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Edited by
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MICROBIOLOGY of
Taylor & Francis
Taylor & Francis Group
MICROBIOLOGY of
Fruits and
Vegetables
MICROBIOLOGY of
Fruits and
Vegetables
Edited by
Gerald M. Sapers
James R. Gorny
Ahmed E. Yousef
@ Taylor & Francis
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Microbiology of fruits and vegetables / edited by Gerald M. Sapers, James R. Gorny, Ahmed E. Yousef.
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1. Fruit— Microbiology. 2. Vegetables—Microbiology. I. Sapers, Gerald M. II. Gorny, James R. III.
Yousef, Ahmed Elmeleigy.
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Preface
Fruits and vegetables represent an important part of the human diet, providing
essential vitamins, minerals, and fiber, and adding variety to the diet. In their
Food Guide Pyramid, the U.S. Department of Agriculture encourages con-
sumption of 3-5 servings of vegetable items, and 2-4 servings of fruit items per
day. In today's global economy, fresh fruits and vegetables are available year
round.
In the U.S. and other technologically advanced countries, high-quality fresh
and processed fruits and vegetables are widely available. Fresh-cut fruits
and vegetables represent a large and rapidly growing segment of the fresh
produce industry. These commodities have an excellent safety record with
respect to incidence of foodborne illness. Nevertheless, surveillance statistics
compiled by the U.S. Centers for Disease Control and Prevention indicate that
significant and increasing numbers of outbreaks have been associated with
fresh fruits and vegetables, or their products. The presence of human patho-
gens in fresh produce is borne out by U.S. Food and Drug Administration
product recall data, and by microbiological surveys of domestically produced
and imported commodities. Increased recognition of a food safety problem
with produce may reflect greater consumption of fruits and vegetables,
more frequent eating out, greater reliance on imports of out-of-season fruits
and vegetables from "third world" producers, and improved surveillance and
reporting methods by public health agencies.
In addition to safety concerns, microbial spoilage of fresh produce repre-
sents a source of waste for consumers, and an economic loss to growers,
packers, and retailers. Post-harvest decay, bacterial soft rot, and microbial
spoilage of fresh-cuts and processed juices are continuing problems.
In recent years, extensive research has been conducted on microbiological
problems relating to the safety and spoilage of fruits and vegetables. Active
areas of research include incidence of human pathogen contamination, sources
of microbial contamination, microbial attachment to produce surfaces, intract-
able spoilage problems, efficacy of sanitizing treatments for fresh produce,
novel interventions for produce disinfection, and methodologies for micro-
biological evaluation of fruits and vegetables.
In this book, we have attempted a comprehensive examination of these
topics, focusing on issues, rather than attempting an encyclopedic compilation
of information about all commodities, classes of microorganisms, or categories
of spoilage. We have not included certain topics, such as preharvest diseases of
produce or production of fermented vegetables, which are adequately covered
Preface
elsewhere. We have selected chapter authors who are active researchers in their
respective fields, and thus bring a working knowledge of current issues,
industry practices, and advances in technology.
The book is divided into five sections: (I) Contamination and State of
Microflora on Fruits and Vegetables; (II) Microbial Spoilage of Fruits and
Vegetables; (III) Food Safety Issues; (IV) Interventions to Reduce Spoilage
and Risk of Foodborne Illness; and (V) Microbiological Evaluation of Fruits
and Vegetables. Within each section we have grouped chapters that cover
specific issues related to the overall topic. For example, Section I contains
chapters on sources of microbial contamination, attachment of microorgan-
isms to fresh produce, internalization and infiltration of microorganisms in
produce, and stress adaptation by microorganisms and safety of produce.
I wish to thank the individual chapter authors for the authoritative
and comprehensive coverage of their respective topics, and my co-editors,
Dr. James R. Gorny and Dr. Ahmed E. Yousef, for their assistance in
developing the concept and organizational structure of the book, identifying
suitable chapter authors, reviewing the completed chapters, and helping me
assemble the manuscripts into a form suitable for publication. I also thank
Susan Lee, Food Science Editor at Dekker/CRC Press and her editorial staff
for their guidance, invaluable help, and patience in working with us on this
project. I thank my employer, the USDA Agricultural Research Service's
Eastern Regional Research Center, for allowing me the time, and providing
the resources, that enabled me to participate in this project. Finally, I must
thank my wife for her unlimited patience and understanding during the
many long hours when I was attached to the computer and unavailable to meet
her needs.
Gerald M. Sapers
Editors
Gerald M. Sapers received his Ph.D. in food technology from MIT in 1961. He
joined the USDA's Eastern Regional Research Center (ERRC) in 1968, after 2
years at the U.S. Army Natick Laboratories, and 6 years in private industry.
He has conducted research on dehydrated potato stability, apple volatiles,
safety of home canned tomatoes, utilization of natural pigments, pigmentation
of small fruits, cherry dyeing, control of enzymatic browning in minimally
processed fruits and vegetables, mushroom washing, and microbiological
safety of fresh produce, which is his current area of research. He has been a
Lead Scientist at ERRC since 1991. Dr. Sapers has published 110 scientific
papers, 3 book chapters and 5 patents. He is an active member of the Institute
of Food Technologists' Fruit and Vegetable Products Division, and the
International Fresh-cut Produce Association.
James R. Gorny received his Ph.D. in plant biology from the University of
California, Davis, and his M.S. and B.S. degrees in food science from
Louisiana State University in Baton Rouge. He is currently vice president of
Technology and Regulatory Affairs for the International Fresh-cut Produce
Association, and has been the author and editor of numerous scientific
publications including: Editor-in-Chief of the IFPA Food Safety Guidelines
for the Fresh-cut Produce Industry and a contributor to the chapter on
"Produce Food Safety" in the recently revised U.S. Department of Agriculture
Handbook 66. His research has focused on the effects of modified atmos-
pheres on the quality and safety of whole and fresh-cut fruit produce. He
has been actively involved in the fresh-cut produce industry since 1986, and
has worked extensively as a consultant on food safety, packaging, quality
assurance, operations, and general management issues, both nationally and
internationally.
Ahmed E. Yousef received his Ph.D. in food science from the University
of Wisconsin (UW)-Madison in 1984. Subsequently, he served as a post-
doctoral researcher at the Department of Food Science and the Department
of Food Microbiology and Toxicology, UW. Dr. Yousef joined The Ohio
State University (OSU) as an assistant professor in 1991. At OSU, Dr. Yousef
investigated food biopreservation using bacteriocins, explored new applica-
tions of ozone in food processing, and addressed the safety of foods processed
by novel technologies such as pulsed electric field, high pressure processing
and ohmic heating. He is currently a professor at the Department of Food
Editors
Science and Technology and the Department of Microbiology, teaching
the main food microbiology course at OSU. Dr. Yousef has published
2 books, 10 book chapters, and 70 scientific papers and review articles, and a
patent. He is an active member of the Institute of Food Technologists, the
American Society for Microbiology, and the International Association of Food
Protection.
Contributors
Bassam A. Annous
Eastern Regional Research Center
Agricultural Research Service
U.S. Department of Agriculture
Wyndmoor, Pennsylvania
Jerry A. Bartz
Department of Plant Pathology
University of Florida
Gainesville, Florida
Robert B. Beelman
Department of Food Science
Pennsylvania State University
University Park, Pennsylvania
Larry R. Beuchat
Center for Food Safety
Department of Food Science and
Technology
University of Georgia
Griffin, Georgia
Maria T. Brandl
Western Regional Research Center
Agricultural Research Service
U.S. Department of Agriculture
Albany, California
F. Breidt, Jr.
Agricultural Research Service
U.S. Department of Agriculture
and Department of Food Science
North Carolina State University
Raleigh, North Carolina
Naveen Chikthimmah
Department of Food Science
Pennsylvania State University
University Park, Pennsylvania
Pascal Delaquis
Food Safety and Quality
Agriculture and Agri-Food Canada
Summerland, British Columbia,
Canada
Mary Ann Dombrink-Kurtzman
National Center for Agricultural
Utilization Research
Agricultural Research Service
U.S. Department of Agriculture
Peoria, Illinois
Elazar Fallik
Department of Postharvest Sciences
of Fresh Produce
ARO-The Volcani Center
Bet-Dagan, Israel
William F. Fett
Eastern Regional Research Center
Agricultural Research Service
U.S. Department of Agriculture
Wyndmoor, Pennsylvania
Daniel Y.C. Fung
Department of Animal Sciences and
Industry
Kansas State University
Manhattan, Kansas
Contributors
Jim Gorny
International Fresh-cut Produce
Association
Davis, California
Lisa Gorski
Western Regional Research Center
Agricultural Research Service
U.S. Department of Agriculture
Albany, California
Dongsheng Guan
Department of Animal and Food
Sciences
University of Delaware
Newark, Delaware
Yingchan Han
Department of Food Sciences
Purdue University
West Lafayette, Indiana
Dallas G. Hoover
Department of Animal and Food
Sciences
University of Delaware
Newark, Delaware
J.H. Hotchkiss
Department of Food Sciences
Cornell University
Ithaca, New York
William C. Hurst
Department of Food Science and
Technology
University of Georgia
Athens, Georgia
Lauren Jackson
Center for Food Safety and
Applied Nutrition
U.S. Food and Drug Administration
Bedford, Illinois
Susanne E. Keller
National Center for Food Safety
and Technology
U.S. Food and Drug
Administration
Summit Argo, Illinois
Michael F. Kozempel
Eastern Regional Research Center
Agricultural Research Service
U.S. Department of Agriculture
Wyndmoor, Pennsylvania
Ching-Hsing Liao
Eastern Regional Research Center
Agricultural Research Service
U.S. Department of Agriculture
Wyndmoor, Pennsylvania
Richard H. Linton
Center for Food Safety Engineering
Purdue University
West Lafayette, Indiana
Robert E. Mandrell
Western Regional Research Center
Agricultural Research Service
U.S. Department of Agriculture
Albany, California
Pamela G. Marrone
AgraQuest, Inc.
Davis, California
Julien Mercier
AgraQuest, Inc.
Davis, California
Arthur J. Miller
U.S. Food and Drug
Administration
Center for Food Safety and
Applied Nutrition
College Park, Maryland
Contributors
J.-M. Monier
Laboratoire d'Ecologie Microbienne
Universite Claude Bernard Lyon 1
Villeurbanne, France
Travis L. Selby
Department of Food Sciences
Purdue University
West Lafayette, Indiana
Philip E. Nelson
Department of Food Sciences
Purdue University
West Lafayette, Indiana
Ynes R. Ortega
Center for Food Safety
Department of Food Science and
Technology
University of Georgia
Griffin, Georgia
Mickey E. Parish
Citrus Research and Education
Center
University of Florida
Lake Alfred, Florida
Luis A. Rodriguez-Romo
Department of Food Science and
Technology
The Ohio State University
Columbus, Ohio
Gerald M. Sapers
Eastern Regional Research Center
Agricultural Research Service
U.S. Department of Agriculture
Wyndmoor, Pennsylvania
Charles R. Sterling
Department of Veterinary Science
and Microbiology
University of Arizona
Tucson, Arizona
Dike O. Ukuku
Eastern Regional Research Center
Agricultural Research Service
U.S. Department of Agriculture
Wyndmoor, Pennsylvania
B.G. Werner
Department of Food Sciences
Cornell University
Ithaca, New York
Ahmed E. Yousef
Department of Food Science and
Technology
The Ohio State University
Columbus, Ohio
Contents
SECTION I Contamination and State of Microflora
on Fruits and Vegetables
Chapter 1
Microbial Contamination of Fresh Fruits and Vegetables 3
Jim Gorny
Chapter 2
Attachment of Microorganisms to Fresh Produce 33
Robert E. Mandrell, Lisa Gorski, and
Maria T. Brandt
Chapter 3
Internalization and Infiltration 75
Jerry A. Bartz
Chapter 4
Microbial Stress Adaptation and Safety of Produce 95
Luis A. Rodriguez- Romo and Ahmed E. Yousef
SECTION II Microbial Spoilage of Fruits
and Vegetables
Chapter 5
Bacterial Soft Rot 117
Ching-Hsing Liao
Contents
Chapter 6
Microbial Spoilage of Fresh Mushrooms 135
Naveen Chikthimmah and Robert B. Beelman
Chapter 7
Spoilage of Juices and Beverages by Alley clobacillus spp 159
Mickey E. Parish
SECTION III Food Safety Issues
Chapter 8
Interventions to Ensure the Microbial Safety of Sprouts 187
William F. Fett
Chapter 9
Microbiological Safety of Fresh Citrus and Apple Juices 211
Susanne F. Keller and Arthur J. Miller
Chapter 10
Microbiological Safety Issues of Fresh Melons 231
Dike O. Ukuku and Gerald M. Sapers
Chapter 11
Fresh-Cut Vegetables 253
Pascal Delaquis
Chapter 12
Outbreaks Associated with Cyclospora and Cryptosporidium 267
Ynes R. Ortega and Charles R. Sterling
Chapter 13
Patulin 281
Lauren Jackson and
Mary Ann Dombrink-Kurtzman
Chapter 14
Safety of Minimally Processed, Acidified, and Fermented
Vegetable Products 313
F. Breidt, Jr.
Contents
SECTION IV Interventions to Reduce Spoilage and
Risk of Foodborne Illness
Chapter 15
HACCP: A Process Control Approach for Fruit and Vegetable Safety... 339
William C. Hurst
Chapter 16
Effect of Quality Sorting and Culling on the Microbiological
Quality of Fresh Produce 365
Susanne E. Keller
Chapter 17
Washing and Sanitizing Treatments for Fruits and Vegetables 375
Gerald M. Sapers
Chapter 18
Gas-/Vapor-Phase Sanitation (Decontamination) Treatments 401
Richard H. Linton, Yingchang Han, Travis L. Selby, and
Philip E. Nelson
Chapter 19
Modified Atmosphere Packaging 437
E.G. Werner and J.H. Hotchkiss
Chapter 20
Hot Water Treatments for Control of Fungal Decay on
Fresh Produce 461
Elazar Fallik
Chapter 21
Surface Pasteurization with Hot Water and Steam 479
Bassam A. Annous and Michael F. Kozempel
Chapter 22
Novel Nonthermal Treatments 497
Dongs heng Guan and Dallas G. Hoover
Chapter 23
Biological Control of Microbial Spoilage of Fresh Produce 523
Julien Mercier and Pamela G. Marrone
Contents
SECTION V Microbiological Evaluation of Fruits
and Vegetables
Chapter 24
Sampling, Detection, and Enumeration of Pathogenic and
Spoilage Microorganisms 543
Larry R. Beuchat
Chapter 25
Rapid Detection of Microbial Contaminants 565
Daniel Y.C. Fung
Chapter 26
Methods in Microscopy for the Visualization of Bacteria and
Their Behavior on Plants 595
Maria T. Brand! and J.-M. Monier
Index 621
Section I
Contamination and
State of Microflora on
Fruits and Vegetables
Microbial Contamination
of Fresh Fruits
and Vegetables
Jim Go my
CONTENTS
1 . 1 Introduction 4
1 .2 Produce Contamination 5
1 .3 Microorganisms of Concern 7
1 .4 Incidence and Association of Human Pathogens
with Produce 8
1 .4. 1 FDA Imported Produce Survey 8
1 .4.2 FDA Domestic Produce Survey 9
1.4.3 USDA Microbiological Data Program (MDP) 10
1.4.4 Produce-Associated Foodborne Illness Traceback
Investigation Results 12
1.5 Potential Sources of Produce Contamination by
Human Pathogens 13
1.5.1 Food Safety Risk Factors Associated with Production
of Fresh Produce 13
1.5.1.1 Land Use 14
1.5. 1 .2 Soil Amendments 14
1.5.1.3 Wild and Domestic Animal Control 14
1 .5. 1 .4 Irrigation Water 15
1.5.1.5 Harvest Operations 15
1.5.2 Food Safety Risk Factors Associated with Postharvest
Handling of Produce 16
1.5.2.1 Employee Hygiene 16
1.5.2.2 Equipment 16
1.5.2.3 Wash and Hydrocooling Water 17
1.5.2.4 Cold Storage Facilities 17
1.5.2.5 Packaging Materials 18
1.5.2.6 Modified Atmosphere Packaging of
Fresh Produce 18
4 Microbiology of Fruits and Vegetables
1.5.2.7 Refrigerated Transport, Distribution, and
Cold Storage 19
1.5.3 Food Safety Risk Factors Associated with
Foodservice, Restaurant, and Retail Food Stores
Handling of Produce 20
1.5.4 Consumer Handling of Produce from Purchase to Plate 21
1.6 Effective Management Strategies: Contamination Prevention
and Intervention 21
1.6. 1 Good Agricultural Practices (GAPs) 22
1.6.2 Current Good Manufacturing Practices (cGMPs) 23
1 .6.3 Hazard Analysis Critical Control Point (HACCP) 24
1.7 Research Needs 25
1.7.1 Microbial Ecology of Human Pathogens in the Agricultural
Production Environment 26
1 .7.2 Agricultural Water 26
1 .7.3 Soil Amendments 26
1.7.4 Proximity Risk of Potential Contaminant Sources 27
1.7.5 Intervention Strategies to Reduce the Risk of
Human Pathogen Contamination of Fresh Produce 27
1.8 Summary 27
References 28
1.1 INTRODUCTION
Fresh fruits and vegetables are perceived by consumers to be healthful and
nutritious foods because of the plethora of scientifically substantiated and
documented health benefits derived from consuming fresh fruits and vegetables
[1]. However, recent foodborne illness outbreaks in the U.S. and throughout
the world have been increasingly linked epidemiologically to consumption of
fresh fruits, vegetables, and unpasteurized juices. These incidents have caused
growers, shippers, fresh-cut produce processors, distributors, retailers, import-
ers, and government public health officials to re-evaluate the risk of con-
tracting foodborne illness from consumption of fresh fruits and vegetables
and to re-evaluate current production and handling practices.
While the probability of contracting a foodborne illness via consumption
of fresh fruits or vegetables is very low, a small probability does exist. Because
fresh fruits and vegetables are often consumed uncooked so that there is
no "kill" step, prevention of produce contamination with human pathogens
is the only practical and effective means of ensuring that these food pro-
ducts are wholesome and safe for human consumption. This means that a
complete supply chain approach to prevent contamination at any point in the
produce continuum is essential to ensuring public health by minimizing the
incidence of foodborne illness associated with produce consumption. Ensuring
Microbial Contamination of Fresh Fruits and Vegetables 5
the integrity of produce from field to fork is the responsibility of everyone in
the produce continuum, including growers, shippers, processors, distributors,
retailers, and consumers. It must also be remembered that the health benefits
derived from eating at least five servings of fresh fruits and vegetables daily
far outweigh the very small probability of contracting a foodborne illness.
A meaningful assessment of the risk associated with contracting a
foodborne illness from consumption of fresh fruits and vegetables involves
understanding the microbiology of fresh fruits and vegetables as well as field
production, processing, and handling practices. As such, the fresh produce
industry is extraordinarily diverse and complex in the number of products
produced, how the products are grown and handled, and the geographic
areas from which these products are sourced. A typical retail grocer in North
America will have available on a daily basis upwards of 300 different
produce items for sale. The morphological characteristics of a produce item
may also contribute to its propensity for contamination, since produce
items may be derived from the leaves, stems, stalks, roots, fruits, and flowers
of plants. Because the produce continuum represents such diversity, it is
only possible to describe broad generalities about current practices of the
produce continuum and the food safety risk associated with them, as an
in-depth analysis of this plethora of products would be encyclopedic in volume.
1 .2 PRODUCE CONTAMINATION
Contamination of fruits and vegetables by human pathogens can occur
anywhere in the farm to table continuum including contamination of seed
stocks and during production, harvesting, postharvest handling, storage,
processing, transport distribution, retail display, and/or preparation (food-
service or home). Produce contaminated with human pathogens cannot be
completely disinfected by washing or rinsing the product in an aqueous
solution, and low sporadic levels of human pathogens can be found on pro-
duce [2,3]. In 2004 the Alliance for Food and Farming [4] analyzed Centers
for Disease Control and Prevention (CDC) data sets [5,6] and summa-
rized information regarding foodborne illness outbreaks that have been
associated with produce consumption. The study's objective was to analyze
likely sources of produce contamination and categorize the most likely place
that the contamination occurred, that being either during production/
growing or during postproduction handling. The *'postproduction" category
included produce-associated foodborne illnesses that were most likely due to
improper handling at the foodservice, retail, or consumer level, while the
"grower" category included foodborne illnesses associated with produce that
were most likely attributable to the farm, packing, shipping, or other
agricultural postharvest handling. Analysis of CDC data indicated that
improper handling of fruits and vegetables at foodservice establishments or
by consumers caused 83% of produce-associated foodborne illness outbreaks,
while "grower"-implicated cases comprised 17% of produce-associated
Microbiology of Fruits and Vegetables
C/)
.*:
03
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100 - pi
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80 - i-i
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J J 1j J 111 I J J
1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001
% of produce outbreaks associated with growing, packing, shipping and/or processing
% of produce outbreaks associated with improper handling after leaving the farm
FIGURE 1 .1 Produce-associated outbreaks due to suspected farm contamination versus
postproduction handling. (Adapted from Analysis of Produce Related Foodborne Illness
Outbreaks, Alliance for Food and Farming, April 2004, www.foodandfarming.info
documents/85876_produce_analysis_604.pdf.)
foodborne illness outbreaks. Data from this report presented in Figure 1.1
show that the percentage of ;c grower"-related contamination incidents as a
percent of all produce related outbreaks has been declining since 1996, and this
trend is most likely due to implementation of good agricultural practices
(GAPs) by grower/shipper/packers.
The Alliance for Food and Farming 2004 report and the CDC [7] both
indicate that about 12% of foodborne illnesses occurring in the U.S. between
1990 and 2001 has been associated with consumption of fresh fruits and
vegetables. This figure of 12% of outbreak cases associated with produce
consumption represents a greater proportion of foodborne illness burden being
represented by fresh fruits and vegetables than was reported in the past
(Table 1.1). CDC data also indicate that produce-related outbreaks have
become larger, involving more individuals and increasing in frequency.
Foodborne illness outbreak reports related to produce consumption have
most likely increased due to:
• Better detection and diagnostic methods for human pathogens which
can epidemiologically associate produce consumption with illness
(PulseNet, SODA salmonella outbreak detection algorithm, etc.).
Microbial Contamination of Fresh Fruits and Vegetables
TABLE 1.1
Trends in Burden: Foodborne Outbreaks Related to Fresh Produce,
1973-1997
1970s 1990s
No. outbreaks per year 2 16
Median cases per outbreak 21 43
Outbreaks of known vehicle (%) 0.7 6
Outbreak associated cases (%) 0.6 12
Adapted from Sivapalasingham, S., Friedman, C.R., Cohen, L., and Tauxe, R.V.,
/. Food Pro t. 67, 10, 2004.
• Increased surveillance for human pathogens by public health
agencies.
• Increased per capita consumption of fresh fruits and vegetables in
North America.
• Increased awareness that produce may be a potential vehicle for
human pathogens, thus leading to increased epidemiological investi-
gations of produce as a potential vector.
• Increased global sourcing of produce items to ensure year around
supply of the broad diversity of produce available in modern grocery
stores.
• Longer postharvest storage and longer shipment times that may
also contribute to increased potential for illness by allowing for
proliferation of an initial low number of human pathogens to an
infectious or disease-causing dosage.
1 .3 MICROORGANISMS OF CONCERN
Harris et al. [8] extensively reviewed outbreaks associated with fresh produce
and reported that the most common human pathogens associated with produce
foodborne illness outbreaks are: E. coli 0157:H7, Salmonella spp., Shigella
spp., Listeria monocytogenes, Crytosporidium spp. [9], Cyclospora spp.,
Clostridium botulinum, hepatitis A virus, Norwalk virus, and Norwalk-like
viruses. These microorganisms can be categorized as follows:
• Soil-associated pathogenic bacteria (Clostridium botulinum, Listeria
monocytogenes).
• Feces-associated pathogenic bacteria (Salmonella spp., Shigella spp.,
E. coli 0157:H7, and others).
• Pathogenic parasites (Cryptosporidium, Cyclospora).
• Pathogenic viruses (hepatitis A, enterovirus, Norwalk-like viruses).
Many of these pathogens are spread via a human (or domestic animal)
to food to human transmission route. Handling of fruits and vegetables by
8 Microbiology of Fruits and Vegetables
infected field-workers or consumers, cross contamination, use of contami-
nated water, use of inadequately composted manure, or contact with con-
taminated soil are just a few of the ways that transmission of human pathogens
to food can occur.
Data from the CDC foodborne outbreak surveillance system show that
from 1988 to 1998 the two most commonly reported microorganisms
associated with fresh produce foodborne illness outbreaks were Salmonella
spp. and E. coli 0157:H7 with 45% and 38% of the fruit and vegetable linked
outbreaks, respectively, being attributed to these two microorganisms. How-
ever, recent foodborne illness outbreaks associated with produce consumption
have been caused by viruses (hepatitis A) and parasites (Cyclospora spp.). CDC
data demonstrate that the majority of reported foodborne illnesses in the
U.S. are of unknown etiology and are most likely caused by viruses such as
Norwalk-like viruses [10,11]. Unfortunately, diagnostic tools for detection
and enumeration of viruses that may cause foodborne illnesses are severely
lacking.
1.4 INCIDENCE AND ASSOCIATION OF HUMAN
PATHOGENS WITH PRODUCE
1 .4.1 FDA Imported Produce Survey
In March 1999 the U.S. Food and Drug Administration (FDA) initiated
a 1000-sample survey of imported fresh produce raw agricultural commodi-
ties from 21 countries and included: broccoli, loose-leaf lettuce (radicchio,
escarole, endive, chicory leaf, mesclun, and others), cantaloupe, celery, straw-
berries, scallions/green onions, tomatoes, parsley, culantro (a herb), and
cilantro [12]. Loose-leaf lettuce products included radicchio, escarole, endive,
chicory and others. These high-volume imported fresh produce raw
agricultural commodities were selected by the FDA for the imported
produce sampling assignment based on the following risk factor criteria:
epidemiological outbreak data, structural characteristics of the produce item,
growing conditions, processing and consumption rates. Raw agricultural
commodities are defined in the Federal Food, Drug, and Cosmetic Act as "any
food in its raw or natural state, including all fruits that are washed, colored, or
otherwise treated in the unpeeled natural form prior to marketing.'' These raw
agricultural commodities were analyzed for the presence of Salmonella
spp. and E. coli 0157:H7. All commodities except for cilantro, culantro, and
strawberries also were analyzed for Shigella spp. Produce imported from
Mexico, Canada, Costa Rica, Guatemala, the Netherlands, Honduras,
Belgium, Italy, Israel, Chile, Peru, Colombia, Trinidad and Tobago, New
Zealand, Nicaragua, the Dominican Republic, France, Argentina, Ecuador,
Haiti, and Korea were sampled. Six countries provided 25 or more samples for
analysis: Mexico, Canada, Costa Rica, Guatemala, the Netherlands, and
Honduras.
Microbial Contamination of Fresh Fruits and Vegetables
TABLE 1.2
Number of Samples Collected and Analyzed and the Number of Samples
Confirmed Positives for Human Pathogens Per Each Type of Imported
Produce
No. human pathogen
No. of
positive
samples
Salmonella
Shigella
Produce item
samples
(% positive per produce item)
spp.
spp.
Broccoli
36
(0.0%)
(0.0%)
(0.0%)
Cantaloupe
151
11 (7.3%)
8 (5.2%)
3 (2.0%)
Celery
84
3 (3.6%)
1 (1.2%)
2 (2.4%)
Cilantro
177
16 (9.0%)
16 (9%)
N/A
Culantro
12
6 (50.0%)
6 (50%)
N/A
Lettuce
116
2(1.7%)
1 (0.9%)
1 (0.9%)
Parsley
84
2 (2.4%)
1 (1.2%)
1 (1.2%)
Scallions
180
3 (1.7%)
1 (0.6%)
2(1.1%)
Strawberries
143
1 (0.7%)
1 (0.7%)
N/A
Tomatoes
20
(0.0%)
(0.0%)
(0.0%)
Total
1003
44 (4.5%)
35 (3.5%)
9 (1.3%)
Note: N/A = not analyzed.
Adapted from FDA Survey of Imported Fresh Produce, U.S. Food and Drug Administration
Center for Food Safety and Applied Nutrition, Office of Plant and Dairy Foods and Beverages,
January 30, 2001, www.cfsan.fda.gov/~dms/prodsur6.html.
Data presented in Table 1.2 show that of 1003 samples that were collected
and analyzed, 35 samples (3.5% of the total number of samples) were found
to have detectable levels of Salmonella spp., 9 samples (0.9% of the total
number of samples) were found to have detectable levels of Shigella spp., and no
samples (0%) were found to have detectable levels of E. coli 0157:H7. These 44
samples positive for the presence of a human pathogen represent approx-
imately 4.4% of the total number of product samples tested.
The three produce items with the greatest incidence of pathogen con-
tamination were cilantro, cantaloupe, and culantro, accounting for 1.6, 1.1,
and 0.6%, respectively, of the overall contamination (4.4%). The remaining
produce items each contributed 0.3% or less to the overall contamination.
1.4.2 FDA Domestic Produce Survey
In March 2000 the FDA initiated a 1000-sample survey of domestic fresh
fruit and vegetable raw agricultural commodities [13]. Cantaloupe, celery,
cilantro, loose-leaf lettuce, parsley, scallions (green onions), strawberries, and
tomatoes were collected and analyzed for Salmonella spp. and E. coli 0157:H7.
Cantaloupe, celery, parsley, scallions, and tomatoes were also analyzed for
Shigella spp. This survey was the domestic complement to the FDA imported
produce survey.
10
Microbiology of Fruits and Vegetables
TABLE 1.3
Number of Samples Collected and Analyzed and the Number of Confirmed
Positives for Human Pathogens Per Each Type of Domestic Produce Sampled
No. human pathogen
No. of
positive samples
Salmonella
Shigella
Produce item
samples
(% positive per produce item)
spp.
spp.
Cantaloupe
164
5(3.1)
4 (2.4%)
1 (0.6%)
Celery
120
(0.0%)
(0.0%)
(0.0%)
Cilantro
85
1 (1.2%)
1 (1.2%)
N/A
Lettuce
142
1 (0.7%)
1 (0.7%)
N/A
Parsley
90
1 (1.1%)
(0.0%)
1 (1.1%)
Scallions
93
3 (3.2%)
(0.0%)
3 (3.2%)
Strawberries
136
(0.0%)
(0.0%)
N/A
Tomatoes
198
(0.0%)
(0.0%)
(0.0%)
Total
1028
11 (1.1%)
6 (0.6%)
5 (0.8%)
Note: N/A = not analyzed.
Adapted from FDA Survey of Domestic Fresh Produce, U.S. Department of Health and Human
Services, U.S. Food and Drug Administration Center for Food Safety and Applied Nutrition,
Office of Plant and Dairy Foods and Beverages, January 2003, www.cfsan.fda.gov/~dms
prodsul0.html.
Data presented in Table 1.3 show that of 1028 domestic samples that
were collected and analyzed, 6 samples (0.58% of the total number of samples)
were found to have detectable levels of Salmonella spp., 5 samples (0.49% of
the total number of samples) were found to have detectable levels of Shigella
spp., and no samples (0%) were found to have detectable levels of E. coli
0157:H7. One or more samples of cantaloupe, cilantro, lettuce, parsley, and
scallions were found to have detectable levels of human pathogens.
Cantaloupes had the highest number of positive samples (5), followed by
scallions (3), cilantro, lettuce, and parsley (1 each).
When adjusted to account for the number of samples of each commodity
collected, scallions had the highest detectable rate of human pathogens (3.2%)
of the total 93 samples collected. Cantaloupe had a 3.1% rate of detectable
human pathogens with 5 out of 164 samples collected testing positive. One of
85 cilantro samples tested positive for the presence of a human pathogen giving
a detection rate of 1.2%. One of 90 parsley samples (1.1%) was found to have
detectable levels of Shigella spp. and one of 142 (0.7%) lettuce samples was
found to have detectable levels of Salmonella spp.
1.4.3 USDA Microbiological Data
Program (MDP)
In 2001 the U.S Department of Agriculture (USDA) implemented a program to
collect information regarding the incidence, number, and species of important
Microbial Contamination of Fresh Fruits and Vegetables
11
foodborne pathogens and indicator organisms on domestic and imported
fresh fruit and vegetable raw agricultural commodities. USDA's Agricultural
Marketing Service (AMS) was appointed to undertake the program that is
currently known as the microbiological data program (MDP). MDP was
primarily designed to provide data on microbial presence in order to establish a
microbial baseline to assess the risks of contamination, if any, in the domestic
food supply.
In 2002 USD A MDP analyzed a total 10,317 samples of five raw agri-
cultural commodities: cantaloupe, celery, leaf lettuce, romaine lettuce, and
tomatoes [14]. Samples were collected in commerce at wholesale and/or
distribution centers, and 86% of the samples came from domestic sources;
11% of the samples were imported; and no country of origin information was
obtained for 3% of the samples.
Samples were analyzed for generic E. coli and Salmonella spp. with E. coli
isolates being further analyzed for the presence of the following virulence
factors: enterohemorrhagic shiga-toxins SLT-1 and SLT-2, hemolysin HlyA,
invasive trait (intimin-eae) enterotoxigenic toxins (heat stable STa, STb; heat
labile LT), enteropathogenic — invasive character (intimin eae-a), enteroag-
gregative — gene associated with the virulent plasmid, necrotizing cytotoxic —
cytotoxic necrotizing factor (CNF-1 and 2), enteroinvasive — IpaH gene
known to be associated with EIEC, and Kl capsular antigen. The presence of
virulence factors does not necessarily mean that the strains isolated from the
produce items are pathogenic to humans, but may have pathogenic potential.
Data presented in Table 1.4 show that of the 10,315 USD A MDP
samples that were collected and analyzed for Salmonella spp., only 3 samples
(0.03% of the total number of samples) were found to have detectable levels
of Salmonella spp. Of the 10,276 USDA MDP samples that were collected
TABLE 1.4
Summary of the USDA MDP Analysis for Salmonella spp. and £. coli with
Associated Virulence Factors for Cantaloupe, Celery, Leaf Lettuce, Romaine
Lettuce, and Tomatoes
No.
of samples
No. and % of
No. and % of samples
tested for
No. of samples
samples testing
testing positive for
Salmonella
tested for
positive for
E. coli with a
Produce item
spp
■
E. coli
Salmonella spp.
virulence factor
Cantaloupe
1,077
1,077
(0.0%)
2(0.19%)
Celery
2,175
2,174
(0.0%)
3 (0.14%)
Leaf lettuce
2,180
2,161
3 (0.14%)
27(1.25%)
Romaine lettuce
2,177
2,158
(0.0%)
29(1.34%)
Tomatoes
2,706
2,706
(0.0%)
3(0.11%)
Total
10,315
10,276
3 (0.03%)
64 (0.62%)
Adapted from USDA,
Microbiological Data Program
Progress Update
and 2002 Data Summary,
www.ams.usda.gov/science/mpo/MDPSumm02.pdf, 2004.
12 Microbiology of Fruits and Vegetables
and analyzed for E. coli, 64 samples (0.62% of the total number of samples)
were found to have detectable levels of E. coli with associated virulence
factors. Twenty-seven (1.25%) of 2161 leaf lettuce and 29 (1.34%) of 2158
romaine lettuce samples were found to have detectable levels of E. coli with
associated virulence factors. Cantaloupe, celery, and tomato had incidence
rates for the presence of E. coli with associated virulence factors of 0.19, 0.14,
and 0.11%, respectively.
Follow-up FDA farm investigations and other information from both
the agency's imported and domestic produce surveys indicated that failure
to follow GAPs was often associated with the findings of pathogen con-
tamination. In particular, inadequate manure management and lack of
appropriate field and transport sanitation practices were most frequently
associated with overall contamination. Specific problems included fields that
were open to domestic animals or were fertilized by untreated animal manure,
equipment and tools that were not being sanitized, unsanitary harvesting
and/or packing equipment or practices (e.g., woven plastic bags to collect
cilantro after harvest), and unsanitary methods of transportation (e.g., trucks
washed with nonchlorinated water and/or cleaned infrequently) [12].
1.4.4 Produce-Associated Foodborne Illness
Traceback Investigation Results
Traceback investigations have yielded no definitive information as to the
causes of recent produce-associated foodborne illness outbreaks. The inability
to identify clearly where contamination occurred and the actual causes of
recent foodborne illness outbreaks associated with produce consumption is
frustrating to the industry and regulators alike and is a significant hurdle to
developing a means of ensuring that similar outbreaks do not recur. Without
science-based data that clearly identify the cause of recent foodborne ill-
nesses associated with produce consumption, only speculation and opinion
can be used to hypothesize about what may have gone wrong. It is imperative
that industry, academia, government, and consumers collaborate and take
an active role by working together on developing and implementing measures
that enhance produce food safety. Guzewich [15] reported in a summary
of produce-related outbreak farm investigations that the practices most likely
to have contributed to numerous recent outbreaks related to produce
consumption are:
• Questionable practices regarding safe water use.
• Inadequate animal management (domestic and/or wild animals).
• Unsanitary facilities and equipment.
• Inadequate employee health and hygiene practices.
It is important that future investigations do not simply focus on the
suspected primary causes of produce contamination in the supply chain, but
allow for identification of hitherto unidentified actual causes of produce
Microbial Contamination of Fresh Fruits and Vegetables 13
contamination. Regulatory agency traceback investigations of facilities
suspected of being involved in a foodborne illness outbreak must focus on
determining the efficiency and effectiveness of the facilities' GAP program
and attempt to identify clearly if the contamination occurred due to non-
compliance with GAPs or due to deficiencies in GAPs as they are currently
formulated.
1.5 POTENTIAL SOURCES OF PRODUCE
CONTAMINATION BY HUMAN PATHOGENS
While produce quality can be judged by outward appearance based on such
criteria as color, turgidity, and aroma, food safety cannot. Casual inspection
of produce cannot determine if it is in fact safe and wholesome to consume.
Most fresh fruits and vegetables are grown in nonsterile environments, and
conventional fruit and vegetable growers have less control over conditions
in the production field as compared to an enclosed production or food
preparation facility. The surfaces of produce have natural microflora
composed of microorganisms that are generally benign. However, low-level
contamination of produce with pathogenic microorganisms may sporadically
occur. Production, harvesting, washing, cutting, slicing, packaging, transport-
ing, and preparation all offer opportunities for produce contamination. While
it is well established from the data presented above that the vast majority of
produce contamination with human pathogens occurs in postproduction
situations (Figure 1.1), if contamination does occur during growing and
initial postharvest handling of produce, the consequences can be far greater.
This is due to the potential for amplification of human pathogens through-
out distribution and the increased risk of cross contamination posed by
handling a food product contaminated with a human pathogen.
1.5.1 Food Safety Risk Factors Associated with
Production of Fresh Produce
Management of growing conditions is of paramount importance in prevent-
ing the contamination of fresh produce by human pathogens. There are risk
factors to consider such as growing conditions, agricultural practices used
by specific growers, the time of year, growing region/environment, and
management practices that may change over the course of a season. Climate,
weather, water quality, soil fertility, pest control, as well as irrigation, and
other management practices are difficult to integrate towards the develop-
ment and implementation of microbial risk prevention and reduction programs
on the farm [16].
Organic foods including organic fresh fruits and vegetables are one of
the fastest-growing segments of the U.S. food industry, and there are
many product claims among organic producers and handlers that organic
products are safer and more nutritious. Only a limited number of studies
14 Microbiology of Fruits and Vegetables
have been conducted comparing conventional versus organic fruit and
vegetable production practices and the effects on product food safety risk.
There is currently no scientific evidence to support claims that organically
grown fruits and vegetables are either safer or pose a greater food safety risk
than conventionally grown produce [17-19].
1.5.1.1 Land Use
The safety of fruits and vegetables grown on any given piece of land is not
only influenced by the current agricultural practices but also by former land
use practices. Human pathogens may persist in soils for long periods of
time [20-22]. There may be increased risk of soil contamination if produc-
tion land was previously used as a feedlot or for animal grazing since fecal
contamination of the soil may be extensive. However, it is difficult to deter-
mine exactly the magnitude of the risk as the persistence of human patho-
gens in soil varies by the pathogen in question, soil type, climate, irrigation
regimes, initial pathogen population numbers, etc. [23].
1.5.1.2 Soil Amendments
Soil amendments are commonly but not always incorporated into agricul-
tural soils used for fruit and vegetable production to add organic and
inorganic nutrients to the soil as well as to reduce soil compaction. Human
pathogens may persist in animal manures for weeks or even months [24,25].
Proper composting via thermal treatment will reduce the risk of potential
foodborne illness. However, the persistence of many human pathogens in
untreated agricultural soils is currently unknown and under extensive
investigation [26-28].
1.5.1.3 Wild and Domestic Animal Control
Wild and domestic animals such as birds, deer, dogs, rodents, amphibians,
insects, and reptiles are known to be potential reservoirs for human patho-
gens and their feces may facilitate the spread of human pathogens in
agricultural production settings, packinghouses, processing, and during
distribution [29-31]. Food processing, warehousing, and distribution facilities
routinely have animal control programs in place to prevent contamination
of fruits and vegetables. However, production agriculture in open fields
is challenged by infestation of wildlife and has only a limited number of
remedies available to deal with periodic infestations by these pests. There
is little or no data available for production agriculture operations to
assess the risks associated with the presence of a particular wild animal
species in production fields, field harvesting equipment, and/or in an adja-
cent field. While a zero tolerance for the presence of wild animals in produc-
tion environs would potentially eliminate the risk of produce contamination,
such operating procedures are simply impractical if not impossible to
implement.
Microbial Contamination of Fresh Fruits and Vegetables 15
1.5.1.4 Irrigation Water
Irrigation water is another potential vector by which contaminants may be
brought in contact with fruits and vegetables. Well water is perceived to be
less likely to be contaminated with human pathogens than surface water
supplies, due to the limited access to sources of potential contamination.
Production agriculture operations routinely test irrigation water sources
for the presence of human pathogens and/or indicator microorganisms.
However, such testing is of only limited value, particularly for flowing
surface water sources, since water tested at any given point in time will not
necessarily be the same water used to irrigate crops in the future. Whenever
water comes in direct contact with edible portions of fruits and vegetables,
particular care should be taken to ensure that the water does not contain
human pathogens. Pesticide application with contaminated water is thought
to be the cause of the 1996 cyclosporosis outbreak associated with fresh
raspberries grown in Guatemala [32-34], and recent research has demonstrated
that commonly used pesticides and fungicides do not significantly affect the
survival or growth of human pathogens [35].
Irrigation water if contaminated with human pathogens may conta-
minate soils, and splashing of soils by irrigation or heavy rain may facilitate
produce contamination [36]. A number of recent studies have also indicated
that fresh produce may be contaminated by root uptake of human pathogens
during irrigation with contaminated water [37,38]. Other research reports
have indicated that this phenomenon does not occur [39,40]. It is currently
unclear if root uptake of human pathogens is a significant source of con-
tamination of fresh produce. However, direct contact of contaminated water
with edible potions of crops is an obvious means of produce contamination by
human pathogens.
1.5.1.5 Harvest Operations
During harvesting operations field personnel may contaminate fresh fruits
and vegetables by simply touching them with an unclean hand or knife blade.
Portable field latrines as well as hand wash stations are routinely made
available and used by harvest personnel. Monitoring and enforcement of field
worker personal hygiene practices such as hand washing after use of field
latrines are critical to reduce the risk of human pathogen contamination on
fresh produce. Due to the potential for contamination, produce once
harvested should not be placed on bare soils before being placed in clean
and sanitary field containers [41]. Field harvesting tools should be clean,
sanitary, and when possible not be placed directly in contact with soil. Harvest
ladders are commonly used to harvest tree fruit and may serve as a potential
source of contamination, if soiled ladder rungs are handled by pickers to
move the ladder. Therefore, ladders should be constructed in a sanitary
manner so as to allow the easy movement of the ladder without the fruit
picker having to grip the ladder rungs. Reusable field harvest containers
16 Microbiology of Fruits and Vegetables
must also be cleaned and sanitized on a regular basis to reduce the potential
for cross contamination.
1.5.2 Food Safety Risk Factors Associated with
postharvest handling of produce
Depending upon the commodity, produce may be field packaged in containers
that will go all the way to the destination market or may be temporarily
placed in bulk bins, baskets, or bags that will be transported to a packing
shed. Employees, equipment, cold storage facilities, packaging materials, and
any water that directly or indirectly contacts harvested produce must be
kept clean and sanitary to prevent contamination.
1.5.2.1 Employee Hygiene
Human beings are a significant reservoir for human pathogens and therefore
gloves, hairnets, and clean smocks are routinely worn by packinghouse
employees and field harvest crews to reduce the potential for contamination
of fresh produce during handling. The cleanliness and personal hygiene of
employees handling produce at all stages of production and handling must be
managed to minimize the risk of contamination. Availability of adequate
restroom facilities and hand washing stations and their proper use are
critical to preventing contamination of produce by employees. Shoe or boot
cleaning stations may also be in place to reduce the amount of field dirt and
potential contamination from field operations that may enter packing sheds,
processing plants, and distribution centers. Employee training regarding
sanitary food handling practices, in a language in which employees are fluent,
is essential to reducing the potential for employees contaminating food
products that they are handling. This is particularly difficult in the produce
industry as employees are often seasonal or temporary contract employees;
thus a strategy of repetitive training is often needed.
