacidraib 288.451
induslnil 58
oqunokptical H>k of 62-3
Glycerol ilckdDiKr 152
Glycolipids 117-8
Glycolysis 54-7. 141-2
Glycoproteins, hcai-stabfc 06
Gh, -osklu.es 289.118.123-4
Clycosyblkm 4-S
i -il_-i .i^p.raliii 12
C«i«i de hooiere 461
GP1 4
Grti
IS'.
301-7
Gape
315-22
oMnipcoinii. aniiiiil 301
Gape beny
anmaiksubsiance* In 256-9
cell wall, change* in 254-5. 346
cokii 240. 255-0
description 242
dcvcbpmenial stages 243-4
fomaibn 242-1
grinding «hok 262
auiiuatbit changes in 248-59
■miiicu ion. charactcibik), of 248
251
format urn of
imilr..-
morpbobgy 244-5
nitrogen compounds in 251-4
oeiank ackt. in 251-3
pnenolk compound accuaubibn in 255-6
rcspintoiy quoikn in 250. 25 1
senior 241
sugataccumubibnin 249-51
measuring 261-2
ublb
218
Gape chisici
coaposinn - uuiuriy 245-8
description of -,«un 245-8
hctcmpcacity In matuic 247
shading of 203
Crape doing 300-1
off-vi
300
Grape anuMnii 241-94.404-7
change* during 248-59
climate effects on 200. 272-7. 282
tkinilna 239
dcscriptiou&om position 242-8
<IB.eavc and 282
cv.laiBn 201-3
index 201-3
Ijtht effect ua 2b3
apl.il 301
iwuiocIa and 277-9
soil i. ft /fe it illation and 279-80
Male of 2S9
Mudy 200- 1
Icapcotuic influence on 203-0
variety and 277-9
vine management and 28D- I
water availably and 200-72
Grape muM
evt act ion by low Icmpcratutc picssing 300-7
Crape pulp
coko 240
components 240-8
maturity 200
Grape quality 3SS-7
improvement of by overripening 300- I
Grape Reaction Product (GRP) 320. 4 19
Grape ripening
impact of water balance 208-9
impact of water deficit 209-71
vine water lock, montoring of 206-8
water availability, effect of 200
Grape iced
component* 245.250
maturity 200
tannin*, in 334. 349. 350
see irfni ^tctilcvtncvi
Grape slin
component* 244-5
infection, response to 285
maturity 200
paimcnt dissolution, potential 350
tannin* in 334. 349
Grape Mall,, components
see tint DcMcmming
Gray ml 283.290-2.401-4.450
steiito Botiytncincica
Grcnaehe 387. 399. 440. 447. 471
climate and 282
a-...,
282
Giowth hormone bbsynthesis inhibioc
Gyropalkt syMcm 403
Hail 282
Himmliaporti spp. 19. 20. 4 1 . 404
acid pot and 292
contamination by 83
idem ilka ion 24
karyotype analyse. 37
sulfating and 214
HmwnuLi spp. 19. 20. 41. 70. 83
Hapanonls 119
Harvest
acidly adjustments of 307- 10
climate and 27 1
date 301.357.405
btc 299
sanitary Mate 292-4
successive 300
Harvest transport 304-5. 329. 408
Hnrvcsleis. btcml Miite 302-4
Harvesting
manual 303.305.300.407
mechanical 302-4.329-34.407
multiple selective 407.450
tcmpcratuic at 407
Heat concent rat km 310
see aim Thcimovinilicaiion
Hedging 270
Heliothermkr index 204
Hctcroihallism 10-9
Hcianoa
Heiolin
70
: 55. 249
bacterial dcgradal.in 158
mctaholnm
hcterofcrmeniativc 143-4
homofcrmcuiativc 141-2
muM 55-7
Hcxasc monophosphate pathway 180-8
Histamine 132. 153. 174.280
Hntidinc 132
decaibovybtkinof 153-5.174.371
II--,
14
Homothulhsm 10-9
Hoppea 303.329.410
Hybridization 120. 130
Hydiomcteo, SI
Hydroponics 280
Hyperoiygenut«.n 418
lee. dry 408
InhibitbncanMani 140
Iodine 205.210
1PP 257
~SN
Iniiulbn 26ft. 282
Irriguloiv mining .til
bopcalcnyl p\ iophi"pkuc. WT IPP
botopomcB 314
Juke ckcirkal conduct nay 408
Juke eitiaclbn 300-1. 122. 408-9.452-5
im-c<lia<c Kuch 409-13
pofc <li> iop In 101
yellow wine of B2.i78-9
Kanoivpc imI)iu 10-7
KclOBC* 186 '
Kcionkackfc, 201-2
KUkr factor 19-22. 108.144
Kilkr phenomenon 19-22
pcaetk* of 19-20
pnyiblonv of 19 -2D
ink in wmcm.liiw 20-2
Klaerlera iff. 19.il. 81
acid M *«l 292
kkmitcuibnof 24,29
karyotype amhin of 17
uilkiiapund 214
icmpccuurc effects on 100
A,7ii>im»irwn *pn. 19
Krcb>.cycle S8-bl.nl. lil
Iji Tour. Charks Cjpninl ifc 2
lacvaie 289.291.293-4.3O0.32O-2.33O.355.