1.5.2.2 Equipment
Recent research has demonstrated that unsanitary packinghouse facility
equipment may play a major role in contaminating fresh fruits and vegetables
if packinghouse facility food contact surfaces such as conveyor belts and dump
tanks that convey produce are not cleaned and sanitized on a regularly
scheduled basis with food contact surface approved cleaning compounds
[42,43]. Sanitizers to be effective should be used only after thorough cleaning
with mechanical action to remove organic materials such as dirt or plant
materials. Food processing plants and equipment associated with them are
normally designed with wash-down sanitation in mind. However, sanitary
design of facilities and equipment used to handle raw agricultural commodities
has received only limited attention. Therefore, there are currently no
universally accepted standards for equipment or sanitary design for
facilities that handle raw agricultural commodities. Rough postharvest
Microbial Contamination of Fresh Fruits and Vegetables 17
handling at packinghouse facilities should be avoided to reduce mechanical
damage and punctures to fruit which may allow for the introduction of plant
spoilage pathogens via these wounds, as this has been demonstrated to enhance
the potential for growth and survival of some human pathogens [44].
1.5.2.3 Wash and Hydrocooling Water
All water that comes in contact with produce for drenching, washing, hydro-
cooling, or vacuum cooling must be of sufficient microbial quality to prevent
contamination. Recirculated water should have sufficient quantities of an
approved wash water disinfectant to reduce the potential for cross contamina-
tion of all produce in the drenching, washing, or hydrocooling system. Wash
water disinfectants are not capable of sterilizing the surface of produce.
Research has demonstrated that washing produce in cold chlorinated water
will reduce microbial populations by two or three log units (100- to 1000-fold),
but complete elimination of microbes is never achieved because microorga-
nisms adhere so tenaciously to the surface of produce and may be present in
microscopic hydrophobic areas on the produce surface [2,3] or in inaccessible
attachment sites (stomata, lenticels, punctures). Rinsing produce with water
that contains a wash water disinfectant will significantly reduce the number of
microorganisms present on the produce but it will not remove or inactivate all
bacteria. Human pathogens cannot be completely removed from produce by
washing in cold chlorinated water [20,45]. (See Chapter 17 for more details.)
It is particularly important that water used for hydrocooling produce
be free of pathogenic microorganisms, as when warm produce is placed in
cold water, intercellular air spaces within fruits and vegetables contract,
creating a partial vacuum (pressure differential). This has been demons-
trated to facilitate infiltration of water, which may contain human patho-
gens, into fresh produce items. While this phenomenon is known to be an
important source of plant pathogen infections during postharvest handling
of fruit and vegetables [46-49], only recently has direct evidence been brought
forward to show that human pathogens may enter produce by this same mecha-
nism. In a follow-up investigation of potential sources of imported mango
contamination, Penteado et al. [50] provided evidence that Salmonella spp.
may be internalized in fresh mangoes during simulated postharvest hot water
insect disinfestation procedures which included a water bath cooling step [51].
However, Richards and Beuchat [52] demonstrated that adhering to or
infiltrating of S. Poona cells into cantaloupe tissue via the stem scar is
not dictated entirely by the temperature differential between the melon and
the immersion solution containing salmonella cells, but it is also influenced
by properties unique to tissue surfaces.
1.5.2.4 Cold Storage Facilities
Cold storage facilities and, in particular, refrigeration coils, refrigeration drip
pans, forced air cooling fans, drain tiles, walls, and floors are potential
18 Microbiology of Fruits and Vegetables
harborages for human pathogens and as such should be cleaned and sani-
tized on a frequent and regular basis. Listeria monocytogenes can proliferate
quite slowly at refrigerated temperatures and may contaminate cold stored
produce if condensation from refrigeration units or the ceiling drips onto
produce. Placing warm produce with field heat into a cold room with
insufficient refrigeration capacity will cause a temperature rise in the room
and, as the room cools, a fog or mist may occur. As the water condenses out
of the air and onto surfaces of walls and ceilings that harbor human patho-
gens, contaminated condensate may end up dripping onto the stored produce.
Therefore, it is imperative that sufficient cooling capacity is available
when cooling produce.
1.5.2.5 Packaging Materials
Since packaging materials come in direct contact with fresh fruits and vege-
tables, they may serve as a potential source of contamination. Packages such
as boxes and plastic bags require storage in such a manner as to protect
them from insects, rodents, dust, dirt, and other potential sources of
contamination. All packaging materials cannot be stored inside enclosed
facilities due to space constraints. However, if packaging materials are
stored outside an enclosed building, sufficient precautions should be taken
to reduce the probability of rodent/animal infestation, and measures should be
taken to allow for easily identifiable indicators of an infestation. Plastic field
bins and totes are preferred to wooden containers, since plastic surfaces are
more amenable to cleaning and sanitizing, which should be done after every use
to reduce the potential for cross contamination. Wooden containers or
field totes are almost impossible to surface sanitize since they have a porous
surface. Cardboard field bins if reused should be visually inspected for
cleanliness and lined with a polymeric plastic bag before reuse to prevent
the potential risk of cross contamination.
1.5.2.6 Modified Atmosphere Packaging of
Fresh Produce
The risk of Clostridium botulinum on ready-to-eat modified atmosphere
packaged (MAP) fresh-cut fruits and vegetables has been investigated exten-
sively by a number of research groups in recent years [53-57]. C. botulinum is
a spore-forming bacterium commonly found in agricultural environs. Under
suitable environmental conditions (temperatures above 5°C, low oxygen
conditions, and a pH above 4.6) this microorganism may produce a deadly
toxin. Recent research efforts have examined C. botulinum risk factors
for various fresh-cut MAP produce. In general, overt gross spoilage of fresh-
cut produce occurs well before toxin is produced on shredded cabbage,
shredded lettuce, broccoli florets, sliced carrots, and rutabaga. The endemic
microflora on fresh-cut produce play an important role in signaling the end
of shelf life and are also believed to suppress toxin production by C. botulinum
Microbial Contamination of Fresh Fruits and Vegetables 19
[58]. However, some products such as butternut squash and onions have
been demonstrated under temperature abuse conditions to have the poten-
tial of appearing acceptable although containing botulinal toxin [53]. The
important interaction between MAP and microbial food safety must always
be considered, and continued research efforts to understand fully these
relationships are currently underway. An in-depth assessment of the risk of
botulism contributed by MAP of fresh-cut produce may be found in Gorny
et al. [59]. Several studies at research institutes have found that MAP
technologies commonly used in the fresh-cut industry have varying effects
on the survival and growth of E. coli 0157:H7, Salmonella spp., Shigella spp.,
and L. monocytogenes [60-63]. While some pathogenic strains may be inhib-
ited, others are unaffected, weakly inhibited, or even stimulated. Because
L. monocytogenes can grow at refrigeration temperatures, there is concern that
low inoculum levels, coupled with extended shelf life obtained by the use
of MAP, may allow L. monocytogenes to proliferate to infectious dosages
late in shelf life. The FDA recently reviewed the risk associated with
consumption of fresh fruits and vegetables as well as 20 other ready-
to-eat food categories and published, as a draft, a risk assessment on the
relationship between foodborne L. monocytogenes and human health
(www.fda.gov). Risk from human pathogens due to the use of MAP must be
assessed on a per product basis. This is due to the complex interactions
between the produce, the indigenous microflora, the pathogen, and its
environment. An excellent example of this interaction is the inhibitory effect
of carrot extract on growth of L. monocytogenes [64]. Due to these complex
interactions, broad generalities cannot be drawn regarding the risk of specific
human pathogens on various fresh-cut fruits or vegetables and interactions
with MAP.
1.5.2.7 Refrigerated Transport, Distribution, and
Cold Storage
Produce is best shipped in temperature-controlled refrigerated vehicles.
Maintaining perishables at their appropriate temperature when being trans-
ported to destination markets will extend shelf life. When appropriate,
holding fresh fruits and vegetables at or below 5°C will significantly reduce
the growth rate of microbes including human pathogens. However, cold
temperatures and high relative humidity conditions which are often optimal
for shelf life extension of fresh fruits and vegetables may actually help favor
the viability of some human pathogens such as viral particles.
Trucks used during transportation are also a potential source of con-
tamination from human pathogens. Therefore, trucks should be routinely
cleaned and sanitized on a regular basis, and trucks that have been used
to transport live animals, animal products, or toxic materials should not
be used to transport produce or used only after effective cleaning and
sanitation.
20 Microbiology of Fruits and Vegetables
1.5.3 Food Safety Risk Factors Associated with
Foodservice, Restaurant, and Retail Food
Stores Handling of Produce
In 2003 the FDA collected data via site visits to over 900 establishments
representing nine distinct facility types including restaurants, institutional
foodservice operations, and retail food stores. Direct observations of produce
handling practices were supplemented with information gained from discus-
sions with management and food workers and were used to document the
establishments' compliance status based on provisions in the 1997 Model
FDA Food Code [65].
Failure to control product holding temperatures, poor personal hygiene,
use of contaminated equipment/failure to protect food handling equip-
ment from contamination, and risk of potential chemical contamination were
the risk factors found to be most often out of compliance with the 1997
FDA Model Food Code. The percentages of "out of compliance" observations
for each of these risk factors were found to be: improper holding time/
temperature (49.3%), poor personal hygiene (22.3%), contaminated equip-
ment (20.5%), and chemical contamination (13.5%). Specifically, for the
improper holding time and temperature risk factor, it was found that
maintaining cold holding temperatures at or below 5°C (41°F) for produce
items that are classified as potentially hazardous foods (PHFs) did not occur
in 70.2% of the observed situations. Holding PHFs at or below 5°C (41°F) is
critical to preventing the potential growth of human pathogens, which may
rapidly proliferate on inadequately refrigerated PHFs. Date marking of
refrigerated ready-to-eat PHFs is also an important component of any food
safety system, and it is designed to promote proper food rotation and limit
the growth of L. monocytogenes during cold storage. However, appropriate
date marking of ready-to eat PHF produce items made on-site did not occur
in 34% of the observations.
The personal hygiene risk factors associated with produce that are most
in need of attention at retail and foodservice operations include adequate,
available, and accessible hand washing facilities. These personal hygiene risk
factors were found by the survey to be not in compliance with the 1997
FDA Model Food Code 33.3, 26.2, and 20.6% of the time, respectively. Hands
are very common vehicles for the transfer of human pathogens to food
products, and food handlers' hands may become contaminated when they
engage in activities such as handling raw meat products, using the lavatory,
coughing, or handling soiled tableware.
Food safety procedures for cleaning and sanitizing food contact surfaces
and utensils for handling produce were found to be not in compliance with
the 1997 FDA Model Food Code in 44.4% of the observations in this
study. Proper cleaning and sanitization of food contact surfaces is essential to
preventing cross contamination. The 2004 FDA report clearly indicates that
foodservice and retail operators must ensure that their produce food safety
Microbial Contamination of Fresh Fruits and Vegetables 21
management systems are designed to achieve active managerial control over
the risk factors associated with handling produce identified in the report.
1.5.4 Consumer Handling of Produce from
Purchase to Plate
Li-Cohen and Bruhn [66] in 2002 published the most extensive consumer
handling study of fresh produce from the time of purchase to the plate. Via a
national mail survey of 624 respondents these researchers quantified
consumer produce handling practices as they relate to food safety risk. Six
percent of consumer respondents replied that they never or seldom wash fresh
produce before consumption, and greater than 35% of respondents do not
wash melons before consumption. Approximately half of all respondents did
not wash their hands before handling fresh produce. Ninety-seven percent of
all respondents reported that they always washed food preparation surfaces
after contact with raw meat products. However, 5% of respondents only dry
wipe, and 24% of respondents wash these potentially contaminated food
preparation surfaces only with water (without soap or a disinfectant). This
survey also found that many respondents did not separate produce from raw
meat, poultry, or fish in their refrigerators. These limited observations clearly
indicate the need for educational outreach to consumers that must emphasize
safe handling practices of produce from purchase to consumption.
1 .6 EFFECTIVE MANAGEMENT STRATEGIES:
CONTAMINATION PREVENTION AND
INTERVENTION
Every foodborne illness outbreak is a tragic event, and an approach that
prevents contamination and possible amplification of human pathogens in the
produce supply chain is the most effective means of ensuring fresh produce
safety. However, the complexity of effectively implementing this strategy is
stated concisely by the FDA [16]:
Although the available scientific literature is adequate to identify sources of
contamination and estimate microbial persistence on plants, the specific influence
and interactions among the production environments and crop management
practices are not sufficiently understood to provide detailed guidance to growers
and shippers. Also, the diversity of cropping systems, scale of operation, use and
design of equipment, regional and local practices, environmental influences,
specifics of on-farm soil related factors, and many other production factors
defy any attempt to develop an encompassing assignment of microbial risk to
commodities or to crop management practices.
Sampling produce is not an effective means of ensuring product safety.
Data from the USDA MDP and FDA domestic and imported produce
sampling surveys indicate that human pathogens are found on fresh produce
22 Microbiology of Fruits and Vegetables
infrequently and in low numbers. Because of this fact increased sampling for
the presence of human pathogens by either private enterprises or government
regulators will not effectively reduce foodborne illnesses associated with
produce consumption because it is simply an ineffective strategy. Increased
produce sampling or surveillance would also potentially take valuable limited
resources away from potentially more productive research efforts that identify
risk factors and mitigation strategies.
Approaches that prevent contamination are warranted and these strategies
include effective management and intervention strategies for growing, handling,
distributing, and preparing fresh produce that include but are not limited to:
• Good Agricultural Practices (GAPs)
• Good Manufacturing Practices (GMPs)
• Hazard Analysis Critical Control Point (HACCP) programs
1.6.1 Good Agricultural Practices (GAPs)
The FDA published Guidance for Industry: Guide to Minimize Microbial Food
Safety Hazards for Fresh Fruits and Vegetables in 1998 which has since come
to be referred to as Good Agricultural Practices (GAPs). Although this
document carries no regulatory or legal weight, due diligence requires
producers to take prudent steps to prevent contamination of their crops.
GAPs have been widely implemented by the fresh fruit and vegetable industry
and as formulated provide the produce industry with an excellent description
of broad prescriptive actions that may be taken to enhance produce food
safety. Numerous retail and wholesale buyers have made compliance to GAPs,
and subsequent independent third-party audits to ensure compliance with
GAPs, a requirement for the purchase of fresh fruits and vegetables.
The guide identifies eight principles of food safety within the realms of
growing, harvesting, and transporting fresh produce and suggests that the
reader "use the general recommendations in this guide to develop the most
appropriate good agricultural and management practices for your operation.''
The application of these principles is aimed at preventing contamination of
fresh produce with human pathogens. The eight principles are listed below
followed by areas of implementation:
1. Prevention of microbial contamination of fresh produce is favored
over reliance on corrective actions once contamination has occurred.
2. To minimize microbial food safety hazards in fresh produce, growers
or packers should use GAPs in those areas over which they have
a degree of control while not increasing other risks to the food supply
or the environment.
3. Anything that comes in contact with fresh produce has the potential
of contaminating it. For most foodborne pathogens associated with
produce, the major source of contamination is associated with human
or animal feces.
Microbial Contamination of Fresh Fruits and Vegetables 23
4. Whenever water comes in contact with fresh produce, its source and
quality dictate the potential for contamination.
5. Practices using manure or municipal biosolid wastes should be
closely managed to minimize the potential for microbial contamina-
tion of fresh produce.
6. Worker hygiene and sanitation practices during production, harvest-
ing, sorting, packing, and transport play a critical role in minimizing
the potential for microbial contamination of fresh produce.
7. Follow all applicable local, state, and federal laws and regulations, or
corresponding or similar laws, regulations, or standards for operators
outside the U.S. for agricultural practices.
8. Accountability at all levels of the agricultural environment (farms,
packing facility, distribution center, and transport operation) is
important to a successful food safety program. There must be quali-
fied personnel and effective monitoring to ensure that all elements
of the program function correctly and to help track produce back
through the distribution channels to the producer.
It is currently unclear if recent outbreaks associated with consumption of
produce are due to lack of compliance with GAPs or if there are deficiencies
in GAPs as they are currently formulated. Little scientifically based data
exist regarding the risk associated with many of the production and post-
harvest handling practices commonly used in production agriculture and in
postharvest handling situations or what the most effective risk management
strategies may be.
1.6.2 Current Good Manufacturing
Practices (cGMPs)
The cGMPs are set forth in 21CFR1 10 and provide guidelines that ensure that
food for human consumption is safe and has been prepared, packed, and held
under sanitary conditions. The cGMPs provide food processors, such as fresh-
cut produce processors, with the core principles of sanitary food handling, and
they serve as well-recognized and agreed upon standards of conduct and
operation. The cGMPs are well written in that they provide general guidance
regarding regulatory expectations of performance and conduct without being
overly specific or prescriptive, and this aspect of the cGMPs accommodates the
many diverse specific situations that are encountered in the food industry
today. The regulations as currently written provide flexibility for the diverse
formats under which these regulations are applied, by use of terminology such
as "adequate facilities," "where appropriate," "necessary precautions,'" and
"adequate controls." This flexibility allows the cGMPs to be applied to the
plethora of situations encountered during the production, handling, and
distribution of food products. Also, and very importantly, by not being overly
prescriptive the cGMPs allow for incorporation of new technologies and
innovation without the need to revise the regulations. The cGMPs are the
24 Microbiology of Fruits and Vegetables
commonly agreed upon and scientifically based standards by which industry
and regulators effectively and harmoniously communicate the standards of
performance and conduct when food products are being prepared, packed, or
held. As such the cGMPs are centrally important in reducing the risk of
product adulteration and food safety risk to consumers.
2 1CFR 110.19 specifically exempts raw agricultural commodities from
compliance with cGMPs, and raw agricultural commodity safe production and
postharvest handling practices are not as clearly defined and commonly agreed
upon as cGMPs and HACCP in the food processing industry. Therefore,
raw agricultural commodities producers and handlers do not have the advan-
tage of simply adopting long-standing food safety programs that exist in the
food processing industry, as they must modify these programs on a site-specific
basis.
1.6.3 Hazard Analysis Critical Control Point
(HACCP)
Hazard Analysis Critical Control Point (HACCP) is a systems approach method
to ensure the safety of a food product. The terms HACCP and food safety
program are often used interchangeably and synonymously. However,
HACCP is not the equivalent of a food safety program, as HACCP is merely
a component of an overall food safety program. The terms food safety
program and HACCP are not interchangeable and should not be used
synonymously. A HACCP plan cannot be established without prerequisite
programs such as GAPs, cGMPs, and sanitation standard operating
procedures (SSOPs) being in place. HACCP is a food safety system pioneered
by the Pillsbury Co. to reduce the risk associated with the food eaten by
astronauts for manned space flights. HACCP is a systems approach that:
• Identifies potential sources of contamination in food production
systems.
• Establishes methods for detecting the occurrence or prevention of
contamination.
• Clearly prescribes what corrective actions will be taken to prevent
consumption of contaminated food items.
The National Advisory Committee on the Microbiological Criteria for
Foods (NACMCF) has clearly defined what HACCP is in a 1997 document
entitled HACCP Principles and Application Guidelines (available on line
at: www.fst.vt.edu/haccp97/). In this document, NACMCF clearly defines
seven criteria that must be met by a HACCP program [67]. The seven basic
principles of HACCP are:
1. Assessment of hazards.
2. Determine critical control points (CCPs) to control the identified
hazards.
Microbial Contamination of Fresh Fruits and Vegetables 25
3. Establishment of limits at each CCP.
4. Establishment of CCP monitoring procedures.
5. Establishment of corrective actions to be taken when CCPs exceed set
limits.
6. Establishment of record keeping systems to document the HACCP
program.
7. Establishment of procedures to verify that the HACCP is functioning
properly.
HACCP is described as a management system — designed for use in all
segments of the food industry from growing, harvesting, processing,
manufacturing, distributing, and merchandising to preparing food for
consumption. The NACMCF committee endorsed HACCP as an effective
and rational means of ensuring food safety from harvest to consumption [67].
However, if all of the above criteria cannot be met, then a HACCP plan cannot
be established and HACCP may not be the appropriate food safety solution for
the process under consideration. This does not mean that process hazards
should be ignored but simply that the risks and hazards associated with a
process need to be dealt with via an alternative mechanism. Another important
aspect of any HACCP program is prerequisite ability to monitor quantitatively
critical control points. If one cannot monitor and control important process
critical control points then HACCP is not appropriate. Food safety programs
such as HACCP and cGMPs are well defined and may function well within the
control environs of a food processing plant; however, these food safety
program components may not be appropriate in production agriculture
situations. For example, as food handling operations move from a confined
four-walled food processing facility to a three-walled packinghouse operation
and/or back to an open agricultural growing operation, it is obvious that not
all cGMPs and/or HACCP requisites could possibly be implemented.
The fresh-cut produce industry strongly believes that HACCP is an effect-
ive means of enhancing food safety by control of chemical, physical, and
biological hazards that are reasonably likely to occur in the absence of
controls. HACCP systems may be considered for intact and fresh-cut produce
only when sufficient information and data have been gathered to establish
appropriate preventive control measures (FDA, 1998).
It is unclear if HACCP can or should be used as a component of a
food safety program for production agriculture. HACCP as currently
formulated by NACMCF cannot be used as a food safety program for pro-
duction agriculture. However, risk reduction and mitigation must be eval-
uated and implemented in production agriculture to enhance produce food
safety. See Chapter 15 for a comprehensive discussion of HACCP.
1.7 RESEARCH NEEDS
Everyone in the produce handling continuum must understand the food
risks that they are facing, because if these risks are not clearly understood
26 Microbiology of Fruits and Vegetables
then they cannot be appropriately addressed and managed. Speculative actions
that attempt to reduce produce food safety risks, if incorrect, potentially
take limited food safety resources away from actual risks which have not been
addressed while adding to the perception that the issue has been addressed, and
raising expectations. Enhanced research efforts and financial support are
needed to identify clearly means of intervention and quantify how much risk
is reduced by specific actions, so that limited food safety resources can
most effectively be deployed.
There are a number of food safety issues related to fresh and fresh-cut
produce production and handling that warrant further investigation to gain
a better basic understanding of how human pathogens and produce interact.
A better understanding of this interaction will aid in the development of
intervention strategies and increase the safety of the food supply. Five areas
of research that are of high priority for the fresh and fresh-cut produce
industries are discussed in the following.
1 .7.1 Microbial Ecology of Human Pathogens in
the Agricultural Production Environment
Human pathogens in agricultural/farm environs are typically present in low
numbers and frequency, making their investigation difficult if not impos-
sible. Preventing human pathogen contamination of produce is currently
the most effective means of reducing foodborne illness risk. However, there is
a significant lack of information regarding human pathogens on the farm
and in postharvest produce environments. Understanding the microbial eco-
logy, persistence, niches, harborages, life cycle, and factors affecting survival
and growth of human pathogens in an agricultural/farm environment,
including water and soil amendments, is essential to developing and imple-
menting intervention and control measures to reduce the risk of contaminating
fresh produce.
1 .7.2 Agricultural Water
GAPs rely on management practices that prevent contamination of produce on
the farm and during postharvest handling operations. Water is a significant
potential source of human pathogens in the farm environment. Ensuring that
agricultural water is of sufficient microbial quality for its intended purpose is
critical in ensuring the safety of produce. Therefore, identification of better
methods to determine the food safety risk associated with a particular irrigation
water source for a particular use warrants further investigation. Potential lines
of investigation include identification of indicator organisms that highly
correlate with the presence/absence of viable human pathogens.
1 .7.3 Soil Amendments
Identification of better methods to determine the food safety risk associated
with a particular lot of composted manure to be used as a soil amendment
Microbial Contamination of Fresh Fruits and Vegetables 27
is warranted. Identification of indicator microorganisms that correlate well
with the presence/absence of viable human pathogens is needed. Determination
of the time/temperature history and other composting variables that affect the
survival of human pathogens in compost is also needed.
1 .7.4 Proximity Risk of Potential
Contaminant Sources
No produce operation is an island unto itself. Therefore it is important to
assess risks posed by adjacent agricultural and nonagricultural operations
that are known to be potential sources of human pathogens. Greater under-
standing and quantification of risk posed by such adjacent operations is
needed to formulate strategies to reduce risk. Simply put, how close is too
close? What factors should be contemplated when assessing the risk of adja-
cent operations to agricultural production and postharvest handling opera-
tions, and what mitigation steps would be effective to reduce risk?
1.7.5 Intervention Strategies to Reduce the Risk
of Human Pathogen Contamination of
Fresh Produce
Aqueous-based wash water disinfectants do not achieve significant reductions
in microbial populations of human pathogens on fresh produce. Investiga-
tion of alternative nonaqueous-based disinfectants on produce, such as the
use of vapor phase ozone and chlorine dioxide disinfection technologies,
warrants further investigation.
1.8 SUMMARY
Produce contamination by a multitude of human pathogens can occur
anywhere in the produce continuum from field to fork, and once contamina-
tion occurs, no effective interventions exist to eliminate human pathogens
from fresh fruits and vegetables. Although there are many potential
scenarios for produce contamination to occur, no science-based risk assess-
ment has clearly identified and quantified the risk associated with various
produce handling steps from field to fork. A better understanding of risk
factors associated with produce handling practices is needed, so that more
effective intervention strategies may be developed to enhance produce
food safety and reduce the incidence of foodborne illnesses associated with
fresh fruit and vegetable consumption. To date, a preventative approach
to contamination of fresh fruits and vegetables by the use of GAPs, cGMPs,
and HACCP has proven to be the most effective means of ensuring
produce food safety. It is imperative that public health officials and
industry establish standardized metrics and baseline data regarding produce-
associated foodborne illnesses and the risks associated with various
28 Microbiology of Fruits and Vegetables
handling practices. Data detailing foodborne illnesses associated with produce
consumption must be indexed and standardized to ensure that the data that
are being reported, accurately reflect actual illness incidence trends, and are
not simply reporting anomalies due to increased surveillance, improved
detection techniques, or increased per capita consumption of a specific
commodity. Without the ability to quantify accurately foodborne illness
and compare data over a prolonged period of time, it will be impossible to
measure accurately progress and the efficacy of enhanced produce safety
activities and tactics that are being implemented to reduce the incidence of
produce contamination with human pathogens.
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2
Attachment of
Microorganisms to
Fresh Produce
Robert E. Mandrel I, Lisa Gorski, and
Maria T. Brand I
CONTENTS
2. 1 Introduction 34
2.2 Basic Anatomy and Biochemistry of Roots and Leaves 35
2.2. 1 Rhizoplane 35
2.2.2 Phylloplane 36
2.3 Microbial Flora of Plants 37
2.4 Attachment by Plant Nitrogen Fixing, Epiphytic, and
Pathogenic Bacteria to Plants 38
2.4. 1 Rhizobium spp. (Rhsp) 38
2.4.1.1 Two-Step Model of Attachment 40
2.4.1.2 Attachment Factors 40
2.4.2 Agrobacterium tumefaciens (Agt) 41
2.4.2.1 Agt and Rhicadhesin 43
2.4.2.2 Pili 43
2.4.2.3 Cellulose 43
2.4.3 Ralstonia (Pseudomonas) solanacearum (Rs) 43
2.4.3.1 EPS and LPS 43
2.4.3.2 Type III Secretion System (T3SS) 44
2.4.3.3 Type II Secretion System (T2SS) 45
2.4.3.4 Rs Lectins, Fimbriae, FHA 45
2.4.4 Erwinia spp 45
2.4.5 Pseudomonas spp 46
2.4.6 Xanthomonas campestris (Xc) 47
2.4.7 Azospirillum spp 48
2.4.8 Klebsiella spp 48
2.5 Fungi and Viruses and Plants 48
2.6 Potential Attachment Factors of Enteric Bacterial Pathogens
for Plants 48
33
34 Microbiology of Fruits and Vegetables
2.7 Attachment of Human Enteric Pathogens to Plants and
other Interactions 52
2.7.1 Lettuce and E. coli 0157:H7 52
2.7.2 Tomatoes and Apples and Salmonella enter ica (Se) 54
2.7.3 Sprouts and E. coli 0157 (EcO\51) and Se 54
2.7.4 Cilantro and Se Thompson (SeT) 56
2.7.5 Produce Samples and L. monocytogenes (Lm) 56
2.7.6 Cantaloupe and Se, EcO\51, and Lm 58
2.7.7 Arabidopsis thaliana and EcO\51 and Se 58
2.7.8 Plant-Microbe Biofilms 59
2.8 Conclusions 60
Acknowledgments 61
References 61
2.1 INTRODUCTION
Microbes are crucial to plant life, and, therefore, to the successful
production of produce as a commodity. Plant microbes can be beneficial as
symbionts [1-3], competitors of plant pathogens for biocontrol [4,5], and for
promoting plant growth [6-8]. Indeed, most of the fundamental knowledge
of the biology, microbial ecology, and genetics of plants has been obtained as
a result of studies to understand and prevent plant disease. However, plants
also are vulnerable during growth to microbial pathogens from the environment
(e.g., soil, water, air, amendments). The links between fresh produce/produce
dishes with more than 300 outbreaks in the U.S. since 1990 [9,10], and the
obvious vulnerability of preharvest produce to pathogens in the production
environment, have stimulated similar basic studies of the biology of enteric
pathogens on produce.
The sources of the microorganisms exposed to plant surfaces may be
from the plant seed itself [11-13], and through the initial contact with soil,
irrigation water and air. The microbial communities of the rhizosphere (roots
and the part of the soil affected by contact with roots) and the phyllosphere
(leaves and the environment in contact with leaves, e.g., water, air) of produce
are in constant change due to factors that affect microbes, such as humidity,
temperature, nutrients, UV radiation, insects, and wild animals. Plant tissues
are in close contact with potentially thousands of different species of bacteria,
viruses, and other microorganisms [14]. Fruit and vegetable crops also have a
rich microbial flora, including in many cases coliforms and fecal coliforms
that are unavoidable considering the presence of domestic and wild animals
near production environments [15-18]. Indeed, 190 produce-associated out-
breaks have been documented in the U.S. for the years 1973-1997 [19]. Plant
bacteria have evolved multiple mechanisms suitable for initiating interac-
tions essential for successful colonization of plants [3,20,21]. However, a major
interest of those working on microbial food safety of produce is whether there
Attachment of Microorganisms to Fresh Produce 35
are equivalent, or similar, mechanisms of attachment used by human patho-
gens that contaminate produce commodities.
Considering the analogous or common secretion systems, outer surface
proteins, and polysaccharides among the plant and human pathogens, and
similarities among disease-associated genes of humans and plants (e.g.,
Arabidopsis thalianci), this area of investigation perhaps offers more promise
than many plant and animal pathogen researchers perceived initially [22].
An ultimate goal of studies of attachment of human pathogens to plants is
the development of intervention methods to minimize attachment and survival
of human pathogens.
Our goal in this chapter is to review current knowledge of perhaps the
most important event that initiates the association between most microorgan-
isms and plants: attachment. We focus our attention on studies that provide
insight into fundamental molecular plant-microbe interactions. The most
definitive knowledge on plant-microbe interactions involving attachment has
been provided in two areas: (1) mechanisms of disease of bacterial plant
pathogens, and (2) molecular mechanisms involved in the symbiotic relation-
ship between nitrogen-fixing bacteria and plants. These studies provide a
context for assessing the potential mechanisms of attachment of enteric human
pathogens to produce.
2.2 BASIC ANATOMY AND BIOCHEMISTRY OF
ROOTS AND LEAVES
2.2.1 Rhizoplane
The rhizoplane is the area of the root interfacing with soil. Root tips and root
hairs are immersed in mucigel, a substance created by the combination of
plant-secreted mucilage (composed of pectins and hemicelluloses) and com-
plex polysaccharides produced by bacteria that also degrade mucilage [23].
Bacteria are exposed to the mucilage when the cuticle covering it on root hairs
is punctured or degraded physically or chemically. Depending on the plant,
root exudates contain a variety of substances that can act as chemoattract-
ants for microorganisms and/or substrates for growth. Sugars, amino acids
and other amino compounds, organic acids, fatty acids and sterols, growth
factors, nucleotides, and other compounds are produced from the aging
epidermal cells [24]. Most natural rhizosphere bacteria attach to specific
regions of the root; root hairs are a common site of attachment of the rhizobial
bacteria involved in nitrogen fixation [25,26], and, possibly, attachment of
human enteric pathogens [27]. Figure 2.1 shows a general schematic of a region
of a plant root illustrating an emerging root hair (Figure 2.1 A). As noted
above, epidermal cells comprise the surface of the root tissue as it develops
with specific epidermal cells becoming root hair cells; cortex cells compose
a second layer under epidermal cells (Figure 2.1G). After attachment of
certain nitrogen-fixing bacteria such as the rhizobia, root hair cells are induced
36
Microbiology of Fruits and Vegetables
FIGURE 2.1 (Color insert follows page 594) Anatomy of a root hair. Schematic
representation of structures that are part of the anatomy of a plant root hair with
attached bacteria. (A) Root hair epidermal cell; (B) nucleus; (C) bacteria bound to
epidermal cell surface in aggregates and as biofilm; (D) rhizobial bacteria; (E) root hair
infection thread initiated by rhizobial bacteria; (F) curling root hair tip; (G) cortex cells;
(H) junction between root epidermal cells with attached bacteria; (I) bacteria binding as
single cells, then aggregating; (J) magnification of I (not drawn to scale): J-l, single
bacterial cell binding by pili/fimbriae; J-2, plant lectins interacting with bacterial
carbohydrate (e.g., EPS, LPS, CPS, cellulose fibrils); J-3, bacterial flagellin interacting
with plant receptor (e.g., polysaccharide); (K) lesion produced by plant pathogen.
to curl, initiating the complex process of thread formation within the root
hair (Figure 2. IE).
2.2.2 Phylloplane
The phylloplane is the interface between the leaf and the environment.
Epidermal cells compose the upper and lower surfaces of a leaf (Figure 2.2B),
and are covered by the cuticle, which is composed of a polymer matrix
(cutin), polysaccharides, and associated waxes. The cuticle acts as a barrier and
prevents water loss from the leaf (Figure 2.2A). The cuticular waxes are
lipophilic long-chain fatty acids (20 to 40 carbons); some fatty acids are
oxygenated forming aldehydes, ketones, sterols, and esters [28,29]. The waxes
in leaves are mostly saturated and, therefore, highly resistant to degradation
by most microorganisms. However, an important part of the microbial ecol-
ogy of plants, and of the phyllosphere in particular, are fungi that secrete
cutinases that degrade leaf waxes [30]. Lesions in the cuticle can expose
potential sites of attachment for other microorganisms (Figure 2. IK and
Figure 2.2L). In addition, some bacteria, including epiphytic bacteria,
probably adhere to the cuticle and interact with the plant and obtain nutrients
Attachment of Microorganisms to Fresh Produce
37
FIGURE 2.2 (Color insert follows page 594) Anatomy of the cross-section of a leaf.
Schematic representation of structures that are part of the anatomy of most plant leaves
and attached microorganisms. (A) Cuticle layer; (B) upper epidermis; (C) palisade
parenchyma; (D) vascular bundle composed of phloem and xylem; (E) biofilm
composed of bacteria and other microorganisms; (F) EPS; (G) stomates within upper
and lower epidermis; (H) trichome; (I) cuticle; (J) free bacteria and other microorgan-
isms within water droplet; (K) recessed area between epidermal cells; (L) biofilm on
underside of leaf forming a lesion into the vascular system.
without damaging the surface [31]. Depressions formed at junctions of
epidermal cells appear to have thinner cuticles, with microorganisms often
residing at these sites (Figure 2.2B).
The upper and lower surfaces of plants are not considered favorable
to microbes because of the cuticle and the rapid and repetitive fluctuations in
the physicochemical conditions (e.g., humidity, temperature, leachates) to
which microbes must adapt [32,33]. Many of the nutrients present in root
exudates noted above have been detected also in leaf exudates [34]. Some of
the chemicals in leaf exudates/leachates are likely chemoattractants, inducing
movement of the microbe towards a closer interaction that may involve
attachment [35].
2.3 MICROBIAL FLORA OF PLANTS
The unique physical and biochemical qualities of each plant surface as a result
of the plant genotype and of responses to environmental stimuli (light,
temperature, humidity, atmosphere, pH, soil) are major determinants of the
plant microbial community. Large differences in the types and numbers of
bacteria can occur on different plants, leaf-to-leaf of the same plant [36], and
leaf-to-leaf seasonally and even daily [32,37]. Bacteria usually colonize
38 Microbiology of Fruits and Vegetables
and/or are present in areas of a leaf that retain water and are protected from
UV light [38].
Early studies to quantify the populations of bacteria on vegetables repor-
ted that >2 x 10 6 predominantly Gram-negative CFU/g could be isolated
from the outer leaves of cabbage, in contrast to 4 x 10 bacterial CFU/g that
were isolated from inner leaves [39]. The results of numerous studies have
been in agreement with these findings, with viable aerobic bacteria ranging
between 32 CFU/g on inner leaves of lettuce to 10 7 CFU/g on spinach or
peas in warm, humid conditions [38]. Washing vegetables in water usually
will decrease the number of bacteria present only marginally (2.5- to 3-fold),
reflecting the relatively tight attachment of bacteria to the surface. Viable
bacteria have been detected in the interior tissue of cucumbers and tomatoes,
locations that had been assumed to be sterile [38]. Thus, bacteria interact
with plants by mechanisms that probably involve significant movement of
bacteria on tissues, and subsequent attachment, and in some cases entry into
tissues (possibly endophytic). These events are in contrast to the infiltration
of bacteria into cut or damaged plant tissue, or into natural openings in
fruits and vegetables like pores, stems, and calyx during food processing [40].
The internalization of human pathogens in produce is of great concern,
regardless of when it occurs (pre/postharvest), because it would decrease the
effectiveness of any disinfection steps to minimize contamination [40].
Many microorganisms are present in the plant rhizosphere and phyllo-
sphere, including bacteria, fungi, protozoa, nematodes, and viruses. In add-
ition, many different bacterial genera have been isolated from fruits and
vegetables (Table 2.1) [17]. Although most of these organisms probably
interact by specific and different mechanisms with plants, little is known about
the interactions involved. As noted, most of the fundamental and defini-
tive studies of plant-microbe interactions involve Gram-negative plant
epiphytic or pathogenic bacteria. A review of the literature related to this
subject provides a context for assessing observations obtained from very
preliminary studies of human pathogens in similar environments. In addition,
it is probable that some of the human pathogens use similar and/or modified
mechanisms of attachment. Thus, attention to any similarities in human
pathogens and plant bacteria location on plants, biochemistry of bacterial
outer surface structures, and possible adhesins, could provide clues for more
fundamental studies.
2.4 ATTACHMENT BY PLANT NITROGEN FIXING,
EPIPHYTIC, AND PATHOGENIC BACTERIA
TO PLANTS
2.4.1 Rhizobium spp. (Rhs?)
The process whereby bacteria communicate with leguminous plants (e.g.,
soybean, bean, pea, peanut, lentil, chickpea, alfalfa, clover) and become
TABLE 2.1
Putative Attachment Factors Described for Epiphytic Bacteria and Plant Pathogens
Attachment factor(s) J
Ref.
Type of bacteria
Nitrogen-fixing
Rhizobium japonicum
Bradyrhizobium japonicum
Rhizobium trifolii
Rhizobium leguminosarum
Rhizobium leguminosarum bv. trifolii
Rhizobium leguminosarum bv. trifolii
Rhizobium meliloti (Sinorhizobium)
Plant pathogens
Agrobacterium tumefaciens
Agrobacterium tumefaciens
Agrobacterium tumefaciens
Agrobacterium tumefaciens
Agrobacterium tumefaciens
Erwinia carotovora
Erwinia chrysanthemi
Erwinia chrysanthemi
Klebsiella aerogenes
Pantoea stewartii
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Pseudomonas jiuorescens
Pseudomonas jiuorescens
Pseudomonas syringae pathovars
Ralstonia solanacearum
Ralstonia solanacearum
Ralstonia solanacearum
Xanthomonas campestris
Xanthomonas campestris pathovars
Xylella axonopodis
Xylella fastidiosa
Xylella fastidiosa
Epiphytic/biocontrol bacteria
Azospir ilium spp.
Azospirillum brasilense
Azospirillum brasilense
Pseudomonas jiuorescens
Pseudomonas jiuorescens
Pseudomonas putida
a EPS, exopolysaccharide; CPS, capsular polysaccharide; LPS, lipopolysaccharide; Hrp, hypersensitive
response and pathogenicity; HecA, homologous to FHA; FHA, filamentous hemagglutinin.
b Gal, galactose; Lac, lactose.
c Secreted from bacteria and binds to carbohydrate on bacteria.
d P. aeruginosa can be both a human and a facultative plant pathogen.
e PA-IIL has high affinity for L-fucose.
FHA, filamentous hemagglutinin; based on gene homologs in different pathovars.
8 RSL has affinity for L-fucose > L-galactose > L-arabinose/D-fructose/D-mannose; RS-IIL has high
affinity for fructose and mannose.
EPS
49
BJ38 lectin (Gal/Lac) b
52
CPS
25
Rhicadhesin
21, 26
LPS
50
RapAl c
53
Nexl8
187
LPS
64
T-pilus, F-conjugation factor
188
Rhicadhesin
21
att-encoded proteins
60
CPS
61
Type 1 fimbriae, Hrp
88, 188
Hrp proteins
188, 189
HecA (FHA)
85
Type 3 fimbriae
111, 188
Hrp
188
Type IV pili
104
PA-IIL lectin 6
190
Type II pseudopilus
81
Type III?
95
Type IVB pili, Hrp
188
Type IVB pili, Hrp
188, 191, 192
Type IVB pili, Hrp
79,188
FHA homologs 1
80
RSL and RS-IIL lectins 8
83
Type IVB pili, Hrp
105, 188
FHA homologs
106
Flagellum, type IV pili
193
Type IVB pili?
188
FHA homologs
194
EPS and CPS
108
Flagellum (polar)
78, 109
MOMP
110
Fimbriae
195
Flagella
97
Agglutinin?
99
40 Microbiology of Fruits and Vegetables
endosymbionts involved in nodule formation and nitrogen fixation on roots
provides the most advanced model of plant-microbe interactions. Research
continues on understanding the fundamental steps involved [3]. It involves
specificity between Gram-negative Rhsp and the host [41], including the site
on roots where it is initiated [42]. The early steps of the process do not
involve attachment; chemical signals are released from the plant, inducing
bacterial genes that encode the release of corresponding signals to the plant
that induce nodule development on roots. Rhsp then adhere tightly to the
surface of the curling tips of root hair cells [21,42].
2.4.1.1 Two-Step Model of Attachment
The general consensus model that describes Rhsp root attachment proceeds
in two steps (Figure 2.1) [21]. Chemicals released by the plant (e.g., flavonoids)
induce movement of bacteria by chemotaxis towards chemicals exuding
from the root (Figure 2. ID) [3]; this results in close contact between the
roots and the bacteria and initiation of attachment [21,43]. The first step in
attachment involves a bacterial Ca -binding protein called rhicadhesin
(~ 14,000 Da) which is responsible for attachment of mostly single (not
aggregated, Figure 2.1C) bacterial cells directly to the root hair (Table 2.1) [43].
Growth of the bacteria under low Ca conditions decreases direct attach-
ment considerably [44], possibly due to the release of rhicadhesin under low
Ca 2+ [21]. The second step in attachment involves bacterial synthesis of
cellulose fibrils that bind rhicadhesin leading to auto-aggregation, and/or
firm binding of other bacteria at the site of infection [45]. Under carbon-
limitation, R. leguminosarum bv viciae cells form aggregates on root hair tips
by attaching to other rhizobia cells (Figure 2.11 and J).
2.4.1.2 Attachment Factors
Rhicadhesin inhibits the attachment of many Rhizobiaceae spp. to pea root
hair tips, including R. leguminosarum biovars, R. meliloti, R. lupini,
Bradyrhizobium japonicum, as well as Agrobacterium tumefaciens (Agt) and
A. rhizogenes, indicating that rhicadhesin or rhicadhesin homologs are part of
a common mechanism of attachment to root hairs [43]. When Mn
concentration is limiting, the bacterial cells attach and form aggregates
also, but apparently this process is accelerated by a pea plant lectin that
binds to a carbohydrate receptor/ligand on the bacteria (Figure 2.1 J) [46].
Transgenic alfalfa plants transformed with pea lectin, bound B. japonicum, and
R. leguminosarum better than did untransformed lines [47]. Expression of
rhizobium exopolysaccharide (EPS) has been reported to be essential for
infection thread entry into the root hairs, possibly due to lectin-carbohydrate
interactions [47]. Earlier studies reported that EPS, and not lipopolysac-
charide (LPS), was the probable bacterial receptor responsible for specific
interactions between R. japonicum cells and soybean root hairs by means of
a soybean lectin (Figure 2.1J-2) [48,49].
Attachment of Microorganisms to Fresh Produce 41
LPS and lipooligosaccharides (LOS) are potential attachment factors as
receptors for lectins expressed by the plant host, since they are prominent cell-
surface glycoconjugates in Gram-negative bacteria. Plant-microbe studies
indicate that EPS and LPS/LOS both are possible receptors for plant lectins,
and can be highly variable within a population of cells depending on the
environment and mechanism of gene expression. In two studies, LPS of
R. leguminosarum was abundant on cells during their attachment to the
rhizoplane of Zea mays compared to cells present in the root cortex [50];
a 38,000 M r cell surface lectin in B. japonicum (BJ38), inhibitable by lactose
and galactose, also was identified [51,52]. Thus, these separate results define a
candidate bacterial carbohydrate receptor and a bacterial lectin (possibly pili)
that are involved putatively in attachment to plant lectins (Table 2.1) [50], or
plant carbohydrates (Table 2.1 and Figure 2.1 J), respectively [51,52].
In an attempt to clone the rhicadhesin gene of R. leguminosarum bv.
trifolii, a unipolar cell surface protein, RapAl, was identified that bound to
cognate carbohydrates on the bacteria [53]. The RapAl protein was pro-
posed to be a bacterial lectin. The unipolar location of the lectin and
activity in agglutination/aggregation of bacteria indicated it is similar to the
B. japonicum BJ38 lectin described above (Table 2.1). In an earlier study, polar
attachment of R. trifolii by the clover root hair lectin trifolin A to bacterial
extracellular microfibrils composed of capsular polysaccharide (CPS) was
described [25]. Thus, it is speculated that these bacterial lectins are capable of
recognizing carbohydrate structures present both on the bacteria and plant
root hairs [53], a binding activity that could result in both aggregation of
bacteria and attachment of bacteria (possibly singly or as aggregates) to root
hairs (Figure 2.11 and J). The expression of RapAl was stable at any growth
phase, but the expression of the bacterial receptors was highest during
exponential phase of growth, corresponding to higher agglutination [53]. These
results reflect the dynamic state of the rhizobial cell surface, which changes
due to growth phase and contact with plants, and affects attachment.