3ft0-2.18i.i5l
an.hib.of 402.418-9
I-nviM j.ii. il'. nail 293
lactkackl 07. 158. 172
otkbikin of by accik acid bacteria IKK
vdv Bacteria, lucik acid
Udic dncue 158. 173-4
diapmunof 173
l-UCtkodchydn>£Cnj»Ci 07
UxlotxAillns »pp. 120. 121.473
cine aeid metabolism by 149-51
classification 125
descriptbn 125
cilmnol influence on Iftft
cvolutkin durinp « incmaklnp 171-2
kkmiSciibn 120
inoculaibn of commercial 378-9
en. I. Mid H-Ji%!'i>iuisli.»i b) lib
pcn<nc mctabotam by Mi
pH Influence Ib4-S
lupxassimilaibnby 144
sulillng ClICCI on 211
leeiito Bacteria, luctk ac*l
Uaf. bbek 283
Leafcmmlinp 281-2
Lcafecnich 283
Ues 07.210.218. 37fi. 421-2. 42ft
clarification of 428
fcrmcalaiiun Udciic* and 105-0
lxeuocnhocL. AMonk 'an I
LeuconoBot spp. I IS. 124
adaptaibn phenomena IA7-8
anilnlnc metaboltMU by 155-ft
citrk acid metabolism by 149-51
classlficaibB 125
description 1 25
ci ki nn I influence on Iftft
CVOBBbn duiinji s.incmuUn,! 171-2
heuue metabolism by 144
heiidtnc decanSo*ylaibB by 153-5
idem ific.it bn 120. 129
inoculaibn of commercial 379-80
laakdetaie caused by 173
malkacklnamfoimailonby 145-9
mablactk termcntuibn awl 377-8
pentose mctabolnm by 144
pH Influence 164-5
polyaaccharidc* I*"" 7
S mriaor, interact »w oil) 175-7
sugar assimiluibn by 144
wilpnurdbvidc effect on 12D. 165
temperature effeci on 166-7
e.tlto Baciei
156
. :,.i.
L.I
Lgvbi
I.**, effect* on malucubn 261
Unabl 258
Upld bllaycr 117
LlpkU 7. 07-73
l.ipo polysaccharide 184
liqueur dr tiritgf 462
liqueur d'e<p*!i!<ai 464
Lyiozymc 229-31
nature of 229-30
pmpeitie*of 229-30
ulncnuikintt. applkaiba* of 230- 1
Maccaheu 399.471
Maccnubn 355-8
In ummjtk .-■ .- n i.- ■ .-ini- 201
ciiibonk 385-94.446
anaciobk mctabolnm 386-8
ua*cou* cichamro. 180
grape tansbrmatbns by 1S8- 1
mkn*bk>g\ 190
principle* 385-6
u*imr 390-3
duratbn 316.348-9
pK-fcrmcnuibn 152
prim-ink* 347-8
ia red wincmakiag 345- 58. 385-94
role 345-7
Mn 414-7
larrnul pwi-fc menial ion 352
lypwof 347
i* wkic wineaaling 394. 398
Mademniikio 194
Magamkim 253.283
MagaClic KW1M0CC 313.314
Mabucrccll 80
Mibn 307
Ma Ik- acid
acidi&catkinand 3D7
aiueiubk dcpndaina 380-8
bacterid laaJormaiba 145-9. 172
bk-degradnbilk) 309
&»ru7tri/«v«i*nd 289.291
dcacklifcalaiaaad 308- 10
dcgadaiiinby ycul 09
la grape pulp 240
ia grape ripening 251-3
ia grape ikia 245
ia grape va reiki 277
Icafihiaaiagto reduce 282
pmauium 4*1 280
wgat coaccalralioa 4 ml 312
Icapcaiure cITccuon 204-0
Malobclkaciivily 164
Maloloeik cu}K 140-7
Maloloeik reracvaiiin ph»« 145
Malobcik t rant formal km 145
Mabbclk ycaal 147
Manaopnxcitt. 3-5. 11.428.433.435
MaiBeng.PeiiiandG(o» 418
Miik/iiIMii 478
Ml:.-. '.)■.-.■. li mclf. 313
Maiucuion. grape 241-94
change* til nag 248-59
ikwriji: inn/t-impmitixG iLiiini: 242-8
ellecu on biochemical pioccucs 203-0
iapjci o( varkno lawoa an 270-83
monitoring 200
Maiucuion index 201-3
Maturity, deliokion of 259
McuuKBCri wain 201-2
Merl* 243. 271. 280. 307. 349
7.90
Ma
i::
MilciTil;,'.'l COadlBW 272-7
Methanol 229. 255
Mclhbaol 423.424
Meihoxypyia/inei 259. 200
Methyl aalbaaihlc 279
Meivtaulauia *pf . * '
418
Mikle*. downy/powdery 283. 404
\lllta,nil,riKr 243
Mitral. 89.253.280
Minchondrin 13-4
Model* 82. 80
MOG 303. 305
Moacotarv acmnn 104
Mm
-rid
Muwai 401.471.472
Alevandrn 301
ainmalk* ia 318.401
grayrtftia 402
staler la 243
teipeaoh ia 24A. 247. 279. 405
Mini
■catty 240. 277. 280
bnmning 320-2.417
cnmpoaibn 240
cooceairalbo 310-3,350
dcmiy.