2.4.2 Agrobacterium tumefaciens (Agt)
Agt is a Gram-negative bacterium that when inoculated on wounded dico-
tyledonous plant tissue causes crown gall tumors by transferring a portion
(T-DNA) of a resident plasmid (Ti-plasmid) into the plant [54]. The essential
nature of Agt attachment to plant wound tissue for Agt root transformation
in a pinto bean leaf model was first reported by Lippincott and Lippincott
[55]. Subsequent studies have identified multiple Agt mutants or strains
defective in attachment to different plant tissues [56-63], although for many
of the mutants the functions of the predicted proteins have not been identified
or characterized.
Whatley et al. reported that both LPS on Agt cells and purified LPS
inhibited specifically Agt tumorigenic activity on pinto bean leaves by > 50%
[64]. Although specific genes or gene products were not identified, the authors
suggested that LPS interacts with the sites of attachment on the leaves.
42 Microbiology of Fruits and Vegetables
Five Agt Tn5 transposon mutant strains unable to attach to carrot cells in
suspension (10 7 bacteria/ 10 5 cells), and non-tumorigenic on carrot disks and
wounded bean leaves, were identified with an associated loss of 33, 34, and
38 kDa proteins [65]. Revertants of the nonattaching mutants were isolated
and shown to have regained virulence and ability to attach, confirming the
involvement of the proteins in attachment. In addition, LPS purified from the
parent and each of the mutant strains, inhibited by 30 to 60% the attach-
ment of Agt to carrot cells, supporting the hypothesis that LPS plays a role
also in attachment [65]. Agt biovar 3 (A. vitis), which is predominantly iso-
lated from grapes and causes root decay, produces a polygalacturonase, that
appears to function by modifying specifically grape root cells in a manner that
increases attachment of the biovar 3 Agt [58].
In attachment (att) mutants characterized by Matthysse et al. [60], open
reading frames (ORFs) were identified that have homology to genes encoding
the membrane-spanning proteins of periplasmic binding protein-dependent
(ABC) transporter systems and ATP-binding proteins of Gram-negative
bacteria, and to an ORF in an operon of Campylobacter jejuni associated
with attachment. These results do not identify a specific attachment factor;
rather they suggest other mechanisms involved in attachment, including
secretion in or out of cells of a substance required to condition the medium
for bacterial attachment, or ATP-transporter-dependent transfer of plant
signals into bacteria with induction of a substance important for attachment.
One of the attachment mutants was mutated in a gene, attR, homologous to
bacterial transacetylase genes [61]. The attR mutant strain lacked an acety-
lated CPS present in the parent strain Agt C58, and consequently did not
attach to wound sites and was avirulent for legumes and nonlegumes [63].
The attR mutant strain also did not attach to root hairs and root epidermis
of nonlegumes, but did attach to these areas on legumes (alfalfa, bean, pea).
These results suggested that attR plays a role in binding of Agt to, and
in colonization of, root hairs on nonlegume plants, but that attR has no role
in colonization of root hairs on legume plants [63]. Thus, two systems for Agt
attachment and colonization are available and may function depending on
the plant species.
A polysaccharide purified from the water-soluble fraction of a phenol-
water extraction of Agt strain C58 cells inhibited the attachment of Agt to
carrot cells. The extracted polysaccharide was acidic, acetylated, and com-
posed of glucose, glucosamine, and an unidentified deoxy-sugar [61]. Inter-
estingly, the ligand in carrot cells that binds the Agt polysaccharide may be
a homolog of vitronectin (S protein), a serum-spreading factor in animals
and part of the extracellular matrix [66]. The vitronectin-like protein was
detected immunochemically as present on the surface of carrot cells [66], and
was detected previously on tomatoes, soybeans, and broad beans [67]. If Agt
is bound to plant cell vitronectin, and vitronectin is linked by integrin to actin
in the cytoskeletal network as it is in animal cells, this would provide an
intimate contact for initiation of the crucial step of transport of Agt T-DNA
and proteins to the nucleus of plant cells [66].
Attachment of Microorganisms to Fresh Produce 43
2.4.2.1 Agt and Rhicadhesin
Rhicadhesin, noted above as a proteinaceous attachment factor for R.
leguminosarum (Table 2.1), was reported to be important in attachment of
Agt to pea root hair tips [59]. However, attachment by rhicadhesin in this
system was dependent upon sufficient expression in Agt of cyclic (3-1,2-glucan,
an osmoregulating molecule synthesized by the chvB encoded protein [59].
2.4.2.2 Pili
Although there are several Agt virulence proteins suspected of interacting
with proteins on different plants [68], a strong candidate for having a role
in attachment of Agt to plants is a pilus, the structure of which in Agt is
composed predominantly of multiple copies of the VirB2 protein (propilin)
[69,70]. The mature pilus is expressed as ~10 nm diameter filaments on the
cell surface and is required for transformation, presumably, by interacting
with plant cell wall or membrane molecules [68]. This conjugative T-pilus has
significant sequence homology to the conjugative F-pilus of E. coli [70].
Mutants produced in Arabidopsis thaliana by T-DNA insertion revealed
plant lines resistant to Agt transformation (rat) and modified in Agt attach-
ment to root hairs: rati, which encodes an arabinogalactan-related enzyme,
and rat3, which encodes putatively a plant cell wall protein [68]. Therefore,
both carbohydrate and proteins of the plant are implicated as receptors for
Agt pili.
2.4.2.3 Cellulose
Previous to these descriptions of the mechanisms of attachment of Agt to
plant cells and their role in transformation, Matthysse et al. described the
synthesis of cellulose fibrils by Agt induced by the attachment of the bacteria
to carrot cells [71]. Although the cellulose fibrils appeared not to be necessary
for initial attachment, they were shown to be important in anchoring Agt and
associated bacteria to the plant cell surface, and enmeshing Agt in aggregates
associated with tumor formation [71,72]. An 11 kb region (celABCDE) was
identified containing two operons involved in cellulose synthesis [73]. Thus,
cellulose is important in establishing a stable and perhaps more complex
interaction of Agt with plant tissue subsequent to attachment.
2.4.3 Ralstonia (Pseudomonas)solanacearum (Rs)
2.4.3.1 EPS and LPS
Rs is a Gram-negative soilborne plant pathogen (PP) that infects more than
200 species of plants including fresh produce-related plants like tomato,
potato, eggplant, banana, and papaya, and causes bacterial wilt disease [74].
Rs enters the root tissue and invades the plant through the xylem, then moves
44 Microbiology of Fruits and Vegetables
through the vascular system into the aerial parts of the plant (Figure 2. IK).
EPS and LPS were identified as major cell surface molecules associated with
virulence of Rs, perhaps functioning by blocking the xylem vessel and pre-
venting water movement [75]. Sugars identified in composition analyses of EPS
include different proportions of ^-acetylgalactosamine, glucose, rhamnose,
basillosamine, and uronic acids [75]. At least one Rs LPS O-antigen was char-
acterized chemically and reported to contain rhamnose, 7V-acetylglucosamine,
and xylose [76]. Mutations in Rs EPS genes (ops gene cluster) modified
unexpectedly the synthesis of both EPS and LPS, and corresponded to a signi-
ficant decrease in the ability of Rs to attach to (presumably), and to infect,
two-week-old axenic eggplant seedlings (inoculated in cotyledon) and three-
week-old eggplant plants (inoculated in leaf stem). Although five of seven
complemented ops mutants had nearly their full virulence restored, no
association of LPS or EPS with attachment to the plant tissue was defined [75].
2.4.3.2 Type III Secretion System (T3SS)
Many of the Gram-negative bacterial plant and animal pathogens described
in this chapter produce a T3SS, which has been shown in multiple systems to
be crucial to the delivery of multiple virulence factors into the extracellular
milieu, but more importantly, directly into plant and/or animal cells. The T3SS
was discovered by characterization of gene clusters present in pathogenicity
islands and in large plasmids with similarities to flagellar assembly genes [77].
The common genes and functions of the T3SS in plants and details regard-
ing the assembly of pilin and avirulence (avr) genes are provided in an
excellent review [77].
The T3SS in plant pathogenic bacteria involve the hrp (hypersensi-
tive reaction and pathogenicity) genes [77]. /*r/?-related genes that are relevant
to attachment of bacteria to plants include those involved in the synthesis
of the novel Hrp pilus. The potential role of the Hrp pilus in delivery of
bacterial proteins into plant cells by direct interaction suggests that it also
is an attachment factor for other plant pathogens and human pathogens
with T3SS (e.g., Pseudomonas, Erwinia, Xanthomonas, Ralstonia, Salmonella,
Shigella, Yersinia). However, experiments with Rs in a tobacco plant cell
co-culture model indicated that T3SS-encoded pili, composed mainly of Hrp Y
protein, had no role in attachment [78]. An interesting finding was the
observation of the HrpY pili and fimbriae concentrated at the same end of the
Rs cells, perhaps indicating that a unipolar location was important
biologically, possibly in attachment with plants other than tobacco. However,
in another study, Rs mutants lacking Hrp pili retained twitching motility
and, by electron microscopy, a different polarly located pili structure was
observed [79]. This Rs pilus is a 17 kDa protein encoded by pilA, and 46%
identical to P. aeruginosa type IV pilin. Rs type IV pili were shown in this study
to have a role in autoaggregation, biofilm formation on plastic surfaces, and
transformation. However, a PilA - mutant retained capability to bind to
tobacco cells and to tomato roots, but in a nonpolar fashion, indicating that
Attachment of Microorganisms to Fresh Produce 45
PilA has a qualitative role in attachment [79]. The lack of a quantitative
effect on attachment of Rs lacking either Hrp pili (type III) or type IV pili
to tobacco or tomato plant cells/roots indicates that their major role may be
more relevant in natural plant environments (e.g., nutrient acquisition, genetic
exchange, and movement and biofilm formation in xylem).
2.4.3.3 Type II Secretion System (T2SS)
Multiple T2SS loci have been identified also in the genome sequence of Rs
strain GMI1000 [80]. In P. aeruginosa, the T2SS produces bundled fibrils
called type II pseudopilins that increase adherence of the bacteria to plastic
surfaces and are involved in production of biofilms [81]. The Rs and other
species T2SS-encoded fibrils may have a role in attachment to plants.
2.4.3.4 Rs Lectins, Fimbriae, FHA
Two Rs protein lectins with potential roles in attachment have been
characterized recently: RSL (9.9 kDa subunit) with activity/specificity for
L-fucose > L-galactose > D-arabinose; and RS-IIL (11.6 kDa subunit) with
activity/specificity for D-fructose and D-mannose [82,83]. The RS-IIL is similar,
but not identical, to the PA-IIL lectin described for P. aeruginosa. The activity
of the RS-IIL lectin for sugars also prominent in plant cell walls has stimu-
lated studies of the role of Rs lectins in attachment to more relevant and
complex plant glycoconjugates [83].
Finally, multiple ORFs identified in the Rs genome strain GMI1000 are
similar to nonfimbrial adhesins or hemagglutinin (e.g., FHA) molecules, some
of which promote strong adhesion to mammalian cells [80,84]. Future work
is necessary to determine whether these proteins have any role in attachment
of Rs to plants in a complex soil environment.
2.4.4 Erwinia spp.
The soft-rot pathogen Erwinia chrysanthemi (Echr) expresses an adhesin
(HecA) that has homology to filamentous hemagglutinins (FHA) expressed
in both plant and animal pathogens (Table 2.1) [85,86]. A hecA mutant of
Echr had decreased virulence in seedlings of a particular tobacco cultivar,
but not other cultivars or plants, indicating a relatively specific attachment
[85]. Observation of green fluorescent protein (GFP)-labeled mutant and
wild-type strains by confocal microscopy illustrated that the mutant cells did
not aggregate by end-to-end attachment, nor attach to seedling roots, nearly as
well as the wild-type cells. Attachment of the mutant strain to the leaf
surface was decreased dramatically and no Echr aggregates on leaves were
observed. Thus, at least with the specific tobacco cultivar described, HecA
appears to function as an important Echr adhesin [85].
Cell suspension cultures of Gypsophila paniculata ("baby's breath") leaf
segments with a pathogenic strain of Erwinia herbicola pv. gypsophilae
(Ehg) resulted in a greater than five-fold increase in plant cell aggregation
46 Microbiology of Fruits and Vegetables
compared to a nonpathogenic Ehg strain, indicating attachment had occurred;
electron microscopy revealed intimate attachment by a possible "'bridge"
between Ehg and the plant surface [87]. However, no attachment molecules
related to this interaction were identified.
Both Echr and E. carotovora (Ecar) possess hrp genes encoding a T3SS,
but their role in virulence of Ecar had been unclear [88]. Recently, mutations
in Hrp system structural genes confirmed that T3SS proteins are required for
full virulence of Ecar for potatoes [89]. Erwinia amylovora, the cause of fire
blight in many plants, was observed by scanning electron microscopy (SEM)
at regions on and in plant leaves, on the epidermis around detachment sites
of leaf hairs, and on stems and roots of apple seedlings [90]. Although inti-
mate interactions between the T3SS Hrp pili and Hrp virulence proteins of
E. amylovora have been observed by transmission electron microscopy
(TEM) [91], no evidence for direct attachment of the pilus to host cells has
been described. Finally, the in vitro specificity of a presumed Erwinia rhapontici
(pathogenic for rhubarb) lectin for 7V-acetyllactosamine (galactose-pl-
4N-acetylglucosamine) would be intriguing if it were related to an attach-
ment factor for plants, but it is apparently nonfimbrial, and no attachment
factor has been described [92].
2.4.5 PSEUDOMONAS SPP.
Pseudornonas syringae (Ps) causes disease in more than 80 plant species,
including many important produce-related plants [32]. Ps has been studied
extensively as both a pathogen and an epiphyte on plant leaves. Attempts
to remove epiphytic populations of bacteria on leaves, including Ps, by
vigorous washing and sonication revealed that: (1) some bacteria were bound
more strongly than others, (2) the phylloplane was heterogeneous relative to
optimal attachment/colonization, and (3) pili-minus Ps strains were washed
more easily from leaves compared to wild-type Ps [32,93,94].
P . fluorescens (Pf) strains colonize roots and are important competitors for
biocontrol of plant pathogens [95], and also possibly of human pathogens [96].
Flagellin was shown to be important in motility of Pf for chemotaxis and
colonization of potato roots [97]. Piliated/flmbriated strains of Pf were
shown to bind to roots of corn seedlings better than a nonpiliated variant strain
[98]. Fimbriae/pili (34 kDa) purified from Pf strains also bound to the roots.
The fibrillar nature of the pili suggests that they may be T2SS pili (see below).
Pf hemagglutination activity was inhibited by all sugars representative of
those present in plant root exudates, thus indicating carbohydrate-specific
binding activity.
Pseudornonas putida (Pp) is common in soil and acts as a plant growth
promoter and suppressor of fungal pathogens. A kidney bean root surface
glycoprotein was described that agglutinated Pp cells; agglutination-
negative Pp mutants were reported to be 20- to 30-fold less effective in
attaching to root surfaces of seedlings [99]. Motility was shown to be associ-
ated with efficient Pp attachment to sterile wheat roots in a simplified model
Attachment of Microorganisms to Fresh Produce 47
system, implicating flagellin as a potential attachment factor [100]. However,
no definitive attachment factor was identified for Pp in these two studies.
P. aeruginosa {Pa) provides a good example of a species that can be a
nonpathogenic or pathogenic organism in plants and animals [101,102]. This
broad pathogenesis for different hosts reflects the conservation of virulence
mechanisms for attacking quite different hosts, including possibly mecha-
nisms of attachment [103]. Pa is very relevant also to fundamental studies of
the biology of enteric human pathogens in produce; it may provide clues
about mechanisms of attachment, communication, and invasion, important
for development of methods for minimizing both human and plant pathogens
on plants.
The type IV pilus has been described as the most important "virulence-
associated adhesin" of Pa [104]. However, this is due to the emphasis on
characterization of attachment of Pa to specific glycosphingolipids of mam-
malian epithelial cells. In plant models, differences in Pa attachment to leaves
of different arabidopsis ecotypes have been reported [102]. Pa cells were
observed attached perpendicularly to, and degrading regions of, the surfaces of
leaves; in other regions, cells bound to trichomes in multiple layers probably
as biofilms. The perpendicular orientation noted for Pa cells on arabidopsis
surfaces is reminiscent of the unipolar location of rhicadhesin, of rhizobium,
and of Hrp pili and fimbriae of Rs (described above), and suggests that an
attachment factor may be localized similarly. The movement of Pa cells
(possibly by type IV pili twitching motility) towards stomatal openings, entry
into them, then attachment to host cell walls, reflects pathogenesis similar
to erwinia and ralstonia pathogens [102].
2.4.6 Xanthomonas campestris (Xc)
Type IV-encoded bundle-forming fimbriae have been characterized in Xc
pv. vesicatoria (Xcv) [105]. The fimbriae are composed mainly of a protein
subunit of 15.5 kDa (FimA). The use of a FimA-mutant strain indicated that
fimbriae had no role in colonization of tomato leaves. However, major dif-
ferences were noted between the wild-type and mutant strains in the amount of
cell-cell aggregation in laboratory cultures, on tomato leaf surfaces, and on
trichomes, with the wild-type always more prevalent [105]. These results
suggest that if the fimbriae assist attachment of Xcv to tomato surfaces, they
may be specific for selected regions of the plant, such as trichomes. Putative
FHA proteins suspected of being involved in attachment in other species also
are present in Xc [106].
2.4.7 Azospirillum SPP.
Azo spirillum spp. have been investigated because of their nitrogen-fixing
capability while in close contact with grass roots [107]. Two different
polysaccharide structures were identified in strains of A. brasilense (Abr) and
A. lipoferum (A lip): a CPS tightly associated with the cell surface, and an EPS
appearing to be less dense and extending from the cell [108]. A wheat lectin,
48 Microbiology of Fruits and Vegetables
wheat germ agglutinin, bound to Abr and A lip cells, and the binding was
inhibited by 7V-acetylglucosamine. Thus, these results provided evidence of
carbohydrate surface structures that are candidate receptors for specific
plant lectins [108].
Abr strains express a polar flagellum [109]. Attachment of a nonmotile Abr
flagellar mutant to wheat roots was reduced dramatically, whereas purified
flagella bound directly to wheat roots. The major outer membrane protein
of Abr was reported to be an adhesin responsible for Abr-Abr aggregation,
and the variable attachment of Abr to root extracts of wheat > corn >
sorghum > bean >> chickpea > tomato [110]. The degree of aggregation
of Abr cells is related possibly to the amount and composition of the EPS and/
or CPS [110].
2.4.8 Klebsiella spp.
Klebsiella spp. are enteric bacteria that can be soilborne, saprophytic, and
cause serious human illness. An associative nitrogen-fixing strain of K.
aerogenes expressing type 3 fimbriae was characterized for attachment [111].
The fimbriae of 23.5 kDa were associated with hemagglutination of human O
erythrocytes, and the adhesion of bacteria to plant seedling roots. In addition,
the purified fimbriae also bound directly to root tissue. Other strains of
klebsiella also were shown to bind to roots by the type 3 fimbriae [111].
Subsequently, type 1 fimbriae were reported to mediate adherence of K.
pneumoniae and Enterobacter agglomerans (Pantoea agglomerans) strains to
plant roots, with binding inhibitable by a-methyl-D-mannoside [112]. Thus,
different types of fimbriae appeared to mediate adherence of different
enteric species to plant roots.
2.5 FUNGI AND VIRUSES AND PLANTS
The invasion of plant tissues by phytopathogenic fungi is likely initiated
by attachment steps [113]. There have been few reports describing this
attachment process [114], but degradation of the cuticle and fungal hyphae
germ tube formation is important [113]. Studies of the survival of gastro-
intestinal viruses in water, soil, fruits, and vegetables have been reported [115],
but there is no definitive information about the mechanisms of virus
attachment to plants. A recent report on the specific attachment of cucumber
necrosis virus (CNV) to fungal zoospores and invasion of cucumbers may be
relevant to the introduction of foodborne gastrointestinal viruses in produce
by similar mechanisms [116].
2.6 POTENTIAL ATTACHMENT FACTORS OF
ENTERIC BACTERIAL PATHOGENS FOR PLANTS
The increasing amount of genetic and molecular data obtained for a variety
of microorganisms, including those that interact with, and/or are pathogenic
Attachment of Microorganisms to Fresh Produce
49
TABLE 2.2
Potential Attachment Factors in Human Pathogens That are Similar to Those
Described for Plant Bacteria
Enteric bacteria/human pathogen
Attachment factor(s)
Ref.
Escherichia coli
G-fimbriae
196
Escherichia coli
Flagellin
197
Enterohemorrhagic E. coli
Fimbriae
198
Enteropathogenic E. coli
Type IV bundle forming pili
199
Enteropathogenic E. coli
Fimbriae
200
Enterotoxigenic E. coli
Fimbriae
201
Enterotoxigenic E. coli
Pili
202
Uropathogenic E. coli
Type 1, P, S pili
203
Enter obacter (Pantoea) agglomerans
Type 1 and 3 fimbriae
112,
188
Klebsiella pneumoniae
Type 1 and 3 fimbriae
112,
188
Listeria monocytogenes
Flagellin
171,
204
Salmonella enter ica
Type 1 fimbriae
205
Salmonella enter ica
Type IVB pili
206
Salmonella enter ica
Thin aggregative fimbriae (curli)
207
Shigella jiexneri
Pili
208
Shigella Jiexneri
Type 1 fimbriae
209
Vibrio cholerae
Fimbriae
210
in, plants and humans, has revealed a remarkable amount of conservation
in the mechanisms available to them for survival and sometimes patho-
genicity [103,117-119]. Table 2.2 lists some of the species of enteric bacteria
that are potential foodborne pathogens, and the corresponding proteins
that are putative factors for attachment to their animal hosts. Clearly, multiple
types of fimbriae (G, types 1 and 3, IVB (curli, bundle forming)), pili (type 1,
P, S), and flagella represent the major known attachment factors in enteric
bacteria. In addition, all of the Gram-negative enteric bacteria shown in
Table 2.2 express LPS, and some express CPS; both are major surface
glycoconjugates that could serve as receptors for plant lectins, similar to the
mechanisms described previously for plant nitrogen-fixing, epiphytic, and
pathogenic bacteria (Table 2.1). Klebsiella spp., Enterobacter spp., and Pseudo-
monas spp. bridge the environments of plants and animals by their capability
to colonize both hosts, presumably with Type 1 and 3 fimbriae, pili, and lectins
involved in attachment (Table 2.2) [111,112,120]. Although certain species of
these three bacterial genera have been recognized to have biologically relevant
interactions with plants (e.g., nitrogen fixation, pathogenesis, competition),
there is no evidence that the major foodborne enteric human pathogens
(Table 2.3), many of which have been associated with produce outbreaks, are
either pathogenic or beneficial for the plant. However, the apparent inter-
actions of human pathogens with plant tissues, and their ability to survive
and grow on plants under certain conditions (e.g., temperature, Table 2.3),
o
TABLE 2.3
Summary of Some Human Pathogen-Plant Models and Observations Related to Attachment
Plant-human
pathogen model
Lettixce-E.coli 0157:H7
(pre- and postharvest)
Tomatoes-5. enter ica
Apples-S. enter ica
Sprouts-S. enter ica
Sprouts-£.co// 0157:H7
Observations and conclusions
Attachment of human pathogen to edges/grooves of
seed coat and root hairs
Attachment to stomates and trichomes
Cells concentrated at leaf epidermal cell junctions
Aggregates on roots and leaves
Strain differences in adherence
Internalization (45 urn below surface)
Attachment to roots, stems, leaves, flowers
Attachment to stem scar > intact fruit skin
Strain differences in survival and growth (adherence?)
Viable cells isolated from stem scars up to 49 days after
inoculation of fruit and at least 9 days after inoculation of roots
Internalization; protection from sanitization
Attachment to stem, calyx, broken skin > intact skin
Sanitization less effective when attached to stem, calyx, broken skin
Human pathogen and aerobic bacteria concentrate on damaged seeds
Attachment to root hairs and edges of seed coats
Tight attachment; hard to sanitize
Human pathogen attached better than 2 of 3 plant epiphytic species
Internalization through emerging root hairs (endophytic?)
Minimal attachment to sprout tissue
Nonpathogenic E. coli isolated from cabbage attached to sprouts
effectively
Grows well with nutrients in sprout irrigation water
Ref.
121, 127, 133, 134, 136, 211
142-144, 212
145
13,27, 120, 148
13, 27, 148
n
o
q_
o
era
=3
Q_
<
era
ex
Cilantro leaf-5. en t erica
Cut radish-L. monocytogenes
Cantaloupe-5. enterica,
E. coli, and L. monocytogenes
A. thaliana—E.coli 0157:H7
and S. enterica
Spinach/radish-C jejuni
Grew on leaves at 30°C > 22°C (3 days)
Tolerated low humidity (60%)
Attachment to leaf veins, senescent and damaged regions
Aggregates with plant pathogen and epiphytic bacteria
Leaf extract compounds bind Se
Attaches well to cut radish
Attachment dependent on temperature
Attachment of flagellar, export, and sugar phosphotransferase system
mutants decreased
S. enterica attaches more strongly compared to E. coli and
L. monocytogenes
S. enterica strains variable in surface hydrophobicity and electrostatic
charge
Attachment correlated with bacterial cell surface hydrophobicity and
charge
Attachment to root hairs, stems, leaves, flowers
Internalization at emerging root hairs
Attached tightly; washing and sanitization ineffective
Competitive epiphytic bacteria identified
Minimal attachment to leaves and roots
Attachment to soil components
Survives best at 10-1 6°C
128
171
125
175
129
>
i— h
sr
n
zr
3
3
n
o
o
C/J*
3
fD
~0
o
Q_
C
n
CD
Ul
52 Microbiology of Fruits and Vegetables
indicate that human pathogens interact with plants/produce by more than
simply physical and nonspecific ways.
2.7 ATTACHMENT OF HUMAN ENTERIC PATHOGENS
TO PLANTS AND OTHER INTERACTIONS
Recent studies of human pathogens in produce models have suggested that
foodborne pathogens, many of which are Gram-negative, may interact with
plants by mechanisms evolutionarily conserved, and at least somewhat similar
to those described above for plant bacteria (Table 2.3). Studies of human
pathogens and produce have involved, generally, assessments of the attach-
ment and survival of human pathogens on postharvest, retail market pro-
ducts [121-126]. Other studies have used human pathogen-plant models to
investigate attachment on seeds and seedlings or young plants that are
contaminated in the laboratory and grown in chambers under various
conditions (e.g., humidity, light, temperature, competitors) [27,127-129]. The
biology of attachment of human pathogens in preharvest (soil, other
microbes, temperature, UV, extended exposure time) and postharvest (rinse
water, shorter exposure time, temperature) environments may be quite dif-
ferent. Samples of human pathogen-contaminated produce/plants have been
examined mostly by conventional culture methods, polymerase chain
reaction (PCR), fluorescence microscopy, or other methods. These studies
have provided no definitive information regarding the molecular interactions
that may be involved in attachment of human pathogens to plant tissues.
However, it is anticipated that future work in this area will provide more
fundamental biochemical or genetic data related to attachment. A few
examples of studies pertinent to the concepts of plant-human pathogen
attachment are presented below and summarized in Table 2.3.
2.7.1 Lettuce and E. coli 0157:H7
In the last decade there have been more than 15 foodborne outbreaks linked
to contaminated lettuce or salad [10,130,131]. This provided the impetus
for initiation of studies of attachment of human pathogens in both pre-
and postharvest lettuce model systems [127,132-137]. Recent studies of E. coli
0157:H7 (£c0157) on store-bought lettuce indicated that cells attached in
a relatively short time period, and that not all cells could be removed by
vigorous washing or treatment with chlorine (Table 2.3) [133,134,138].
EcO\51 ', and other human pathogens and microbes, often are most con-
centrated at cut surfaces since there are vast nutrient resources released that
can be metabolized by human pathogens. Cut lettuce leaves immersed in
a suspension of a strain of Ec0157 (up to 10 8 CFU/ml) were exposed
to fluorescent anti-jE'c0157 antibody and observed by confocal microscopy.
EcO\57 attached predominantly to the cut edges of leaves; fewer cells
attached to the intact cuticle of leaves, but were observed attached
Attachment of Microorganisms to Fresh Produce 53
near stomates, on trichomes [133], and concentrated on vein areas of the leaf
[133]. Strains of Ec0157 ', pseudomonas, salmonella, and L. monocytogenes
(Lm) attached to different regions of cut lettuce leaves, indicating different
and specific mechanisms of attachment for different species or strains [134].
Recent studies of £"60157 and postharvest lettuce have addressed the
general nature and force of the plant-isc'0157 interactions by measuring the
effect of surfactants and other treatments. More hydrophobic surfactants
were the most effective in detaching EcO\51 from the leaf cuticle, but cells
at cut edges remained attached [137]. Attachment of £c0157 to the lettuce leaf
surface was 0.8 log 10 higher after treatment with CaCl 2 ; treatment with
NaCl had no significant effect [137]. Neither CaCl 2 nor NaCl, however, had
any significant effect on attachment to the cut edges.
Interestingly, it was reported that the medium in which EcO\51 cells were
grown affected attachment. Cells grown in tryptic soy broth were more
hydrophilic, produced more CPS, and attached better to edges of lettuce
(0.4 log 10 ) and to the surface of both lettuce and apple (0.8 to 1.0 logio) than
those grown in nutrient broth, suggesting that CPS may be involved directly
in attachment [139]. These studies suggest that .Ee'0157 has different mecha-
nisms for attaching to different regions of lettuce leaves, possibly involving
hydrophobic interactions, surface carbohydrates (CPS/LPS), neutralization
of ionic charge, or bridging of anionic moieties by divalent cations.
In sprout models of £6'0157-lettuce attachment, strains of ^c'0157
implicated in produce outbreaks attached to lettuce roots approximately one
log 10 better than did two of five nonpathogenic E. coli strains, indicating
variability in attachment among strains [127]. Attachment of EcO\51 strains
was highest to seed coats and roots compared to the shoots.
GFP-labeled EcO\51 was observed under a fluorescence stereomicro-
scope to bind in aggregates to the grooves and edges of the seed coats, and to
small root hairs of sprouted seedlings [127]. High concentrations of EcO\51
added to soil prior to growth of lettuce seedlings resulted in pathogen bound
to all parts of the plant. Aggregates of £c0157 cells were observed on
cotyledon and root tissue of lettuce seedlings grown for 5 days in spiked soil
[127]. These studies illustrate the potential for E. coli to colonize both pre- and
postharvest lettuce.
In similar studies, an £'c0157-GFP strain spiked in manure-contaminated
soil (10 4 to 10 8 CFU/g) was monitored by confocal microscopy for presence
on lettuce plants grown in the soil and then treated with chlorine and HgCl 2
[136]. £c0157-GFP remained attached to the edible portion of treated
lettuce seedlings grown in soil spiked with the highest concentration of
o
££'0157 (10 CFU/g). In addition, cells were observed as aggregates on
three-day-old leaf surfaces, with some cells present 45 urn below the outer leaf
surface. There are multiple potential routes of entry for human pathogens
in plants (lateral roots, stomates, pores, cuts, lesions, "invasion"), but whether
specific mechanisms of attachment are involved during internalization on
preharvest produce is not known (Figure 2.1 and Figure 2.2).
54 Microbiology of Fruits and Vegetables
2.7.2 Tomatoes and Apples and Salmonella
enterica (se)
Outbreaks of salmonella illness associated with raw tomatoes have been
reported [140,141]. Tomatoes inoculated with high doses of S. Montevideo
(SeM) and stored for 3 days retained viable cells on skin and stem scars [142].
However, SeM survived at 2 to 4 logio higher concentrations in scars and
cracks compared to skin, both after washes in water and 100 u.g/ml aqueous
chlorine. Tomato plants inoculated at stems or flowers with a combi-
nation of five different S. enter ica serovars including SeM were analyzed
to determine the incidence and length of time salmonella survived in fruit
[143]. SeM was isolated from stem scar tissue up to 49 days after inoculation,
but S. Poona, S. Michigan, and S. Enteritidis also were isolated at 22 to 39
days from pulp and stem scar tissue. Further studies with the five strain
combination with hydroponically grown tomato plants reported the uptake
and survival of salmonella for at least nine days on hypocotyls, cotyledons,
stems, and leaves of plants inoculated at intact or cut roots [144]. These
studies confirmed the capability of Se to survive and grow on and in tomato
plants and fruit, and indicated the possibilities of strain differences in
attachment, and multiple types of attachment involved in the interaction of
Se with a variety of plant tissues.
Apple fruit provides a surface and environment for human pathogens
similar to that of tomato fruit. The intact skin is composed of a waxy cuticle
less conducive to attachment by human pathogens than other regions of the
fruit [145]. Apple fruit immersed in 10 8 CFU/ml of a strain of S. Chester
and dried for 10 minutes retained human pathogen on broken skin, and the
calyx and stem, at 20° C better than at lower temperatures; also, more human
pathogen cells in these regions survived chemical sanitization than those
on intact skin.
2.7.3 Sprouts and E. coli 0157 (fcO!57) and Se
Numerous outbreaks of EcO\57 and Se associated with contaminated
sprouts have occurred since 1995, and a number of the outbreaks have been
traced to seeds contaminated with relatively low levels of pathogen [146].
Although calcium hypochlorite at 20 mg/ml has been recommended for
sanitizing seeds [147], it does not remove the entire natural microbial flora
on the seed, suggesting that bacteria are attached in sites inaccessible to
chemical treatments [13]. A comparison of the growth of multiple Ec0157 and
Se strains on alfalfa sprouts revealed major differences in attachment among
the strains [27]. Six strains of EcO\57 grew an average of 1.5 log 10 less on
sprouts compared to five strains of Se (Table 2.3). An 2fc0157-GFP strain
attached poorly to sprout roots and shoots, whereas individual cells and
aggregates of Se Newport-GFP were observed adhering to sprout seed coat
edges and root hairs (Figure 2. 3D). The 10- to 1000-fold difference in
attachment to sprout tissues by EcO\57 compared to Se strains was confirmed
Attachment of Microorganisms to Fresh Produce
55
FIGURE 2.3 (Color insert follows page 594) Confocal micrographs of bacteria on plant
leaf, stem, and root tissues, and bacteria bound to material extracted from leaves. (A)
Natural microorganisms, mostly bacteria, bound to junction of epidermal cells on a
lettuce leaf. The bacteria were stained with LIVE BacLight Gram stain (Molecular
Probes, OR). (B) GFP-labeled S. enter ica and dsRed-labeled P. agglomerans cells bound
singly and in aggregates after their inoculation and incubation on the leaves of cilantro
plants. Natural epiphytic bacteria were stained with SYTO 62 (Molecular Probes) and
were detected in the close vicinity of the inoculated strains. The SYTO 62 signal was
assigned the pseudocolor blue. (C) GFP-labeled £c0157:H7 bound in the region of a
lateral root emerging from an Arabidopsis thaliana plant. The arrow points to a region
where the EcO\51 cells have become internalized. (D) GFP-labeled S. enterica bound to
the root hairs (Rh) of an alfalfa sprout. (E) A thick biofilm of natural microorganisms
colonizing the root of an alfalfa sprout and stained with LIVE BacLight Gram stain. (F)
GFP-labeled S. enterica cells attached to a dried compound extracted from cilantro
leaves and identified as stigmasterol. (Brandl and Mandrell, unpublished data.).
in subsequent studies [148]. Interestingly, a Se Newport strain appeared to be
as fit in sprouts up to at least three days as three epiphytic strains isolated
from sprouts. In addition, four nonpathogenic E. coli strains isolated from
field-grown cabbage (Table 2.3) attached as well as the epiphytic strains, but
less than Se Newport. The results of these and other studies of Se on
sprouts [149] indicated that specific interactions occur between human patho-
gens and plants, and suggested that enteric bacteria and human pathogens
isolated recently from plant surfaces may retain fitness and attachment
capability for plants.
56 Microbiology of Fruits and Vegetables
2.7.4 ClLANTRO AND SE THOMPSON (SeT)
The investigation of an outbreak of SeT associated with cilantro suggested
that the cause of the outbreak was a result of preharvest contamination of
imported cilantro [150]. The outbreak strain of SeT increased on cilantro
leaves ~1.0 logio and 2.0 log 10 at 24 and 30°C, respectively, 18 hours after
inoculation [128]. Observation by confocal microscopy of SeT-GFP incubated
on the leaves of cilantro plants revealed that SeT cells localized to the vein
area of leaves. Small microcolonies of cells were observed on the leaf veins, but
larger concentrations of both individual and aggregated cells were observed
on senescent portions of the leaf and in lesions, suggesting that the release
of nutrients from leaky or damaged plant cells enhanced growth of the
pathogen (Figure 2.2L) [128]. Co-inoculation of cilantro with SeT-GFP and
P. agglomerans, a plant epiphyte isolated from cilantro and containing a red
fluorescent protein, revealed that SeT cells (Figure 2.3B, green cells) were
attached to the leaf in aggregates with P. agglomerans (red/pink cells) and other
natural epiphytic bacteria (purple cells) [128]. These results suggest that SeT
interacted with the plant and native bacteria after prolonged exposure to the
leaf surface.
In an attempt to assess the mechanism of attachment of SeT to cilantro
leaves, a chloroform-methanol (2:1) extract of cilantro leaf surfaces was
prepared and fractions obtained by separation by thin-layer chromatography
(TLC). Multiple TLC-purified bands were applied to glass slides, exposed to
a suspension of SeT-GFP cells and the slides were incubated. Unbound cells
were washed from the slide and the slide was observed by confocal micros-
copy. Figure 2.3F is representative of the results observed with one of the
samples that bound SeT cells most effectively. This sample was analyzed
by mass spectrometry and shown to be composed of >90% stigmasterol,
a sterol compound that is present in the cuticle and has been detected in
other leaf extracts [151]. Interaction with cuticular waxes or sterols in regions
of the leaf where nutrients are more available is a reasonable strategy for
bacteria. A recent study reported the growth of epiphytic Pseudomonas spp. on
apple cuticle membranes without disrupting the membrane, and the release
of a variety of bacterial proteins (e.g., flagellin, porin, ABC transporter binding
component) through the membrane [31].
2.7.5 Produce Samples and
L. MONOCYTOGENES (L\i)
Lm is a Gram-positive, facultative intracellular pathogen acquired most
often through the consumption of contaminated food. Listeriosis is a
serious illness that can cause a variety of symptoms including septicemia, liver
failure, meningitis, and spontaneous abortion and death [152,153].
Lm can survive as a saprophyte on decaying plants and grows at a wide
range of temperatures [154]. Outbreaks have occurred due to produce
contaminated with Lm [155]. Lm has been isolated from market produce
Attachment of Microorganisms to Fresh Produce 57
such as cabbage, corn, lettuce, peppers, sprouts, radishes, potatoes, cucumbers,
grains, parsley, and watercress [154-162]. It has been reported to grow on
asparagus, broccoli, cantaloupe, cauliflower, and leafy vegetables [163-166],
and attach to cut potato tissue [167].
A 60-minute exposure of whole cucumber to 10 8 CFU/ml of Lm followed
by washing resulted in 10 to 10 CFU/g of Lm remaining attached [168].
Lm attached to unwaxed cucumber better than to waxed cucumber, in contrast
to the decrease in attachment of Se typhimurium and Staphylococcus aureus
to unwaxed cucumber, indicating either that the Lm cell surface is relatively
more hydrophilic [168] or that openings on the cucumber surface (stomates,
pores, cracks) are sealed by wax. Similarly, 80% of the Lm cells added to
whole cantaloupes at approximately 10 CFU/cm remained attached;
however, higher concentrations of Se and £60157 attached initially to the
fruit surface compared to Lm [125].
In a study comparing the attachment of multiple foodborne human
pathogens to cut lettuce with that of P. fluorescens (Pf) by observations under
a confocal scanning laser microscope, Lm and EcO\51 attached preferen-
tially to the cut edge of lettuce, Pf attached to the uncut surface, and Se
Typhimurium attached to both locations [134]. The nutrient-rich and
hydrophilic nature of a plant cut surface compared to the hydrophobic waxy
cuticle, is consistent with Lm, but not Pf attaching and concentrating in
this location [134,169].
There have been numerous studies of Lm attachment to postharvest
produce, but few studies of Lm attachment to preharvest plants/produce.
In one study, a 100- to 1000-fold difference was reported in the attachment
and colonization of different strains of Lm to alfalfa sprouts grown from
inoculated seeds [170]. No association of attachment with any known Lm
surface characteristic (serotype) or genotype could be discerned. The same
investigators reported minimal differences in the attachment of seven differ-
ent strains of Lm to radish tissue after 2 hours (4.76 to 5.39 CFU/g tissue),
indicating that the mechanism of attachment in this system was relatively
conserved among strains. A screen of a library of Tn977-LTV3 Lm mutants
of one of the strains with fresh-cut radish tissue (4 hours, 30°C) resulted
in identification of three attachment-defective mutants [171]. Two mutations
were in genes of unknown function within an operon-encoding flagellar
biosynthesis; only one of the mutants lacked flagella and was nonmotile.
A third mutant carried an insertion in an operon necessary for the transport of
arabitol. All three mutants attached at least 10-fold less compared to the
parent strain, which bound to the radish tissue at levels as high as 5 logio
CFU/g at 30°C. However, none of the mutants attached less than the parent
strain when the samples were incubated at 37°C. Incubation temperatures of
10 and 20° C affected the attachment of the single motility mutant negatively,
whereas the arabitol transport mutant was decreased in attachment at 10
and 30°C. Changes in the Lm cell surface at low temperatures (e.g., 10 versus
37°C) have been shown previously to occur, including decreased chain lengths
and branching of membrane fatty acids [172], and up-regulation of three
58 Microbiology of Fruits and Vegetables
genes predicted to encode cell surface proteins: fbp (putative fibronectin
binding protein), flaA (flagellin), and psr (putative penicillin binding protein)
[173]. These variable results associated with temperature suggest that Lm
might express different attachment factors in different environments (e.g.,
temperature) [171].
2.7.6 Cantaloupe and Se, £c0157, and Lm
Ukuku and Fett studied the type and strength of attachment of multiple
strains of Se, E. coli, and Lm, including outbreak- or food-associated strains,
to the surface of cantaloupes [125]. The bacterial cell surface charge and
hydrophobicity of each of the strains were determined and compared to
the strength of the interaction, as measured by the number of cells retained
on the cantaloupe surface after immersing whole melons in water. Attachment
was measured both on melons spiked with individual strains and mixtures
of strains. Se had the highest and most variable surface hydrophobicity, and
the highest negative and positive surface charge; E. coli, EcO\57, and Lm
strains were similar in hydrophobicity, but Lm had a much higher negative
surface charge compared to E. coli. Although more E.coli cells attached
initially to the melon surface compared to Se and Lm, Se attached more
strongly than either E. coli or Lm after storage at 4°C up to 7 days, regard-
less of whether strains were added individually or as mixtures [125]. The
strength of attachment of each of the species was correlated significantly
with the hydrophobicity and the negative and positive surface charge of the
strains, indicating that all of these parameters were important in attachment.
2.7.7 Arabidopsis thaliana and Fc0157 and Se
Many genetic tools are available for studying A. thaliana (thale cress) [174].
Thus, it provides an opportunity to gain insight into the response of a plant
to human pathogens. Single strains of £60157 and Se Newport were assessed
in an A. thaliana model for attachment and growth characteristics [175]. In
initial experiments, the human pathogens, applied to sterile roots under ideal
humidity, remained attached at high concentrations (10 9 CFU/g tissue) with
eventual migration to the stems/shoots (2 x 10 CFU/g). Examination of the
roots by confocal microscopy revealed that £c0157-GFP and Se Newport-
GFP strains appeared to have "invaded'' the plant interior specifically at
locations where lateral roots emerge (Figure 2.3C, Figure 2.1H). A similar
result was obtained recently with a Se typhimurium strain in an alfalfa seed-
ling model using relatively low numbers of cells (~10 CFU) [120]. Single
cells and cell aggregates of EcO\57 and Se Newport were observed also on
shoots and flowers [175]; surprisingly, EcO\51 was isolated also from seed
and chaff harvested from contaminated plants, and from plants grown
from contaminated seed (unpublished results). The interaction of these
two important human pathogens with multiple plant tissues suggests that
multiple attachment mechanisms are involved [175].