201
Muu c Urine* ion IDS. 322-3
Mida& 210. 220. 227. 450. 457
NAD* 55.57.59
NADII habnecoib 02.03
NAD--GDH 70
NADH 57-8
NADP' 70
NADPH 55. 04
Ncphclomelcr 427
Nkutinaakk 90
Nkmiuaiklc-adcnlnc dinucleotidc.m- NAD
reduced form, in NADH
N (cot iaaaklc-adc nine dinuclcoiidc pmnphatc. n
NADPH
5-NHiolurylacrylic acid 224
Nitrogen
45S
i 87
hik. 107
87
grape 85.240.253-4
.'1. ■ v ■:.- ciatwta and 289
djRcreat varkfic* 277
mclabolnm of coapouad* 09-70
m>iI fcitili.-.iiMii ■■lib 279-80
*»iree*of«ipply 85-9
Morauc under 237-8
Noble m 285-90. 300. 398. 402. 450-S
acciicackl ia grape* 00-7
change* in grape cheakaK 287-90.451-2
eryoareaervatto.ofcrop 300
lenueaaiioa pioccu, 455-7
bar. cm ing. ma null 302.300
aifccnliaaioQ 452-5
nvnlw Boir \tis rinawi
AVuiia'nvi 243
Nuclcinoaci 14
Oak 399
Oaanok acid 227-8
OtlOB
aceikac.l 404
animal 324
boliytk MO. 402
cooked rMiap 423
dincMifcc 20b
foxy 279
waur
Mcdwaa 132. ISfi-7
kin hv 144
22a
318
a klu h
i IS
Ufawkm 279
Oech»k. degree 201.202
0n«»«< *pp '""■ 123
OIV recommendation* 190. 307
Okanolk ackl 91.92. 240
Open* 121
OammU. icvcec 310-3. 356
OtlancI 243
Ovetrifeninp 299- 301. 4DS. 440
Otatouccialc 71
O.kkukm
en/ynuik 235.318-22.342
mcchanka* of juice 418-20
pnHccibaiguiM 212-3. 218.420- I
Otklaik-n- reduction poicMnl 191. 192.235.236
Ovkkiik-n- reduction icacikin 59. 139.419.436
Ovklorciiicuic 319
0«>sen S8. 102-4. I ID. 107
PAL 255-6
Palomino 475
Paniothenkacid 90. 110
"ha*
irefleci DI-2
ir.LaiB 2.89. 151.369.385
iriaiton 224.231-4
III
unisof 232-3
PCR 30-S. 126. 132-5
del* ftCtfJCIKt* ,1-MKUlid ■■ lk W 411
»«h mkrovuelliiei 40
RFLP 31-5
Pectin -el hylcMenuc.BV PME
Pectin -ethyl cMcisiut (PME) 255
Pectiioicj, 427.455
Pectin* 246.254-5.284.422
Petliaenccai *pp. 117. 120
clarification 125
deKiiptbn 125
Clkinol mlUiCiHt on 166
evolution duriaa wuwldng 171-2
hiMklioc decanSoxylation by 154
kkntihcuikin 126. 132
pcMmc mctabolkm by 144
Jilphurdioiidc clitct on 105.211
nv.Vi.i Bactciii. bcik ackl
Pt.lru Xi-coi. 300.476
Pcduncukir an 284
Penicillinm *pp. 291. 294
Pentose •eijbolum 144
I'caknc phosphate pathway 143
Peptkkiglvean 116.184
Perception thir*hoUI 174
PetmeaM* 9. 10. 72
peaeal amino acid. «v GAP
Pin. villi wo 321
Peukkk resklue* 9S. 279
Pel. Veiuot 387
Petit Arvioe 401
pH
hii.1in.il 164-5. 180
mu*t 240. 253. 206
*oibk ackl and 225
threshold 104
Pbcaolk compound*
climate and 355
pope 246.255-0
a* jhcu.miit— en of maturity 20.
noble/gray «* and 289
temperature and 265
variety/* intake difference* 277
ink. of 408
l-.iL ."i..kki.iu ill,.! on 167
oxidation of 320
volatile wood 437
Pnenoiype 122
Phenylalanine ammoniilyuc. *v PA1.
Phcavkthawl 70
Phospholipid*
mitochondrial 13
pkinuk ■cmhoiK
in bacteria 1 18
in veaM 7
Pbo*phork acid 246
Pbo*phorylnlioa 55-7
oxidative 54.59
*ub v. rale- level 54
tun*- 54
Phototroph* 53
Phytogeny 122
Phytoakxin* 285
FJeMfl *pp. 19. 20. 41. 43. 83
Pigaitf 338. 349-50
Pint
224
Pin kino 434
Pinal 277.459
Pine* Blanc 243
Pino Gib 401
Pint* Noif 277.279.350.356.397.418
Plaaiingdcmily 280
Phomkb. 121
Pboienag 470
PME. grape JIO-8
Pokirogriphk ■c.ixiicmcni 293
Pbllinaibo 242-3
PbhckuuUnifcixlks 294
Pt>lyg4laciun.n«e* 310-8
Puh ■cri.iMn. chala reactba. tee PCR
Polyo*ide* 254
Polyphenol* 107.434
Pbhiatvkiodcb 156-7.434
Pbawce 330
kicking «l 342-3
removal of 3)9
see iito Cap
ftirkt coHteiUi 475
pope 240. 247. 253. 2W1. 280.283.313
mil fcailinibn 279-80
PiMauaim bkarbunaic 108
PiMauaim uaraic 309. 375
Pour li plem Mage 280 . 288 . 450
ton rtff u age 280 . 300 . 450
Pottriiutrc iiffe 292
Prelcnncaialba (icalmcnu. 299- 324
Picm .liaei 363-7
coapotHkinof 305-7
me of 305-7
hma
hukci 4 ID
Bucher 412
Champtgnc hydraulk 459
cb*ed<anl 411-2
membrane laak 412
moving-head 410-1.453
pacumaik 411-3.453
verlkal cage hydnulk 453
VCflkfllKKW 410
cokl. tee Civoeuewibn
dirca 447
ia i Ii.nii|i.ii- ■ miking 458-9
bo- temperature 300
noble hh grapes, 452-3
la ml v.ine making 303-5
.low 414
whole ckMcr 409-13
I'rmuic. aaiimkrobial high 224
Pfbe <k mom*e 458
Piwyaaidk pmlilc* 278
Proline 254. 277
Prophage 179
Pnxcucs.gnpc 310.322
Proieim
miaiMihibk 240. 254
(.bock 120. 168
Proton motive force 102. 104
Pteiilamimia %pp. 202
Pumping-over 81.103.341-4.349-50
■utomalk 338-9
Pump*. pomace 303
Puachiag dnu n. tee Cap punching
Piilfiuiu)!- 458
PYPPeokimn 293
Fyadac 279.282
Pyrkknal phmphaie (PLP) 70. 73-4
Pviidoiiae 90
Pyaivale 55.71. 189
gKccropyruvk fcrmcmaiba. products formed by
63-4
Pyaivale decaaxuyUwclPDC) 58
Pyaivkackl 02.251
V.:n,
289. 32D. 418-20
Racking 19 1 . 239. 439. 472
RaUiaing 398.402
RiiouhVi, bv. 453
RCM 314-5
RctiihCdtiiiKcnraicdmuM.uv RCM
Refugee* bn 100
Rnpiralbn 58-01
Rcurkibn Fragmeai Itigih Polymorphism (KIll'i
129
Rev. cm n. I 285
Rhiztipus ipp. 322
Rlniuiitiitulll \pp. 41
RlHiflavio 90
Riddling 403
Ruling 399.402
climnc and 200
clonal «kclbn in 279
nabk am is 402
*huier in 243
Rbp 273
Roouiocla 277-9
Ropuicu, 150-7
xtv aim PcdioeiKxiB. %pp.