Attachment of Microorganisms to Fresh Produce 59
2.7.8 Plant-Microbe Biofilms
Intact plant surfaces, especially those of leaves, are relatively inhospitable
environments for microorganisms, providing limited sites for attachment,
surface retention of water, and nutrients. Nevertheless, many microorganisms
have developed mechanisms to attach, survive, or grow in microniches on
different plants. The micrograph shown in Figure 2.3A demonstrates the
localization and high density of epiphytic bacteria on a lettuce leaf. Both
Gram-positive and Gram-negative bacteria are interacting in aggregates and
possibly competing for the limited nutrients available in the microniche at
the junction of epidermal cells where cuticular waxes are less dense, water
accumulates, and nutrients are more available than in other sites. Although
biofilms with classic structures described in recent studies are rarely found
on plants, thick three-dimensional biofilms have been observed on sprouts
sampled from a commercial sprout facility (Figure 2.3E). The image reveals
the potential for complex interactions to occur between Gram-positive and
Gram-negative resident bacteria under ideal conditions of plentiful water,
exuded nutrients, and warm temperatures during food production or pro-
cessing. A thick mat of mostly aggregated bacterial cells was detected on the
root hairs of the sprouts (Figure 2.3E, *'Ep"). Although plant tissue was
likely present within the biofilm (Figure 2.3E, arrow), it appears that multi-
ple layers of cells compose the biofilm, and that the presence of EPS at the
surface of, or within, the biofilm is possible. Similar aggregates of bacteria
have been observed using SEM on roots of alfalfa, broccoli, clover, sunflower,
and mung bean sprouts [176,177]. Following attachment of bacteria as indi-
vidual cells on leaf surfaces, aggregation is crucial as a strategy to ensure
survival under environmental stresses such as water or nutrient depletion, UV
irradiation, unfavorable temperatures, or predation [178,179]. In many enteric
bacteria, fimbriae composed of curli protein interact with a cellulose poly-
saccharide resulting in aggregation and either pellicle formation or biofilms
(Table 2.2) [180]. T3SS-encoded proteins in other bacteria (Table 2.1 and
Table 2.2) are analogous to curli. In a recent report, T3SS-encoded protein in
Echr was shown to interact with P-glucanlike (noncellulose) carbohydrates,
and this interaction was crucial for pellicle and biofilm formation in vitro [181].
Thus, T3SS proteins, and possibly type 1 pili, conjugative pili, and curli
(fimbriae), are important in aggregation leading to biofilm formation.
Biofilm formation is thought to be a major reason for the persistence of
microorganisms, including pathogens, for long periods in food processing
environments [182]. Bacteria, filamentous fungi, yeasts, and even viruses
may be represented within biofilms on a plant surface. Therefore, the mecha-
nisms of initiating bacterial autoaggregation and mixed-species aggregation,
and the attachment of bacteria singly or as aggregates to plant surfaces or
to microorganisms/EPS in preexisting biofilms on plant surfaces, could
involve attachment factors such as those described in this review (Table 2.1
and Table 2.2). Understanding the mechanisms could yield intervention
strategies for decontamination of produce.
60 Microbiology of Fruits and Vegetables
2.8 CONCLUSIONS
The many years of difficult and labor-intensive studies on plant-microbe
interactions involved in plant symbiosis and disease have begun to yield
fundamental molecular information regarding bacterial attachment to plants
(Table 2.1). The attachment factors designated in Table 2.1 can be grouped
essentially into five categories: polysaccharides (EPS, CPS, LPS), outer
membrane proteins, flagella, pili, and fimbriae. In some systems, bacterial
protein factors have been identified that bind to plant carbohydrates (e.g.,
rhicadhesin), and in others, a bacterial polysaccharide is bound by a plant
lectin (e.g., Rhsp EPS/CPS/LPS). It is probable that attachment for some
bacteria will involve both strategies ("dual bridge") simultaneously, or with
different hosts and/or in different environments. The attachment factors
identified in human pathogens mostly relate to studies with animal cell lines
or animal models (Table 2.2). However, it is very likely that flagella, pili, and
fimbriae might have roles as attachment factors for human pathogens
on plants, considering their prominent outer surface location and length.
Absent from Table 2.2 are EPS (e.g., colanic acid), CPS (e.g., K-antigens), and
LPS (e.g., O-antigens), all very important complex carbohydrate-containing
molecules synthesized by human pathogens; these molecules are surface-
expressed and often regulated by environmental cues [183-185]. Surface
complex carbohydrates are excellent candidates for possible interactions
with plant lectins of the appropriate specificity [186]; a precedent is the well-
defined rhizobiaceae EPS interaction with pea plant lectin (Table 2.1).
The model studies of enteric human pathogens with plants/produce
indicate the general fitness of human pathogens in these environments. Similar
to plant bacteria, human pathogens appear to possess multiple specific
mechanisms of attachment and growth (Table 2.3). The interactions of
human pathogens with host plants probably will involve many unique
factors depending upon the plant and the pathogen. It is probable that events
occur preceding the direct interaction of a human pathogen with a plant that
are important for attachment. For example, the environment in which the
pathogen has remained viable (water, manure, soil, eukaryotic micro-
organisms, insects, animals) will dictate what surface molecules are expressed
and the metabolic state of the human pathogen prior to interaction with the
plant host. Also, the human pathogen may be associated with other
microorganisms in aggregates or in a detached biofilm. The plant may release
chemicals that are signals and/or chemotaxis factors for some human
pathogens. The availability of different types of plant receptors (specific and
nonspecific) will determine the efficiency of attachment. After the human
pathogen cell or cells make direct contact with the potential host plant, the
human pathogen attaches either specifically or nonspecifically by weak or strong
interactions depending upon the site of attachment. Flagellated cells may
move (e.g., twitching motility) along a surface until an optimal attachment
site is recognized. Initial attachment likely occurs by biochemical forces or
Attachment of Microorganisms to Fresh Produce 61
by human pathogen proteins extended from the surface (pili/flmbriae, flagella),
with tighter attachment established later by other surface molecules. Based
on other plant-microbe interactions (Table 2.1), a possible strategy for
attachment may combine human pathogen protein-plant receptor (e.g.,
carbohydrate) and plant lectin-human pathogen polysaccharide (e.g., EPS,
CPS, LPS) interactions. The human pathogen may then be further secured by
human pathogen cell-cell aggregation (possibly involving T3SS) or human
pathogen-plant microbe aggregation, both of which likely require expres-
sion of different attachment factors.
The presence of putative attachment factors in enteric human patho-
gens that are similar to those of plant-related bacteria, point to obvious
approaches for identifying fundamental mechanisms of human pathogen
attachment to produce. Fimbriae, pili, flagella, polysaccharides, and porin
proteins are all candidates for direct attachment to and aggregation of human
pathogens on plant tissue. Recent advances by researchers in studies of how
native microbes attach and interact with the rhizoplane and phylloplane
provides inspiration and guidance for researchers studying the biology of
human pathogens in similar environments.
ACKNOWLEDGMENTS
The authors thank Dr. Jeri Barak for providing information prior to
publication and Dr. Amy Charkowski for an image of GFP-labeled S. enterica
on sprouts. This work was supported by the U.S. Department of Agriculture,
Agricultural Research Service CRIS project 5325-42000-040.
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Internalization and
Infiltration
Jerry A. Bartz
CONTENTS
3.1 Overview of Internalized Microorganisms 75
3.2 Location of Internalized Organisms in Plants 77
3.3 Structures that Enable Internalization 78
3.4 Process of Internalization 80
3.5 Internal Structures of the Plant Involved in Internalization 80
3.6 Types of Internalization 82
3.6. 1 Aerosols 82
3.6.2 Water Channels and Water Congestion 82
3.6.3 Internalization in Wounds 83
3.6.4 Infiltration of the Plant Surface by Aqueous
Cell Suspensions 84
3.6.5 Events in Plant Development 85
3.7 Implications and Control 87
References 90
3.1 OVERVIEW OF INTERNALIZED
MICROORGANISMS
Microorganisms embedded in plant tissues may be defined as "internalized,"
derived from "internal," meaning located inside the plant surface. Function-
ally, internalized microbes cannot be washed off the plant, they are protected
from environmental stresses, and they cannot be inactivated by contact bio-
cides or other surface disinfectants. Inside the plant, most microorganisms are
located in spaces between cells called intercellular spaces, whereas plant viruses
and certain other pathogens are inside host cells. Microbes in the intercellular
spaces are bathed in nearly saturated relative humidity with a gas composition
that enables aerobic metabolic activities [1]. The main threat to the survival of
internalized microorganisms appears to be mechanisms that protect the plant
against microbial attack [1]. As such, the microbe must either evade, counter-
act, or not induce its host's defenses. Plant pathogenic microorganisms, which
by nature harm plant tissues, have developed ways to cope with host defense
75
76 Microbiology of Fruits and Vegetables
reactions. In contrast, nonplant pathogens usually do not harm living tissues
and, as a consequence, appear unlikely to stimulate plant defenses. Moreover,
the absence of tissue damage reduces the likelihood that nonpathogens will be
exposed to preformed antimicrobial chemicals, which would be compartmen-
talized in the cytoplasm or specialized cells.
Microorganisms that are resistant to washing, surface disinfectants, or
environmental stresses are not always internalized. Romantschuk et al. [2]
noted that washing leaves, with or without sonication, does not remove all
bacteria that live entirely on the plant surface, perhaps because portions of
this population may embed in surface biofilms or other attached aggregates
(see Chapter 2). Additionally, bacteria have been observed partially buried
in surface waxes [3] and in cracks in the cuticle [4]. Microorganisms embedded
in aggregates, biofilms, surface waxes or ruptures in the cuticle are somewhat
protected against environmental stresses [5] and surface treatments. However,
truly internalized organisms, which are located beneath layers of plant cells,
would have much greater protection.
Proof that microorganisms exist inside healthy, unblemished fruits and
vegetables was provided by Samish et al. [6]. Using special surface sterilization
procedures, her group isolated Gram-negative, motile, and rod-shaped bacteria
frequently from tomatoes, cucumbers, English peas, and green beans sampled
from farm fields. Populations were found less frequently in melons and
bananas, whereas successful isolations were infrequent in grapes, citrus fruits,
olives, and peaches. Internal populations of microorganisms would likely be
highest in root tissues [7] and lowest in the acidic environment within certain
fruit tissues [6].
Internalized microorganisms are part of a complex microbial ecosystem
associated with plants [8,9]. Epiphytic microorganisms survive and multiply
on the plant surface, whereas endophytes colonize the interior of plants
without causing noticeable damage [9]. Those that grow on or in plants and
cause damage are plant pathogens [10]. Epiphytes, endophytes, and plant
pathogens may be considered resident microorganisms because they compose
the plant-associated microbial ecosystem. Individual species that fail to
establish a presence in this ecosystem despite one or more introductions are
called casual microorganisms [11]. Casual microbes are usually ill-suited to
survive on the plant surface. Once inside the plant, however, casuals can
survive for prolonged periods of time depending on their ability to adapt to an
environment that is high in humidity but low in available nutrition. However,
under certain conditions, internalized casuals multiply. For example, Dong
et al. [12] observed endophytic growth of Escherichia coli and Salmonella
enterica (strains of Cubana, Typhimurium, and Infantis serovars) in alfalfa
and barrel medic seedlings grown in test tubes. Young [13] noted that water
congestion of leaf tissues enabled a wide range of bacteria to multiply. King
and Bolin [14] reported that severe tissue water congestion caused plant cell
membranes to leak minerals and metabolites, which supported the growth
of saprophytes. Furthermore, the development of large populations of
Internalization and Infiltration 17
microorganisms on fresh-cut vegetables could produce nonspecific spoilage,
likely because plant defense mechanisms were compromised by anoxia.
3.2 LOCATION OF INTERNALIZED ORGANISMS
IN PLANTS
Plants are covered by a protective layer made up of cutin polymers embedded
in waxes [1,3,15-17]. This layer is relatively impervious to water penetration
or loss, gas exchange, and penetration by particulates. Various structures in
the plant surface enable the gas exchange required for vital metabolic and
photosynthetic processes occurring in the underlying cells. Therefore, to inter-
nalize, a microorganism must either directly penetrate the surface layer or enter
through a surface opening (aperture) or wound.
The surface coating of plants and structures beneath it may be cate-
gorized as either symplast or apoplast [16,17]. The symplast or living matter
includes the cytoplasm of cells, whereas the apoplast includes the surface layer,
cell walls, air spaces between cells and in the cell wall matrix, and the pri-
mary water-conducting tissues (xylem) [17]. Sieve tube elements, which are
primarily devoted to movement of the products of photosynthesis and other
cellular processes, accompany the xylem vessels [16]. However, the sieve
tube elements are filled with a cytoplasm-like material. As such, whether they
should be included in the apoplast is unclear. In certain parts of the plant,
sieve tubes transport water, whereas in other parts, xylem vessels carry
sugars, etc.
The xylem, composed of specialized vessels, tracheids, and associated
parenchyma, connects the water-absorbing tissues in the root system with the
rest of the plant [18]. Vessels and tracheids are filled with water contain-
ing dissolved minerals and occasionally organic solutes. The general structure
of these water-conducting elements tends to exclude microorganisms such
that only a few specialized types are able to enter and move through the
system. Individual vessel cells connect through perforation plates that would
appear to allow passage of suspended particulates such as bacteria [18].
However, Pao et al. [19] observed multiple, helical perforations in the walls
and ends of the vessels in the stem scar of orange fruits that blocked move-
ment of bacteria. Whether these were xylem vessels or tracheid cells, which
do not possess perforation plates [18], is unclear. Both types of water-
conducting cells attach to adjacent cells through pits in their secondary walls.
The pits are paired with those in walls of an adjacent parenchyma or vessel
cell. The base of each pit pair contains a membrane composed of the initial
primary cell wall of the adjacent cells and the middle lamella. Pit mem-
branes contain pores that are slightly larger than plasmodesmata. At a
reported 0.3 urn in diameter [1], such pores would not allow passage of
bacteria. However, the pits are freely permeable to water and solutes. Microbes
that can enzymatically digest the pits, such as the wilt pathogens, inhabit
xylem vessels [1]. Moreover, microbes that are able to weaken pit membranes
78 Microbiology of Fruits and Vegetables
that interface with adjacent parenchyma could egress from the vessel as well as
obtain nutrition from the parenchyma cells.
A large portion of the apoplast of most plants consists of intercon-
nected intercellular air spaces, which are linked with openings in the plant
surface [18]. Less than 1% of the volume of potato tubers is devoted to
intercellular spaces, whereas up to 66% of certain leaves is air space [20].
3.3 STRUCTURES THAT ENABLE INTERNALIZATION
Naturally occurring surface apertures and wounds are keys to the internaliza-
tion of microbes. Two apertures, stomata and lenticels, function in gas
exchange, whereas hydathodes provide relief of excessive internal water
pressures. Stomata occur in the epidermis of all above-ground parts of plants.
Specialized stomata function as nectaries (secrete nectar) in certain types
of flowers [1]. Stomata are apertures in the plant's epidermis that are created
by two specialized cells called guard cells. The turgor of the guard cells
changes with exposure to sunlight, darkness, or moisture stress [21]. The guard
cells swell during daylight opening the pore, and shrink during darkness or
with water stress, closing the pore. Epidermal cells adjacent to the guard
cells may grow under the stoma forming a substomatal chamber [18].
Schonherr and Bukovac [22] suggested that stomata be viewed as narrow
capillaries having inclined walls.
Lenticels are specialized portions of a periderm, which is an impervious
secondary surface layer that replaces the epidermis or forms on the surfaces
of wounds [18]. The periderm is composed of a phellogen (cambium),
phellum (corky cells), and phelloderm (resembles parenchyma cells formed
inside the phellogen). A lenticel is similar in organization to the surrounding
periderm, except that the lenticel phellogen is more active and contains
intercellular spaces [1,18]. It produces a phellum that is loosely organized
with many intercellular spaces. Thus, gases readily diffuse through lenticels
into the underlying tissues of the plant organ. Phellum cells in lenticels may
or may not be suberized (cell walls infiltrated with and coated by a poly-
meric organic chemical complex that is a barrier to moisture diffusion) [15],
whereas the phellum of the regular periderm is nearly always suberized.
Lenticel-like structures may form on certain types of fruit [18]. In certain
types of apple fruit, a periderm-like structure forms under stomata but a
phellogen is usually absent. Certain types of melons crack as they approach
maturity. Living cells beneath the crack develop into a phellogen that pro-
duces the characteristic net common to cantaloupes and certain other fruit. The
net resembles a lenticel in structure. Certain lenticels respond to changes in
the environment around them. For example, cells in lenticels on potato tubers
proliferate when the soil becomes moist [23,24]. These proliferated cells are
thin-walled, surrounded by large intercellular spaces, and highly susceptible
to microbial attack.
Internalization and Infiltration 79
Hydathodes, apparently designed to release excessive water pressure in
the plant, vary in complexity among different plant species but all provide
a connection between the water-conducting elements and the external envi-
ronment [18]. Certain ones resemble stomata except for not closing during
darkness. Others are specialized for water release and may be better termed
"water glands.'' Gas exchange could occur through hydathodes that are
not water congested. Water congestion develops in above-ground tissues of
plants when the roots absorb water more rapidly than above-ground parts
lose it to evapotranspiration [25]. The excess water can pool under the
epidermis causing edemas or, more often, water moves from the ends of the
vascular strands through the leaf mesophyll and then into and out of hyda-
thodes in a process called guttation [21]. Guttation droplets, which are derived
from xylem sap, appear on the edges of leaves and are often confused with dew.
However, guttation may occur at any time of the day, particularly if the soil
is moist, plants are growing rapidly, and evapotranspiration is low [26].
Lawn grasses and corn have been observed to excrete water in bright sunlight.
Guttation may be part of a natural detoxification method in certain plants,
particularly when rainfall, fog, or dew cause the droplets to fall from the plant
surface [17].
Fruit attachment structures on certain plants contain natural openings
involved with gas exchange. Most of the gas exchange required by the inter-
nal cells of tomato fruit occurs through the stem scar [27]. If the stem scar is
covered with wax, the carbon dioxide levels in the intercellular spaces
increase two to four times above normal, evidence that the wax layer blocks
equilibration of respiratory C0 2 with the external environment. If the rest of
the fruit is waxed and the stem scar is not, the CO2 level in the fruit
remains similar to that in nonwaxed fruit. Air injected into a tomato fruit
submerged in water bubbles from cracks in the edges of the stem scar [28].
Only rarely are any bubbles observed at the blossom end of the fruit. If
the stem is still attached, the air bubbles from the area between the stem and
fruit.
Wounds also connect a plant's intercellular air-space network with the
surrounding environment. Wounds can arise from various biotic and abiotic
factors including insects, storms, wind-blown particles, harvest crews, etc.
Excessive water uptake or even normal growth may produce cracks in the
surface of plant organs. Trichomes, defined as outgrowths of the epidermis
[18], are easily damaged and are a frequent site for infection by plant
pathogenic bacteria and growth of epiphytes [9]. Whether broken trichomes
enable the internalization of resident or casual microbes is unclear. The
porosity of wounds to gases and moisture often changes over time due to
healing processes involving the formation of closing layers such as a periderm,
or suberization and lignification of cell layers [15]. These changes usually
quickly restore the wound to an imperviousness to water loss and penetration
by particulate matter similar that of the intact surface layers [18].
80 Microbiology of Fruits and Vegetables
3.4 PROCESS OF INTERNALIZATION
The internalization process whereby microorganisms enter the plant apoplast
is either active or passive. During active internalization, microbes grow
through the plant surface into intercellular spaces, which is consistent with the
activity of various plant pathogens [10]. During active internalization, plant
pathogens penetrate directly through the cuticle or indirectly through
stomata, lenticels, hydathodes, or wounds. Passive internalization implies that
microbes are carried into the apoplast due to contact with an object causing
injury or by a penetration of apertures by water, aerosol, or particulate that
contains microbes. Plant viruses may internalize in plant tissues that are being
fed upon by insects [10]. Aerosols may enter open stomata during a mass
flow of gases into leaves [29]. Aqueous suspensions of microorganisms may
infiltrate surface apertures or wounds either spontaneously [22] or because
of pressure differentials between the apoplast and the external environment
[30-32]. Suspensions also may diffuse or be drawn into plants through water
channels, which are a direct liquid connection between a plant's intercellular
spaces and its exterior environment [25].
Most surface apertures of plants are large enough to allow passage of
bacteria and smaller particulates, whereas fungal spores would likely be
excluded. The stem scar of tomato fruit may allow the passage of spores of the
sour rot fungus, Geotrichum candidum [33], although the evidence was not
conclusive. Lesions of Rhizopus stolonifer and Geotrichum candidum developed
around and beneath the stem scar of tomato fruit that had been previously
treated to cause an internalization of the spores of these fungi [28]. Vigneault
et al. [34] reported that tomatoes cooled with water containing spores of
R. stolonifer usually decayed during subsequent storage. However, whether
the spores in these examples internalized through the stem scar is unclear.
In contrast, wounds involving tissues with large intercellular spaces appear
likely to internalize fungal spores.
3.5 INTERNAL STRUCTURES OF THE PLANT
INVOLVED IN INTERNALIZATION
The morphology of the surface pores and interconnected intercellular
spaces has a direct influence on how readily particulate matter moves through
the plant surface as well as the size of particles admitted. Intercellular spaces
are delimited by the walls of the surrounding cells. The spaces form when
cells dissolve (lysigenous), tear (rhexigenous), or separate (schizogenous) [18].
Cell walls are primarily composed of cellulose existing as microfibers bound
to hemicellulose, specialized structural proteins, and pectins [1,16-18]. The
walls of adjacent cells are initially cemented together by pectic compounds
that compose a middle lamella. As these cells mature, they assume a more
rounded as compared with an initial square or rectangular shape. The
rounding splits the middle lamella apart at cell-to-cell contact points leaving
Internalization and Infiltration 81
a pectic sheath on the exposed walls [1]. Micropores, sometimes called micro-
capillaries, exist in the lattice of microfibers and associated carbohydrates.
These pores may be partially filled with pectic compounds or other wall
material. Additionally, the microcapillaries contain water in a pectin gel or
as free water, such that the relative humidity in the intercellular spaces ranges
from 98 to 100%. Sakurai [16] suggested that a plant's symplast is surrounded
by a liquid medium.
The precise environment within apertures and intercellular spaces is
unclear. Internalized, nonplant pathogenic microbes are not likely to be in
direct contact with plant cell membranes due to the thickness and structure
of the plant cell walls, which was referred to as a matrix by Sattelmacher et al.
[17]. Apoplastic fluid containing an array of solutes exists in the wall matrix.
However, the fluid's solute concentration and pH is not likely to equal
those reported to make plant tissues a favorable nutritional environment for
growth of bacteria [35]. The pH of the apoplastic fluid, which varies with
the location in the plant, the nutrition of the plant, and even the time of day
[17], would seldom be as low as that reported for macerated plant tissues,
where cell vacuoles have been ruptured. Xylem sap generally has a pH between
5 and 7, whereas the average pH of all apoplastic fluid ranges from 4.5 to 7.0.
In ripening fruit, the apoplast pH is reduced due to leakage of organic acids
through the plasmalemma and exposure of carboxyl groups from the
hydrolysis of pectin [16]. However, the contents of cell vacuoles normally
have a much lower pH than does the xylem sap or apoplastic fluid [21]. The
pH of vacuoles in lemon fruit was measured down to 2.4, whereas a pH of 0.9
was reported for fluid in the cell vacuoles of a species of begonia.
Certain reports conclude that the cell walls bounding intercellular spaces
have a coating of water, whereas others have suggested the exposed wall is
actually hydrophobic due to an incrustation of cutin [17]. The plant cuticle
has been observed to cover the guard cells and pore of a stoma and to extend
partially into the substomatal chamber [1,3,15]. Schonherr and Bukovac [22]
noted that the chemical characteristics of the surfaces of the cuticle on the
plant surface were similar to those within the substomatal chamber. The
cuticular complex, however, contains both polar carbohydrates and relatively
nonpolar cuticular components [15]. Cutin has been described as a poly-
ester with polar properties and an affinity for water [1]. The thickness of cutin
on the plant surface increases with light intensity and exposure to moisture
stress, which seem related to a restriction in water loss [3]. The thickness of
an internal cuticle in the succulent tissues of fruits and vegetables could be
quite different from that in leaf tissues where water loss through transpiration
can be a major stress on the plant. Thus, an incrustation of cutin might
not make cell walls hydrophobic, particularly in fruits and vegetables.
A combination of waxes and epidermal hairs help keep stomata from being
clogged with water as a result of dew formation or rainfall [2]. Such a plug
of water might substantially impair gas exchange [20]. The waxes on the
stomata surfaces repel water, whereas the stomatal pore contains a bubble of
air [1]. Thus, during dew formation, water droplets would bead up over the
82 Microbiology of Fruits and Vegetables
surface waxes and air bubble associated with the aperture. Goodman et al.
[1] suggest, however, that changes in temperature or leaf movement could
create pressure differentials that would draw surface water into stomata. By
contrast, a wind and rainstorm during daylight hours would produce
substantial water soaking of leaves through open stomata [36].
3.6 TYPES OF INTERNALIZATION
3.6.1 Aerosols
A mass flow of air through open stomata on leaves [29] could internalize
floating aerosol-sized particles including bacteria and viruses. Such aerosols
can disperse long distances from sources. Fattal et al. [37] detected aerosol-
ized enteric bacteria and viruses as far as 730 m downwind of wastewater
sprinkler irrigated field plots (note that the authors did not attempt detec-
tion at greater than 730 m). Gottwald et al. [38] concluded that in the spread
of citrus canker, the pathogenic bacteria could be dispersed as an aerosol,
in leaf debris, or wind-driven rainfall more than 5 miles by a single severe
rainstorm.
3.6.2 Water Channels and Water Congestion
Free water in surface apertures such as stomata constitutes a "water channel"
that connects a plant's apoplast with its external environment. Microorgan-
isms can internalize through water channels in various ways. Additionally,
persistent congestion of the apoplast by water may restrict oxygen availability,
which could compromise the resistance of the cells to microbial attack
[1,13,14]. Burton [20] noted that cells in respiring plant tissues become
anaerobic if water congestion blocked them from direct contact with air in
intercellular spaces. Tissues in a potato tuber covered with a film of water and
stored at 20°C become anaerobic within 2.5 hours [39]. Wet tubers are
susceptible to bacterial soft rot [40]. The loss of natural resistance to the disease
associated with tissue anaerobiosis occurs relatively quickly. Bartz and Kelman
[41] reported that freshly harvested and then washed tubers developed
soft rot during subsequent storage at 20°C if their surfaces remained wet for
20 hours, whereas if the tuber surfaces dried within 16 hours the disease did not
develop.
Water channels in leaf tissues have been associated with a large-scale
internalization of plant pathogenic bacteria. Massive wildfire and blackfire
lesions developed on field-grown tobacco only if leaf tissues were water con-
gested at the time the plants were exposed to inoculum [36,42]. In the absence
of water congestion, lesions tended to be small and of little consequence. By
contrast, in the absence of inoculum (disease absent from the field), water-
congested leaves recovered from a water-soaked appearance without evidence
of necrosis or other damage.
Internalization and Infiltration 83
Experimentally, water-soaked areas on leaf surfaces were correlated with
rapid internalization of bacteria [36,42,43-46]. Leaves of various plant
species were water-soaked by applying water under pressure to the root
system or cut surface of petioles [42,43] or as a water stream from a syringe or
sprayer [36,45,46]. Bacteria misted or poured on such surfaces were rapidly
internalized as were the carbon particles in India ink, solutions of water-soluble
dye, and suspensions of plant viruses. In the absence of water congestion,
a similar application of aqueous cell suspensions or India ink led to little or
no evidence of internalization. Cocci of Staphylococcus aureus penetrated
rapidly into water-congested leaf tissues providing clear evidence that
internalized bacteria need not be motile [46] or from a plant-associated
ecological niche. Johnson [43] concluded that bacterial suspensions were
pulled into water-congested tissues by capillary forces, which is inconsistent
with the concept that intercellular spaces are bounded with hydrophobic
surfaces [17]. Even with established water channels, however, water does not
totally flood intercellular spaces on submerged or partially submerged leaves.
Partially flooded intercellular spaces should function like closed capillary
tubes. Water would enter until pressure on trapped air balanced the capillary
forces. Thus, aqueous suspensions or solutions could penetrate quickly
through water channels but would move only a few cell layers due to a devel-
oping back pressure.
The guttation of water through hydathodes creates water channels where
bacteria and similarly sized microbes can passively internalize in plant leaves
[26]. Under normal conditions, guttation disappears when leaves begin to
transpire. Curtis [26] concluded that most guttation droplets are sucked back
into the leaf at this time. The drying of guttation moisture may concen-
trate solutes such that certain ones may damage the leaf surface. Mechanical
movement of guttation moisture back into hydathodes could passively
internalize bacteria and any other particulates that are small enough to pass
through the pore.
Mild water congestion of leaf tissues, which would not be visible as
water soaking also appears to enhance microbial internalisation [25]. For
example, preinoculation incubation of plants under high humidity leads to
more disease than postinoculation incubation [45,47]. Citrus leaves become
infected by Xanthomonas citri only if the substomatal chambers are filled with
water [48]. This level of water congestion would not be visible to the unaided
eye. A bacterial disease of cucumber, angular leaf spot, progressed most
rapidly when the soil was warm and contained high moisture despite
daytime air temperatures that inhibited pathogen development [50]. It is
precisely this type of environment that favors guttation.
3.6.3 Internalization in Wounds
Fresh wounds feature an immediate release of fluid from ruptured vacuoles
and plasmalemma. This "cell sap'' congests the intercellular spaces in and
beneath the damaged cells creating instant fluid channels [25]. Within seconds
84 Microbiology of Fruits and Vegetables
of contact, particulate matter or aqueous suspensions may be transported up
to 1 cm laterally from a puncture wound in a leaf [43]. This concept of rapid
internalization in wounds is supported by tests on the disinfection of wounds
on tomato fruit. Bartz et al. [50] observed that within 5 seconds of application
of an aqueous cell suspension of E. carotovora subsp. carotovora to the flat
surface of a fresh wound on a tomato fruit, a portion of that population could
not be completely eliminated when the fruit was washed for 2 minutes in
lOOppm free chlorine at pH 7.0 in a scale model flume. Gently rubbing the
submerged wound surface with a soft bristle brush or with a gloved finger
did not improve disinfection efficacy. In contrast, lOppm free chlorine present
over similar wounds on fruit in the same flume prevented inoculation by a
similar suspension, whereas just 5 ppm prevented most wounds from becom-
ing inoculated. A water-soluble dye could be completely rinsed from these
wound surfaces if the fruit was rinsed under running tap water within 6 seconds
of dye application. If the wash was delayed more than 6 seconds a portion
of the dye could be observed embedded in intercellular spaces beneath the
wound.
3.6.4 Infiltration of the Plant Surface by
Aqueous Cell Suspensions
Water or aqueous cell suspensions of bacteria may infiltrate apertures as well
as wounds on fresh fruits and vegetables during harvest and handling [30,34,
51-53]. This infiltration can directly internalize microbes and can be either
spontaneous or pressure driven. Schonherr and Bukovac [22] observed
spontaneous penetration of stomata on leaves by water that was amended
with a surfactant. A biosurfactant produced by Pseudomonas fluorescens alters
the wax crystals on the surface of broccoli florets and may aid in spon-
taneous penetration of that structure by plant pathogenic strains of this
bacterium [3]. Fresh wounds also appear susceptible to spontaneous
infiltration by surface moisture [43,50].
Pressure-driven infiltration of fruits and vegetables means that pressure
on water covering plant surfaces forces water into surface apertures despite
air bubbles and the waxy nature of the pore surfaces. The cooling of fruits
and vegetables leads to a reduction of gas pressures in the apoplast [54],
particularly if the surface apertures are clogged with liquid. This pressure
differential would persist until internal temperatures and gas pressures
equilibrate with the external environment. Tomatoes allowed to cool while
submerged in water may increase in weight due to water uptake [30,51].
Hydrocooled tomatoes increased in weight as they cooled [34] as did
hydrocooled strawberries [52]. If the water contained cells of Erwinia
carotovora subsp. carotovora or spores of Botrytis cinerea, water uptake
correlated with a rapid development of internal lesions when the tomatoes or
strawberries, respectively, were subsequently stored. When submerged in
an aqueous cell suspension of E. coli at 2°C, fruit of four different apple
cultivars initially at 22° C internalized the bacterium in the outer core region
Internalization and Infiltration 85
of the fruit during a 20-minute exposure [53]. A water-soluble dye was
observed to internalize in similar treatments. However, evidence for the
penetration of the skin, likely through open lenticels, appeared to be limited to
injuries to the surface. Kenney et al. [55] observed E. coli cells up to 24 um
deep in open lenticels on "Delicious" apple fruit that had been cooled in an ice
bath. Bruising the surface increased the number of internalized bacteria,
particularly with respect to those embedded in cracks in the surface waxes.
However, washing the apples in distilled water prior to examination led to
an apparent reduction in penetration to depths no greater than 6 |im. Cooling
hot water-treated mango fruits (46° C) in water (22° C) for 10 minutes led
to infiltration of the fruit by a dye solution or by a suspension of Salmonella
enterica [56]. The dye and bacteria primarily entered through the stem scar.
Direct injury to plant tissues may be caused by an infiltration by water,
likely because the congestive water is absorbed by the cells causing them to
swell. Tomato fruits that absorbed water equal to 3% or more of their
original weight developed visible cracks, usually near the shoulders [30]. Studer
and Kader [51] reported that a high percentage of freshly harvested tomatoes
submerged for 15 to 120 minutes in water of various temperatures devel-
oped splits (breaks in the surface), whereas those stored overnight before the
water treatment did not. Warming the water to above the fruit temperatures
reduced but did not prevent the splits.
Hydrostatic pressure also can force water into apertures on fruits and
vegetables [32]. Fruit or vegetables at the bottom of containers of submerged
products would be exposed to a hydrostatic pressure on product surfaces
equal to the total depth of submersion. Hydrostatic pressures would be
additive to pressure differentials associated with cooling but counteract those
associated with warming. However, water depth pressures would be exerted
more rapidly than those associated with temperature changes. Hydrostatic
pressures not only directly force water into surface apertures but also tend to
squeeze submerged products and may cause air to bubble out of openings.
When the hydrostatic pressure is removed, the product is likely to expand to its
original volume leading to an internal pressure differential that will draw water
into the product.
An abrupt impact with water can cause microbial internalization and
water channels in fruits and vegetables. Water impact forces occur when field
containers of freshly harvested fruits or vegetables are emptied into water or
when a pile of a product is dispersed into a packinghouse flume by a heavy
stream of water. Pressure washing systems in packinghouses also are likely
to produce water congestion in surface apertures.
3.6.5 Events in Plant Development
In the field, plant root systems and hydathodes appear most likely to
internalize microorganisms. Plant roots are likely to internalize soil
microbes because wounds form during root growth. The development of
86 Microbiology of Fruits and Vegetables
lateral roots in plants usually begins at the pericycle, which underlies the
endodermis [18]. The endodermis is a tightly packed cell structure located
several cell layers below the root surface. As the root tip forms and then
emerges, it breaks through the endodermis and cortex creating an open
wound, which is a frequent site for colonization by soilborne bacteria [1,12].
Even casual bacteria can grow in wounds created by the emergence of
lateral roots. Escherichia coli 0157:H7 internalized in lettuce apparently
through the root system when the plants were fertilized with contaminated
manure or irrigated with contaminated water [57]. In controlled studies with
seedlings of several plant species grown in test tubes, strains of Salmonella
enterica were able to colonize the lateral root emergence wounds and then
colonize intercellular spaces in the interior of the root [12]. Certain bacterial
types applied to these plantlets were observed in xylem vessels, whereas others
were rarely found in such cells. All applied bacteria were observed in the cortex
of the root. Populations of a known endophytic bacterium, Klebsiella
pneumoniae, exceeding log 8.0 CFU/g fresh weight were found in the root
system of seedling rice plants grown in test tubes [7]. Endophytic popula-
tions were successfully initiated by the inoculation of seedlings with as few as
1 CFU per seedling [12]. Whether populations multiplied on the rhizoplane
prior to entering the plant or found sufficient nutrition to multiply totally
inside plants could not be determined; however, endophytic populations
were correlated with those on the rhizoplane.
The soilborne, bacterial wilt pathogen Ralstonia solanacearum (Pseudo-
monas solanacearum) was observed to penetrate tobacco roots through
epidermal cells that were damaged by lateral root emergence [58]. After
penetration, the bacteria moved intercellularly in the cortex. The entrance of
bacteria into xylem vessels appeared to occur where the endodermis was not
fully developed or as a consequence of hypertrophy of xylem parenchyma
cells, which appeared to disrupt young xylem vessels.
Growth cracks in plant surfaces during maturation processes could, at least
temporarily, provide microbe internalization sites. Wide temperature
changes, rainfall, the planting of crack-susceptible cultivars, and fertilization
programs featuring high nitrogen and low potash have been associated with
growth cracks in tomatoes [59]. Growth cracks in tomato fruit surfaces are
a frequent site of microbial attack and predispose the fruit to pre- and
postharvest decay [60]. Such cracks could enable internalization of a wide
range of microorganisms. Many other crops have cultivars designated as
crack resistant. In any fresh fruit or vegetable, the development of cracks or
punctures in surfaces leading up to harvest, at harvest, or after harvest could
enable various microbes to internalize. While most plant organs with growth
cracks are culled during the packing process, items with minor cracks or
punctures could be shunted to fresh-cut processing and lead to a contami-
nated product. Alternatively, microbes could internalize in plant organs that
naturally crack during development such as cantaloupes [18].
Internalization and Infiltration 87
3.7 IMPLICATIONS AND CONTROL
Many of the examples of the internalization of microorganisms by fruits
and vegetables cited above involve situations occurring during crop production
or harvest that cannot be controlled. Many internalization hazards can be
controlled. The results of internalization can range from poor shelf life due
to decay to unwholesomeness due to contamination by hazardous micro-
organisms. The list of human pathogens that can be internalized by fruits and
vegetables is extensive [4]. For crops intended for consumption as raw
products, such contamination is, at present, irreversible. With the globaliza-
tion of agriculture and the consumer demand for fresh crop items all year
round [61], there are nearly endless opportunities for microorganisms
originating in the fields and surface waters of underdeveloped countries to
end up in salad or fresh fruit items served in homes and restaurants in
developed countries.
The inability of even the strongest surface disinfectants to eradicate
completely human pathogens from contaminated fresh fruits and vegetables
and yet be compatible with a product appearance that meets marketing
requirements is well documented [4]. The failure of chlorinated water
treatments, even at concentrations exceeding 5000 ppm, to eradicate plant
pathogens from inoculated wounds has been known since 1945 [62]. Much
conjecture has been focused on the inability of chlorine, a strong oxidizer, to
disinfect contaminated wounds. Often authors suggest that active chlorine
reacts with wounded tissues such that pathogen structures are not exposed to
a critical dose. However, with contaminated wounds on tomato fruit,
increasing doses and mechanical scrubbing of the wound surface have not
led to significant increases in efficacy [63]. With contaminated cantaloupes,
however, Ukuku and Fett [64] observed an increase in efficacy of 200 ppm
chlorine (pH 6.4) or 5% H 2 2 if the fruits were rubbed during the 2-minute
immersion treatment, although not all contamination was eradicated. Based
on Johnson's [43] theories on capillary movement of suspensions into leaf
tissues and observations on dye movement and suspensions of soft rot bacteria
into wounds on tomato fruit, Bartz et al. [50] suggested that solutions of
active chlorine applied to inoculated wound surfaces on tomatoes displaced
the pathogen cells further into the underlying intercellular spaces.
Internalization risks can be minimized through use of HACCP-type
(hazard analysis critical control point) analyses and practices in
production systems [65]. The ultimate goals of such a program are to mini-
mize water penetration of plant tissues, crop contact with hazardous
microorganisms, open wounds on plant surfaces, and situations likely to
cause fluid penetration of plant surface apertures. Particularly, crops
intended for raw consumption should never be treated, irrigated, washed, or
cooled with poor-quality water [66]. Improperly composted manures should
never be used in fields intended for production of fresh fruits and vegetables.
In a recent survey of fruit and vegetable producers in Minnesota, one grower
88 Microbiology of Fruits and Vegetables
spread untreated manure throughout the growing season and 90% of the
fruit and vegetable samples from that farm were positive for E. coli [67].
Fields should be fenced to keep out domestic or wild animals and should
be located at least 5 miles from the nearest feed lots or other concentrations
of animals [65]. Field workers should not be allowed to work with or harvest
a crop if they are ill or have recently been ill. Working with water-congested
plants creates a special hazard and should be avoided. Cultivars selected for
production should resist the development of growth cracks or other
characteristics that enable penetration by microorganisms.
Certain handling steps after harvest can reduce the internalization hazard.
For example, the porosity of tomato stem scars to water is greatest immedi-
ately after harvest and then decreases over time [32]. Leaving a stem attached
until just before water treatment only slightly reduces this characteristic. Studer
and Kader [51] observed that tomato fruits split readily (from water uptake) if
they were submerged in water immediately after harvest but did not if stored
overnight before treatment. Additionally, warm fruit is more likely to absorb
water than cool fruit during exposure to hydrostatic pressure as well as during
exposure to water cooler than the fruit [30,31,51]. Thus, allowing tomatoes
to cool overnight before packing them should decrease the likelihood of
water infiltration during the unloading and washing processes at packing-
houses. Although this prepacking storage would allow pathogen growth on
damaged fruit (which otherwise would have been culled), small wounds would
begin to heal and the stem scar would dry, thereby reducing the number of
water channels. Additionally, the loss of a small amount of water from each
fruit should decrease the likelihood for handling injuries to the tomato surface.
With citrus, Eckert [62] noted that a standard practice in California was to
"wilt" the fruit before washing and packing to reduce susceptibility to surface
injuries.
The water used to handle or wash fruits and vegetables must be conti-
nually sanitized during the workday, particularly if the water is recycled.
Moreover, the sanitizer must be present where the unwashed product enters the
water system to minimize the chances for an internalization of hazardous
microorganisms at the initial contact point. Highly reactive chemicals such
as ozone [68] may be too unstable for maintenance of adequate residuals.
Currently, hypochlorous acid from solutions of sodium hypochlorite, liquefied
elemental chlorine, or solid powder or pellets of calcium hypochlorite best
combines efficacy, speed of action, and stability for minimizing internalization
hazards at packinghouses. Moreover, residues from the chlorinated water
treatment either quickly dissipate from treated products or are harmless salts.
Unfortunately, water chlorination cannot make badly contaminated surface
waters safe to use for handling and washing produce as it is not effective
against the resting stages (cysts, oocysts) of certain human parasites [68].
Additionally, where high chlorine demand exists, such as with shredded
vegetables or with certain root crops, maintenance of adequate residuals is
difficult.
Internalization and Infiltration 89
Whether water chlorination eliminates the need to suppress completely
water infiltration during postharvest handling is unclear. The infiltration of
tomatoes with chlorinated water failed to prevent the development of
postharvest decay when submerged fruits were treated with hydrostatic
pressure at room temperature [69], but did prevent such decays when fruits
were hydrocooled [34]. The presence of chlorine in the water appeared to
increase the porosity of tomato stem scars [69]. The chlorination of the water
used to hydrocool strawberries led to a significant reduction in botrytis fruit
rot [52]. As noted above, however, chlorinated water treatments have
consistently failed to eradicate completely microorganisms from fruit or
vegetables likely to have internalized a portion of the contamination. For
example, the washing of contaminated wounds on tomato fruit with over
500 ppm free chlorine at pH 7.0 reduced the subsequent development of soft
rot by 50% in one test and had no effect in two [70]. In two separate reports
on tomatoes that had been contaminated in the laboratory, washing wounds
or stem scars with 100 ppm or more of free chlorine failed to eliminate
Salmonella Montevideo [71,72].
When fruits or vegetables are unloaded into or washed by water, infiltra-
tion of natural apertures due to a temperature related pressure differential may
be controlled by maintaining water temperatures above those of the incoming
fruits and vegetables [30]. Current recommendations for water handling steps
with tomato fruit are to keep water temperatures about 5°C (10°F) above those
of incoming fruit and to limit fruit contact with water to 2 minutes [73]. This
handling recommendation also includes provision for maintaining 100 to
1 50 ppm free chlorine in the water. The pH of chlorinated water should be
in the range of pH 6.5 to 7.5 to ensure ample concentrations of the killing
agent, HOC1, and minimal corrosion [65]. Warming the water increases
chlorine's efficacy and decreases its stability [68]. In cooler weather, use of
warm water to handle tomatoes has been associated with a reduction of surface
injuries [74].
Selection of crop cultivars may also help reduce internalization hazards.
The relative tendency of a tomato stem scar to absorb water appears to be
a varietal characteristic [75]. Certain varieties consistently absorbed more
water than others over different harvests of the same crop or the same cultivars
in different fields and seasons. Heggestad [76] reported that leaves of
certain tobacco cultivars were less likely to develop water congestion than
others. In naturally occurring outbreaks of wildfire disease, lines that were
less prone to water congestion had less disease. McLean and Lee [48] noted
that structural differences in stomata were responsible for the resistance of
mandarin orange to a bacterial disease, citrus canker. Therefore, the ten-
dency of plant tissues to resist water intrusion and microbial internalization
might be enhanced by selection and breeding.
Encouraging tissue respiration has been suggested as an internali-
zation reduction treatment during preparation of fresh-cut lettuce. Takeuchi
and Frank [77] reported that a high respiration rate in minimally processed
lettuce produced a "counterforce" that reduced the internalization of cells of
90 Microbiology of Fruits and Vegetables
E. coli. Thus, warming lettuce to encourage respiration during sensitive
stages of fresh-cut lettuce preparation might decrease the potential for
internalization of bacteria from wash water. Subsequently, a group of authors
noted that reducing the 2 over the lettuce to 2.7% reduced the internali-
zation associated with low-temperature incubation [78]. Ostensibly, the
counterforce was CO2 released from respiration. The methodology used in
these reports, however, raised questions about the validity of the authors'
conclusions [79]. The lettuce was purchased from local stores and stored at
4°C. Tissue sections were prepared and submerged in water or an aqueous
cell suspension of E. coli for 24 hours at 4, 10, 22, or 37°C [77,78]. The authors
did not indicate if the tissues and fluids were equilibrated to these tem-
peratures prior to the incubation. In fact, Takeuchi et at. [78] commented in
their discussion that ". . . subsequent infiltration of the bacteria into the lettuce
as it cooled during the inoculation period." If the lettuce tissues cooled during
the incubation, then a pressure differential would occur in the intercel-
lular spaces, as discussed in Section 3.6.4. This would lead to an infiltration
of the bacterial suspension into the cut edges. Conversely, if the tissue
sections warmed, internal gases would expand and tend to prevent infiltration.
Without knowledge of tissue and fluid temperatures at the beginning of the
test, it is impossible to interpret the results. Additionally, the relatively
high cold-water solubility of C0 2 as compared with 2 may be involved.
Due to respiration, 2 in intercellular spaces would be absorbed by the
lettuce cells. If C0 2 production matches 2 absorption, gas pressures in the
intercellular spaces should not change. However, at low temperatures, a
significant portion of the C0 2 released by mitochondria is likely to remain
dissolved in cell sap. As such, the uptake of 2 could contribute to a reduc-
tion in internal gas pressure. (Note that this would be much like the standard
laboratory exercise on measuring plant tissue respiration with a Warburg
apparatus where the C0 2 produced is scrubbed by an alkali solution and
oxygen uptake is measured with a manometer.) In the absence of significant
respiration, changes in the partial pressures of 2 and C0 2 should not be
factors in pressure differentials developing within the tissues.
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4
Microbial Stress Adaptation
and Safety of Produce
Luis A. Rodriguez-Romo and Ahmed E. Yousef
CONTENTS
4.1 Microbial Stress Adaptation Phenomenon 95
4.1.1 Stress 95
4. 1 .2 Stress Response 96
4.1.3 Stress Adaptation and the General Stress Response 96
4. 1 .4 Regulation of the General Stress Response 97
4.2 Produce Microbiota as Influenced by Stress History 98
4.2. 1 Preharvest Stress 98
4.2. 1 . 1 Temperature Fluctuation 99
4.2.1.2 Ultraviolet Radiation 100
4.2.1.3 Osmotic Stress 101
4.2.2 Postharvest Stress 102
4.2.2.1 Cold Stress 102
4.2.2.2 Acid Stress 104
4.2.2.3 Oxidative Stress 105
4.2.2.4 Minimal Processing 106
4.3 Microbial Stress Adaptation on Produce 106
4.4 Assessing Stress Adaptation and Associated Risks 107
4.5 Summary 108
References 108
4.1 MICROBIAL STRESS ADAPTATION
PHENOMENON
4.1 .1 Stress
Microbial stress can be defined as any deleterious physical, chemical, or
biological factor that induces modifications in the physiology of micro-
organisms (i.e., changes in the genome or proteome) that adversely affect
microbial growth or survival [1-3]. The application of this broad definition
of stress in food processing implies that many preservation treatments (e.g.,
heat, cold, and acid) are considered stresses, and, as a result, these may
95
96 Microbiology of Fruits and Vegetables
significantly influence the behavior of foodborne pathogenic and spoilage
microorganisms. Depending on the severity, stresses affect a microbial popu-
lation in a number of ways. Exposing microorganisms to a sublethal stress
(simply, this will be referred to as "stress") affects their metabolic activities
unfavorably, leads to cell injury, and consequently retards or temporarily
arrests their growth. When a microorganism is exposed to a severe adverse
condition (i.e., lethal stress), this causes irreversible cell damage and,
consequently, a decrease in population viability.
4.1.2 Stress Response
The microbial cell has the means to sense stresses such as those leading to
ribosomal disruption (e.g., heat stress) or modification in cell membrane
fluidity (e.g., cold shock). Response to these stresses is presumed beneficial
to the cell, but it occasionally has detrimental consequences. Protective
responses require physiological adaptations to compensate for stress damage
and permit the cell to continue its growth and ensure its survival. Similarly,
the bacterial cell responds to stress induced by inherent physiological change.
Entry of a cell population into the stationary phase, for example, triggers
a general stress response, which results in microbial resistance to multiple
stresses. Adaptive stress response involves the induction of a number of genetic
and physiological mechanisms, as well as morphological events, which include:
(1) synthesis of protective proteins that participate in damage repair, cell
maintenance, or suppression of stress agents, (2) temporary increase in
resistance to lethal factors, (3) transformation of cells to a latent state, e.g.,
spore formation or induction of viable-but-not-culturable state, (4) evasion of
the host's defense mechanisms, and (5) adaptive mutations [1,2,4-7].
Environmental or physiological conditions may hinder a cell's ability to
respond to stress. Chilled or metabolically exhausted cells, for example, may
not respond to radiation stress. Similarly, when dormant bacterial spores
are exposed to an injurious stress they are incapable of responding until
conditions are favorable for germination and outgrowth. Lack of response to
a stress may sensitize a microbial cell to subsequent stresses that are other-
wise innocuous. Response to a stress also may exhaust a cell's ability to cope
with subsequent stresses, causing a stress-sensitizing effect.
4.1.3 Stress Adaptation and the General
Stress Response
Exposure of a microorganism to stress triggers a series of metabolic responses
that may adapt the cell to subsequent lethal levels of the same type of stress
or to multiple lethal stresses. The cell's adaptive response is generally referred
to as stress adaptation. Stress, ensuing adaptive response, and the mani-
festation of this phenomenon in food preservation are collectively described as
stress hardening. Food microbiota are regularly subjected to stress hardening.
Therefore, the stress adaptation phenomenon is of paramount importance
Microbial Stress Adaptation and Safety of Produce 97
when evaluating the efficacy of intervention strategies to achieve food safety
and to preserve food quality. Although stress adaptation is usually asso-
ciated with the undesirable acquired resistance of foodborne pathogens to
processing, this phenomenon also plays a key role in the survival of beneficial
microorganisms used as probiotics or fermentation starters.
The microbial mechanisms to survive adverse environmental conditions
can be divided into two classes, consisting of limited and multiple adaptive
responses [8]. A limited or specific adaptive response results when micro-
organisms are exposed to a sublethal dose of a physical, chemical, or biologi-
cal stress, which protects cells against subsequent lethal treatment with
the same stress [8,9]. A multiple adaptive response, also known as cross
protection, occurs when microbial cells adapt to an inherent physiological
condition or to an environmental factor, which results in protection against
subsequent lethal treatments, including stresses to which the micro-
organism had not been previously exposed [8,10-12]. This cross-protective
response requires the induction of the general stress response, and it is triggered
by stresses relevant to produce, both preharvest and postharvest, including
cell starvation, exposure to high or low temperatures, high osmolarity, and
low pH [12,13]. The activation of the general stress response is characterized
by reduced growth rate or induced entry into stationary phase. The regulation
of the general stress response has been well characterized in several
microorganisms. This regulation is under the control of the alternative sigma
factors, which bind to core RNA polymerase, mediating cellular responses
through redirection of transcription initiation. Sigma S (a , also known
as RpoS) and a B regulate the general stress response in Escherichia
coli and other Gram-negative bacteria, and in Bacillus subtilis and other
Gram-positive bacteria, respectively [13,14].
4.1.4 Regulation of the General Stress Response
The general stress response is regulated by the rpoS, a gene that encodes the
a in E. coli and other bacteria such as Shigella flexneri and Salmonella enterica
serovar Typhimurium [15-17]. Although the regulation of the general stress
response has been studied in a variety of microorganisms, the regulation
mechanisms covered in this section will refer to E. coli, an organism in which
these mechanisms have been well characterized. During rapid growth,
microbial cells, not exposed to any particular stress, have hardly detectable
levels of a . Exposure of these cells to stress (e.g., entry into stationary phase,
high osmolarity, high or low temperature) results in rapid a accumulation
to high levels, and subsequent expression of more than 50 genes involved
in stress adaptation [16]. The regulation of rpoS, which determines the cellu-
lar concentration of a , occurs at multiple levels, including transcrip-
tion, translation, and post-translational modifications (i.e., a proteolysis),
with the level of control being dependent on the type of stress affecting
the cells [15,16,18]. In general, sudden exposure of bacteria to lethal
stresses, which requires a rapid response (i.e., a shocking stress), involves a
98 Microbiology of Fruits and Vegetables
proteolysis-mediated regulation, while gradual exposure to stress usually
requires stimulation of rpoS expression at the transcription or translation
level [16,19].
Enhanced cellular accumulation of a occurs during microbial growth in
rich media, while cells are transitioning from late exponential phase to station-
ary phase [15,19]. At the transcriptional level, the two-component system,
cAMP and its receptor protein, the catabolite regulatory protein (CRP), act
as negative regulators of rpoS. Conversely, small molecules such as guanosine-
3 / ,5 / -bispyrophosphate (ppGpp), homoserine lactone, and polyphosphate may
enhance rpoS transcription [16,18]. Translational control involves a series
of complex mechanisms in which stress conditions such as high osmolarity,
low temperature, or entry into late exponential phase stimulate the trans-
lation of rpoS mRNA [13]. It has been suggested that these stresses can play
an important role in stabilizing the mRNA secondary structure, allowing
its accessibility to ribosomes, and therefore enhancing its translation [16].
Activation of rpoS mRNA translation requires the presence of Hfq, a small
mRNA binding-protein that stabilizes the secondary structure of the
polynucleotide. Translation of rpoS can also be enhanced by the stabilization
of the mRNA with a small RNA fragment (DsrA RNA) in cells stressed by
temperature downshifts [13]. Control at the post-translational level involves
regulation of the sigma-factor proteolysis rate. In cells growing exponentially,
the levels of a are very low because of its continuous proteolysis. Sudden
stresses, including carbon starvation, shift to low pH, high temperature, and
high osmolarity, prevent a proteolysis and permit its accumulation in the
cells to trigger the general stress response. Proteolysis of a requires ClpXP
protease, which is regulated by the RssB protein. The level of phosphoryla-
tion or dephosphorylation of RssB, influenced by the stresses already ment-
. . . . c
ioned, determines its affinity for a and the subsequent recognition of the
a -RssB complex by the ClpXP protease [13,16].
The activation of the general stress response, mediated by a , results in
the expression of stress-adaptive genes, including bolA (involved in control-
ling cell morphology), cfa (involved in cyclopropane fatty acid synthesis),
uspB (involved in ethanol resistance), and katE and katG (encoding
catalases), among many others [15,20]. Sensitivity of bacteria, defective in
the rpoS gene, to a series of stresses such as heat shock, oxidative environment,
starvation, acid, ethanol, and ultraviolet radiation provides additional, and
indisputable, evidence of the role of a in the control of the general stress
response [21,22].
4.2 PRODUCE MICROBIOTA AS INFLUENCED BY
STRESS HISTORY
4.2.1 Preharvest Stress
Fruits and vegetables can be contaminated with bacterial pathogens and
spoilage microorganisms by contact with feces, soil, irrigation water,
Microbial Stress Adaptation and Safety of Produce 99
improperly composted manure, air-carried dust, wild and domestic animals,
and human handling [23-28]. Survival and potential proliferation of
contaminants on produce depends on the type of microorganism, the type
and condition of produce, and the environment (e.g., temperature, humidity).
Examples of environmental stresses that may affect microorganisms on
fresh produce include nutrient restrictions, temperature and pH fluctuations,
water availability limitations, exposure to ultraviolet radiation, presence of
organic compounds (e.g., pesticides) and metal contaminants, inhibitory plant
tissue reactions, and microbiota competition, among many others.
Many foodborne pathogens have enteric origin, which could limit their
ability to survive in other environments, colonize plant tissues, and compete
with plant-associated microorganisms [23]. However, it is known that
Salmonella can survive, adapt, and proliferate in soil, a nonhost environ-
ment characterized by its thermal variability, high osmolarity, pH fluctuations,
and variable nutrient availability [28]. Brandl and Mandrell [29] reported
that Salmonella Thompson was able to colonize the surface of cilantro
leaves and proliferate when plants were incubated at warm temperature (30° C).
In addition, it was observed that the microorganism tolerated plant dry
conditions (60% relative humidity) at least as well as usual bacterial plant
colonizers (e.g., Pantoea agglomerans and Pseudomonas chlor or aphis). There is
evidence that secretions of plant seeds can induce microbial stress. Miche et al.
[30] studied the response of E. coli, containing luxCDABE reporter genes,
to germinating rice seed exudates, and reported that these secretions enhanced
the expression of microbial genes involved in general stress, heat shock, and
oxidative stress responses.
4.2.1.1 Temperature Fluctuation
Temperature variations at different stages of preharvest can affect the
behavior of microbial populations present on produce. Besides influencing
microbial growth, sudden fluctuations of temperature can cause heat or cold
shock, and consequently may enhance the tolerance of foodborne pathogens
to subsequent stresses. In this section, the effect of heat-induced stress will
be discussed; cold stress will be addressed in another section of the
chapter. Sublethal heat stress refers to the stress resulting from exposing a
microbial population to temperatures higher than the maximum for growth
and lower than that causing considerable cell death. Response to heat stress
is most obvious when this stress causes minimal (less than one log) reduction
in cell population.
Sublethal heat stress causes damage in the macromolecular structure of
bacterial cells (e.g., protein denaturation), causing disruption of metabolic
activities, which consequently affects microbial growth [31]. Microbial cells
react against heat by inducing a universal protective response, generally known
as the heat-shock response. This response involves the transient overexpres-
sion of heat-shock proteins that protect the cells against heat damage and
other stresses. Heat-shock proteins include molecular chaperones (e.g., DnaK
100 Microbiology of Fruits and Vegetables
and GroEL) which repair cell injury by refolding the denatured proteins
[2]. Other heat-shock proteins have protease activity (e.g., ClpP), dependant
on ATP, and are involved in the degradation of heat-damaged proteins
[32]. Additionally, microorganisms may adapt to mild heat by modifying
the fluidity of their cell membranes; this is accomplished by increasing the
length or level of saturation of the membrane's fatty acids [11].
The transcription of the majority of the heat-shock proteins in E. coli
is controlled by the alternative sigma factor, a [33]. Additionally, a is
involved in the regulation of heat-induced genes of this bacterium [34,35].
Induction of the heat-shock response in B. subtilis requires several regulatory
groups including the HrcA-CIRCE system, which controls the major
chaperone genes. The general stress response, controlled by a B , and the genes
encoding Clp protease system are also involved in regulating the heat-shock
response of this bacterium [3,33].
In addition to heat, several other stresses may trigger the synthesis of
heat-shock proteins, and, as a result, induce multiple stress-protective
responses. These stresses include changes in pH or osmolarity, ultraviolet
irradiation, and the presence of substances such as ethanol, antibiotics,
aromatic compounds, and heavy metals [36]. Synthesis of heat-shock proteins
after exposure to other stresses may be attributed to the presence of a common
stress sensing mechanism in the cells, which detects accumulated abnormal
proteins in the cytoplasm [36-37].
There is substantial evidence confirming that exposure to sublethal heat
increases the resistance of microorganisms to single or multiple lethal stresses.
Seyer et al. [38] observed that E. coli, heated at 55°C for 105 minutes and
permitted to recover, had enhanced tolerance to subsequent lethal, thermal
treatments (60°C for 50 minutes). In addition, the investigators reported that
internal cell concentration of the DnaK chaperone played a key role in
microbial recovery and stress tolerance. Lou and Yousef [39] indicated that
stressing Listeria monocytogenes with heat (45° C for 60 minutes) protected the
cells to subsequent exposure to lethal concentrations of ethanol, hydrogen
peroxide, and sodium chloride. Lin and Chou [40] observed a similar behavior
in the same microorganism under comparable sublethal stress conditions.
These researchers also indicated that thermal stress at a higher temperature
and shorter time (48°C for 10 minutes) than those used by Lou and Yousef
[39], protected L. monocytogenes against sodium chloride but decreased the
resistance of the pathogen to lethal concentrations of hydrogen peroxide.
4.2.1 .2 Ultraviolet Radiation
Microorganisms are exposed to ultraviolet (UV) radiation from sunlight
while present on the surface of fruits and vegetables. The main fraction of
solar UV radiation that reaches the Earth's surface consists of long-
wavelength UV (320-400 nm), which is usually designated as ultraviolet A
(UVA). This type of radiation affects the microbial cell membrane and causes
oxidation of unsaturated fatty acids. In addition, UVA participates in an
Microbial Stress Adaptation and Safety of Produce 101
oxygen-dependent reaction that involves the photosensitization of pigments,
which results in the generation of reactive oxygen species (e.g., O^) with
antimicrobial activity [41]. In E. coli, UVA induces lethal and sublethal
stress that may cause temporary growth inhibition, loss in phage sensitivity,
and inhibition of tryptophanase induction [42,43]. There is evidence that
when E. coli is treated with sublethal UVA radiation while in the stationary
phase the microorganism recovers rapidly and acquires resistance to sub-
sequent lethal irradiation treatments; however, this tolerance is not associated
with the general stress response involving rpoS [41,42].
Short-wave UV radiation (200-280 nm), designated as ultraviolet C (UVC),
causes damage to microbial DNA and RNA by inducing formation of
pyrimidine-base dimers and DNA-protein crosslinks, which results in cell
growth cessation, decreased viability, or cell death [44]. This microbicidal
UVC radiation, particularly at 254 nm, has been implemented as a preservation
treatment in a variety of foods; however, its application as an intervention
strategy to be used alone is not recommended [11,45]. Sublethal doses of
UVC may induce mutations and render cells tolerant to lethal irradia-
tion and other stresses [46,47]. Hartke et al. [48] reported that irradiation of
Lactococcus lactis with sublethal UVC (at 254 nm) induces the production of
numerous protective proteins and enhances the tolerance of the micro-
organism to subsequent lethal treatments with heat, acid, and hydrogen
peroxide. Bacterial cells can recognize damage caused to DNA as a con-
sequence of UVC exposure and trigger a series of mechanisms to repair
deleterious nucleic acid modifications. These processes require the parti-
cipation of enzymes, which can be induced in the absence or presence of visible
light, and these are named dark-repair and photoreactivation mechanisms,
respectively [44]. Damage caused to microbial DNA can be counterbalanced
by induction of the SOS response, which regulates the expression of genes
involved in DNA repair [49].
4.2.1.3 Osmotic Stress
Although osmotic stress of microorganism on produce surfaces is not
common at the preharvest stage, the following scenarios are likely to occur.
Microorganisms may experience osmotic stress when they are exposed to
saps released from bruises and wounds on produce surfaces. Dryness of micro-
organisms on produce surfaces also may result in osmotic stress. Expo-
sure of surface microbiota to salts may occur with some commodities if
brine flotation is used in conveying, sorting, or sizing operations. In order to
survive osmotic stress, microorganisms must keep a balance between the
water inside the cells and the concentration of solutes in the environment.
Generally, bacterial cells use two protective mechanisms to survive hyper-
osmotic stress: (1) discharging the excess of solutes within the cells to the
outside and (2) accumulating compatible solutes or osmolytes. In addition,
microorganisms adapt to this stress by modifying their cell membranes, e.g.,
by increasing the ratio of trans to cis unsaturated fatty acids [2,4]. Microbial
102 Microbiology of Fruits and Vegetables
accumulation of compatible solutes is a mechanism that has been well
characterized. Compatible solutes are small, polar, organic molecules that
remain water soluble at relatively high concentrations without affecting
intracellular structures or metabolic activities. These solutes include com-
pounds such as carnitine, trehalose, glycerol, sucrose, proline, mannitol,
glycine-betaine, and small peptides, among others [4,15]. The accumulation of
compatible solutes, as a result of osmotic stress, requires the expression of
proteins involved in the synthesis of the osmoprotectants or their transport
systems [2,50]. The synthesis of several proteins that participate in osmolyte
accumulation is under the control of the general stress response sigma factors
a and a in E. coli and B. subtilis, respectively, which regulate the expres-
sion of chaperones and proteases [3,15]. Therefore, adaptation of micro-
organisms to osmotic stress may render them resistant to subsequent stresses of
different types. Pretreatment of B. cereus with NaCl (1%) caused microbial
resistance to subsequent lethal treatments with heat, ethanol, hydrogen
peroxide, and acid. However, stress adaptation failed to protect the
microorganism against a medium containing 12% NaCl [51,52]. Periago et at.
[53] observed that pre-exposure of the same microorganism to osmotic stress,
with 2.5% NaCl for 30 minutes, induced cell tolerance to lethal heating
at 50°C. Osmotic stress of L. monocytogenes, by previous exposure to 3.5%
NaCl for 2 hours, increased microbial tolerance to acid (pH 3.5), but the
adaptation was strain-specific [54].
4.2.2 POSTHARVEST STRESS
Many sources of microbial contamination of produce at the postharvest
stage have been identified. These include humans (i.e., workers and
consumers), wild and domestic animals, insects, improperly sanitized har-
vesting equipment, transportation vehicles and containers, air-carried dust,
wash, rinse, and cooling water, ice, processing and packaging equipment, and
storage facilities, among many others [23,24,26,55-57]. Foodborne pathogens
can survive on the intact outer surface of fresh fruits and vegetables, but
they may not proliferate due to restriction of nutrients and water, or as a
result of their inability to synthesize degradative enzymes against protective
barriers covering produce. Survival and proliferation of pathogens on
produce are enhanced by physical damage (e.g., punctures and bruises) of
the protective epidermal barrier or the infection of the produce with pests
and microorganisms [26]. Microbial stress adaptation may occur at various
postharvest stages, and can involve transportation conditions, use of wash
and rinse water at variable temperature, application of intervention strategies
(e.g., use of sanitizers), pH fluctuations, and storage and packaging conditions.
4.2.2.1 Cold Stress
Microorganisms respond to cold stress by undergoing an adaptive response
known as the cold-shock response. Adaptation to cold stress involves the
Microbial Stress Adaptation and Safety of Produce 103
expression and accumulation of cold-shock proteins, which could protect
the cells to subsequent freezing or against other lethal stresses [53,58,59].
Broadbent and Lin [58] observed that cold shocking L. lactis at 10°C for 2
hours increased its resistance to freezing (— 60°C for 24 hours) and
lyophilization. Bollman et al. [60] reported that stress-adapted E. coli
0157:H7, previously cold-shocked at 10°C for 1.5 hours, had enhanced
survival in several foods including milk, whole egg, and sausage when
compared to the nonadapted bacterium. A previous study indicated that
cold shock of B. cereus (7°C for 2 hours) increased the survival of the
microorganism to subsequent lethal thermal treatment [53]. In a different
study, cold shocking Clostridium perfringens at 15°C for 30 minutes increased
the thermotolerance of the bacterium at 55°C [59].
The cold-shock response involves a number of physiological adjust-
ments, which include modifications in the cell membrane fluidity via increas-
ing the unsaturation of membrane lipids or decreasing the chain length of its
fatty acids, synthesis of protective proteins that bind to DNA and RNA, and
importation of compatible solutes [4]. The cytoplasmic membrane, nucleic
acids, and ribosomes participate in sensing temperature variations in microbial
cells, and temperature downshifts induce the synthesis of up to 50 different
cold protection-associated proteins [61,62]. Microbial response to cold stress
involves the overexpression of two types of proteins, the cold-shock proteins
(Csps) and the cold-acclimation proteins (Caps). A sudden drop in tempera-
ture induces the rapid, and transient, synthesis of Csps. Conversely, Caps
are synthesized for extended time periods under continuous microbial growth
at low temperatures; the expression of both protein types, however, can overlap
during stress adaptation [63,64].
The cold-shock response has been well characterized in E. coli, and its
Csps fall into two classes, I and II. Class I Csps are expressed at very low levels
at 37° C, and are induced and overexpressed after a temperature downshift
to 15°C. These class I proteins include the major cold-shock protein,
CspA (a RNA- and DNA-binding chaperone), ribosomal binding factors
(e.g., RbfA, CsdA), and the transcriptional termination and antitermination
factors (e.g., NusA) [4,61,65]. Class II Csps are present in cells at 37°C, and
are induced at moderate levels (< 10-fold) after the cold shock. Among the
induced Csps, there are recombination factors (e.g., RecA), a subunit of
DNA gyrase (GyrA), and energy-generating enzymes (e.g., dihydrolipoamide
transferase and pyruvate dehydrogenase) [61,64,66].
In spite of the evidence of the protective effects of cold shock against
multiple stresses, other researchers indicated that previous exposure to low
temperatures sensitized L. monocytogenes [67,68] and Vibrio parahaemolyticus
[69] to subsequent thermal treatments. As discussed earlier in this chapter,
exposing microorganisms to a stress may lead to their adaptation or
sensitization to more severe stresses. This variable behavior of pathogens
in response to cold stress should be considered when treating produce that
has been previously refrigerated to antimicrobial processes such as surface
pasteurizing.
104 Microbiology of Fruits and Vegetables
4.2.2.2 Acid Stress
Foodborne bacteria usually encounter drastic pH variations in the environ-
ment, and are exposed to acidic conditions while present in foods, during
processing, and when they invade the gastrointestinal tract of animals
and humans [70]. Acidification is a common food preservation method, in
which organic acids (e.g., acetic, propionic, and lactic) are produced during
fermentation or added as preservatives to foods. These weak acids, in their
nondissociated form, are capable of diffusing into microbial cells; once inside
the cytoplasm, they dissociate and decrease the intracellular pH, which results
in disruption of metabolic activities. Acid stress of foodborne microorga-
nisms results from the combination of the biological effect of low pH and the
direct effect of weak acids [15].
Microorganisms have developed strategies to respond to acid stress by
inducing a protective response known as the acid-tolerance response (ATR)
[71]. Microbial cells develop an ATR when exposed to a moderately low pH
(e.g., 4.5 to 5.5), and this results in the induction of proteins that protect the
cells against lethal acid conditions (e.g., pH < 4). In addition, cells respond
to acid environments by modifying their membrane composition, increasing
proton efflux and amino acid catabolism, and by synthesizing enzymes
involved in DNA repair [3,4].
In Salmonella Typhimurium, two different acid adaptation systems are
recognized: these are the log-phase and the stationary-phase ATR [71]. Log-
phase ATR is triggered when cells are grown under moderately acid condi-
tions, and involves the synthesis of acid-shock proteins under the control of
a , the signaling protein, PhoP, and the iron regulator, Fur [15,70]. The
stationary-phase ATR consists of a -independent and a -dependent
mechanisms. The response independent of a requires acid induction, and
involves the participation of the response regulator, OmpR, to control the
synthesis of acid-shock proteins. The induction of the ATR dependent on a
does not require previous exposure of the microorganism to acid, and it is
triggered by entry of the cells into stationary phase [3,4,71]. Therefore, the
latter ATR involves the induction of the general stress response, which is
associated with multiple stress adaptation. Wong et al. [72] reported that V.
parahaemolyticus, pretreated in acid medium (pH 5.0 to 5.8), showed increased
resistance to treatments with low salinity and heat (45°C). In a different
study, Rowe and Kirk [73] indicated that exposing pathogenic E. coli to acid
shock (pH 4 for 1 hour) enhanced microbial tolerance against subsequent
lethal treatments with osmotic stress (20% NaCl) or heat at 56°C.
Microorganisms grown under mild acid conditions are more resistant
to lethal acid environments, as well as to other lethal stresses, than those grown
at neutral pH [71]. Tosun and Gonul [74] indicated that Salmonella
Typhimurium, grown at pH 5.8, developed tolerance to lethal doses of
heat, salt, and organic acids, but not to cold shock. Ryu and Beuchat [75]
observed that acid-adapted E. coli 0157:H7, grown under gradual pH
reduction in a medium containing 1% glucose, showed enhanced tolerance
Microbial Stress Adaptation and Safety of Produce 105
to thermal treatments (52°C) in apple cider and orange juice. In a different
study, Bacon et at. [76] reported that stress-adaptation of Salmonella spp.,
grown under gradually increasing acid conditions (i.e., in a medium containing
1% glucose), caused cross-protection against lethal heat treatments. Listeria
monocytogenes, growing under similar gradually increasing acid conditions, or
previously treated at pH 5.0 to 5.5 for 90 minutes, showed enhanced survival
to a lethal acid medium (pH 3.5). Nonetheless, exposure to other stress con-
ditions including high osmolarity, heat, and cold was unable to protect
the microorganism against acid [77]. These results may have implications in the
washing of fruit since many of the commonly used washing agents are
acidic in nature. Application of these agents in a manner that sensitizes, rather
than hardens, the pathogens to other stresses would improve the safety of
produce.
4.2.2.3 Oxidative Stress
Foodborne microorganisms are exposed to oxidative stress, which may be
induced endogenously as a result of microbial metabolism or exogenously due
to treatments that increase the levels of reactive oxygen species, i.e., hydrogen
peroxide (H 2 2 ), superoxide anion (O^), hydroxyl radical (HO*), and
singlet oxygen ( 2 ). Similarly, microbial oxidative stress can be triggered by
conditions that lead to depletion of protective antioxidant molecules or
enzymes. Reactive oxygen species can be generated during processing as a
result of radiation, presence of heavy metals, or treatments with oxidizing
sanitizers. Reactive oxygen species are deleterious to microorganisms, and can
cause extensive damage to their cellular components such as lipids, proteins,
and nucleic acids; this negatively affects cell functionality and reduces its
viability [78-80]. Microorganisms respond to oxidative stress by synthesizing
(1) protective proteins (e.g., glutathione reductase, thioredoxin 2) and other
organic molecules (e.g., methylerythrol, cyclopyrophosphate) with antioxi-
dant capacity or (2) proteins that participate in repairing oxidative damage
(e.g., exonuclease III and endonuclease IV), specifically repairing deleterious
modifications affecting nucleic acids [2,81,82].
In E. coli, response to oxidative stress caused by H 2 2 and 2 ~ is under the
control of oxyR and soxRS regulons, respectively [79,81,83]. Genes controlled
by oxyR include those encoding the hydroperoxidase I (HPI), glutaredoxin,
glutathione reductase, NADPH-dependent alkyl hydroperoxide reductase,
and a protective DNA-binding protein (Dps) [83,84]. The regulon soxRS
controls the expression of genes encoding Mn-superoxide dismutase
(Mn-SOD), endonuclease IV, glucose-6-phosphate dehydrogenase, fumarase,
aconitase, and ferredoxin reductase, among others [80,83]. In unstressed
cells, both proteins OxyR and SoxR are present in an inactive form. During
oxidative damage, e.g., by exposure of cells to H 2 2 , OxyR senses the
stress and is activated by the formation of intramolecular disulfide bonds
[81,84]. There is evidence that the colanic acid polysaccharide produced
by many strains of E. coli 0157:H7 protects the microorganism against
106 Microbiology of Fruits and Vegetables
oxidative stress and other environmental conditions such as acid, heat, and
osmotic stresses [85]. Van der Straaten et al. [86] reported that RamA, a protein
synthesized in response to oxidative stress in Salmonella Typhimurium, could
be involved in antibiotic resistance and virulence.
Produce microbiota are often exposed to oxidative stress. Sanitizers
that may be used in washing produce (e.g., chlorine, chlorine dioxide, and
ozone) undoubtedly lead to oxidative stress, which may trigger stress adap-
tation among these microorganisms. Metal ions in washing water and oxygen
in package headspace are additional factors that may contribute to the
oxidative stress adaptation of microorganism on produce.
4.2.2.4 Minimal Processing
Recently there has been an increase in consumer demand for high-quality and
safe foods with fresh-like attributes. Minimally processed fruits and vegetables
can be defined as products that are processed with methods (e.g., low-level
irradiation and active packaging) that achieve food preservation and safety
while causing minimal quality modifications or alteration of the fresh
characteristics compared to produce treated by conventional food preservation
treatments [87]. Applying minimal processing involves using preservation
factors singly or in combination. Therefore, minimal processing may be con-
sidered an implementation of the "hurdle concept" which refers to the
application of mild preservation factors (i.e., hurdles) in combinations, either
in sequence or simultaneously, to enhance microbial inactivation by additive
or synergistic effects [88,89].
Combination of sublethal stresses, although potentially acting syner-
gistically to inactivate microorganisms in foods, could lead occasionally to
stress adaptation and cross-protective responses [11,90]. Examples of cross
protection were reported by Lou and Yousef [39] who observed that stressing
L. monocytogenes with heat (45° C for 60 minutes) protected the cells to
subsequent exposure to lethal concentrations of ethanol, hydrogen peroxide,
and sodium chloride. During food processing, microorganisms are treated
with sublethal stresses sequentially rather than simultaneously. Therefore,
microbial exposure to a sublethal stress could harden the microorganisms
and protect them against subsequent treatment factors or hurdles. Con-
sequently, stress hardening could pose limitations to the possible benefits of
the hurdle concept. However, careful application of minimal processing could
alleviate the consequences of stress adaptation of microbiota in produce.
4.3 MICROBIAL STRESS ADAPTATION ON PRODUCE
Information in published literature regarding microbial stress adaptation on
produce is scarce. However, greater processing resistance of natural microbial
contaminants on produce surfaces compared with those inoculated onto
these products may support the hypothesis that most produce microbiota are
adapted to stresses encountered in the field and throughout the production
Microbial Stress Adaptation and Safety of Produce 107
chain. Readers are cautioned that apparent processing resistance of produce
microbiota also could be attributed to their inaccessibility to treatments,
or the inability of the analyst to recover these microorganisms using
common sample preparation and processing techniques. Association of
microbial contaminants with pores, stem scars, wounds, and other surface
irregularities could protect microorganisms and make them appear resistant
to processing.
Attachment of microorganisms to fruit and vegetable surfaces could
initiate stress adaptive response against physical and chemical treatments.
Gawande and Bhagwat [92] reported that Salmonella spp. attached to apple,
tomato, or cucumber had enhanced surface contact-mediated acid tolerance
and increased survival, by 4 to 5 log, to acid stress induced by exposure to
sodium citrate (50 mM, pH 3) for 2 hours when compared to cell suspens-
ions treated under the same conditions. When these investigators inoculated
Salmonella Typhimurium on the surface of fresh-cut apples, and stored them
at 4°C for 2 hours, the tolerance of the pathogen to acid stress increased.
Han et al. [93] treated green pepper, contaminated on the surface with E. coli
0157:H7, using chlorine dioxide gas (0.2 to 1.2mg/l), and reported that
attachment of the microorganism to injured pepper surfaces protected the
cell against the gas when compared to cells attached to uninjured surfaces.
Francis and O'Beirne [94] inoculated acid-adapted L. monocytogenes,
previously exposed to pH 5.5 for 60 minutes, on lettuce, swedes, dry coleslaw
mix, and bean-sprouts, which were packed under modified atmosphere and
subsequently stored at 8°C for 14 days. The researchers observed that the
stress-adapted microorganism had enhanced survival compared to unstressed
controls, under relatively high (25 to 30%) carbon dioxide atmospheres.
Hsin-Yi and Chou [95] stressed E. coli 0157:H7 in acidified medium (pH 5 for
4 hours) and inoculated the microorganism in acidic mango or asparagus
juice with subsequent storage at 7°C. The investigators reported that acid
adaptation and low temperature increased microbial survival in both fruit
juices.
4.4 ASSESSING STRESS ADAPTATION AND
ASSOCIATED RISKS
Safety of food may be achieved using treatment factors that halt the growth
of pathogens. The optimization of these treatments requires an understand-
ing of the limits between conditions that support growth and those in
which growth is not possible, also known as the growth-no-growth interface
[96]. The growth-no-growth interface can be defined as the boundary at
which the microbial growth rate is zero and the lag phase is infinite [97]. The
behavior of foodborne pathogens at the growth-no-growth interface has
been assessed using models that take into account combinations of tempe-
rature, pH, a w , and concentrations of chemical compounds [77,98-100].
These and other predictive and risk assessment models should consider the
108 Microbiology of Fruits and Vegetables
contribution of stress adaptation to the survivability and behavior of
pathogens in food. The majority of models available to evaluate survival or
inactivation of chemically stressed bacteria are based on primary models,
which describe the fate of microbial populations as a function of time [96].
To develop reliable microbial inactivation models, researchers should con-
sider the physiological state of the organism and the potential induction of
stress-tolerance responses [97]. However, including stress adaptation in these
models depends greatly on researchers' ability to monitor accurately and
quantify the stress adaptation phenomenon experimentally.
Rapid and quantitative assessment of microbial adaptive response to a
predefined stress remains a great challenge. Advances in this area would
improve our understanding of how the microbial cell responds to multiple
stresses, or its ability to exhibit multiple responses to a single stress, leading
to cross protection. These techniques would also enable researchers to
measure the response of microbial cells to a complex battery of stresses.
Advances in genomic and proteomic research may bring the scientific com-
munity closer to this goal [20,33]. Although genome-wide microarray anal-
ysis enables researchers to identify genes expressed in response to stress, the
technique does not distinguish between expressions leading to adaptation
and those that are not directly related to this phenomenon. Fluorescence
staining is a promising technique for rapidly assessing stress response.
Instrumentation advances may enable researchers to monitor the effect of
stress on membranes in real time with the use of fluorescent dyes. Reliable,
quantitative measures of stress adaptation should facilitate the efforts to
develop mathematical predictive models of stress-, adaptive-, and cross-
protective responses. Change in these responses as a function of stress type and
intensity, for example, would help predict the behavior of pathogens during
food processing and storage, and their virulence in infected individuals.
4.5 SUMMARY
Adaptation of foodborne pathogens to environmental and processing
stresses is a potential risk that may greatly compromise the safety of food.
Only anecdotal evidence is available, supporting the notion that microbiota
on produce owe their processing resistance to the stress adaptation
phenomenon. Repeated demonstration of stress adaptation under laboratory
conditions provides indirect proof that the phenomenon is of paramount
importance to the safety of food. Modern and efficient techniques are needed
to assess and monitor accurately the adaptive response in foodborne
pathogens.
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Section II
Microbial Spoilage of Fruits
and Vegetables
Bacterial Soft Rot
Ching-Hsing Liao
CONTENTS
5.1 Introduction 117
5.2 Diversity of Soft-Rot Bacteria 118
5.2. 1 Pectolytic Erwinia spp 119
5.2.2 Pectolytic Fluorescent (PF) Pseudomonads 119
5.3 Factors Affecting the Survival of Soft-Rot Bacteria in Nature 120
5.3.1 Plant Vegetation 120
5.3.2 Temperature and Atmospheric Conditions 121
5.3.3 Latent Infection and Internalization 121
5.4 Enzymatic and Molecular Mechanism of Tissue Maceration
by Soft-Rot Bacteria 122
5.4.1 Biochemical Characterization of Pectate Lyase (PL) 122
5.4.1.1 Analysis of PL Isozymes 122
5.4. 1 .2 Production of Other Pectic Enzymes 122
5.4.2 PL as the Principal Tissue-Macerating Factor 123
5.4.2. 1 Transposon Mutagenesis 123
5.4.2.2 Cloning and Analysis of PL Genes 123
5.4.3 Control of PL Production and Pseudomonas Rot 123
5.4.3.1 Two-Component Regulatory Gene System 123
5.4.3.2 Role of Calcium Ions 124
5.4.3.3 Use of Ion-Chelating Agents for Control
of Pseudomonas Rot 125
5.5 Interactions Between Soft-Rot and Human Pathogens on
Fresh Produce 125
5.5.1 Synergistic Interactions 126
5.5.2 Antagonistic Interactions 126
5.6 Selected Farm Practices for Control of Both Soft-Rot
and Human Pathogens 127
References 128
5.1 INTRODUCTION
Global production and international trade of fresh fruits and vegetables
have increased very sharply during the past two decades, mainly because of
117
118 Microbiology of Fruits and Vegetables
consumers' awareness of the health benefit expected from this popular diet [1].
More than 300 fresh and fresh-cut produce items are available for sale at
supermarkets throughout the U.S. [2], largely due to the advanced postharvest
technologies, improved crop varieties, and efficient distribution systems.
To meet the market demand, new strategies are required to increase the
production of fresh produce in farms and to reduce the postharvest losses
caused by biotic and abiotic factors. It has been estimated that between 10 and
30% of fresh fruits and vegetables produced in the U.S. are wasted, mainly due
to three factors: mechanical injuries, physiological decays, and microbial
spoilage [3].
Microbial spoilage accounts for a substantial proportion of postharvest
losses of fresh produce, which can be caused by a wide variety of microorganisms
including bacteria, fungi, or yeasts [4,5]. In general, the spoilage of acidic fruits
such as apple, orange, and berries is caused by molds, lactic acid bacteria, or
yeasts. The spoilage of fresh produce with neutral pH such as salad vegetables
and edible roots or tubers is caused by bacteria capable of producing pectolytic
enzymes required for degradation of plant cell walls. Bacterial spoilage of
fresh produce is usually found in the form of soft rot, which is characterized by
water-soaking and total disintegration of plant tissues [6,7]. As reported in the
literature, bacterial soft rot has been identified as the leading cause of disorders
in many types of produce, including potato [8], lettuce [9,10], bell pepper [11],
cucumber [12], and tomato [13]. This disorder can cost the fresh produce
industry and consumers hundreds of millions of dollars annually [3].
In addition to its economic impact, soft-rotted plant tissue may serve as a
carrier or reservoir for foodborne human pathogens and pose a potential threat
to the safe supply of fresh produce. Wells and Butterfield [14,15] reported that
the rotted plant tissues were more likely to harbor salmonella than the
apparently healthy counterparts. They found a 5- to 10-fold increase in the
population of Salmonella Typhimurium in potato slices co-inoculated with
soft-rot bacteria [14]. Therefore, an integrated approach to control the
proliferation of both soft-rot bacteria and foodborne human pathogens on
fresh produce is required.
In this chapter, the diversity of soft-rot bacteria associated with postharvest
losses of horticultural commodities and the factors affecting their survival in
nature are discussed. In addition, the enzyme and molecular genetic mechanism
by which soft-rot bacteria (especially fluorescent pseudomonas) cause
maceration of plant tissues is reviewed. Furthermore, the synergistic and
antagonistic interactions between spoilage microorganisms and human
pathogens on fresh produce are discussed. Farm practices that are useful for
controlling the dissemination and proliferation of both soft-rot and human
pathogens on fruit and vegetable crops are presented.
5.2 DIVERSITY OF SOFT-ROT BACTERIA
Soft rot of fresh produce can be caused by diverse groups of bacteria including
erwinia, pseudomonas, xanthomonas, Clostridium, bacillus, and cytophaga [4].
Bacterial Soft Rot 119
The characteristics of these bacteria and their association with the spoilage of
fresh produce under different conditions have been briefly described [7]. As
strict anaerobes, Clostridium spp. cause soft rot of potatoes under oxygen-
depleted conditions, especially when a more aggressive plant pathogen such as
erwinia is present [16,17]. Pectolytic Clostridium spp. also play a role in the
spoilage of fresh-cut produce that is packaged using an impermeable film [4].
Pectolytic bacillus including Bacillus polymyxa and B. subtilis have also been
shown to be associated with soft rot in a wide variety of crops including
potatoes, tomatoes, carrot, onion, and cucumber grown at elevated temper-
atures from ambient to 37°C [18]. Like Clostridium and bacillus, pectolytic
cytophaga [19] and xanthomonas [20] are generally considered the secondary
pathogens which invade plants following the attack of a more aggressive patho-
gen such as erwinia or pseudomonas. Based on a series of studies previously
conducted in our laboratory [21], erwinia and pseudomonas combined account
for over 90% of soft rot of fresh produce while in storage or at markets. Less
than 10% of soft rot of fresh produce found at the markets could be caused by
xanthomonas, cytophaga, bacillus, or other unidentified genera [4,21].
5.2.1 Pectolytic Erwinia spp.
The soft-rot erwinia group, consisting of three species or subspecies, E.
carotovora subsp. carotovora (Ecc), E. carotovora subsp. atroseptica (Eca), and
E. chrysanthemi (Ech), is the major single cause of microbial spoilage of
vegetables. The losses due to soft-rot erwinia cost tens or hundreds of millions
of dollars yearly [3,7]. Ecc has the broadest host range causing diseases in almost
every species of vegetable crops grown in temperate and subtropical regions [7].
Eca is present at cooler regions and is more often associated with black leg of
potatoes [22] in the field than with soft rot of fresh produce after harvesting. In
contrast, Ech causes diseases of crops grown in subtropical or tropical regions
[23,24]. Both Ecc and Ech grow poorly and fail to induce soft rot of fresh
produce at 10°C or below. At 20°C or higher, Ecc is considered the most
destructive soft-rotting pathogen of fruits and vegetables. The soft-rot erwinia
group is widespread in nature and very closely associated with plant vegetation
and can be readily isolated from weed, plant debris, rhizosphere soil, and
lenticels of potato tubers [25,26]. However, soft-rot erwinia is rarely detected on
the surfaces of plant leaves or true seeds [22]. In addition to erwinia, other
enteric bacteria including enterobacter, klebsiella, and serratia are commonly
present on the surfaces of many different types of vegetable crops [4,5]. A vast
majority of enteric bacteria are nonpectolytic and not expected to cause the
spoilage of fresh produce. However, they may play a critical role in maintaining
the quality and safety of fresh produce by enhancing or suppressing the growth
of spoilage and pathogenic microorganisms on the surfaces of plants.
5.2.2 Pectolytic Fluorescent (PF) Pseudomonads
For fruits and vegetables that are stored at refrigeration temperatures,
pectolytic fluorescent (PF) pseudomonads are responsible for a substantial
120 Microbiology of Fruits and Vegetables
proportion of soft-rot disorder observed in markets. PF pseudomonads as a
group are physiologically and taxonomically heterogeneous, mainly consisting
of P. viridiflava and five biovars of P. fluorescens [21,27-29]. The latter was
often designated as P. marginalis in the plant pathology literatures [21,28,29].
These pseudomonads are widespread in nature and can be isolated from diverse
ecological niches including soils, irrigation water, rhizosphere, and surfaces
of fruits and vegetables [27-29]. PF pseudomonads account for over 40% of
total bacterial rot found at retail and wholesale produce markets [21]. They are
especially abundant on the surfaces of leafy or salad vegetables including
spinach [30], lettuce [31-33], cabbage [34], potato lenticels [25], tomatoes
[35,36], and bell pepper [37]. On salad vegetables including lettuce, cabbage, and
spinach, PF pseudomonads account for over 30% of total native bacteria
recovered. Because of their prevalence in nature, PF pseudomonads are
expected to play an important role in maintaining the safety and quality of
refrigerated or ready-to-eat vegetables or fruits. They could be readily isolated
from very diverse ecological niches including soil [38], rhizosphere [39], surfaces
of fresh vegetables [30-37], and wash water from produce processing plants [38].