R« 213
\eriiiui Botrytu eincica: individual rot*
Row. \pKiog 280
Ruauiagoff 358-07
chuoviag momem fo. 358-00
piemaiuic 300- 2
SimxIiiuhii fits Btnrittii
cbiifccaiion ami 95
clauiacaiba 20-9
dclimlatbn 30-5
Smxiiirom jm bnutnii tavttintied )
■.lll..l-it
29
hex mbUacc 232
karyotype imlysn 37
PCR piottc* 39
phcnykiknul piuduciba by 70
nrifyoagmpc* II
iV*-ry«ro«» <m trmiiiiie 3
«'■" «kl penned 72
aucn«ii»D ..ml 385
cell -all
cfciin
chM>m<iu>mn 14
cbuincaiioii »-9
cbiul dwemky wnhin *3. 46
dcliaiMbn 13
•kimiiual urain 45.40
ecotagy 43-8
glyiolyiis 55
glvccny lit n n 12
apnpe* 41
I.:.-; i.
231
higher ikcbihtiml 70
kkntihcuibn of vim Moliu 35-41)
inocubibnufc-h 429.402
karyotype imbiB 30-7
killer 6ctoi is 19
MET1 gene 30-S
htfkaeklhacicro.iiiieraeibir. with 175
miDNA »hjI>sU 35-0
PCR pnttto, IS 40
pbiak •emhr.-e 7
icpinductioii 15
lempeaiurc effects an 100
winery cobal/Mbn by 42
Skmluramycet *pp.
cbuificMioa 20-9
»rly i
170
hybrid* 32
h.-.u- «klb«lerH. intend DIP ■■ .1, 175
Saxiimmyminmm 20. 29. 31-2
S€Kxiurom\xode\ *pp. 24. 43
S»cch»n»e 54.202.240.249-50.314.471
Silific-dtnn 313
S«»eme* 450-2
S»uvtg*>ii 399.400.451
•m« .vegetal 282
KiiK-hMIB nc<-nnn in 283
climate and 205
gr*y «. i" 402
ntacceubn 414.417
■natural bn 405
nnbk mi 402
*haltcr 243
uigiicoaccnttatbn. nluny 404
Saugnia 301.478
Scdiii
322
SccdkttKM 243
Sckctba. grape 300-7
Scmllb* 287. 399. 400. 405. 414. 415
ShMlei 243. 279. 282
Sherry 02. 399
Shlkimk acid put bitty 255
'...!.:
240
Sodbm chloride, wl 280
Soib. 279-80
Soleo procc** 301.470
Solera *y*icm 477
Solid*. *u*pe«kd 421-2.425
So*ic nckl 223-7
amimkitibhIPiiipcaictor 225-0
che«k»lpiopcoie*of 224
gust alive impMi of 220
physical paipcftk*t>f 224
Mobility or 220
u*e*of 220-7
So*i*g
h»rvc*t 305.328.407
*uccc**ive 287
Soiobo 290.452.477
Soot rot 292. 404
Souring 98
Spitibg 238.239
Speck*. dcRnUnn of 23.24
Spoilage, acetic 190-2
Spoilage ycnii 43
Spontbiion..vri- Vea*t icpniihKibn
Subiliraibn 224. 370. 457
mkufebbgkal 231
Sukd 370-80.429.472
M.-.
457
Ste*>U 91-3. 103
Siibcne*yntha*c 285
S/iepiocaccus muum 150
Sim* protein* 121
Sublraclivc lcchnk|UC* 310-3
Succink acid 307
Smliiiinl and Chauvcl fomib 105
SugatbcidHy Rilb 201
Sugariakohol laB<DmlDi cub 2
Sugardcgadubn pathway* 54-01
'■I I'.l I! ir.' . R-1* ' L. '1.1 ll.-.ll I ' h
Sugat*
addition of 3I3-S
detection 314
bacterial metabob.ni
acetkackl 180-8
belle Mid 140-4
coKcamlov. 272. 420
pope yield and 281
high 88. 101. 106-?. 110
measuremcni of grape 261-2
lechnique* to Increase 310-S
energy potential 99
grape aatuniikin 249-51
In grape pulp 246
noble mh and 287-9
wHurdiovideand 202
Sugur-ulilifing mdaholk puihuuyi. regulation of
01-9
Safatmoit 219
Sulliting
combination, empirical bws of 209
drawing olfnnd 300-2
during Murage 220- I
effeti of 351
equilibrium n-actioM during 200-8
)• fortified wincmakiug 470
he-lory 375-0
bctk'acidbadcria.clfcd on 109-70
belie (b.n»c nnd 174
malobdk fcracnlalkinaad 375-0.401
noble tot 455
by Mtlluring banxk 220-1
le-peature und 208-9
)■ white » inc-almg 408. 415. 420- 1 . 424. 455
yea** awl 210.213-4
irr irfm Sulphur dioiide
Sulfiir combustion 220
Sulfur compound*, volatile 439-41
SulnJt dioxide 353.473
alkrigks und 190
nnunieiobbl prnpeoics 2D9- 1 1
amfradernl 211
amihingal 210-1
binding
dkaaxinyl gmup* 203-4
riihydmtyacdoac 205
dhaatil 200-1
IxtonkackU 201-2
sugar
202
chemiury 197-200
bound 198-200
fax 197-8