The importance of PF pseudomonads as the leading cause of spoilage of
refrigerated fresh produce is primarily due to their psychrotrophic nature,
nutritional versatility, and predominant presence on the surfaces of fresh
produce. PF pseudomonads are responsible for a very large proportion of
decay of fresh fruits and vegetables stored at low temperatures [21]. In
addition, some P. marginalis and P. viridiflava strains can also cause soft-rot
disease of horticultural crops in the field, e.g., the "pink eye" of potato tubers
[39]. A few reports also showed that other fluorescent pseudomonads including
P. aeruginosa [40], P. tolasii [7], and P. chorii [9] were involved in postharvest
spoilage of vegetables or mushrooms.
5.3 FACTORS AFFECTING THE SURVIVAL OF
SOFT-ROT BACTERIA IN NATURE
5.3.1 Plant Vegetation
Soft-rot erwinia and pseudomonas are widespread in nature and can be readily
isolated from decayed tissue, plant debris, rhizosphere soil, and weeds
[25,26,38,41,42]. De Boer [41] reported that Eca was isolated more often from
soil in which potatoes had been grown in previous years than from soils in
which other types of crops had been grown. Thus, survival and over-wintering
of erwinia in soil can be greatly affected by the type of crop grown in the
previous season. Soft-rot erwinia is rarely found on the surfaces of leafy
vegetables and true seeds and survives poorly in sterilized soil [22]. Plant
vegetation appears to be important for long-term survival of erwinia and
pseudomonas in soil [26,28,41]. Because of the widespread distribution in plant
and nonplant environments, it is impossible to eliminate completely soft-rot
erwinia and pseudomonas from propagation materials, irrigation water, or
soils in the field.
Bacterial Soft Rot 121
5.3.2 Temperature and Atmospheric Conditions
Refrigeration is the most convenient and effective means to maintain the
organoleptic properties, to reduce the spoilage, and to extend the shelf life of
fresh produce. The International Fresh-Cut Produce Association (IFPA) [42]
recommends minimally processed produce be stored at 1 to 4°C to maintain
the quality and safety. Refrigeration of fresh produce at between 4 and 10°C is
commonly used by the industry to extend the shelf life and to prevent the soft
rot caused by bacteria (such as Ecc and Ech) and fungi. At this temperature
range, the development of soft rot by Eca and PF pseudomonads will occur.
The minimum temperature for growth of Eca has been estimated to be between
3 and 6°C [7] and the minimal temperature for growth of PF pseudomonads
estimated to be 4°C or below [21].
The seven genera of soft-rot bacteria mentioned above require somewhat
different optimal atmospheric conditions for growth and induction of spoilage.
For instance, Clostridium spp. are strictly anaerobic and PF pseudomonads
(with the exception of nitrate-denitrifying strains) are strictly aerobic.
Induction of soft rot in potatoes by erwinia and Clostridium is greatly
enhanced by the depletion of oxygen [16]. Reduction in oxygen concentration
or increase in carbon dioxide concentration in the atmosphere reduced the
growth of PF pseudomonads [43] and their ability to induce soft rot on fresh
produce [44].
5.3.3 Latent Infection and Internalization
Although the internal parts of plant organs are generally considered sterile
[6], many different types of bacteria including soft-rot erwinia, pseudomonas,
and serratia can be detected within apparently healthy tomatoes [45] and
cucumber fruits [46]. These bacteria presumably exist in a commensalistic or
quiescent state, which can be activated only when the stressed conditions in
fruits are removed. A large proportion of storage rot of fruits is due to
external contamination by soft-rotting microorganisms and a small propor-
tion of them may be caused by the activation of latent bacteria inside the
fruits. The route by which soft-rot erwinia penetrates into the internal parts
of apparently healthy tomato fruits is unclear but possibly may be through
the connective tissue at the stem end of the fruits [47]. Bartz and Kelman [48]
also reported that the bacterial soft-rot potential in potato tubers was affected
by difference in temperature between tubers and suspensions of erwinia at
the time of inoculation by immersion. A series of laboratory experiments have
conclusively demonstrated that human pathogenic bacteria including E. coli
0157:H7 and salmonella can be infiltrated into apple [49,50], orange [51],
tomatoes [51], and lettuce [52-54] if the temperature of bacterial suspension
is lower than that of fruits. Surface cleaning and sanitization treatments
are not expected to eliminate completely the undesirable bacteria that become
internalized [55] and those attached to the surfaces of intact or injured fruits
[56,57].
122 Microbiology of Fruits and Vegetables
5.4 ENZYMATIC AND MOLECULAR MECHANISM OF
TISSUE MACERATION BY SOFT-ROT BACTERIA
5.4.1 Biochemical Characterization of Pectate
Lyase (PL)
5.4.1.1 Analysis of PL Isozymes
Soft-rot erwinia including Ech, Ecc, and Eca are characterized by their ability
to produce an array of pectolytic enzymes including pectin methylesterase
(PME), polygalacturonase (PG), pectin lyase (PNL), and pectate lyase (PL).
These enzymes can be readily detected in filtrates of bacterial cultures and
assayed by the standard biochemical procedures [58]. The PLs produced by
Ech, Ecc, and Eca are usually present in multiple (three to five) isozymic forms
in culture filtrates, which can be readily identified by isoelectric focusing (IEF)
gel electrophoresis and overlay enzyme staining techniques [59]. In Ech, a
second set of PL isozymes, inducible only in the presence of plant constituents,
have been identified using molecular genetic and enzyme analyses [60].
The biological and pathological function of each pectic enzyme produced by
soft-rot erwinia has not been fully determined. It is also unclear if production
of certain pectic enzymes is restricted to specific tissues or organs or limited
to specific stages of plant development. With the aid of molecular genetic
technologies, experimental results [61-63] have shown that no single pectic
enzyme produced by soft-rot erwinia is absolutely required for the pathogen to
initiate disease development. However, the PL isozymes, especially alkaline
PLe, usually display the highest degree of tissue macerating ability in vitro [64]
and are assumed to be the principal enzymes required for development of soft
rot by erwinia in vivo [62].
Because of their complex pectic enzyme system, it is difficult to purify a
single PL isozyme from culture filtrates of Erwinia spp. However, due to
the simplicity of the pectic enzyme system in other spoilage bacteria, including
P. fluorescens, P. viridiflava, and Xanthomonas campestris, it is relatively easy to
purify the PLs from their culture filtrates [65]. Normally, following two simple
steps (ammonium sulfate precipitation and anion exchange chromatography),
the PL can be purified from culture filtrates of these two PF pseudomonads
to near homogeneity [65,66]. Enzymological properties of PLs purified from
culture filtrates of P. fluorescens and P. viridiflava have been characterized, and
a minute amount of purified enzyme was capable of causing total maceration
of potato tuber tissue even in the absence of live bacteria [67].
5.4.1.2 Production of Other Pectic Enzymes
In addition to PLs, soft-rot erwinia produces an array of other pectic enzymes
including pectin methyesterase (PME), polygalacturonase (PG) and pectin
lyase (PNL). Production of PME, PG, and PNL by soft-rot pathogens does not
seem to play a significant role in initiating the maceration of plant tissues.
However, they may be required for interactions with host plants or coping with
Bacterial Soft Rot 123
adverse environments [68]. It has been reported that purified PG, but not
purified PME or PNL by itself, is sufficient to induce soft rot of potato tuber
slices. Production of PNL by soft-rot Erwinia spp. [69] and Pseudomonas spp.
[70] is inducible only after exposing the bacteria to DNA-damaging agents
such as ultraviolet radiation and mitomycin C. The ecological and pathological
significance of producing PNL by erwinia and pseudomonas remains obscure.
The role of PME in soft-rot pathogenesis is minimal and probably not
required. However, it has been suggested that a coordinated action between
PME and PL may be necessary for complete degradation of native pectins in
plant cell walls. More information about the enzymatic mechanism of soft-rot
pathogenesis by Erwinia spp. can be found in earlier reviews [61-63,68].
5.4.2 PL as the Principal Tissue-Macerating
Factor
5.4.2.1 Transposon Mutagenesis
The notion that PL is the principal or sole pathogenicity factor of soft-rotting
pseudomonads can be supported by a series of molecular genetic studies. By
using transposon (7>z5)-mediated mutagenesis, Liao et al. [66] isolated several
types of P. viridiflava mutants that became defective in production or secretion
of PL. When assayed on plants, nonpectolytic P. viridiflava mutants were
unable to induce soft rot on potato tuber slices. The loss in the ability to
produce or secrete PL is accompanied by the loss in the ability to induce soft
rot. This result provides the first unequivocal evidence that PL is the sole
enzyme required for the induction of soft rot by P. viridiflava [66].
5.4.2.2 Cloning and Analysis of PL Genes
The gene encoding PL has been cloned from the genomes of P. fluorescens [71],
P. viridiflava [72], and Xanthomonas campestris [73]. When cloned PL gene
was mobilized into nonpectolytic mutants of P. viridiflava or P. fluorescens, the
PL-producing and soft-rotting ability of nonpectolytic mutants was restored
[74-76]. These results provide direct genetic evidence that the gene coding
for PL is the principal or sole pathogenicity or virulence determinant of soft-
rotting P. viridiflava or P. fluorescens.
5.4.3 Control of PL Production and
Pseudomonas Rot
5.4.3.1 Two-Component Regulatory Gene System
The enzymatic and molecular genetic mechanism of soft-rot pathogenesis
caused by erwinia has been extensively investigated and reviewed [61-63,68].
However, very little is known about the mechanism by which PF pseudo-
monads regulate the production of PL and induction of tissue maceration
in plants. Pleotropic mutants of P. fluorescens and P. viridiflava showing
124 Microbiology of Fruits and Vegetables
simultaneous loss of production of both pectolytic and proteolytic enzymes
have been identified by transposon mutagenesis [74-76]. Results from Southern
Blot analysis revealed that mutants were derived from the insertion of Tn5 into
one of two distinct genomic fragments. Two genes regulating the production
of pectolytic enzyme and induction of soft rot, designated as gacS (=repA
or lemA) and gacA (=repB), have been identified in these two fragments and
subsequently cloned and confirmed by complementation studies [74-76].
Based on the nucleotide sequence analyses, the gacS and gacA genes
were respectively predicted to encode a sensory and a regulator protein in the
two-component regulatory protein family [74,76]. The gacS/gacA genes were
predicted to act in pairs to mediate the production of an array of extracellular
compounds including PL, protease (PRT), exopolysaccharide (EPS), and ion-
chelating siderophores [74-76], possibly in response to environmental signals.
The gacS/gacA genes in biological control strains of P. fluorescens have also
been shown to regulate the production of phospholipase C [77], lipase [78],
and antibiotics [79-81]. Proper function of the gacS/gacA gene system is also
required for the formation of disease lesions on snap beans by Pseudomonas
syringae pv. syringae [82]. This two-component gacS/gacA gene system can
also interact with the stationary-phase factor 5 s (encoded on rpoS) in a biolog-
ical control strain of P. fluorescens to control the responses of this strain to
environmental stimuli [83]. In P. aeruginosa, the activator GacA will interact
with two quorum sensing proteins (LuxR, Luxl) to regulate the production of
an autoinducer (butylhomoserine lactone) [84]. It has not yet been investigated,
however, if RpoS, LuxR, and Luxl would act in concert to regulate the
production of PL and other extracellular compounds in soft-rotting P.
fluorescens and P. viridiflava.
A group of P. viridiflava mutants failing to excrete PL and Prt across the
outer membrane have also been generated by transposon mutagenesis during
the isolation of nonpectolytic mutants [66]. These secretion-defective mutants
(designated Out - ) were assumed to result from the insertion of Tn5 into a gene
belonging to the Type II secretory gene family [85,86]. Out" mutants were
also unable to induce soft rot on potato tuber slices and bell pepper fruits [66].
This indicates that the synthesis and the secretion of PL are two critical
steps required for induction of soft rot.
5.4.3.2 Role of Calcium Ions
Production of PL in certain strains of P. fluorescens is inducible by
pectic substrates [87,88] or plant tissue extracts [89-91]. However, in other
P. fluorescens strains production of PL is not affected by the type of carbon
source included in the medium [91]. Recently, we investigated the mode of PL
production in 24 strains of P. fluorescens and found that production of PL in
certain P. fluorescens strains (4 out of 24) was not induced by pectic substrates
9-1-
but by Ca [92]. These four strains produce ten times more PL in medium
containing 1 mM CaCl 2 than in one containing no CaCl 2 supplement.
Supplement of CaCl 2 in the medium not only affects the amount but also the
Bacterial Soft Rot 125
final destination of PL. Over 86% of total PL produced by strain CY091 in
CaCl2-supplemented medium was excreted into the culture fluid. By
comparison, only 13% of total PL produced by this strain in CaCi2-deficient
medium was detected in the extracellular fraction. The effect of Ca" + on PL
production is concentration-dependent and can be replaced by Sr 2+ , but not by
Zn 2+ , Fe 2+ , Mn 2+ , Mg 2+ , or Ba 2+ [92].
5.4.3.3 Use of lon-Chelating Agents for Control of
Pseudomonas Rot
Because of the indispensable role of Ca in the production, secretion, and
catalytic activity of PLs, the potential of using ion-chelating agents such as
EDTA for control of pseudomonas rot has been investigated [92]. Application
• ••••0 _i_
of ion-chelating agents such as EDTA to limit the availability of Ca to P.
fluorescens infecting the plants thus offers a potential strategy for control of
soft rot caused by pseudomonads. We have demonstrated that application of
0.05 uM (or 40ppm) of EDTA, alone [92] or in combination with a bacteriocin
(nisin) [93], suppresses the induction of soft rot by P. fluorescens. Zucker and
Hankin [94] also reported that EDTA treatments reduced the soft rot potential
of potato tubers.
It should be noted, however, that the EDTA treatment would not be
effective for control of soft rot caused by erwinia, because Erwinia spp.
O _i_ O _l_
produce not only Ca -dependent PL but also Ca -independent PG.
However, infiltration of potato tubers or apple fruits with CaCl 2 can enhance
their resistance to attack by Ecc or Eca [95] or Penicillium expansum [96].
Changes in calcium fertilization in potato fields could also affect the
susceptibility of potato tubers to bacterial soft rot [97]. Infiltration of potato
tubers and fruits with Ca 2+ was thought to strengthen the cell walls and
consequently increase their resistance to postharvest rot pathogens [98]. None
of the above control strategies have been applied on a large scale for commer-
cial operations.
5.5 INTERACTIONS BETWEEN SOFT-ROT AND
HUMAN PATHOGENS ON FRESH PRODUCE
Despite the lack of a known mechanism for attacking plants, the gastro-
intestinal human pathogens including salmonella, E. coll 0157:H7, and
L. monocytogenes can survive and even grow on fruits or vegetables over a
long period of time [99]. Their survival and growth can be affected by the
indigenous microflora and by the storage conditions [4]. The dynamics of
the interactions between native microflora, spoilage bacteria, and human
pathogens, especially under modified atmospheres, have been investigated [5].
The results obtained thus far indicate that the effect of spoilage or saprophytic
microorganisms on the proliferation of human pathogens on fresh produce
could be either synergistic or antagonistic, largely depending on the type of
pathogens, fresh produce, and storage conditions examined.
126 Microbiology of Fruits and Vegetables
5.5.1 Synergistic Interactions
The interactions between soft-rot and human pathogens on fresh produce
began to catch the attention of public health officials when Wells and
Butterfield [15] reported that the rotted plant tissue more often harbored
salmonella than the healthy counterpart. They also demonstrated that the
population of salmonella increased by 5- to 10-fold on potato or carrot slices
that were co-inoculated with soft-rotting E. carotovora or P. viridiflava. Carlin
et al. [100] later showed a positive correlation between the number of
L. monocytogenes and the extent of soft rot observed with endive leaves. These
studies indicate that the rotted plant tissues may provide extra nutrients to
enhance the growth of human pathogens. Contaminated plant tissues can then
serve as a reservoir or vehicle for the dissemination of clinically important
pathogens in farms or food processing facilities.
Surveys of salmonella contamination on rotted fruits and vegetables
induced by molds or fungi have also been reported [16]. The incidence of
salmonella contamination on rotted tissue induced by molds or fungi was
about one tenth of that induced by soft-rotting bacteria. Nevertheless, the
fungi-induced rotted tissues are three times more likely to contain salmonella
than the healthy counterpart. In spite of this, the investigators [16] concluded
that rotted tissues pose little or no greater safety risk than the healthy tissues.
Gastrointestinal pathogens including salmonella and L. monocytogenes usually
do not grow, or grow very poorly, on acidic fruits (pH < 4.0) such as apple and
orange [101]. Conway et al. [102] demonstrated that these bacterial pathogens
were able to multiply in rotted tissues induced by specific groups of fungal
pathogens such as Glomerella cingulata but not in rotted tissues induced by
other groups of fungal pathogens such as Penicillium expansum. They found
that the population of L. monocytogenes increased in apple fruits infected with
G. cingulata but declined in fruits infected with P. expansum. Conway et al.
[102] revealed that the pH in P. expansum-'mduced rotted apples decreased
from 4.7 to 3.7 as opposed to the increase in pH from 4.6 to 7.7 in G. cingulata-
induced rotted fruits. Riordan et al. [103] also showed that the population of
E. coli 0157:H7 increased 1 to 3 logs on apple infected with G. cingulata
but continued to decrease in rotted tissues infected with P. expansum. The pH
change in rotted tissues induced by different groups of fungi thus plays a
critical role in the fate of human pathogens on fresh produce.
5.5.2 Antagonistic Interactions
In contrast to the synergistic effect, a number of studies have shown that the
growth of human pathogens on fresh produce could be suppressed by the
presence of postharvest rot pathogens. For examples, the growth of L. mono-
cytogenes on potato slices [104], spinach [30], and endive [105] could be
markedly reduced by diverse strains of fluorescent pseudomonads. The inhibi-
tion was thought to be caused by the production of iron-chelating fluorescent
siderophores or antimicrobials by the pseudomonads [106]. Carlin et al. [105]
Bacterial Soft Rot 127
reported that more growth of L. monocytogenes was detected on endive leaves
that were rinsed with disinfectants than those rinsed with water. Two
pseudomonad antagonists possibly responsible for inhibiting the growth of
L. monocytogenes on endive leaves have been identified [105]. Additional
strains of fluorescent pseudomonads capable of inhibiting the growth of
L. monocytogenes or L. innocua on different types of produce including carrot,
lettuce, bell pepper, and sprouting seeds have been isolated [106,107].
L. monocytogenes is in general more susceptible than salmonella or E. coli
0157:H7 to the antagonists naturally present on the surfaces of fresh produce
[106]. In addition to the saprophytic antagonists, postharvest rot pathogens
including P. fluorescens and P. expansum can also inhibit the growth of human
pathogens such as E. coli 0157:H7 and L. monocytogenes [102-104]. It has
been suggested that elimination of native microflora (including bacterial and
fungal rot pathogens) from fruits and vegetables may create a less competitive
environment for the proliferation of human pathogens on fresh produce [4].
5.6 SELECTED FARM PRACTICES FOR CONTROL OF
BOTH SOFT-ROT AND HUMAN PATHOGENS
To minimize the dissemination and proliferation of the harmful microorgan-
isms on growing plants, it is necessary to take preventive measures to intervene
in the introduction of contamination sources in the field. Sources of soft-
rotting erwinia and pseudomonas [22,28,38] and foodborne human pathogens
that may contaminate or infect growing plants in the field have been previously
reviewed [99,108]. Preventive measures and good agricultural practices (GAPs)
for reducing the contamination of field crops with foodborne pathogens have
been suggested in several guidance references including one published by the
U.S. Food and Drug Administration [109] and another one by the IFPA [1 10].
Preventive control strategies for bacterial soft rot have also been reviewed
by Eckert and Ogawa [111] and by Lund [6]. A few practices useful for control
of both soft-rot bacteria and foodborne human pathogens in the fields are
indicated as follows:
• Use seeds and propagation materials that are free of soft-rot bacteria
and human pathogens for planting. Although soft-rot erwinia are
generally not considered seedborne [22], long-term survival of salmo-
nella and soft-rot pseudomonas in water [112] and on alfalfa seeds
destined for sprouting has been reported [113].
• Properly dispose of the decayed plant materials in the field, which can
become the inoculum source of soft-rot bacteria [26] and serve as a
fertile ground for the proliferation of foodborne human pathogens
such as salmonella [14,15].
• Avoid the use of improperly treated manure or compost in the field.
Long-term survival of salmonella and E. coli 0157:H7 in feces and in
partially composted manure or biosolids has been documented [99].
128 Microbiology of Fruits and Vegetables
Application of improperly treated compost possibly containing
decayed materials may also serve as the inoculum source of soft-rot
bacteria.
• Monitor and ensure that water to be used for irrigation, washing, and
preparation of protective chemicals is devoid of harmful micro-
organisms. Both soft-rot bacteria and human pathogens have been
known to survive in water for several years [112].
• Harvest the crop at the optimal stage of maturity and with the
minimal mechanical injury. It has been reported that the mature crop
exhibits a higher level of resistance to attack by soft-rot bacteria [22]
and to the colonization by human pathogens [99]. Injured plant
surfaces can serve as the points of entry for soft-rot bacteria and as
the sites for attachment by human pathogens [56,57].
• Maintain sanitary conditions and enforce good worker hygiene in the
field to prevent the contamination of growing or harvested crops with
pathogens carried by farm workers. Outbreaks of foodborne illness
due to the contamination of fresh produce with foodborne pathogens
originating from farm workers have been previously reported [108].
• Use clean and sanitary vehicles for transporting produce from farms
to processing plants.
• Keep the orchards and vegetable farms away from domestic and wild
animals and far away from poultry and dairy farms. Feces and
animal wastes are believed to be the two most important carriers or
reservoirs of foodborne human pathogens [109].
• Remove weeds grown in the field, which may become alternative
inoculum sources for soft-rot erwinia [42] and human pathogens
[100].
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6
Microbial Spoilage of
Fresh Mushrooms
Naveen Chikthimmah and Robert B. Beelman
CONTENTS
6. 1 Fresh Mushrooms 135
6.1.1 Introduction 135
6. 1 .2 Commercial Growing Practices 136
6.1.3 General Composition 137
6.2 Microbiology of Fresh Mushrooms 138
6.3 Spoilage of Fresh Mushrooms 139
6.3.1 Sources of Microorganisms Causing Spoilage 142
6.3.2 Cultural (Growing) Practices Favoring Spoilage 142
6.3.3 Cultural Practices to Suppress Spoilage of
Fresh Mushrooms 142
6.3.4 Postharvest Conditions Favoring Spoilage of
Fresh Mushrooms 146
6.3.5 Postharvest Practices to Suppress Spoilage of
Fresh Mushrooms 147
6.3.5.1 Packaging 148
6.3.5.2 Washing Treatments 149
6.3.5.3 Irradiation 150
6.3.5.4 Pulsed Ultraviolet Light Treatment 151
6.4 Conclusions 1 52
References 153
6.1 FRESH MUSHROOMS
6.1.1 Introduction
Based on phylum classification, fungi are classified as Ascomycota, Basidio-
mycota, Chytridiomycota, Deuteromycota, and Zygomycota [1]. While edible
fungi such as truffles and morels belong to the phylum Ascomycota, most
commercially cultivated edible fungal genera including agaricus, lentinula,
and pleurotus belong to the phylum Basidiomycota.
135
136 Microbiology of Fruits and Vegetables
Mushrooms, the common name for a large group of edible fungi, are
a common and popular food product. The reproductive portion or the fruit-
ing body of the mushroom usually lies above the growing substrate. It is the
portion that is commonly used for consumption. Because of their unique
earthy aroma and taste, many wild mushroom species have been tradition-
ally consumed. However only a few mushroom species have been extensively
cultivated on a commercial basis [2].
Agaricus bisporus (J. Lge) Imbach (button mushroom) is the most
widely cultivated species of edible mushroom, representing approximately
32% of world production in 1997 [3]. China, the U.S., and the Netherlands
are the top three producers of A. bisporus in the world [4]. Lentinula edodes
(Berk.) (shiitake) and Pleurotus ostreatus (Jacq.:Fr) Kumm. (oyster mush-
room), the second and third most cultivated edible mushrooms, account for
approximately 25 and 14% of world production, respectively [3,4]. Commer-
cial mushroom production makes a significant contribution to the total agri-
cultural output of the U.S. In 2002-2003 the U.S. mushroom crop totaled
844 million pounds, valued at $889 million. White and off-white A. bisporus
mushrooms still have by far the largest market share, in particular in the
western hemisphere, accounting for about 80% [5].
Since agaricus is the major genera of cultivated mushrooms around the
world, this chapter mainly describes the microbiology and microbial spoilage
of the cultivated button mushroom, A. bisporus.
6.1.2 Commercial Growing Practices
The agaricus mushroom growing process is unique in that it requires
decomposed organic matter as both a substrate for growing and as a source
of essential nutrients. A typical growth substrate contains straw-bedded horse
or chicken manure, hay, corn cob, brewer's grain, cotton seed, cocoa seed
hull, and water. The substrate mixture is aerobically fermented under
semicontrolled conditions [6,7], a process known as Phase I composting.
Ingredients are mixed and placed in aerated bunkers or formed into long
rows that are periodically turned, watered, and reformed. Rapid microbial
growth over a 15- to 25-day period causes the substrate (compost)
temperatures to reach as high as 175°F (80°C). During the Phase I process
substrate nutrients are converted into forms efficiently assimilated by the
mushrooms.
Phase II composting begins when the finished substrate is transferred
in bulk into controlled atmosphere tunnels, or in trays into controlled
atmosphere rooms where further microbial activity and nutrient conversion
occur. Phase II includes a controlled pasteurization step designed to eliminate
mushroom and human pathogens [8], weeds, and insect pests. A successful crop
requires that the compost temperature reach 130 to 140°F (60°C) for at least
2 hours [9].
Agaricus mycelial starter cultures grown on cereal grains, commonly
known as mushroom spawn, are then mixed into the substrate and allowed to
Microbial Spoilage of Fresh Mushrooms 137
► Agaricus fruiting body
► Casing layer
► Mycelia colonizing compost
► Compost
FIGURE 6.1 Schematic diagram of the mushroom substrate (compost), the peat-based
casing layer, and fruiting bodies.
grow throughout the compost for 14 days. Following complete colonization
of the substrate by A. bisporus mycelia, a two-inch casing layer (consisting of
peat soil amended with calcium carbonate and water) is applied on top of
the colonized substrate bed (Figure 6.1). The casing soil enhances retention
of irrigation water on the growing beds, and promotes mushroom fruit
body formation.
Mycelial growth occurs throughout the substrate and into the casing
layer. After 14 to 21 days, mushroom primordia are formed at the fruiting
stage known as pinning. The primordia develop into mature fruiting bodies
over a one-week period. During the development process, the growing beds
are irrigated to maintain substrate moisture, prevent disease, and maintain
postharvest mushroom quality [2,10,11]. At maturity, the mushrooms are
harvested, stipe-trimmed, packaged, and moved into cold storage.
6.1 .3 General Composition
Edible mushrooms, especially A. bisporus (button), tend to be high in moisture.
Mattila et al. [12] reported that the dry matter (percent solids) content of
A. bisporus grown in Finland was 7.7%. These values for A. bisporus mush-
rooms are similar to those normally experienced in North America, but
moisture can be as high as 95% when mushrooms are excessively irrigated [13].
Mushrooms contain large amounts of carbohydrates including polysac-
charides (such as glucans and glycogen), monosaccharides, and disaccha-
rides (such as trehalose), sugar alcohols (such as mannitol), and chitin. Mattila
et al. [12] reported that A. bisporus contained 4.5% (fresh weight) total
carbohydrates. Most of the polysaccharides are structural components of the
cell walls — chitin and glucans — and are indigestible by humans and can be
considered as dietary fiber. The A. bisporus mushroom species is also known
to contain significant amounts (20 to 30%, dry weight) of the sugar alcohol
mannitol, and 1 to 3% of the disaccharide trehalose [14].
While mushrooms contain only low levels of crude fat (0.31 to 0.35%, fresh
weight) [15,16], they contain a significant amount of protein, vitamins, and
minerals. Mattila et al. [12] found that A. bisporus mushrooms contained
about 2.0% net protein (fresh weight). These mushrooms are also known to be
138
Microbiology of Fruits and Vegetables
high in the B-complex vitamins: niacin, folate, pantothenic acid, and ribo-
flavin [17]. It was found that A. bisporus mushrooms contained almost 0.4%
riboflavin (fresh weight) [12]. With respect to minerals and trace elements,
A. bisporus mushrooms contain relatively high concentrations of potassium
(0.36% fresh weight) [12], copper (0.22% fresh weight) [18], and selenium
(3.2 and 1.4mg/kg, dry weight for brown and white A. bisporus strains,
respectively) [12].
From the standpoint of nutrients, fresh mushrooms are capable of sup-
porting growth of microorganisms. Agaricus mushrooms have a neutral pH
value, and fall in the category of foods with a water activity of 0.98 or higher.
These factors favor the growth of microorganisms, leading to the microbial-
induced quality degradation and spoilage of fresh mushrooms.
6.2 MICROBIOLOGY OF FRESH MUSHROOMS
Doores et al [19] demonstrated that normal healthy mushrooms have
high bacterial populations. Total bacterial numbers ranged from 6.3 to
7.2 log CFU/g of fresh mushroom tissue. The majority (54.0%) of bacteria
isolated from the mushrooms were identified as fluorescent pseudomonads
with flavobacteria comprising the second largest group (10.0%). Recent
experiments in our laboratory have confirmed this pattern, but we have
also been able to isolate the chryseobacterium genus (5.5 log CFU/g) and the
coryneform bacterial genus (5.6 log CFU/g) from freshly harvested mush-
rooms. Halami et al. [20] isolated lactic acid bacteria belonging to the
Lactobacillus sp. and Pediococcus sp. from fresh mushrooms by incubating
agaricus mushrooms in deMan Rogosa and Sharpe (MRS) broth for
enrichment of resident lactic acid bacteria. However, the bacterial counts
were not enumerated in their study.
Mushrooms also contain significant levels of yeasts and molds. Studies
in our laboratory have shown that freshly harvested mushrooms harbor
approximately 3 log CFU of molds and 6 log CFU of native yeast per gram of
fresh tissue (Figure 6.2).
2,
LL
o
O
8
7
6
5
4
3
2
1
Casing layer
Fresh
mushrooms
Aerobic plate
count
Yeast
Mold
FIGURE 6.2 Microbiology of the mushroom casing layer and fresh mushrooms (dw, dry
weight). Error bars represent standard deviation of the mean.
Microbial Spoilage of Fresh Mushrooms 139
6.3 SPOILAGE OF FRESH MUSHROOMS
Quality is the single most important factor affecting retail mushroom sales
[21]. Whiteness, cleanliness, and brown blotches on fresh mushrooms are
the principal factors determining mushroom quality. Consumers prefer to
purchase mushrooms that are bright white, free of casing material or other
unwanted particulate contaminants clinging to the mushroom surface, and
free of brown blotches. The brown blotch discoloration of mushrooms is
perceived as a symptom of decreased freshness or microbiological deteriora-
tion (spoilage).
Enzymatic browning catalyzed by the enzyme tyrosinase (polyphenol
oxidase) [22] is the most important factor involved in quality deterioration of
fresh mushrooms. The browning reactions are initiated by tissue break-
down due to either mechanical damage or bacterial activity [23]. It has been
suggested that the role of tyrosinase in mushrooms is to function as a stress
metabolite [24]. Tyrosinase naturally occurs at high levels in the mushroom
surface tissue, and is normally found in a latent form [5]. When activated
during senescence [22] the enzyme oxidizes mushroom phenolic compounds
into brown melanins [25-27] resulting in brown discoloration. In fresh
mushrooms, tyrosinase and its substrates have been hypothesized to be
located in separate subcellular compartments [22]. When mushrooms are
mishandled or bruised the cellular membrane is damaged, and rapid brown-
ing of the mushroom cap is observed. It has been hypothesized that the loss of
membrane integrity provides greater access of tyrosinase to its substrates,
resulting in formation of brown compounds [22,28] and associated brown
discoloration of fresh mushrooms.
The presence of high bacterial populations in fresh mushrooms is a major
factor that significantly diminishes quality by causing a brown, blotchy
appearance [23] (Figure 6.3). The rate of postharvest deterioration of fresh
mushrooms has been directly related to the initial microbial load [23]. Doores
et al. [19] found that bacterial populations during postharvest storage at 13°C
increased from an initial load of 7 log CFU/g to almost 1 1 log CFU/g over a
10-day storage period. The authors also reported that deterioration of
mushroom quality as indicated by maturity and color measurement appeared
to be concomitant with increase in bacterial numbers. Pseudomonas spp. and
Flavobacterium spp. were the two main groups that predominated during
agaricus mushroom postharvest storage. Similarly we have observed that
bacterial populations tend to increase from 7.3 to 8.4 log CFU/g during a 6-
day storage period at 4°C (Figure 6.4). Populations of yeast increased from 6.9
to 8.0 log CFU/g during the storage period. Populations of molds remained
constant (3 log CFU/g) during the storage period [29,30].
A majority of mushrooms of good quality and color, harvested and
marketed, develop blotches at retail or in consumer homes, even while kept
at refrigeration temperatures. Symptoms of brown blotch disease are
sunken, dark, and brown spots [31] on the mushroom fruit body surface.
Pseudomonas is the major spoilage genus associated with blotch
140
Microbiology of Fruits and Vegetables
(A)
(B)
(C)
FIGURE 6.3 Scanning electron micrographs of mushroom cap surfaces: (A) healthy
tissue (x3000); and blotched tissue showing invading bacteria (B) (x3000) and
(C)(x 10,000).
9
8.5
8
.o> 7.5
Z>
u- 7
O
g> 6.5
_i
6
5.5
5
— ▲
94
— *— Mushroom
—
X.
92
90
whiteness
(L-value)
-^0-^*
X7"
CD
88 -i
k CO
86 J
84
■ Bacterial
population
(Log CFU)
—
,
82
QO
DayO Day 2 Day 5 Day 8
FIGURE 6.4 Increase in aerobic bacterial populations and a concomitant decrease in
the whiteness (measured by L-value) of fresh Agaricus bisporus mushrooms during
postharvest storage at 12°C. The solid line represents aerobic bacterial populations (log
CFU/g fresh mushroom tissue). The broken line represents the L-value of the
mushroom cap during postharvest storage. Data are the average of four independent
samplings.
Microbial Spoilage of Fresh Mushrooms 141
formation of fresh mushrooms [32-34]. Paine [35] attributed Pseudomonas
tolaasii as the causative organism of the classic bacterial blotch disease of
cultivated mushrooms. Application of P. tolaasii cells as low as 20 CFU/cm of
growing beds resulted in blotch formation in mushrooms [36]. Symptoms
of mushroom blotch became visible when 5.4 x 10 CFU/cm were detect-
able in the mushrooms [36]. When P. tolaasii was placed directly onto
7 9
caps, 6x10 CFU/cm" were necessary to produce a blotch lesion (though only
3.5xl0 6 CFU could be recovered). The researchers of the study [36]
concluded that the number of cells of P. tolaasii present in the early primordial
stages of mushroom growth controls the extent of blotch disease seen at
harvesting. It has also been shown that tyrosinase is activated during infec-
tion by the bacterium P. tolaasii or exposure to its toxin, tolaasin, causing
brown blotch disease symptoms of fresh mushrooms [37]. Wells et al. [38],
by isolating and reinoculating the bacteria on freshly harvested healthy
mushrooms, confirmed that postharvest blotch formation and associated
discoloration was caused by three phenotypic groups (pathotypes) of
fluorescent pseudomonads. Severe infections with darkened or yellowed
lesions were caused by strains of pathotypes A or B, respectively. Mild
infections with superficial discoloration were caused by the pathotype C. Based
on cellular fatty acid analysis, the authors concluded that each pathotype
corresponded to one or several mushroom-related pseudomonads reported
in the literature as follows: pathotype A = Pseudomonas tolaasii, pathotype
B = Pseudomonas "gingeri", and pathotype C = Pseudomonas "reactans".
Isolates from mushroom casing material yielded all three pathotypes.
Fluorescent pseudomonads also produce exopolysaccharides (EPSs) asso-
ciated with the sliminess accompanying spoilage of mushrooms. Fett et al.
[39] isolated, partially purified, and characterized acidic EPSs from 63 strains
of mushroom-associated fluorescent pseudomonads. The strains were origin-
ally isolated from discolored lesions on mushroom caps, or from commer-
cial lots of mushroom casing soil. An acidic galacto-glucan named marginalan
was produced by mucoid strains of the saprophyte Pseudomonas putida and
the majority of mucoid strains of saprophytic P. fluorescens isolated from
casing medium. Other strains produced EPSs that included alginate, and
unique EPSs containing neutral and amino sugars and glucuronic acid.
There has been a long and complex association between the fungal genus
trichoderma and mushroom cultivation since Beach [40] first reported disease
symptoms on caps of agaricus mushrooms. In a study by Sharma et al. [41]
colonization assessments confirmed that Trichoderma harzianum biotypes Thl,
Th2a, Th2b, and Th3 inoculated into the mushroom substrate became
established in the mushroom substrate. The extension rate of two Th2 isolates
in the substrate was over 1000 times that of Thl and Th3. Results confirmed
that while Thl and Th3 did not significantly affect yield, Th2 could reduce
mushroom quality and productivity by as much as 80%. In vitro studies by
Mumpuni et al. [42] suggested that the growth of T. harzianum biotypes
could be related to the release of metabolites by A. bisporus into the com-
post substrate. Dilute aqueous solutions of ??-butanol extracts of A. bisporus
142 Microbiology of Fruits and Vegetables
culture filtrates and fruit bodies inhibited Thl and Th3 but stimulated Th2
isolates, suggesting that the active compound(s) may be constitutive com-
ponents of the A. bisporus species.
6.3.1 Sources of Microorganisms
Causing Spoilage
It has been demonstrated that the casing microflora have a vital role in the
sporophore (fruit body) formation of mushrooms from the mycelia stage
[43-45]. The requirement for biotic agents in the initiation of fruit body
formation [45] excludes the possibility of mushroom cultivation on a com-
mercial scale under axenic conditions. This factor, combined with the intensity
of production within a confined area, results in the introduction of micro-
organisms on fresh mushrooms that contribute to spoilage during postharvest
storage.
The casing layer on which the mushroom fruiting bodies develop is a
significant reservoir for the microflora of fresh mushrooms [19]. Doores et al.
[19] found that aerobic bacterial populations from casing material ranged
between 8.2 and 8.5 log CFU/g. In a study conducted by Wong and Preece
[46], the primary sources of Pseudomonas tolaasii on a mushroom farm were
the peat and limestone used in the casing process. This mushroom pathogen
could not be detected in the farm soil, water supply, the mushroom spawn
used, or in compost after spawning, but was isolated from the casing
(peat/limestone mixture) layer of symptom-free mushroom beds and both
the casing layer and compost of beds bearing blotched mushrooms. Secondary
sources were numerous once the pathogen was present in mushroom beds.
These included symptomless and blotched mushrooms, the fingers and shoes
of people handling the crop, their baskets, knives, and ladders. P. tolaasii was
also isolated from dust in the air of infected houses. While spores of infected
mushrooms may transport the bacterium, sciarid flies can act as vectors
contributing to bacterial transfer.
6.3.2 Cultural (Growing) Practices
Favoring Spoilage
The extent of irrigation significantly affects the bacterial populations and the
quality of the mushroom crop. Wong and Preece [36] concluded that very
frequent irrigation, resulting in over-watering, increased blotch symptoms on
mushrooms during growing.
6.3.3 Cultural Practices to Suppress Spoilage of
Fresh Mushrooms
Significant efforts have been directed to improve mushroom quality by add-
ing calcium salts or antimicrobial treatments to irrigation water during
Microbial Spoilage of Fresh Mushrooms 143
cultivation. Barden et al. [47] demonstrated that the postharvest shelf life
of fresh mushrooms increased by 2 days when mushrooms were irrigated with
0.5% calcium chloride. The increase in shelf life was mainly due to a decreased
rate of postharvest bacterial growth and a concomitant reduction of surface
browning. Solomon et al. [48] demonstrated a significant improvement in
quality and shelf life when mushroom crops were irrigated with tap water
containing 50ppm stabilized chlorine dioxide and 0.25% calcium chloride.
Initial and postharvest bacterial counts and degree of browning were lower
in these mushrooms as compared to mushrooms irrigated with water without
chlorine dioxide or calcium chloride. Irrigation treatments involving the
addition of calcium salts to irrigation water to reduce bacterial populations
and improve initial and postharvest mushroom quality have been extensively
studied [10,24,48,49], and are now a common commercial growing practice.
Kukura et al. [11] conducted a study to examine the influence of 0.3%
CaCl 2 added to irrigation water on mushroom tyrosinase activity and
postharvest browning. With the addition of CaCl 2 to the irrigation water,
the calcium content of mushrooms significantly increased, accompanied by
reduced postharvest browning. Irrigation with CaCl 2 had no effect on inher-
ent tyrosinase activity. The CaCl 2 irrigation treatment had even more pro-
nounced improvement on mushroom shelf life following a standard bruising
treatment, as indicated by reduced browning. Based on transmission electron
micrographs, the authors speculated that increased levels of calcium in mush-
rooms irrigated with CaCl 2 may have decreased browning by increasing
vacuolar membrane integrity, thereby reducing the opportunity for tyrosinase
to react with its phenolic substrates.
In other studies in our laboratory [29] we evaluated irrigation with
modified acidic electrolyzed oxidizing (EO) water in combination with 0.3%
calcium chloride on the reduction in bacterial populations of fresh mushrooms.
Crops were grown using standard growing practices except for the experi-
mental additions to the irrigation water of acidic EO water (diluted with 2 parts
of regular irrigation water) and/or 0.3% calcium chloride. Compared to the
control, all treatments reduced bacterial populations on the fresh mush-
rooms. While no significant differences in color were observed between the
treatments on the day of harvest, irrigation with modified acidic EO water and/
or calcium chloride resulted in enhanced whiteness, point-of-sale appearance,
and quality after a 7-day holding period of the fresh mushrooms. Recently
we investigated the effect of irrigation with water containing 0.75% hydrogen
peroxide on reduction in bacterial populations on fresh mushrooms. Irrigation
with 0.75% hydrogen peroxide in combination with 0.3% calcium chloride
added to the irrigation water consistently reduced the bacterial populations on
fresh mushrooms by 85% (compared to bacterial populations on mushrooms
irrigated with water without hydrogen peroxide and calcium chloride). This
irrigation combination treatment shows promise as an effective preharvest
method to enhance the quality of fresh mushrooms.
Research has been conducted to investigate the effect of natural anti-
microbial secondary metabolites added into the irrigation water. In a study
144 Microbiology of Fruits and Vegetables
by Geels [50], a 1% aqueous solution of kasugamycin, an antibiotic produced
by Streptomyces kasugaensis, was evaluated for reducing bacterial blotch
after artificial infection of the mushroom crop with P. tolaasii. An artificial
infection was established in the first flush (harvest) by inoculating the button-
sized mushrooms with a suspension of P. tolaasii. A 1 % aqueous solution of
kasugamycin supplied through irrigation water on the second-flush mush-
rooms drastically reduced bacterial blotch symptoms on these mushrooms at
picking stage. Disease incidence in the second flush in the control treatment
(inoculated with P. tolaasii) was composed of 18% lightly, 29% moderately,
and 10% heavily affected mushrooms, which totaled to 57% affected. The
1% kasugamycin treatment significantly reduced total disease incidence to
only 9% (lightly) affected. In the same study, a sodium hypochlorite-based
irrigation treatment showed no beneficial results.
Studies with canned products processed from mushrooms grown under
experimental cultural conditions indicated that canned product spoilage was
reduced significantly by employing peat versus soils as the casing material
[51]. While this study has no implication on the spoilage of fresh mushrooms,
it does indicate that casing type may have an effect on the microbiology and
microbial spoilage of fresh mushrooms.
Aerated steam treatment is sometimes employed to treat thermally
(pasteurize) the casing layer. Though steam treatment of casing material is
not a common cultural practice, some commercial growers employ past-
eurization (60° C, 140°F) of the casing layer to control diseases associated
with some materials they employ. However, most growers do not heat-treat
their casing material because of the additional cost involved and anecdotal
evidence that crop yield will be reduced.
It has been speculated that reducing the microbial load in the casing
layer may result in reduced bacterial populations associated with the mush-
rooms and improve postharvest quality [23]. Hence, we conducted an
experiment to evaluate casing pasteurization on reduction in bacterial
populations in fresh mushrooms and its effect on crop yield and quality. The
crop was grown at the Mushroom Test Demonstration Facility (MTDF) on
the Penn State University campus using standard growing practices used at
the MTDF except for the pasteurization treatment to the mushroom casing.
Unpasteurized casing served as the control. For pasteurization, the casing
material was held in a steam vault designed for direct steam injection. Steam
was generated on-site. Pasteurization of the casing was conducted by forcing
a mixture of air and steam into the vault to increase the temperature of the
casing material to 60° C (140°F). The casing material was held at 60° C for at
least 2 hours. Following the application of the pasteurized and untreated
(control) casing layers to the colonized compost the rest of the growing,
irrigation, and harvesting procedures were conducted as per standard
MTDF practices. Pasteurization of the casing layer (Figure 6.5) resulted in a
2.9 log CFU/g reduction in total bacterial populations (reducing the total
population in the pasteurized casing from 5.9 to 3 log CFU per gram of
casing layer material). However, bacterial numbers of the pasteurized casing
Microbial Spoilage of Fresh Mushrooms
145
8
7
6
D) O
3
LL A
o 4
D) o
x
o o
_l
X
2
1
Pasteurization/
pre-irrigation
Pasteurization/
1-week irrigation
^
Unpasteurized
casing
Pasteurized
casing
FIGURE 6.5 Effect of pasteurization at 60°C followed by irrigation on total bacterial
populations in the mushroom casing soil.