complements u> 223-39
concentrations 210-21
adjustment 194-0
stnragc.bottling 217-8
ifbr
194
■UssoKing power 215
eartkst use 193
EC legisblkin concerning 197
cllcd on bacteria 105
l-.rm« 194.219-20
Ibrms. properties of 209- 10
molecule* binding 200-0
muu modllicaibn and 3IS
OIV reeommendiikin* concerning 190
f ■ >Ti I ■ 1' i ■ '■' ■ ni'-- .i ri.. 3 17
pH and 197
ph-sBkigkiil effect 190-7
pK and 197
properties 193-4
pn*cusk activity and 310
recommended drily nlktwancc 190
suhstiutcs for 190
lempcmuic influence 208-9
lo«kiy 190
tyrosinase and 320
live in mu*l/wiac treatment 193-221
pnidkal consequences 200-9
wiacmaking. role in 211-0
advantages 211-2
(faadi a mages 211-2
yea* activation 213-4
yeast inhibit no 213-4
winery. u%c in 210-21
wines, combination bnbncc in 205-0
Sulfur treatments, vine 279
Sun-diving 301
Sunught 250. 203. 282
Sunught flavor 403
Supracilradaia 417.453
Survival factor 91-3
Svedbeig values 121
Sweetening reserve 457
Symport 72-3
Syringakbrine 293.403
T<»lle 459
Tanks
capacity 337
lilting 334-9.421
principle system* 335-0
hern.dk 237.330.390
man I 238
openArkiscd 103. 104
Mltc-ptying 339
shape 337
sterilization 233
sulhting effects on 210
temperature control in 330.351-2
tacrmk exchange capacity of 100
.wr i4so Fermcnioi*
iDibh-ICi
10-
in uibtiadive techniques 313
In vine, red 357-8
ricuemming and 332-3
Taaain» (rontuMnO
good/bad' 328. JS7
in gape 240. 2S5. 334. 348-9
■ inhfcioB, 98
i><ktaik>nof 358-00
pni.inih-H.jjnk; 205
ii row 'in 440-7
wood 414.437
Tadaric acid 375
ack)i6cala>n<vlh 307
bacterial *ICIaholi%N of 151
Jfe» ifi* rromvi and 289. 29 1 . 45 1
ckackliflcaiba and 308- 10
evaporalk-naad 311
in gape 245. 247. 251. 253. 250
■ arktal diffcicncci 277
iiaohfcili/aibnol 280
temperature cllccls on 205
-iter M .cm and 200
Taste
hcftticcom 328.354
light-induced 403
malolaclk fer»e«talaincllcctt. ■>• 372
wrbk acid and 220
lulfcting ellecu on 215-0. 219
Tea pc am it
abmpi changes in 102
active 204
akohulk termemation and 99-102.431-2
ambknt . ellecu nn fcimcntation 339
aumataikcnntiolor 81
Ki.it ii.il acmbatm influenced by 120
hctk acid bacteria, cllccl on 100-7
maceration 350-2
malolaclk tcracKMainaad 375
maturation influenccdhy 203-0
ninogcn aMimilatioa and 88
in red»inemal;ing 00.81
wtKk teracntaibw, and 107. 339-45
yeaM giowih and 99- 102
Tcaiuricr 240
Terpcne* 205.402
Teipenk compound* 289. 318. 324
Terpcnoh 240.257-9
Terror t 280. 340. 357
Thctmk exchange coelnckni 100
Thcnnk »bock 102
Thermoperbd 205
Thermoiwiuance 231-3
Thomovinilkaikn 00. 383-5
Thaainc 03.90-1. 190.201-2.455
Thaainc phyrophmphate I I'll') 58. 189
Thsainc pyrophosphate <TPP) 57
Thinning, berry 282
Toasting 437
Tokay 281.287.458
Topping oil 191
Topping. limit 282
TnriiUaparn %pp. 20. 28. 29
Tnriitopiis *fp. 19.24
T,mme <u»ca*C 1 5 1 . 1 74 . 370
Trace ekmenu 89
lor bctk ackl bacteria 102
mcial. in grape pulp 240. 253
Tralko 304.329.410
Tiansphosphoiylaikin 54
Transposons 15
Trkaiboxy Ik ackb. cycle 58
Triihmlei fn.it.tr- 4.322.323
Triamiaa 282
Turtklay 100. 421-2. 427. 455. 400
■iota. nephelometric iVlli 07
Tyndaleflect 421
Tyrosinase 320-2.418-20
I'gni Bla
283.399.419
Vacuole 12-3
Vanillin 437
Varietal dilTeicnces. grape 277-9
Vailing tiacs 339. 348-9. 357-00
IWrtjnn 248. 251. 258. 310
characterwio. 244.254.200
mclaholk pathuay changes at 250- I
o ¥ ank ackl evolution 251-3
waterMicu, and 200
IBftC.