E
o
o
^_
m
=3
E
=>
LL
O
O
Unpasteurized
casing
Pasteurized
casing
Casing treatments
FIGURE 6.6 Aerobic bacterial populations on fresh Agaricus bisporus mushrooms
grown using either pasteurized or unpasteurized casing soil.
increased by 3.9 log CFU/g (from 3 log to 6.9 log) following 1 week of irriga-
tion. At the same time the bacterial numbers increased by 1.4 log CFU/g in
the unpasteurized casing (from 5.9 log to 7.3 log). Interestingly, there was no
significant difference in bacterial numbers in mushrooms grown using
unpasteurized or pasteurized casing (Figure 6.6). Mushrooms grown on
steam-treated casing material showed improved postharvest shelf life.
However the crop yield decreased by 10% when the pasteurized casing
was used.
From a food safety perspective, a recommendation to steam-treat
mushroom casing soils to reduce pathogenic bacterial populations should
be delayed, since steam treatment may negatively affect a hurdle (beneficial
soil microflora) to inhibit foodborne pathogens introduced into the soil (via
irrigation water or cross contamination). Preliminary research in our
146 Microbiology of Fruits and Vegetables
laboratory has indicated that survival of Listeria monocytogenes is enhanced in
pasteurized casing soil (60°C, 2 hours), compared to untreated soil. Under
mushroom growing casing conditions (80% moisture, 22°C), 6.8 log CFU/g
of L. monocytogenes was reduced to undetectable levels in 10 days in untreated
casing soil. During this time period, populations of L. monocytogenes
remained unchanged in pasteurized casing soil. So far, we have been able
preliminarily to identify that the Penicillium sp. present naturally in casing
soils may play a vital role in the destruction of L. monocytogenes. It is possible
that thermal pasteurization of casing soil may destroy the penicillium and
other beneficial microbial populations, thereby allowing survival of L.
monocytogenes in the casing soil. Hence practical nonthermal methods are
urgently required to destroy selectively the foodborne pathogens without
significantly affecting the beneficial microbial populations in casing soils.
Interestingly, L. monocytogenes demonstrated enhanced survival in casing
soils colonized with the agaricus mycelia than in soils without the mycelia
present in it. This situation warrants research on casing soil handling and
disinfecting crop irrigation procedures to achieve preharvest food safety and
quality goals.
Biocontrol has been evaluated as an alternative cultural practice to reduce
bacterial populations and subsequently enhance quality and postharvest
shelf life. Nair and Fahy [52] reported the isolation of three bacteria
antagonistic to P. tolaasii from soil and peat. These were a nonfluorescent
Pseudomonas species from soil, and strains of P. fluorescens and Enter obacter
aerogenes from peat. When the antagonists and the pathogen (Pseudomonas
tolaasii) were added in the ratio of 7.9:6 log CFU/ml to unsterilized peat
and applied to mushroom trays, infection of mushroom sporophores by the
pathogen was effectively controlled. In vitro studies failed to show lysis or
growth inhibition of P. tolaasii by the antagonists. While biocontrol-based
products have been introduced into the market in the recent past for con-
trolling bacterial brown blotch of mushrooms, they have not been a significant
commercial success.
6.3.4 Postharvest Conditions Favoring Spoilage
of Fresh Mushrooms
Postharvest storage conditions significantly contribute to mushroom quality
and shelf life. Pai [53] evaluated the effect of storage temperature (5, 10, and
15°C), and relative humidity (RH) (91, 94, 97, and 99%) on weight loss,
whiteness change, and microbial activity of A. bisporus mushrooms. Weight
loss of tested samples was correlated highly with storage time at each RH level.
Increasing storage temperature and decreasing RH significantly enhanced
(p < 0.05) the rate of weight loss. Mushroom whiteness values were not
affected (/? > 0.05) by changes in RH. Microbial growth increased with
increasing storage temperatures. It was concluded that the use of clean
mushrooms with low initial microbial counts, an environment of high RH, and
Microbial Spoilage of Fresh Mushrooms 147
minimal condensation in packages are important factors for maximizing the
shelf life of mushrooms under refrigerated storage.
Temperature abuse during storage is an important factor contributing
to the spoilage of fresh mushrooms. Tano et al. [54] evaluated the effects
of temperature fluctuation on the atmosphere inside modified atmosphere
containers and their impact on the quality of fresh mushrooms within the
containers. Mushrooms were packaged in 4-liter modified atmosphere (MA)
containers, and an atmosphere of 5% O2 and 10% C0 2 was maintained at 4°C.
Temperature was fluctuated from 4 to 20° C during a 12-day storage period
in cycles of 2 days at 4°C followed by 2 days at 20°C. The severity of
bacterial blotch on mushrooms was assessed using a rating of 1 to 4, with
1 =no bacterial blotch and 4 = above 25% of the mushrooms cap area with
symptoms of blotch disease. Temperature increase during fluctuations
caused anoxic atmospheres both in O2 (1.5%) and CO2 (22 to 10%). The
quality of mushrooms stored under temperature fluctuating regime was
severely affected as indicated by extensive browning, loss of firmness, and
a high level of ethanol in the tissue compared to mushrooms stored at
constant temperature. For the control group, the bacterial blotch index was
negligible over a 6-day storage period, whereas with mushrooms stored
under temperature abuse conditions, the index increased rapidly from 2.6 to
3.6 after 4 days. This study clearly demonstrated that temperature abuse and
temperature fluctuation seriously compromise the benefits of MA packaging
of fresh mushrooms.
Condensation of water in packages can severely affect the quality of
fresh packaged mushrooms. Apart from making the appearance of the
mushroom packs unattractive, condensation is not desirable since a water
layer on mushroom caps supports the growth of Pseudomonas tolaasii [55].
Gormley and MacCanna [56] studied the effect of overwrapping mush-
rooms with different types of perforated and nonperforated films on changes in
mushroom quality during storage. They found that water conden-
sation occurred on the underside of the nonperforated film. At the same time
excessive water loss through the perforated films caused wrinkling and
brown patches on the mushroom caps [56]. Hence it is important to select
packaging material taking into consideration the high respiration rate of
mushrooms and the potential fluctuating storage temperatures during
warehouse storage and retail display.
6.3.5 postharvest practices to suppress spoilage
of Fresh Mushrooms
Various postharvest treatments have been investigated in order to impede
browning and reduce rate of spoilage of fresh mushrooms. While proper cold
storage is a primary requirement during postharvest storage, new or novel
packaging techniques, washing treatments, and irradiation of mushrooms
can further contribute to spoilage suppression [2].
148 Microbiology of Fruits and Vegetables
6.3.5.1 Packaging
Overwrapping mushrooms with plastic film improves their quality as
observed by rate of cap opening, color, and weight loss [56-58]. Since
mushrooms respire heavily (500 mg C0 2 /kg fresh weight/hour at ambient
temperature) [59], it is important to ensure proper ventilation of the packages
to maintain a high O2 environment within the packages. Freshly harvested
mushrooms were found to induce a near anaerobic environment (<2% 2 )
in unventilated, PVC-overwrapped packages within 2 to 6 hours when
incubated at 20 to 30° C [60]. To prevent in-package atmospheres from turning
anaerobic which can increase risk of Clostridium botulinum growth, con-
ventional mushroom packages are also perforated at the top with 2 mm
holes in accordance with a U.S. Food and Drug Administration (FDA)
recommendation [61].
New technologies such as modified atmosphere packaging (MAP) have
been developed in order to delay quality loss and to extend storage life of
mushrooms [62-64]. The MAP method changes the mixture of gases
surrounding a respiring product to a composition other than that of air. The
gas composition of a storage atmosphere may reduce both microbial and
physiological spoilage of fresh mushrooms [65] Lopez-Briones et al. [66]
demonstrated that while up to 2.5% C0 2 seems to benefit mushroom
whiteness, C0 2 concentrations higher than 5% enhanced mushroom dis-
coloration during storage. The authors suggested that a desirable modified
atmosphere for mushrooms storage should contain 2.5 to 5.0% C0 2 and 5 to
10% o 2 .
Water persisting on mushroom caps after irrigation supports the growth
of Pseudomonas tolaasii [55] and subsequent appearance of blotch. Roy et al.
[67,68] evaluated sorbitol as a moisture absorber in mushroom packages
at 12°C. Surface moisture content of mushrooms decreased in the presence of
a sorbitol pouch. Mushrooms packaged with 10 g sorbitol pouches had con-
stant surface moisture content and those packaged with 15 g sorbitol pouches
had the best overall color. Lowering the in-package relative humidity did
not affect the maturation rate of mushrooms but reduced bacterial growth,
suggesting that improvement in color was probably due to reduced bacterial
activity.
Martin and Beelman [60] evaluated the potential of Staphylococcus
aureus to grow and produce staphylococcal enterotoxin in ventilated and
unventilated fresh mushroom packages when stored at 25 to 35°C. Mushrooms
were inoculated with an enterotoxigenic strain of S. aureus and incubated
in overwrapped trays at different temperatures. S. aureus grew and produced
staphylococcal enterotoxin (SE) in unventilated PVC-overwrapped mush-
room packages when inoculated at levels of 3, 4, and 5 log CFU/g of
mushroom after 4 days of incubation at 30°C. Growth of S. aureus was
observed at all levels of inoculation at 25°C, but no SE was detected after
7 days of incubation. When mushroom packages were ventilated, S. aureus
growth was suppressed and no SE was detected after 7 days at 25°C and 4 days
Microbial Spoilage of Fresh Mushrooms 149
at 30°C. However, S. aureus growth in ventilated packs exceeded growth
in unventilated packages when the incubation temperature was increased to
35°C; SE was detected within 18 hours of incubation at this temperature,
even in mushrooms inoculated at a low level (2 log CFU/g). These results show
the extreme importance of proper sanitation and worker hygiene during
mushroom harvesting and packaging, ventilation of fresh mushroom pack-
ages, and use of proper storage temperatures for fresh mushrooms at all
points of the food chain since SE is extremely thermotolerant and can even
survive the rigorous thermal process used in canning mushrooms [69].
6.3.5.2 Washing Treatments
Washing mushrooms has recently gained commercial popularity as a means
of removing casing soil particles and for the application of browning and
microbial inhibitors. Prior to 1986, aqueous solutions of sulfite, particularly
sodium metabisulfite, were used to wash mushrooms for the purpose of
removing unwanted particulate matter and to enhance mushroom whiteness.
While sulfite treatment yielded mushrooms of excellent initial whiteness and
overall quality, it did not inhibit the growth of spoilage bacteria. Therefore, the
quality improvement brought about by sulfite use was transitory. After 3 days
of refrigerated storage, bacterial decay of sulfited mushrooms becomes
evident. In 1986 the FDA banned the application of sulfite compounds to
fresh mushrooms due to severe allergic reactions to sulfites among certain
asthmatics. Following the ban on sulfite compounds for washing fresh
mushrooms, there have been several efforts to develop wash solutions for use
as a suitable replacement for sulfites.
McConnell [70] conducted a review of potential wash additives for
mushrooms including sodium hypochlorite, hydrogen peroxide, potassium
sorbate, and sodium salts of benzoate, EDTA, and phosphoric acids. The
researcher concluded that effective antioxidants, in addition to antimicrobial
compounds, were required to enhance shelf life of fresh mushrooms by
washing. A fresh mushroom wash solution containing 10,000 ppm hydrogen
peroxide and 1000 ppm calcium disodium EDTA was developed. Hydrogen
peroxide present in the wash solution acts as a bactericide. Copper is a
functional cofactor of the mushroom browning enzyme tyrosinase. EDTA in
the wash solution binds copper more readily than tyrosinase, thereby
sequestering copper and reducing tyrosinase activity and associated enzymatic
browning of mushroom tissue.
Beelman and Duncan [71] developed a mushroom wash process (U.S.
Patent 5,919,507). The method employed a first-stage high pH (pH of 9.0 or
above) antibacterial wash followed by a neutralizing wash containing brown-
ing inhibitors. The neutralizing wash contained a buffered solution of
erythorbic acid and sodium erythorbate. Other browning inhibitors such as
ascorbates, EDTA, or calcium chloride were identified as suitable ingredi-
ents for addition to the neutralizing solution. The process also helped remove
debris and delayed microbial spoilage of fresh mushrooms.
150 Microbiology of Fruits and Vegetables
Sapers et al. [72] developed a two-stage mushroom wash process employ-
ing 10,000 ppm (1%) hydrogen peroxide in the first stage aqueous solution,
and 2.25 to 4.5% sodium erythorbate, 0.2% cysteine-HCl, and 500 ppm to
1000 ppm EDTA in aqueous solution in the second stage. The two-stage
washing typically yielded mushrooms nearly as white as sulfited mushrooms
initially, and whiteness surpassed that of sulfited mushrooms after 1 to 2 days
of storage at 12°C [73,74]. The treatment was effective in reducing bacterial
populations in wash water and on mushroom surfaces [75] and had minimal
effects on mushroom structure and composition [76]. The process was
further modified and optimized [72] to include a prewash step using 0.5%
(5000 ppm) to 1% (10,000 ppm) hydrogen peroxide. Mushrooms washed by
this process were free of adhering soil, less subject to brown blotch than
conventionally washed mushrooms, and at least as resistant to enzymatic
browning as unwashed mushrooms during storage at 4°C. However, storage
at 10°C accelerated development of brown blotch and browning.
6.3.5.3 Irradiation
In 1986 the FDA approved gamma irradiation doses up to 1 kGy on fruits
and vegetables for the purpose of insect and/or growth and maturation
control. Low-dose gamma irradiation has been reported to be a very effective
method of controlling deterioration and improving quality and shelf life of
fresh mushrooms [77-79]. Radiation, usually from a cobalt-60 source, is
most effective when applied to the mushrooms shortly after harvest. A dose
of 1 kGy, an FDA-approved dose, greatly reduced bacterial counts and
slowed the rate of senescence [78]. A dose of 0.25 kGy was ineffective in
controlling senescence, while 2 kGy showed no significant improvement over
1 kGy in terms of postharvest quality [78]. Cap opening, stipe elongation,
surface darkening, and tissue softening were either delayed or prevented by
the application of irradiation [78]. Sensory data comparing irradiated
mushrooms with unirradiated controls showed that the former had equal or
superior flavor and texture scores for both raw and cooked samples [78].
In another study, Ajlouni et al. [14] concluded that low-dose gamma
irradiation (1 kGy) was an effective method for improving quality and
extending the shelf life of mushrooms under commercial retail conditions,
but it would need to be coupled with refrigerated storage to be most
effective. Commercial application of irradiation for enhancing the quality of
mushrooms has not yet been used in the U.S. However, cultivated mush-
rooms appear to be a good candidate for irradiation because of their high
market value and short shelf life.
Recently, electron-beam irradiation was evaluated for its application to
fresh sliced mushrooms [80]. The effects of electron-beam irradiation on
microbial counts, color, texture, and enzyme activity of mushroom slices were
evaluated at dose levels of 0.5, 1, 3.1, and 5.2 kGy. Irradiation levels above
0.5 kGy reduced total plate counts, yeast and mold, and psychrotrophic
bacteria counts to below detectable levels, and prevented microbial-induced
Microbial Spoilage of Fresh Mushrooms 151
browning. Firmness of all samples was similar during storage except for the
5.2 kGy sample. Color was not affected by the irradiation treatments. Electron-
beam irradiation at the levels tested did not affect the polyphenol oxidase
activity. Irradiation at 1 kGy was most effective in extending shelf life of
mushroom slices [80].
6.3.5.4 Pulsed Ultraviolet Light Treatment
Ultraviolet (UV) light is a portion of electromagnetic spectrum ranging
from 100 to 400 nm wavelengths. UV light in the wavelength range 100 to
280 nm has germicidal properties due to DNA damage in microorganisms.
Several researchers have demonstrated that the UV light can be used for the
inactivation of foodborne pathogens without adversely affecting the quality
of food. UV light treatment of foods can be accomplished using a pulsed UV
system, whereby the energy is stored in a high-power capacitor and is released
periodically in short pulses (often in nanoseconds). The pulsed UV light
system reduces the temperature buildup as compared to that obtained with
a continuous UV light, due to short pulse durations and cooling periods
between pulses. Thus, the pulsed UV light process may be considered
a nonthermal process.
Beelman et al. [81] conducted an experiment to evaluate the pulsed
UV light sterilization system to reduce bacterial populations in/on fresh
mushrooms. Pulsed UV light treatment was carried out with a laboratory
scale, batch, pulsed light sterilization system (SteriPulse®-XL 3000, Xenon
Corporation, Woburn, MA). The system generated 5.6J/cm per pulse on
the strobe surface for an input voltage of 3800 V and with 3 pulses per
second. The output from the pulsed UV light system followed a sinusoidal
wave pattern, with 5.6 J/cm per pulse being the peak value of the pulse. The
pulse width (duration of pulse) was 360 jas. Packed mushrooms were placed in
the pulsed UV light sterilization chamber and treated with pulsed light. The
first study used a 30-second treatment at a distance of 8 cm from the UV
strobe. The control samples did not undergo any pulsed UV treatment. In the
second study, treatments with varying treatment time (2 or 4 seconds) and
distance from UV strobe (8 or 13 cm) combinations were evaluated. Treated
mushrooms were analyzed for total aerobic bacteria, yeast/mold, and
coliform populations.
The microbiological results from the first experiment are shown in
Table 6.1. The 30-second pulsed UV treatment at 8 cm distance demon-
strated a greater than 1 log (90%) reduction for yeast and mold and aero-
bic bacterial populations. The UV treatment did not significantly affect
coliform populations. On visual analysis, the color of the mushrooms as a
result of the pulsed UV treatment was negatively impacted due to surface
browning.
The microbiological results of the second experiment are depicted in
Table 6.2. In general, the UV treatments of 2 or 4 s duration and 8 or 13 cm
distance from the UV strobe resulted in 0.9 to 1.6 log reduction in total
152 Microbiology of Fruits and Vegetables
TABLE 6.1
Microbiological Populations of Fresh Agaricus bisporus Mushrooms Treated
with Pulsed UV Light (30 Second Application at a Distance of 8 cm from
the UV Strobe)
Microbiological test Control Pulsed UV
Coliforms 6 x 10 2 3 x 10 2
Yeast and mold 6 x 10 3 /2 x 10 3 < 1 x 10 2
Aerobic plate count 1 x 10 7 4.5 x 10 5
TABLE 6.2
Microbiological Populations of Fresh Agaricus bisporus Mushrooms Treated
with Pulsed UV Light at Varying Treatment Times and Distances from the
UV Strobe
Microbiological test Control 2 s/8 cm 4 s/8 cm 2 s/1 3 cm 4 s/1 3 cm
Coliforms 5 x 10 2 4 x 10 2 < 1 x 10 2 < 1 x 10 2 < 1 x 10 2
Yeast and mold 1.6 xlO 4 2.1 x 10 4 3.4 xlO 3 1.3 xlO 3 1.2 xlO 3
Aerobic plate count 1.6 x 10 6 4 x 10 4 1.7 x 10 5 8 x 10 4 4 x 10 4
aerobic populations. Also, increasing treatment time improved reduction in
microbial populations. However, all pulsed UV treatments had a negative
impact on the color of the mushrooms due to surface browning.
The results from the pulsed UV study indicate little potential use for
pulsed UV treatments with white strains but could be useful with crimini
or portabella mushrooms, since the surface discoloration resulting from the
treatments would most likely not be observable by consumers due to the
inherent brown color associated with those types of mushrooms. Also, treat-
ment with UV light could be useful to increase the vitamin D2 content of
mushrooms [15].
6.4 CONCLUSIONS
This chapter mainly describes the microbiology and microbial spoilage of
the white button mushroom Agaricus bisporus. Cultural and postharvest
practices to enhance the quality of fresh white button mushrooms have also
been reviewed. Since the casing layer largely influences the microbiology
of fresh mushrooms, it is possible that the microbiology of mushrooms
grown using casing from the similar sources is largely similar. Cultural and
postharvest practices that enhance agaricus quality may also be applicable
Microbial Spoilage of Fresh Mushrooms 153
to other commercial varieties such as crimini, portabella, shiitake, oyster,
maaitake, and other exotic mushrooms varieties commonly seen in retail
outlets.
While we have noted significant increases in yeast populations during
postharvest storage of fresh mushrooms, the role played by yeast in the
microbial spoilage of fresh mushrooms is largely unknown. Hence, as a start-
ing point, the predominant yeast varieties in fresh mushrooms need to be
characterized.
While it is our understanding that mushroom growers strive to maintain
refrigeration temperatures during storage prior to shipping, temperature
fluctuations and abuse can be commonly encountered during transpor-
tation and retailing. This seriously compromises the quality of fresh
mushrooms. Hence it becomes essential for food transportation companies
and retailers to understand the implications of postharvest storage conditions
on the quality and shelf life of fresh mushrooms.
HACCP (hazard analysis critical control point) is increasingly being
adopted by mushroom growers as a system to enhance the safety of fresh
mushrooms. Studies at Penn State have been conducted to validate critical
control points to ensure the safety of irrigation water [29,30] and the
mushroom compost substrate [8]. Since heat pasteurization is not a practical
method to disinfect the casing layer, research is needed to understand the
microbial ecology of this material and thereby identify and validate other
practical casing or mushroom disinfection procedures.
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Spoilage of Juices and
Beverages by
Alicyclobacillus spp.
Mickey E. Parish
CONTENTS
7.1 Introduction 160
7.2 Taxonomic History 1 60
7.3 Physiological and Phenotypic Characteristics 164
7.3.1 Distinguishing Features 164
7.3.2 Thermoacidophilic Growth 164
7.3.3 Alicyclic Fatty Acids in Membrane 165
7.4 Thermal Resistance Characteristics 165
7.4. 1 D- and z- values 165
7.4.2 Factors Affecting Thermal Resistance 166
7.4.3 Other Control Measures 171
7.5 Industrial Importance 172
7.5.1 Emergence as Spoilage Organisms 172
7.5.2 Types of Spoilage 172
7.5.3 Sanitation 173
7.6 Detection and Identification 174
7.6. 1 Controversy 1 74
7.6.2 Media 175
7.6.3 Heat Shock Conditions 177
7.6.4 Enumeration 177
7.6.5 Detection by Enrichment 178
7.6.6 Identification and Confirmation 178
7.7 Significance of Detection/Isolation from Foods 179
7.8 Future Direction 1 79
Acknowledgment 180
References 180
159
160 Microbiology of Fruits and Vegetables
7.1 INTRODUCTION
The versatility and diversity of the microbial world often lead to unique
and valuable discoveries that expand our knowledge and yield advancements
for humankind. Antibiotics, fermented foods, health and beauty aids, and
other commonly utilized items in our everyday lives were discovered or
produced based upon unusual physiological and phenotypical character-
istics of microorganisms. In the past five decades the study of microbial
extremophiles in geothermal sites with high temperatures and high acidity has
advanced our understanding of spore-forming bacteria and led to the
establishment of the new genera alicyclobacillus and sulfobacillus.
Until the mid-1980s, the presence of bacterial spore-formers in low pH
foods was thought to be insignificant. The reigning dogma of the time declared
that Gram-positive, sporogenous bacteria could not outgrow to any great
extent at pH levels below 4.5. Therefore, the first report of spoilage in shelf-
stable, low pH fruit juices by Gram-positive, spore-forming bacteria [1] was
met with some skepticism. However, by the mid-1990s spoilage of acidic
juice products by members of the recently named genus alicyclobacillus [2]
was well established and the impact of this situation began to clarify. Fruit
juice and juice-containing beverages, bottled tea, isotonic drinks, and other low
pH, shelf-stable products were at risk of spoilage by a widespread
thermotolerant-to-thermophilic organism that could survive pasteurization
and hot-fill treatments, and was, surprisingly, acidophilic in nature. At present,
roughly 20 years after the initial reports of spoilage in fruit juice, concerns by
food processors about these thermoacidophilic, spore-forming bacteria remain
strong, economic losses continue, and effective commercial protocols to
address the situation are limited.
7.2 TAXONOM1C HISTORY
Ecological studies of extreme environments, such as geothermal hot springs,
during the latter half of the 20th century amplified scientific awareness of
unique, spore-forming, acidophilic bacteria with the ability to survive and
reproduce at high temperatures (40 to 100°C). This awareness, coupled with
characterization studies of various isolates, led to the discovery of one such
group, now recognized as the genus alicyclobacillus. The alicyclobacilli
have optimal growth conditions in warm to hot, acidic, low-nutrient environ-
ments and have been isolated from a variety of sources. These bacteria are
rod-shaped, approximately 2 to 4 urn in length and < 1 urn in width. Cells
produce swollen, terminal to subterminal sporangia with refractile
endospores (Figure 7.1) that are significantly heat resistant and capable of
surviving typical pasteurization and thermal concentration conditions of
juice/beverage manufacturing. On agar, colonies are usually a white to cream
color, slightly raised, with smooth to irregular margins (Figure 7.2). Older,
larger colonies take on a translucent character and may have slightly raised
edges.
Spoilage of Juices and Beverages by Alicyclobacillus spp.
161
FIGURE 7.1 (Color insert follows page 594) Gram stain of Alicyclobacillus acidoterres-
tris. Note swollen sporangia at arrows. (Magnification xlOOO.)
FIGURE 7.2 (Color insert follows page 594) Colonies of Alicyclobacillus acidoterrestris
ATCC 49025 on Ali agar after 24 hours at 45°C. Key characteristics: white/cream color,
smooth to irregular edges. Older, larger colonies may develop translucent quality with
slightly raised margins.
In 1967 Uchino and Doi [3] reported the isolation of thermoacidophilic
bacteria from geothermal hot springs in Japan. Similar organisms were
subsequently isolated from other geographically distinct geothermal sources
[4,5]. Brock and Darland [4] isolated thermophilic bacteria in about 300 hot
springs of various pH levels in the western U.S., New Zealand, Japan, and
Iceland. Microbial populations were found in "virtually every spring in the
neutral and alkaline pH range" despite extreme temperatures up to 100°C.
Bacterial isolates in acidic hot springs were temperature dependent and
162 Microbiology of Fruits and Vegetables
were not apparent at 90°C in springs with pH < 4.0 or at 70°C in springs
with pH < 2.0. Darland and Brock [5] named the species Bacillus acido-
caldarius to represent the unusual thermoacidophilic nature of these bacteria.
Hippchen et al. [6] isolated thermoacidophilic bacilli from common soil
samples, and Cerny et al. [1] reported the first account of a thermoaci-
dophile, later identified as B. acidoterrestris, isolated from spoiled fruit juice.
De Lucca et al. [7] reported the first isolation of B. acidocaldarius from
another agricultural source, sugar refineries. Other research has established
the presence of these organisms in high-fructose corn syrup (HFCS) commonly
used as a sweetener in beverages (R. Worobo, personal communication).
De Rosa et al. [8,9] reported the presence of several forms of co-cyclohexane
fatty acids in the membranes of B. acidocaldarius. Poralla et al. [10] suggested
a "cholesterol-like'' function for hopanoids in the membranes of B.
acidocaldarius. Poralla and Konig [11] isolated strains with co-cycloheptane
fatty acids that were later designated B. cycloheptanicus by Deinhard et al.
[12]. Deinhard et al. [13] also reported on the new species, B. acidoterrestris,
the thermoacidophile reportedly most involved in fruit juice spoilage.
Wisotzkey et al. [2] suggested reclassification of B. acidocaldarius,
B. acidoterrestris, and B. cycloheptanicus, as species of alicyclobacillus based
upon their thermoacidophilic phenotype and alicyclic fatty acids in the cel-
lular membranes. In a review of classification schemes for endospore-forming
bacteria, Berkeley and Ali [14] reported high homology (98.8%) between
DNA from A. acidocaldarius and A. acidoterrestris and suggested that these
might belong to one species rather than two. At the present time, the species
remain separate due largely to their differences in optimum growth
temperature and range.
Species of alicyclobacillus (Table 7.1) have been identified in a variety of
samples from six continents, including Antarctica [15]. Differentiation of
isolated strains from known species has been based largely upon genotypic
and phenotypic characterization with phylogenetic and chemotaxonomic
analyses. Albuquerque et al. [16] isolated thermoacidophilic strains from
volcanic soil in the Azores Islands and subsequently designated one as
A. hesperidum. Matsubara et al. [17] isolated a new species, A. acidiphilus, from
acidic beverages based upon phylogenetic analysis of the 16S rRNA gene
sequence and phenotypic differences related to spore morphology, growth
temperatures, and acid production from carbon sources.
Goto et al. [18] isolated the species, A. herbarius, from hibiscus-flavored
herbal tea. This strain contained co-cycloheptane fatty acid as the major
membrane lipid component and could be distinguished from other species
by phylogenetic analysis of the 16S rDNA sequence. In 2003 Goto et al.
[19] isolated a new species from fruit juice and named it A. pomorum. This
novel species does not contain alicyclic fatty acids but clusters among
the alicyclobacilli based upon phylogenetic analysis of the 16S rDNA sequence
with a level of similarity between 92.5 and 95.5%. Tsuruoka et al. [20]
isolated a collagenase positive strain that was closely related to species of the
alicyclobacilli based on 16S rDNA sequence analysis but had less than 33%
"O
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TABLE 7.1
Current Species of Alicyclobacillus and Corresponding pH and Temperature Characteristics
Species
Alicyclobacillus acidocaldarius subsp.
acidocaldarius
A. acidocaldarius subsp.
rittmannii
A. acidiphilus
A. acidoterrestris
A. cycloheptanicus
A. herbarius
A. hesperidum
A. pomorum
A. sendaiensis
A. vulcanalis
Source
Acidic hot spring, U.S.
Geothermal soil, Antarctica
Range
2.0-6.0
2.5-5.0
pH
Optimum
3.0-4.0
4.0
Acidic beverages, Japan
2.5-5.5
3.0
Garden soil and apple juice, Europe
2.5-5.5
3.5-4.0
Soil, Europe
2.5-5.5
3.5-4.0
Hibiscus herbal tea, Japan
3.5-6.0
4.5-5.0
Solfataric soil, Azores
2.5-5.5
3.5-4.5
Fruit juice, Japan
3.0-6.0
4.0-4.5
Soil, Japan
2.5-6.5
5.5
Geothermal pool, U.S.
2.0-6.0
4.0
Temperature (°C)
Range
45-70
45-70
20-55
42-53
42-53
35-65
40-55
30-60
40-65
35-65
Optimum
60-65
63
50
45-50
45-50
55-60
50
45-50
55
55
Ref.
3, 13, 14
10
2, 4, 8, 14
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164 Microbiology of Fruits and Vegetables
DNA-to-DNA reassociation homology with known alicyclobacillus-type
strains. This organism was deemed a new species and designated A. sendaiensis
in recognition of the Japanese city where it was isolated. Simbahan et al. [21]
reported a new species, A. vulcanalis, isolated from a geothermal pool at Coso
Hot Springs in the Mojave Desert, California, U.S.
Rodgers et al. [22] described growth enhancement of Acidiphilium cryptum
by two "alicyclobacillus-like" strains isolated from liquor (pH 2.1) of uranium
leaching operations. These strains had 94 to 96% homology to the alicyclo-
bacillus 16S rDNA sequence but were differentiated from other alicyclo-
bacilli in their ability to oxidize iron. These strains might represent new
alicyclobacillus species.
Taxonomy of alicyclobacillus and other thermoacidophilic genera/species
remains fluid and continued additions and revisions to species and genus
names should be expected for the foreseeable future. As more species-specific
hypervariable DNA regions are discovered in bacterial genomes, it is likely
that changes in taxonomy will continue.
7.3 PHYSIOLOGICAL AND PHENOTYPIC
CHARACTERISTICS
7.3.1 Distinguishing Features
The alicyclobacilli are Gram-positive, spore-forming bacteria having ther-
moacidophilic characteristics. Key diagnostic traits of the alicyclobacillus
phenotype include the presence of co-alicyclic fatty acids in cell membranes,
growth and endospore production under aerobic to facultative conditions
at 45°C and pH 3.0, limited, if any, spore production under anaerobic
conditions, and, to differentiate from sulfobacillus, no utilization of ferrous
iron, sulfide, or sulfur as energy sources under any conditions [14].
It should be noted that some publications refer to the alicyclobacilli as
Gram variable. This is due to their propensity to destain very rapidly during
the Gram stain procedure, which yields a visual appearance that can be
interpreted as indeterminate.
7.3.2 Thermoacidophilic Growth
The acidophilic and thermophilic nature of this genus is generally well charac-
terized although specific growth conditions appear to be species and strain
dependent [5,12,13]. Farrand et al. [23] conducted response surface analyses
to investigate growth of B. acidocaldarius over a range of temperatures and
pH, thereby establishing the extremes at which growth can occur, and con-
firming their thermoacidophilic character. Growth optima and upper/lower
limits for temperature and pH are shown in Table 7.1. In general, A. acido-
caldarius is the most tolerant of very high temperatures with optimum growth
at 60 to 65°C. A. herbarius has a slightly lower optimum growth tempe-
rature (55 to 60°C) while all other species have temperature optima generally in
Spoilage of Juices and Beverages by Alicyclobacillus spp. 165
the 45 to 50°C range. Most species have similar pH optima for growth (pH 3.0
to 4.5) although A. herbcirius and A. sendaiensis optima are higher at 4.5 to
5.0 and 5.5, respectively.
Darland and Brock [5] described the strain-dependent nature for growth
requirements for 14 strains of A. acidocaldarius. Two of the 14 strains had
a minimum pH for growth of 3.0 while the remaining 12 were capable of
growth down to pH 2.0. The lower temperature limit for all 14 strains was 45°C
while the upper limit was 65 and 70° C for eight and six strains, respectively.
Sinigaglia et al. [24] modeled the effects of temperature, water activity, and
pH on germination of A. acidoterrestris spores. Their results support the
thermoacidophilic characteristics of these organisms. Confirmation of their
model by other laboratories is needed.
7.3.3 Alicyclic Fatty Acids in Membrane
In all alicyclobacillus species except one, alicyclic fatty acids are the major
lipid components in the cell membrane. While this is a key distinguish-
ing feature, it should be noted that all species of sulfobacillus, as well as
Curtobacterium pusillum and Propionibacterium cyclohexanicum, also contain
significant quantities of these fatty acids [25—27]. Additionally, one recently
named species, A. pomorum, does not contain significant quantities of
co-alicyclic fatty acids [19].
Since there is no consistent correlation between the thermophilic/
acidophilic phenotype and the presence of co-alicyclic fatty acids, the purpose
of these lipids in cell membranes is not clearly understood and requires further
study.
7.4 THERMAL RESISTANCE CHARACTERISTICS
7.4.1 D- AND Z-VALUES
Although juice spoilage from heat-resistant molds has been known for
decades, the thermal resistance of spore-forming bacteria in low pH fruit
juice and beverages was of little concern to fruit juice manufacturers prior to
the 1990s. Despite the isolation of thermoacidophilic spore-formers from
apple juice in 1982 [1], the importance of this genus to juice stability and
consumer acceptance received little attention until reports of spoilage surfaced
in the early 1990s from Europe and the U.S. Since that time, considerable
research has been published illustrating the abundance of heat-resistant
alicyclobacilli in low pH juices and beverages.
Kinetic parameters of A. acidoterrestris have been elucidated by
several research teams using various techniques with different controlled
conditions and heating menstrua. Early research by Splittstoesser et al. [28,29]
produced D- and z-values that supported previous empirical observations
of spore survival in thermally treated juices. Z)-values (the time necessary at
a specific temperature to reduce the overall microbial population by 90%)
166 Microbiology of Fruits and Vegetables
reported by these researchers in apple and grape juices ranged from almost
60 minutes at 85°C to between 2 and 3 minutes at 95°C (Table 7.2). Since
typical commercial thermal process conditions are in the range 85 to 100°C
for 10 to 30 seconds, Splittstoesser's results demonstrated conclusively that
spores of the alicyclobacilli could survive traditional pasteurization and hot-fill
processes to cause spoilage in shelf-stable products.
Similar kinetic results for A. acidoterrestris have been reported in other
juices, beverages, model broth systems, and distilled water by various
laboratories (Table 7.2). Reported D- values range from 81 minutes at 88°C
to about 1 second at 125°C. Although specific D- and z-values from the
various studies differ, there are general similarities in magnitude. Average
D-values from Table 7.2 are 47 minutes (81 to 85°C), 24 minutes (86 to 90°C),
17 minutes (91 to 95°C), 7 minutes (96 to 100°C), 3.8 minutes (at 110°C), and
0.025 minutes (at 125°C). This is illustrated in Figure 7.3, which shows
an overall thermal death time curve for data points in Table 7.2.
A z- value is the temperature increase needed to reduce by 1-log cycle the
time necessary to produce a 90% reduction in cell populations. This is a
valuable tool when attempting to alter commercial processing conditions
to either decrease the time needed to achieve product safety and stability, or
decrease the temperature to enhance product quality. In essence, when process
time is decreased, the z-value is used to determine the new target processing
temperature. Likewise, if a lower temperature is desired to improve product
flavor, the z-value provides the increased time needed to achieve the same
product safety and stability as with the previous process conditions.
Except for one study that will be discussed below, z-values in Table 7.2
for A. acidoterrestris in juices, beverages, model systems, and water are rela-
tively similar with an average value of 8.3 ± 1.9°C. This means that on average
the time needed to inactivate a specific population of spores will decrease
by a factor of 10 if the pasteurization temperature is increased by 8.3°C.
7.4.2 Factors Affecting Thermal Resistance
It is important to remember that the inactivation kinetics of wild-type
alicyclobacillus strains may differ from those obtained from laboratory
strains that have been subjected to long-term cultivation. Many factors
affecting inactivation kinetics for other microorganisms have been studied
and may provide insights into thermal inactivation of the alicyclobacilli.
Factors include, among others, culture and inoculum incubation temperatures,
sporulation temperature, nutrient composition and pH of the growth medium,
nutrient composition and pH of the heating menstruum (e.g., test juice),
presence or absence of divalent cations, storage temperature of inoculum
stock, osmolarity of test juice matrix, or presence of antimicrobial compounds.
It is also well documented that specific strains within a species can vary
considerably in D- and z-values.
While it might be assumed that various factors would affect thermal
resistance of the alicyclobacilli in a similar manner, studies on this topic
TABLE 7.2
D- and ^-Values Reported for Alicyclobacillus acidoterrestris
Strain
A licyclobacillus acido terrestris
A licyclobacillus acido terrestris
A licyclobacillus acido terrestris
A licyclobacillus acido terrestris
A licyclobacillus acido terrestris
A licyclobacillus acido terrestris
strain VF
A licyclobacillus acido terrestris
strain VF
A licyclobacillus acido terrestris
strain VF
A licyclobacillus acido terrestris
strain VF
A licyclobacillus acido terrestris
strain VF
A licyclobacillus acido terrestris
strain VF
D (minutes)
z(°C)
Z) 85 o C 56
7.7
D 9 q c 23
^95 c 2.8
^85 C 57
7.2
D 90 c 16
D 95C 2.4
^88°C 11
7.2
Dgyc 3.8
D 95C 1.0
D95 c 5.3
Not reported
D 95 o C 2.2-3.3
6.4-7.5
D 9VC 31.3
10.0
#97 c 7.9
#88 c 81.2
5.9
-^iooc 0.8
D 9VC 54.3
7.7
Z) 97 c 8.8
£> 9 i c 46.1
8.5
D 9TC 8.2
/) 91 o C 57.9
8.2
Z) 9 7°c 10.8
D 9VC 49A
7.8
D 9 j c 8.4
Heating menstruum
Apple juice (11.4°B, pH 3.5)
Grape juice (15.8°B, pH 3.3)
Berry juice
Orange juice drink (5.3°B, pH 4.1)
Clear apple juice
Malic acid (0.4%) model broth (12°B, pH 3.1)
Malic acid (0.4%) model broth (12°B, pH 3.4)
Malic acid (0.4%) model broth (12°B, pH 3.7)
Citric acid (0.58%) model broth (12°B, pH 3.1)
Citric acid (0.58%) model broth (12°B, pH 3.7)
Tartaric acid (0.45%) model broth (12°B, pH 3.1)
(-n
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(Continued )
TABLE 7.2
Continued
Strain
A licyclobacillus acido terrestris
strain VF
A licyclobacillus acido terrestris
A licyclobacillus acido terrestris
A licyclobacillus acido terrestris
A licyclobacillus acido terrestris
A licyclobacillus acido terrestris
A licyclobacillus acido terrestris
A licyclobacillus acido terrestris
A licyclobacillus acido terrestris
D (minutes)
z(°C)
Dg\o C
69.5
7.1
D 97 o C
10.0
£*88°C
24
7.1
^95°C
2.7
^88°C
29
6.6
A)5°C
2.7
^88°C
26
6.4
A)5°C
2.3
^85°C
50
7.9
D<)o° c
17
A)5°C
2.7
£*80°C
41
12.2
D 9 QoQ
7.4
A)5°C
2.3
^80°C
24
13.8
£>9o°C
4.6
^95°C
2.0
^80°C
37.9
11.6
D 9 q° c
6.0
A)5°C
1.8
^80°C
54.3
12.9
£) 90 o C
10.3
D 9 5°C
3.6
Heating menstruum
Tartaric acid (0.45%) model broth
(12°B, pH 3.7)
Ref.
33
Mcllvaine buffer, pH 3.0
64
Mcllvaine buffer, pH 5.0
64
Mcllvaine buffer, pH 8.0
64
Orange juice
65
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Apple juice (pH 3.5)
Apple juice (pH 3.5) with 50 IU nisin/ml
34
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Alley clobacillus acldoterrestrls
NCIMB 13137
Alley clobacillus acldoterrestrls
NCIMB 13137
Alley clobacillus acldoterrestrls
NCIMB 13137
Alley clobacillus acldoterrestrls
STCC 5137
Alley clobacillus acldoterrestrls
STCC 5137
Alley clobacillus acldoterrestrls
Alley clobacillus acldoterrestrls
NCIMB 13137
A$s°c 17.5
2)970 c 0.6
D%$° q 65.6
D 9rc 11.9
D 9 \c 3.8
D 9lc 24.\
Duo c 3.9
,0,25^ 0.03
Z>iio» c 3.7
D X25C 0.02
Z/90 c 20.8
Awe 19.3
D 90 c 15.5
Awe 14.8
D 95 » c 5.3
Z/95 c 3.8
9.0
7.8
Not reported
7
7
Not reported
Cupuacu extract (11.3°B, pH 3.6)
Orange juice (11.7°B, pH 3.5)
Blackcurrant concentrate (26.1°B, pH 2.5)
Blackcurrant concentrate (58. 5° B, pH 2.5)
Orange juice
Distilled water
Clear apple drink (no nisin added)
With 50 IU nisin/ml
With 100 IU nisin/ml
With 200 IU nisin/ml
7.8 (no storage)
29 (spores stored at -18°C)
Cupuacu nectar (18°B, pH 3.2)
66
66
66
35
35
35
32
Note: D- value is the time at a specific temperature necessary to reduce the microbial population 1 log cycle (90%); z-value is the temperature increase necessary to
reduce by 1 log cycle the time needed to achieve a 1 log reduction.
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170
Microbiology of Fruits and Vegetables
CD
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2
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°1
-1 -
-2-
-3
y = -0.0691 x + 7.3848
Rsq = 0.7787
70 80 90 100 110
Degrees celcius
120 130
FIGURE 7.3 Compiled literature values for log/) vs. temperature, n = 98. Dashed lines
represent the 95% mean prediction confidence boundary.
are limited and generally not comprehensive in scope. However, there are
interesting results to consider. Two studies [30,31] determined that the presence
,2+
2+
2 +
2+
.2+>
of divalent cations (Ca , Mg , Ba , Mn , and Sr ) in the sporulation
medium did not affect the heat resistance of A. acidoterrestris. They also noted
that techniques to demineralize and remineralize spores with calcium had no
effect on their calcium content or thermal inactivation even though these
techniques were capable of decreasing the thermal resistance of Bacillus subtilis
spores. Vieira et al. [32] concluded that extended cold storage of spores at
— 18°C substantially increased z-values. Freshly prepared spores had a z-value
of 7.8°C while spores stored at — 18°C for 4 and 8 months had z-values of 22
and 29°C, respectively. Since juice concentrates are typically stored at —5 to
— 10°C in large cold-stored tanks (380,000 L or more per tank) or — 18°C in
55 gal drums, establishment of time/temperature processing parameters may
require knowledge of the concentrate storage history prior to use by juice and
beverage manufacturers.
Of three A. acidoterrestris strains tested by Pontius et al. [33], one was
less heat resistant than the other two at both 91 and 97°C in a model juice
system. However, the type of organic acid (citric, malic, or tartaric) in the
model system had no significant effect (P > 0.05) on results. These researchers
determined that results were significantly (P > 0.05) influenced by pH at 91°C
but not at 97°C. Their results were similar to those of Komitopoulou et al.
[34] who showed a decrease in Z)-values when the pH was changed from 4 to 3
in grapefruit juice. This effect was more apparent at 80° C (32 minutes at
pH 3.0 and 52 minutes at pH 4.0) than at 95°C (1.5 minutes at pH 3.0 and
1.7 minutes at pH 4.0).
Under test conditions used by Palop et al. [35], Z)-values at 110 to 125°C
were not different for spores suspended in orange juice, distilled water, or
citrate— phosphate buffers at pH 4 and 7. These researchers noted that a
sporulation temperature of 65°C yielded spores with increased heat resistance
compared to those produced at 45°C. A linear correlation was noted between
Spoilage of Juices and Beverages by Alicyclobacillus spp. 171
sporulation temperature and logZ) 110 °c- No differences in z-values were
observed in this study among spores in the juice, water, and buffer test
matrices.
Further research into processes and relevant parameters for control of
the alicyclobacilli is warranted. While it is obvious that the quality of most
juice and beverage products would not tolerate typical thermal condi-
tions needed to control these organisms, alternative processes or combined
processes may provide a solution.