influcm
:si
279
climate and choke of 277-9
Vine*. Aaetkan 240. 279. 285
Vincyan! Management 357
lullUDcoppcrlrcaiacau in 279
vborcomrol 281-2.355-0
ViniCcaibN 471-2
' ml
■ OCH
Mat i/ou.t naurelx < V DS ) 47 1 - 3
Vintage* 272-7. 288. 340. 357
Viogakr 401
ViiuvliLc pankki. (VLP) 19-20
Vmaaiy, ntuu 322
Viamin* 89-91. 190
itertto AKOibkackl
Vkkuhuic macs 204
\llismtiava 284
Vffs Uibniu-a 257.279
W/ii rintmdifOlia 279
Miis mpeerh 284
Vffs tinifrru 244 . 28 1 . 284
Voliuk acidity 104. 173
Voliiin 121
dcMcmming 331-4
'. n Ipii ■>* 292
Waterbalniioe 271
Walcrc.ipon.lioa 310. .ISO
Water reojjiremem*. vine 279
Waterstress 200.208
bn« reccplbn 329-31
higher akohoh. 70
iaocubinn 370-80
maecratioa 327. 345-58
caaSonic 385-94
principle* 347-8
Weaihcr.advco* 282
oik 345-7
Wake TirU hype Oxygenation 321
WiacMotage." inert u.scs in 237-8
mustaeatkm 341-4
muu cbritcaiioa 323
Wiacmaking. Bordcauiityk .
'.'■ i.-.-m •king, h. . ,- i ..,■! sweel
.w rfio Nobk rot
144
449-58
pomace. mecbinkal effects on 353-5
premium 328.331.340.340.347.357.359.300
press wine* 303-7
Wiacmaking. chumpagac 458
aging. on-ke* 402-3
bascwiao, 458-01
clarification 458
-70
pressiag 303-5
primeur 327. 340. 350. 394
pumpiog-over 103.341-4.349-50
mnniag off 358-07
corking 404
(wiw- 459-00
premature 300-2
sodikackland 225
■Uigoiging 404
lerm eolation 458
champagac method bottle
459-04
step), in 329
Muck fermentation 109- 1 1
sulfating 215-7. 353. 375-0
malobctk 401
pres.s.ag 458-9
riddling 403
Wiacmaking. dry whke
cryoprei.er.ai ion and 300
grape Maturity 260
must clarification 0b
lank lilting 334-9
Unaia coMcmmikim 357-8
temperature 00. 100. 102. 109. 350-2. 375
IhcraxxinificMaia 383-5
win tag time 348-9
WinemaLing. rose 353. 394. 445-9
color 440-7
■-..Hii in.- during storage 217
Wiacmaking. dor 475-9
sherry 475-9
-8
ill mi pressing 447
drawing-oil method 448-9
muu cbriaealioa 00
yellow. Aim 478-9
Wiacmaking. fortified 470-5
French 471-3
sibling 210
WipeMtiog.sparl.liag
Aati S puma me method 409
dor 473-5
translcr method 408
Wiacmaking. ml 327-94
acidification 328
aeration 375
Wine making. sweel
acttfc aekl 04
flrvr iii. Etom 283. 294
anaeiobiosis 380-8
cbhoratkin method 233
automated 380-5
keionk acids 201
bknding 328.300-7.377.
n i
refermentalkin 210
cap-|«inching 349-50
color 205. 215.209.301. 3
!3. 327. 383-5
stabilizers 227
«.to rage 224
coatinuous 3SI-2
titling 211.217.231
crop hctciogcncky 328
Icrmenlation
seerfw Nook nil
Winemaking. whkc
muUv 374-5
akoholic 339-45
acidly 399
acoiun 103. 108.430
malobcik 328. 307-80
Icnnentoa 330-9
aging
barrel 398-9
gaseous etc ha ago. 380
grape aaiurky 200
on ko 400
reduction odors 439-41
grape oualky 355-7
grape sorting 328
Hanoi treatments, met tank"
il 329-34
aramoniimsaks.addkioaof 430-1
annua 398.399.401
bentoake treatmem.s 428-9
conking 331
bknding 429
'iacmakiBg. while iciMitmucil )
clarification 398.422-5
boHytivedsvveei 91.208.219.230.398
champagne
effects on fcimcouikm kinetics
42J
i-O
chemical analysis of 404-0
effect* on wine compositim
104-0.
408
effervescence or 400-8
kes 429
foaincd
methods 426-8
lactk disease « 173
ciushing 413-4
ciyoseketion 417
fresh/fiuky 230.357-8
ice 300. 450
dcacklificatbn 310
ik.wi.BJw 414
fermcnuikm
akoholk 432
foil 473-5
rancn 417.473
sparkling 237
while
barrel 398. 433 -5
a<umai k 400
nraloluctk 432-3
dweniy in 399
grape ipialhv 398
-bscasc Male 401-4
neutral 399-401)
siaincd 440
maiuriy 404-7
harvesting for 407-8
higher a kebab. 70
•ike extraction 397.408-17
seeiilw Chaidonnav. Sauvjinon
yellow.- 478-9
immcdiitc batch 409-13
iamedute coKi«ious 409
Yeast autolysis 174. 170-7
VcaM cclK 3
kike uvkbiH>n
mcchanftms 418-20
piotccling from 417-21
ka
formal do 421-2
Olkfation reduction ptcjiiiaiiu
nraccratk-n 398.414-7
■be)
430
counts 80
cytoplasm 11-4
memhrjne. phimk 7-11
link! mosak model 7
functkinsof 10-1
miochondrii 13-4
nucleus 14-5
mini cbcakal dcvclofmcM during
; 247
wall 3-7.433
mu>i piutcins in 310
f icfeimcniaiion upcratkms. csscnti
397-9
ilmkc
r
YeaM colloicb 433
VcaM tkiov* . separation of 404
YeaM development. coadiionsor 79-112
in ■" icd grapes 327
n: I'litKial Km during I IXI
solids
asactivaioisia 94
format ■■ afo a postf kin ol suspended 421 -
uucfc terBkcvnlkim 107. 1 10-2
suliiing 213.215.217
sulfcir Com pounds, voblilc 439-41
supracxtraction 417
lank filliag 421
temperature com nil 431-2
thertnk shock 102
wood vokitiks 437
yeast colloids 433
yeaM inoculaibn 429-30
AlflB
aroaatkuhilc 400
banel-aged 230
Mine tie bliwic 327.440
Miih :tte notr 397
blush 440
Yeast ecology, grapc/wiac 40-8
YeaMgrowih 83-4.99-100
phases of 83
YeaM hull. 94.90-8. I()0. 112
YcaM inocubukm 30.40-8.429-30.403
YeaM mciabnlic pathways 53-76
Yeast nutritive needs 84-9
YeaM populai»Ds 41.42.80.218
vkibk 80. 233
YcaM reproduction 15-9
YeaM respiration 102-4
YcaM species
ctaiDOlra 22-35
co bin nil no b; akohol-scnskivc 42
dMraSukm 40-1
gB.peA.ine. succession 40-3
■demiheatkm systems for 23-4. 38
taxonomy principles 2. 23-6
molecular 20
YeaM Mane. 94
YeaM Mttricis. active
uv.tfm 5ta iter*
YcaM m rairn
antagonistic 108
:o*rcifeiv
21-2
14,45
citowhmbuu 110
hclcm.'Vimotli.illi. 10-9
idrniilimi»n»f»i*c 35 -40
killer 19-22
leapeoiuit ellect* on 100
> IncjanS-ipccilU' 40
Ye***aciem inteocilo*. 174-7. 189
Ycuu
jdlviilnoiif 213-4
aerobic 83
amino acid uiU'Mion by SO
apKul*cd SI
cell nail
parUul oi=Mili.-iiii .1-0
rale of 3
Cokioy 80
cuckis. hydnthtd 93
hcAldcMtucibnar 231-4
oxidative 102.292
Dxygc* requirement* ol 02
icfcrntcMalba 43
(.election of 213-4
Kami icpioductbll 10-9
Muhioekland 225-0
spoibpc 43.102.429
MilMinjj 4nd 210. 213-4
lempcnriuic and 3X4
vegetative mukiplkatfon 15-0
woe, hail ic<.»iiiKC of 231
vrr iifiu Icimtiilil mn
ZMterpilz 2
Z,)ton*rlur"m.eei *pn. 28- 45, 176
/ymjfloic 40
ZymalyuG 4
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