7.4.3 Other Control Measures
Processors of 100% juices often choose to rely on thermal treatments as
the only hurdle for the production of a safe and stable juice. For those pro-
cessors selling refrigerated, pasteurized products, there is little cause for
concern since the alicyclobacilli do not grow under common refrigeration
conditions of 4 to 8°C. These organisms cause problems in shelf-stable, low pH
products. In some of these products, sodium benzoate, sorbate or some
other preservative may be added to inhibit growth of potential contaminants,
but their effects on the alicyclobacilli are not clearly understood. While
sorbates have been shown to inhibit germination of spores in some food
products [36], this preservative has not been the subject of public, refereed
research in relation to Alicyclobacillus spp.
The addition of the antimicrobial peptide nisin to juice was found to
enhance the lethality of thermal treatment [34,37]. These references report use
of a commercial nisin compound containing 2.5% active nisin with 10 6
international units (IU) per gram. Growth of A. acidoterrestris (Z CRA 7182)
at 25°C was controlled in apple, grapefruit, and orange juices by as little as
5 IU/ml nisin [34]. In the same study, Z>8o°c of spores in apple juice decreased
from 41 minutes without nisin to 24 minutes with 50 IU nisin/ml. As with pH
effects, the effect on Z)-values was more obvious at the lowest (80°C) process
temperature tested than at the highest (95°C). Although citrus juices are
known to be susceptible to spoilage by the alicyclobacilli, it is interesting to
note a greater degree of stability for citrus juice in this study when compared
to apple juice. Natural oils in citrus juices are known to have antimicrobial
activity, which might partially explain this observation.
When stored at 44°C, growth was observed in apple juice containing 50 IU
nisin/ml but was not seen at the 100 IU/ml level over a 6-day period [34].
In contrast, Yamazaki et al. [37] reported substantial growth of A. acido-
terrestris (AB-5) over a 12-day storage trial in a clear apple drink with up to
600 IU nisin/ml. In that study, D 90 o C values were reduced from 20.8 to 14.8
with the addition of 200 IU nisin/ml of apple drink. Yamazaki et al. [30]
reported that lysozyme increased thermal sensitivity of A. acidoterrestris spores
in a citrate buffer (pH 4.0).
Alternative processes other than thermal treatment could provide another
avenue of research to investigate alicyclobacillus inactivation. Lee et al. [38]
reported reductions greater than 5 log in spore populations from the
172 Microbiology of Fruits and Vegetables
application of high-pressure processing (HPP) with thermal treatments.
Although HPP potentially reduces the heat resistance of bacterial spores,
high pressure alone would not provide adequate kill to prevent spoilage. Other
alternative processes, such as pulsed electric field or ultraviolet light treat-
ment, are generally not very effective against bacterial spores. At present, there
are no adequate alternatives to stringent sanitation operating procedures
coupled with current good manufacturing practices and appropriate raw
material specifications for control of alicyclobacilli in beverages. This makes
the establishment of specifications difficult. Many customers of juice/beverage
ingredients desire to set a zero tolerance for the presence of the alicyclobacilli
in concentrates and sweeteners; however, this standard may be unachievable
with current processing and sanitizing technology.
7.5 INDUSTRIAL IMPORTANCE
7.5.1 Emergence as Spoilage Organisms
Food manufacturers continually investigate innovations in product develop-
ment, processing aids, and processing equipment to meet changing consumer
demands in order to maintain adequate profit margins. Changes in products
and manufacturing procedures frequently result in unique quality and stability
challenges. For example, in the 1980s and 1990s the use of oxygen barrier
films in gable-top cartons allowed orange juice packers to increase refrige-
rated shelf life of orange juice from 35 to 60+ days. This increased shelf life
provided enough time for fungal propagules within the paperboard matrix of
the carton to germinate and outgrow into the product thereby increasing
consumer complaints of mold spoilage and leading to use of a different
package design.
Between the mid-1980s and 1990s juice and beverage manufacturers
became aware of spoilage in low pH, shelf-stable products by spore-
forming bacteria. Processors of shelf-stable apple juices noticed occasional
development of strong off-aromas in finished product after a few weeks or
months of storage. Also, a few citrus juice processors reported the slow
growth of a spore-forming bacterium in aseptic juice samples thermally abused
at warm temperatures during quality control testing. Since the final product
was marketed as a refrigerated product, these spore-formers did not result
in spoilage of the finished product.
7.5.2 Types of Spoilage
The earliest documented fruit juice spoilage event related to alicyclo-
bacillus was reported by Cerny et al. [1] and involved an antiseptic off-aroma
in apple juice. The causative organism was described as "related to" Bacillus
acidocaldarius but would likely be classified today as A. acidoterrestris.
Splittstoesser et al. [28] reported growth characteristics of two thermoaciduric
spore-forming isolates that resulted in strong off-aromas in finished apple
Spoilage of Juices and Beverages by Alicyclobacillus spp. 173
juice products. Other reports also documented the isolation of thermoacido-
philes from juices, other low pH beverages, soil, fruit surfaces, and recycled
water [39—46]. Based on reports such as these, investigations were conducted
into the type of spoilage observed and the necessary conditions for such
spoilage to become apparent.
Prior to the recognition of alicyclobacillus as a spoilage agent of juices
and beverages, microbial stability problems were limited to yeasts, lactic acid
bacteria, acetic acid bacteria, or heat-resistant molds. Typical juice spoilage
involves production of C0 2 and off-flavors by fermentative organisms
resulting in bulging or exploding containers. Spoilage events caused by the
alicyclobacilli are different. Since there is no C0 2 to indicate microbial growth,
the powerful antiseptic and medicinal aromas may not be detected until
consumers open the package. The only visual clue to the presence of this
organism is the possible presence of a slight haze in clear liquids such as
clarified apple juice. Therefore, food manufacturers must implement new
quality assurance measures to detect the presence of these spoilage organisms.
The alicyclobacilli produce at least three odiferous phenolic compounds
with low detection thresholds. It was established in early research that guaiacol
is the principal off-aroma produced by the alicyclobacilli although 2,6-
dibromophenol (DBP) and 2,6-dichlorophenol (DCP) were also isolated
from products with large populations of Alicyclobacillus spp. Pettipher et al.
[42] found detectable levels of guaiacol in fruit juices, including orange and
apple juices. Baumgart et al. [43] and Borlinghaus and Engel [47] reported
the presence of DBP in tea and juice products. Orr et al. [48] reported that
guaiacol content in apple juice was not correlated with numbers of cells,
and that the best estimate threshold for guaiacol added to apple juice was
2.23 ppb. Jensen and Whitfield [49] described the production of DCP by the
alicyclobacilli. Gocjnen et al. [50] reported production of guaiacol, DBP, and
DCP by five strains of alicyclobacilli in orange juice.
7.5.3 Sanitation
For some manufacturers it is not feasible to consider the addition of
preservatives, such as nisin discussed above, to juice and beverage products
as a microbial control measure due to regulatory reasons. In these cases,
processors must reduce alicyclobacillus in the product through intensive
sanitation of the processing environment, and through strict specifications
and current good manufacturing practices for the product ingredients. Greater
emphasis on appropriate use of cleaners and sanitizers to control these
organisms on food contact surfaces should reduce the potential for product
contamination.
It is also important for processors to examine carefully all potential sources
of contamination since these organisms are widespread in nature and are
common inhabitants of soil. As thermoacidophiles, alicyclobacilli would be
expected to proliferate under warm acidic conditions in a manufacturing
facility. These conditions can occur in juice/beverage processing plants,
174 Microbiology of Fruits and Vegetables
especially during the warm summer months. (Empirical evidence of this exists in
the large amount of spoilage observed in Europe during the unusually hot
summers of 1994 and 1995.) Some facilities that produce concentrated
fruit juices reclaim water that is released from the juice during the evaporation
process, and utilize it for cleaning purposes within the plant. Since the
recovered water is warm to hot and slightly acidic, it provides an appropriate
environment for proliferation of the alicyclobacilli.
Wisse and Parish [44] and Eguchi et at. [45] reported the presence of
significant alicyclobacillus populations in "condensate water" recovered for use
in citrus concentrate facilities. Not only was the water used to wash equipment
and incoming fruit, in some cases pulp extracted from the juice was washed
with this water to recover sugar solids. This wash water was then diverted to
the evaporator and mixed with juice just prior to the concentration process
thereby guaranteeing a constant source of contamination in juice concentrates
produced by those facilities. Since that discovery, efforts to address the alicy-
clobacillus issue at citrus processing facilities have emphasized the cleanliness
of the condensate water recovery system and treatment of water used in the
facility.
Considering the substantial economic losses sustained due to growth of
the alicyclobacilli in juices and other low pH beverages, there are few refereed
publications discussing cleaning and sanitation requirements to control these
organisms. Orr and Beuchat [51] exposed five strains of A. acidoterrestris
spores to sodium hypochlorite, acidified sodium chlorite, trisodium phos-
phate, hydrogen peroxide, and Tsunami® sanitizer (Ecolab Inc., St. Paul, MN),
for 10 minutes at 23°C. A 5 log reduction in spore population was observed
after treatment with 1000 ppm sodium hypochlorite or 4% hydrogen peroxide.
At lower concentrations, significant (jP<0.05) reductions of 2 log, 0.4 log,
and 0.1 log were observed with 200 ppm hypochlorite, 500 ppm acidified
sodium chlorite, and 0.2% hydrogen peroxide, respectively. Based on these
results, these researchers continued with practical experiments to determine
chemical effectiveness against A. acidoterrestris on apple surfaces. Reductions
in spore populations after 1 minute exposure to 500 ppm hypochlorite or
1200 ppm acidified sodium chlorite were statistically significant (.P<0.05) but
did not inactivate spores more than 1 log as compared to 5 and 2.5 log in the
earlier direct challenge experiments.
7.6 DETECTION AND IDENTIFICATION
7.6.1 Controversy
Methods for accurate and sensitive detection, isolation, identification, and
quantification of the alicyclobacilli in foods have developed slowly and
remain somewhat controversial. In general, detection of these organisms in
juices and beverages has relied upon their thermoacidophilic character and
their ability to produce odiferous phenolic compounds. No standard method
Spoilage of Juices and Beverages by Alicyclobacillus spp. 175
detection/recovery protocols have yet been developed that are universally
accepted.
Research has been conducted on the use of DNA and PCR-based tech-
nologies to detect the alicyclobacilli in food samples. Specific primers for
detection have been developed [52] and a real-time PCR-based detection
method has been developed [53]. Rapid test kits for detection or identifi-
cation of Alicyclobacillus spp. are commercially available from Vermicon AG,
MicroBio Corporation, and BioSys. As with any rapid test methods, cust-
omers should verify and validate these products for their usefulness in the
specific commodity or environmental sample of interest. Customers should
further question the manufacturers for information on false negative and
false positive tests in order to make an informed decision on the usefulness of
such products for a particular application.
7.6.2 Media
There are two general types of media commonly used for the isolation or
detection of the alicyclobacilli. The first type contains four specific mineral
salts (ammonium sulfate, magnesium sulfate, calcium chloride, potassium
phosphate) with minimal amounts of carbon and/or nitrogen sources
(Table 7.3). Most of these media are similar in composition and are based
upon early reports by Uchino and Doi [3] and Darland and Brock [5].
Modifications of these media have been published by Farrand et al. [23],
Deinhard et al. [13], Yamazaki et al. [40], Wisse and Parish [44], and the
Internationale Fruchtsaft-Union (IFU) [54]. Some of these media require
addition of a trace mineral solution although the need for the full complement
of minerals in a recovery medium is unclear.
The second class of media does not contain a complement of minerals
and may be based on more traditional nutrient media with reduced pH.
Examples are acidified versions of potato dextrose agar, orange serum agar,
and plate count agar. YSG agar, recently advanced by Japanese organizations
as part of a universal method for detection of the alicyclobacilli in juices,
contains only yeast extract, soluble starch, and glucose. One particular
nonmineral-containing medium, K agar, was developed specifically for the
isolation of alicyclobacillus [55,56]. While nonmineral media may provide
adequate recovery of alicyclobacilli in certain situations, some research
suggests that mineral-containing media are more effective for enumeration or
for situations where detection of small cell populations via enrichment is
required. A recent study of more than 1500 environmental samples indicates
that a minimal mineral medium, Ali agar [44], recovered significantly (a = 0.05)
more alicyclobacillus strains than two nonmineral media, acidified potato
dextrose agar and K agar, under the conditions of that study (Parish,
unpublished data). Continued research to compare isolation media or
investigate alternative media is warranted.
The official method of the IFU for detection of alicyclobacillus in fruit
juices [54] depends upon the organisms' thermoacidophilic trait. A juice sample
TABLE 7.3
Composition of Minimal Mineral Media for Isolation of the Alicyclobacilli J
Ingredient
Ref. 3
Ref. 5
Ref. 1
Refs. 13, 23
Ref. 40
Ref. 44
Refs. 45, 54
CaCl 2 -2H 2
0.1 g
0.25 g
0.25 g
0.25 g
0.25 g
0.25 g
0.25 g
FeS0 4 -7H 2
0.025 g
—
0.28 mg
—
—
—
—
KH 2 P0 4
l.Og
3.0g
3.0g
3.0g
0.6g
3.0g
3.0g
MgS0 4 -7H 2
0.5g
0.5 g
0.5g
0.5 g
0.5g
0.5g
0.5g
MnCl 2 4H 2
—
—
1.25 mg
—
—
—
—
MnS0 4 4H 2
0.025 g
—
—
—
—
—
—
NaCl
l.Og
—
—
—
—
—
—
Na 2 HP0 4 12H 2
2.5g
—
—
—
—
—
—
(NH 4 ) 2 S0 4
—
0.2 g
0.2 g
0.2 g
0.2 g
0.2 g
0.2 g
NH 4 C1
l.Og
—
—
—
—
—
—
ZnS0 4 -7H 2
—
—
0.4 mg
—
—
—
—
Glucose
5.0g
0. 1-1.0 g
l.Og
5.0 g
l.Og
l.Og
5.0g
Glycerol
—
0. 1-1.0 g
—
—
—
—
—
2
n
Peptone
—
—
—
—
—
—
—
Ribose
—
0. 1-1.0 g
—
—
—
—
—
Soluble starch
—
—
2.0 g
—
—
2.0 g
—
cr
0'
Tween 80
—
—
—
—
—
—
—
5"
Yeast extract
—
0.1 g
2.0 g
l.Og
l.Og
2.0 g
2.0 g
era
Deionized water
1000 ml
1000 ml
1000 ml
1000 ml
1000 ml
1000 ml
1000 ml
a.
Trace elements b
—
—
—
1ml
—
—
1ml
Biotin
10 ng
—
—
—
—
—
—
c
l-H
r r\
Agar
—
—
—
15g
—
15-20g
15-20g
=3
a Media are typically
acidified with organic or mineral acids to pH 3.5 to 4.0 after
autoclaving.
Q_
<
b Solution of 0.66
g/1 CaCl 2
•2H 2 0, 0.30
g/1 Na 2 Mo0 4 -2H 2 0,
0.18
g/1 ZnS0 4
•7H 2 0,
0.18 g/1 CoCl 2 -6H 2 0,
0.16 g/1 CuS0 4 -5H 2 0, 0.15 g/1 MnS0 4
•4H 2 0, 0.10 g/1
05
era
H3BO3.
05
r+
a-
Spoilage of Juices and Beverages by Alicyclobacillus spp. 177
is diluted, heat shocked for 10 minutes at 80°C, incubated at 45°C for 7 days,
and plated onto an acidic, low-nutrient medium (BAT agar reported by
Eguchi et al. [45]; see Table 7.3). Growth at 45°C after 5 days is microscopi-
cally examined to exclude yeasts, followed by restreaking onto BAT and
PCA. After incubation at 45°C, strains that grow on BAT but not PCA are
considered probable alicyclobacillus although "further bio- and genotyping of
suspect alicyclobacillus" is encouraged to ensure the identification.
The most recent edition of the Compendium of Methods for the
Microbiological Examination of Foods describes a direct plating and enrich-
ment procedure [57]. While the protocols are fundamentally sound, the use of
a mineral-containing medium in addition to, or in place of, K agar is sug-
gested. Additionally, direct plating of concentrate is not recommended since
higher Brix levels may inhibit colony formation. In the enrichment protocol,
the heat shock at 90° C is too severe and should be reduced to a range of 70
to 80°C.
7.6.3 Heat Shock Conditions
There are two purposes for using a heat shock during recovery protocols.
First, it eliminates vegetative cells (including vegetative alicyclobacillus) to
allow germination of spores without competition from other organisms.
Second, it activates spores to germinate and outgrow although the actual
increase in spore recovery is not well established. Unpublished results by
Parish show as much as a 400% increase in recovery of alicyclobacillus from
orange juice by use of a mild heat shock. However, heat shock may not be
necessary in finished, shelf-stable, low pH products that do not contain
competitive microflora. Further research is warranted to determine the effect
of heat shocks on spore viability.
Heat shock regimes should be of appropriate duration to eliminate com-
peting microflora but not so stringent as to inactivate spores. Baumgart et al.
[43] suggest 20 minutes at 70°C as an effective heat shock regime. Parish and
Goodrich [58] investigated various times and temperatures from 60°C for
30 minutes to 90°C for 5 minutes, but ultimately recommended 75°C for
10 minutes to recover alicyclobacilli from diluted orange juice. They reported
optimal recovery over a range of times at specific temperatures: 10 to
30 minutes at 60 and 65°C; 5 to 25 minutes at 70 and 75°C; and up to 5 minutes
at 80 and 85°C. Recovery at 90°C was inadequate compared to results at
the other temperatures. Additionally, the heating menstruum may affect
recovery. Several studies show that percent recovery increases as samples
are diluted to lower sugar content.
7.6.4 Enumeration
Enumeration of the cell population may provide important information
related to spoiled packaged products having obvious medicinal off-aroma.
Basic plating techniques for enumeration are spread plating, including spiral
178 Microbiology of Fruits and Vegetables
plates and pour plating [59,60]. In the case of clear liquids, a filtration
technique may be used to concentrate cells from a large sample volume, which
increases test sensitivity. For liquids containing particulate matter, such as
cloudy juices, a most probable number (MPN) technique is appropriate [60].
Enumeration by the MPN technique would be conducted using an appropriate
broth medium such as filter-sterilized juice, or a low pH minimal nutrient broth
such as Ali Broth [44] or BAT Broth [45,54].
7.6.5 Detection by Enrichment
Enrichment of samples is of considerable importance for the recovery of
small numbers of alicyclobacillus from concentrated juices, purees, and
nectars. Although the general concept of enrichment is consistent from one
study to the next, conditions for assaying different samples vary and are
not applicable to all situations. Protocols typically involve incubation of
a sample that is diluted in either a minimal broth medium that contains
minerals with nutrients, or plain water. The level of dilution varies from single-
strength to as little as 2° Brix. Sample sizes typically range from 1 to 100 ml of
concentrate, puree, or nectar.
After dilution, samples are heat shocked followed by incubation at 40
to 55°C for 3 to 7 days. A yeast/mold inhibitor may be added in cases where
a heat shock is not used or is inadequate. At the end of incubation, samples
are streaked onto appropriate media and plates are incubated aerobically at
45 to 50°C for several days. Development of colonies suggests the possible
presence of alicyclobacillus although confirmation requires further analysis.
7.6.6 Identification and Confirmation
Colonies produced by enrichment or direct plating on low pH media must be
subjected to appropriate analytical techniques to confirm their identification
as alicyclobacillus. Deciding which analytical techniques are considered
appropriate is the basis of controversy that has yet to be adequately addressed.
A microscopic examination is necessary to confirm that the isolate is not
fermentative yeast or another type of acidophilic organism, and that it is a
Gram-positive spore-forming bacterium. The sporogenous nature of the
isolate may require further plating and incubation on other media followed
by spore staining and microscopic observations.
Confirmation activities include growth on low pH media at elevated
temperatures (45 to 55°C) with concurrent lack of growth on media of neutral
pH, such as traditional plate count agar or nonacidified minimal media, and
lack of growth at reduced temperatures. One possible protocol is to streak
suspect colonies onto duplicate plates of low pH and neutral pH media.
One plate of each is incubated at 45 to 50° C is incubated and the remaining
plates at 20 to 25°C for appropriate time periods. Observation of growth at
45°C and little or no growth at 25°C on low pH agar with no growth on the
Spoilage of Juices and Beverages by Alicyclobacillus spp. 179
remaining plates at either temperature is consistent with the genus alicyclo-
bacillus. Further testing is suggested for final confirmation.
The most solid proof of identity is obtained from sequence analysis of
species-specific DNA or RNA segments, such as for the 16S ribosomal sub-
unit, coupled with observations of basic phenotypic characteristics (ther-
moacidophilic, Gram-positive bacterial spore-former). Ribotyping has been
investigated by the National Food Processors Association for identification of
the alicyclobacilli with limited success. A protocol for use of randomly
amplified polymorphic DNA was developed by Yamazaki et al. [61] to identify
A. acidoterrestris. Further research is required by other laboratories to verify
the use of these technologies.
7.7 SIGNIFICANCE OF DETECTION/ISOLATION
FROM FOODS
Information regarding the storage conditions and number of alicyclobacilli
necessary to cause spoilage is sparse. Since these organisms can be routinely
isolated from unspoiled products, the question of their significance has
been raised. A few researchers suggest that 10 cells/ml of A. acidoterrestris
can produce enough phenolic compounds to cause an antiseptic/medicinal
off-aroma within a few days to weeks. On the other hand, a publication by
ABECitrus [62] states "Spore counts in the average range of 10 to 10 CFU/ml
in the concentrated juice may be at an acceptable level which does not
compromise further utilization and processing of juice given that adequate
processing practices are employed, particularly after heat treatment or
pasteurization. '' Further, Eguchi et al. [45] reported that only 2 of 13 wild-
type strains produced spoilage when inoculated into orange juice. The
differences of opinion among researchers regarding the presence of alicyclo-
bacillus in foods indicate that questions regarding conditions that lead to
product spoilage are unresolved and require additional investigation.
7.8 FUTURE DIRECTION
Due to their ubiquitous nature in the environment, the alicyclobacilli can be
routinely isolated from liquid sugars, such as HFCS, concentrated juices,
purees, and other agricultural products. Although there is no doubt that
the alicyclobacilli have been involved in spoilage of juice and beverage prod-
ucts, the significance of detecting them in an unspoiled food sample remains
in question. It is unlikely that the detection of a single spore in lOOg of
concentrated juice by quality control testing would correlate with the potential
for spoilage in the final product.
A substantial amount of research is needed to ascertain the significance
of finding these organisms in food products. It is understandable that
beverage-manufacturing companies wish to obtain ingredients (juice, HFCS,
granulated sugar, etc.) that do not contain alicyclobacillus. This should be
180 Microbiology of Fruits and Vegetables
tempered with the knowledge that the significance of finding small numbers of
these organisms in raw ingredients is not fully understood, and that current
technology does not allow HFCS or concentrate/puree facilities to completely
eliminate the alicyclobacilli from their products. Research to determine
the probability of spoilage in products that contain specific numbers of spores
and are stored under various conditions is lacking. Additionally, it would
be helpful to investigate the existence of processing and storage parameters
that might prevent outgrowth of alicyclobacilli in packaged products. Further
discoveries may provide the answers needed to establish acceptable
standards for alicyclobacillus in foods.
ACKNOWLEDGMENT
This document was approved by the Florida Agricultural Experiment
Station for publication as Journal Series No. N-02531.
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25. Suzuki, K. et al., Occurrence of co-cyclohexyl fatty acids in Curtobacterium
pusillum strains, J. Gen. Appl. Microbiol., 27, 261, 1981.
26. Dufresne, S. et al., Sulfobacillus disulfidooxidans sp. nov., a new acidophilic,
disulfide-oxidizing, gram-positive, spore-forming bacterium, Int. J. Syst.
Bacteriol, 46, 1056, 1996.
27. Kusano, K. et al., Propionibacterium cyclohexanicum sp. nov., a new acid-
tolerant co-cyclohexyl fatty acid-containing propionibacterium isolated from
spoiled orange juice, Int. J. Syst. Bacteriol., 47, 825, 1997.
28. Splittstoesser, D., Churey, J., and Lee, C, Growth characteristics of
aciduric sporeforming bacteria isolated from fruit juices, J. Food Prot., 57,
1080, 1994.
29. Splittstoesser, D., Lee, C, and Churey, J., Control of Alicyclobacillus in the juice
industry, Dairy Food Environ. San., 18, 585, 1998.
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Alicyclobacillus acidoterrestris, in acidic beverages, Nippon Shokuhin Kagaku
Kaishi, 44, 905, 1997.
182 Microbiology of Fruits and Vegetables
31. Yamazaki, K. et al., Influence of sporulation medium and divalent ions on the
heat resistance of Alicyclobacillus acidoterrestris spores, Lett. Appl. Microbiol.,
25, 153, 1997.
32. Vieira, M.C. et al., Alicyclobacillus acidoterrestris spores as a target for Cupuacu
(Theobroma grandiflorum) nectar thermal processing: kinetic parameters and
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33. Pontius, A., Rushing, J., and Foegeding, P., Heat resistance of Alicyclobacillus
acidoterrestris spores as affected by various pH values and organic acids, J. Food
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34. Komitopoulou, E. et al., Alicyclobacillus acidoterrestris in fruit juices and its
control by nisin, Int J. Food Sci. Technoi, 34, 81, 1999.
35. Palop, A. et al., Heat resistance of Alicyclobacillus acidocaldarius in water,
various buffers, and orange juice, J. Food Prot., 63, 1377, 2000.
36. Sofos, J., Busta, F., and Allen, C, Sodium nitrite and sorbic acid effects on
Clostridium botulinum spore germination and total microbial growth in chicken
frankfurter emulsions during temperature abuse, Appl. Environ. Microbiol., 37,
1103, 1979.
37. Yamazaki, K. et al., Use of nisin for inhibition of Alicyclobacillus acidoterrestris
in acidic drinks, Food Microbiol., 17, 315, 2000.
38. Lee, S., Dougherty, R., and Kang, D., Inhibitory effects of high pressure and
heat on Alicyclobacillus acidoterrestris spores in apple juice, Appl. Environ.
Microbiol., 68, 4158, 2002.
39. Mclntyre, S. et al., Characteristics of an acidophilic Bacillus strain isolated from
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Alicyclobacillus acidoterrestris from acidic beverages, Biosci. Biotech. Biochem.,
60, 543, 1996.
41. Pinhatti, M. et al.. Detection of acidothermophilic bacilli in industrialized fruit
juices, Fruit Processing, 1 , 350, 1997.
42. Pettipher, G., Osmundson, M., and Murphy, J., Methods for the detection and
enumeration of Alicyclobacillus acidoterrestris and investigation of growth and
production of taint in fruit juice and fruit juice-containing drinks, Lett. Appl.
Microbiol., 24, 185, 1997.
43. Baumgart, J., Husemann, M., and Schmidt, C, Alicyclobacillus acidoterrestris:
Vorkommen, Bedeutung und Nachweis in Getranken und Getrankegrundstof-
fen, Flussiges Obst., 64, 178, 1997.
44. Wisse, C. and Parish, M., Isolation and enumeration of sporeforming,
thermoacidophilic, rod-shaped bacteria from citrus processing environments,
Dairy Food Environ. San., 18, 504, 1998.
45. Eguchi, S. et al., An ecological study of acidothermophilic sporulating bacteria
(Alicyclobacillus) in the citrus industry, Ann. of the 23rd IFU Symposium,
Havana, 2000, p. 257.
46. Jensen, N., Alicyclobacillus in Australia, Food Australia, 52, 282, 2000.
47. Borlinghaus, A. and Engel, R., Alicyclobacillus incidence in commercial apple
juice concentrate (AJC) supplies: Method development and validation, Fruit
Processing, 1, 262, 1997.
48. Orr, R. et al., Detection of guaiacol produced by Alicyclobacillus acido-
terrestris in apple juice by sensory and chromatographic analyses, and
comparison with spore and vegetative cell populations, /. Food Prot., 63,
1517, 2000.
Spoilage of Juices and Beverages by Alicyclobacillus spp. 183
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species in orange juice using GC-Olfactometry and GC-MS, Lett. Appl.
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of Alicyclobacillus acidoterrestris and performance of media for supporting
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based method for rapid and specific detection of spoilage Alicyclobacillus spp. in
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bacillus in fruit juices, in Microbiological Methods, Internationale Fruchtsaft-
Union Microbiology Working Group, Bischofszell, Switzerland, 2004.
55. Walls, I. and Chuyate, R., Alicyclobacillus'. historical perspective and
preliminary characterization study, Dairy Food Environ. San., 18, 499, 1998.
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juices, /. AOAC Int., 83, 1115, 2000.
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Methods for the Microbiological Examination of Foods, American Public Health
Association, Washington D.C., 2001.
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Alicyclobacillus Species and Other Spore-Forming Thermotolerant Acidophilic
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Microbiological Examination of Foods, American Public Health Association,
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identification of the spoilage bacterium Alicyclobacillus acidoterrestris, Biosci.
Biotech. Biochem., 61, 1016, 1997.
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detection methods, ecology, and involvement in the deterioration of fruit juices,
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frutta, Industria Conserve, 72, 353, 1997.
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Alicyclobacillus acidoterrestris spores in different buffers and pH, Food
Microbiol., 15, 577, 1998.
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occurrence and heat resistance of spores, /. Food Prot., 62, 883, 1999.
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Section III
Food Safety Issues
8
Interventions to Ensure the
Microbial Safety of Sprouts
William F. Fett
CONTENTS
8.1 Introduction 187
8.2 Foodborne Illness Associated with Sprouts 188
8.3 Interventions: Seeds 191
8.3.1 Chemical and Physical 191
8.3.2 Biological 197
8.4 Interventions: Sprouts 198
8.5 Reducing the Risk of Future Outbreaks 200
8.6 Research Needs 201
Acknowledgments 202
References 202
8.1 INTRODUCTION
Sprouts are considered a natural healthy food by many consumers in the
U.S. and elsewhere. The North American sprouting industry has grown
rapidly from only a very few commercial growers in 1970 to approximately
300 growers today with a total product market value of approximately
$250,000,000 [1]. Over 20 seed types are used for sprouting in commercial
operations and in the home [2]. Commercial sprouting operations are indoor
facilities and in the U.S. are usually small in size with less than 10 employees
[3]. Distribution of sprouts to retail outlets is local or regional.
Sprouts can be classified as either green sprouts or bean sprouts. Green
sprouts such as alfalfa, clover, broccoli, radish, and sunflower have been
subjected to light at some point in the growing process to allow for chlorophyll
development. Bean (mung bean and soybean) sprouts are propagated under
continuous dark and thus do not produce chlorophyll. Mung bean sprouts
make up the major portion of the market for sprouts in the U.S. Green sprouts
Mention of trade names or commercial products in this chapter is solely for the purpose of
providing specific information and does not imply recommendation or endorsement by the U.S.
Department of Agriculture.
187
188 Microbiology of Fruits and Vegetables
are consumed raw while bean sprouts are most often, but not always, served
after at least light cooking.
Unfortunately, since 1995, both in the U.S. and in other countries, there
have been numerous outbreaks of foodborne illness due to the consumption of
sprouts contaminated with the bacterial pathogens salmonella and Escherichia
coli 0157 [4,5]. Raw sprouts were identified as a special food safety problem
due to the potential for bacterial human pathogens to multiply from low levels
on contaminated seed to high levels on sprouts due to favorable conditions
of moisture, temperature, and nutrient availability during the sprouting
process [4]. The U.S. Food and Drug Administration (FDA) has released a
number of consumer advisories informing the consuming public about the risks
associated with eating raw sprouts, the latest occurring in November 2003
[6], and raw sprouts are considered a ''potentially hazardous food'* in the
FDA Food Code [7]. The consumer advisory states: c 'Those persons who wish
to reduce the risk of foodborne illness from sprouts are advised not to eat raw
sprouts." Particularly vulnerable to foodborne illness are the young, the
elderly, and the immunocompromised.
This chapter provides an overview of the incidence and causes of sprout-
related foodborne illness, interventions that have been tested for eliminating
human pathogens from seeds and sprouts, means for reducing the risk of future
outbreaks, and finally, further research needs.
8.2 FOODBORNE ILLNESS ASSOCIATED
WITH SPROUTS
Several foodborne human pathogens have been isolated from sprouts and
consumption of contaminated sprouts has been associated with numerous
outbreaks of foodborne illness in the U.S. (Table 8.1). Some of these outbreaks
have been international in scope due to the international distribution of sprout
seed [10,12,24,25]. In addition to those in the U.S., sprout-related outbreaks of
foodborne illness have been reported in several other countries including
Canada, Japan, Sweden, Denmark, Holland, Finland, and the U.K. [4,5]. The
earliest documented outbreak in the U.S. occurred in 1973 and was associated
with consumption of raw sprouts grown with home sprouting kits containing
soybean, cress, and mustard seed contaminated with enterotoxigenic Bacillus
cereus [8]. There were no additional sprout-related outbreaks of foodborne
illness recorded in the U.S. until 1990. Since 1995 there have been many
outbreaks due to contamination of alfalfa and clover sprouts with various
serovars of salmonella or E. coli 0157. The first foodborne outbreak due to
mung bean sprouts in the U.S. occurred in 2000 due to contamination with
salmonella [9]. Previously, the only documented mung bean-associated
outbreak of salmonellosis took place in England and Sweden in 1988 [26].
The number of culture confirmed cases in the U.S. has ranged from less than
10 to over 400 per outbreak. The actual number of cases was most likely
much higher due to the significant underreporting normally encountered for
Interventions to Ensure the Microbial Safety of Sprouts
189
TABLE 8.1
Incidence of Foodborne Illness Due to Contaminated Sprouts in the U.S.
No. of
culture
confirmee
1
Year
Bacterium
Location
Sprout type
cases
Ref.
1973
Bacillus cereus
TX
Soybean,
cress, mustard
4
8
1990
Salmonella Anatum
WA
Alfalfa
15
9
1995
Salmonella Stanley
17 states/Finland Alfalfa
242
10
1995
Salmonella Newport
OR
Alfalfa
69
9
1995-1996
Salmonella Newport
7 states/Canada/
Denmark
Alfalfa
> 133
5, 11,
12
1996
Salmonella Stanley
VA
Alfalfa
30
9
1996
Salmonella Montevideo/
Meleagridis
CA/NV
Alfalfa/clover
492
13
1997
Salmonella Infantis/ Anatum
KS/MO
Alfalfa
109
5
1997
Escherichia coli 0157:H7
Multistate
Alfalfa
85
9, 14
1997-1998
Salmonella Senftenberg
CA/NV
Alfalfa/clover
60
13
1998
Salmonella Havana/Cubana
Multistate
Alfalfa
40
13, 15
1998
Escherichia coli 0157:NM
CA/NV
Clover/alfalfa
8
13
1999
Salmonella Mbandaka
Multistate
Alfalfa
87
9, 16
1999
Salmonella spp.
MI
Alfalfa
34
9
1999
Salmonella Typhimurium
CO, CT
Alfalfa/clover
119
9, 17
1999
Salmonella Saint Paul
CA
Clover
36
9
1999
Salmonella Muenchen
Multistate
Alfalfa
-157
18
2000
Salmonella Enteritidis PT33
Multistate
Mung bean
75
9, 19
2001
Salmonella Kottbus
Multistate
Alfalfa
31
20
2001
Salmonella Enteritidis PT1
HI
Mung bean
26
21
2001
Salmonella Enteritidis PT913
i FL
Mung bean
35
9
2002
Escherichia coli 0157:H7
CA/NV
Alfalfa
5
18
2002
Salmonella Enteritidis
ME
Mung bean
16
22
2003
Salmonella Saint Paul
OR/WA
Alfalfa
8
22
2003
Escherichia coli 0157:NM
CO/WY
Alfalfa
13
22
2003
Escherichia coli 0157:H7
MN
Alfalfa
5
23
2003
Salmonella Chester
OR
Alfalfa
24
22
Salmonella
Bovismorbificans
Multistate
Alfalfa
28
22
Escherichia col
i 0157:NM
GA
Alfalfa
5
22
foodborne illnesses [27]. The first recognized sprout-related outbreak due to E.
coli 0157:H7 occurred in Japan in 1996 and was associated with contaminated
Daikon radish sprouts. To date this is the largest recorded foodborne outbreak
due to contaminated sprouts worldwide with well over 7000 confirmed cases
[28,29]. The first recorded sprout-related outbreak of foodborne illness in the
U.S. due to contamination with E. coli 0157:H7 was in 1997 [14]. Contam-
inated sprout seed is thought to be the primary source of the pathogens
responsible for most sprout-related outbreaks of foodborne illness [4,5]. This
conclusion is based on direct isolation of pathogens from seed of implicated
lots and/or epidemiological evidence.
190 Microbiology of Fruits and Vegetables
Several studies have indicated that salmonella and E. coli 0157:H7 present
initially on artificially as well as naturally contaminated seed have the potential
to increase up to 10,000-fold on sprouts propagated at 20 to 30°C. The majority
of growth of salmonella and E. coli 0157:H7 on sprouting seed occurs during the
first 48 hours. For sprouts grown from artificially inoculated seed, maximum
populations of salmonella and E. coli 0157:H7 ranging from 5 to 8 log 10 colony-
forming units (CFU)/g have been reported [30-39]. The maximum pathogen
population obtained was not dependent on the initial inoculum level present on
the seed [36]. For comparison, populations of total aerobes reported for sprouts
typically range from 7 to 9 log 10 CFU/g [30,40-42]. For salmonella on alfalfa,
the doubling time was estimated at 47 minutes during the initial rapid growth phase
and growth was not dependent on pathogen serovar, isolation source, or virulence
[33]. Populations of salmonella and E. coli 0157:H7 were stable from 48 hours
to harvest at 3 to 5 days and then declined only slightly during subsequent storage
of contaminated alfalfa sprouts at 5 to 9°C for 6 to 10 days [33,34,37]. Populations
of B. cereus on sprouts grown from naturally contaminated alfalfa and mung
bean seed reached approximately 4 logio CFU/g [43]. The maximum pathogen
populations attained during germination and growth of naturally contaminated
seed under commercial practice may be several logio units less than that for
artificially inoculated seed [44]. Maximum populations of salmonella attained on
alfalfa sprouts grown from two different lots of naturally contaminated seed were
only 2 to 4 logio MPN/g for salmonella. The reduced growth may be due to several
factors. The first is the much lower overall contamination levels on naturally
contaminated seed when compared to even the lowest initial pathogen popu-
lations utilized for laboratory studies. Second, pathogen populations on naturally
contaminated seed may contain a higher percentage of injured cells. Third, differ-
ing methods of irrigation and increased irrigation frequency employed in
commercial operations may affect the final pathogen populations attained.
Interestingly, salmonella serovars attach more tightly to surfaces of alfalfa sprouts
than do strains of E. coli 0157:H7 and the difference in strength of attachment
was proposed to explain, at least in part, the greater number of outbreaks of
foodborne illness associated with contaminated sprouts due to salmonella [39].
Studies in several independent laboratories have indicated that bacterial
human pathogens can be internalized in sprouts. By use of immunofluores-
cence and scanning immunoelectron microscopy, E. coli 0157:H7 was located
in stomata and the vascular system of radish sprouts grown from inoculated
seed [45]. Bioluminescent Salmonella Montevideo and various salmonella
serovars expressing the autofluorescent green-fluorescent protein were also
located in the internal tissues of mung bean and alfalfa sprouts, respectively,
after inoculation of seed or roots [38,45,46]. The mode of entry of bacterial
human pathogens into plants remains unknown, but it is likely due to passive
uptake at the site of injury where lateral roots emerge [46,48], as salmonella
and E. coli 0157:H7 have not been reported to excrete cell-wall-degrading
enzymes (e.g., pectinases or cellulases) that might facilitate active entry.
Pathogens may form biofilms on sprout surfaces and/or become part of
biofilms produced by native microorganisms [49,50] (Figure 8.1).
Interventions to Ensure the Microbial Safety of Sprouts
191
FIGURE 8.1 Biofilm consisting of native bacteria on the surface of an alfalfa sprout
hypocotyl.
8.3 INTERVENTIONS: SEEDS
8.3.1 Chemical and Physical
Sanitizing sprout seed presents a unique challenge in the arena of produce
safety in that even a low residual pathogen population remaining on contami-
nated seed after treatment appears capable of growing to very high levels (up to
8 logio CFU/g) due to favorable conditions of moisture, relative humidity,
temperature, and nutrient availability during seed germination and subsequent
sprout growth [51,52]. In addition, after a sanitizing procedure seed germina-
tion as well as sprout yield and quality need to be maintained at commercially
acceptable levels. In 1999, based on research available at the time, the FDA
published guidance documents recommending that commercial sprout growers
treat sprout seed with one or more antimicrobial treatments such as 20,000
ppm of Ca(OCl) 2 that have been approved for reduction of pathogens on seeds
or sprouts, with at least one approved antimicrobial treatment applied
immediately before sprouting [53]. Also, in 2000 the FDA and the California
Department of Health Services, Food and Drug Branch jointly released a food
safety training video [54] for use by commercial sprout growers. The video,
based on the FDA guidance documents, contains a recommendation to treat
sprout seed with 20,000 ppm available chlorine from Ca(OCl) 2 for 15 minutes
(continuous mixing) with potable water rinses both before and after seed
treatment. Since sprout seed is considered a raw agricultural product, chemical
seed treatments are subject to approval by the U.S. Environmental Protection
Agency and not the FDA.
192 Microbiology of Fruits and Vegetables
Population reductions reported after treatment of alfalfa seed artificially
inoculated with salmonella or E. coli 0157:H7 using 16,000 to 20,000 ppm of
available chlorine has varied considerably among different laboratories, but
usually are in the range of 2 to 4 log 10 (Table 8.2). Lesser reductions were
achieved after treatments with lower amounts of chlorine. A number of factors
likely contribute to the variability in results. Such factors include the percen-
tage of treated inoculated seed with broken, cracked, or wrinkled seed coats
[81], differences in the initial pathogen population on the seed, the extent of
mixing of sanitizer during treatment, the initial organic load on the seed, and
the use of rinse steps before and after seed treatment. Some studies have been
done with relatively low initial pathogen populations on the seed allowing
for maximum population reductions of 2 to 3 log 10 . One consistent finding
among the various laboratories is that the two pathogens when artificially
inoculated onto sprout seed are not eliminated even by treatment with 16,000
to 20,000 ppm of available chlorine for 10 to 15 minutes.
The findings for similar studies with naturally contaminated seed are not
consistent among laboratories (see below).
Investigations of recent foodborne outbreaks of salmonellosis due to
contaminated sprouts indicates that treatment of sprout seed with high levels
of chlorine by commercial growers reduces, but may not always eliminate,
the risk of human illness [16-18,20]. The inability of seed treatments with high
levels of chlorine to always ensure a pathogen-free seed under commercial
practice may be due to several factors including the use of differing protocols
for administering seed treatments at grower locations. Also, the particular seed
treated, if naturally contaminated, may differ in the level of contamination
present and the location of the pathogens on the contaminated seed (e.g., deep
in cracks, crevices, and/or natural openings) (Figure 8.2). The ability of bac-
terial human pathogens to be internalized in seed under natural conditions
in the field is not known, but seeds in general can harbor internalized native
bacteria [82]. If present in internal tissues of the seed, pathogens may escape
contact with chemical sanitizers.
Numerous chemical treatments in addition to chlorine as well as several
physical treatments have been tested individually or in combination for elimi-
nating pathogens from artificially inoculated sprout seed. To date there are few
reports of stand alone chemical or physical interventions capable of eliminating
pathogens from artificially inoculated sprout seed or consistently achieving the
recommended 5 log 10 reductions [4] without significant adverse affects on seed
germination and/or sprout yield (Table 8.2). Most of the interventions included
in Table 8.2 have been tested using more than the single set of conditions listed.
Additional chemicals tested in the references cited, but not included in Table 8.2,
are aqueous acetic acid, calcinated calcium, carvacrol, cinnamic aldehyde, citric
acid, Citricidal® (NutriTeam, Inc., Reston, VT), CitroBio™ (= Pangermex)
(CitroBio, Inc., Sarasota, FL), Environne Fruit and Vegetable Wash™
(Consumer Health Research, Inc., Brandon, OR), ethanol, eugenol, linalool,
methyl jasmonate, sodium carbonate, sodium hypochlorite, thymol, trans-
anethole, trisodium phosphate, Tsunami 200® (Ecolab, Mendota Heights,
TABLE 8.2
Chemical and Physical Interventions for Reducing Pathogens on Inoculated Sprouting Seeds
=3
rD
— i
<
13
Treatment
Acetic acid, vapor
Acetic acid, vapor
Acetic acid, vapor
Acetic acid, vapor
Acidic EO water
Acidic EO water
Acidic EO water
Allyl isothiocyanate
Ammonia, gas
Ammonia, gas
Ammonia, gas
Ammonia, gas
Ca(OH) 2
Ca(OH) 2
Ca(OCl) 2
Ca(OCl) 2
Ca(OCl) 2
Ca(OCl) 2
Ca(OCl) 2
Ca(OCl) 2
Chlorine dioxide,
acidified
Conditions
242 ul/1 air, 45° C
242 ul/1 air, 45° C
242 ul/1 air, 45° C
300 mg/1 air, 50° C
1081 mV, 84 ppm chlorine
1150 mV, 50 ppm chlorine
1079 mV, 70 ppm chlorine
50 ul/950 cm 3 jar, 47°C
300 mg/1
300 mg/1
300 mg/1
300 mg/1
1%
1%
20,000 ppm
20,000 ppm
18,000 ppm
18,000 ppm
16,000 ppm
16,000 ppm
500 ppm
Log reduction
— <
in
Time
Seed type
Bacterium
(CFU/g)
Seed germination
Ref.
o
m
12 h
Mung bean
Salmonella
>5, no survivors
No effect
55
en
C
12 h
Mung bean
E. coliO\51:Hl
>6, no survivors
No effect
55
- 1
12 h
Mung bean
L. monocytogenes
4.0
No effect
55
24 h
Alfalfa
Salmonella
0.8
No effect
56
2
n'
10 min
Alfalfa
Salmonella
1.5
No effect
57
64 min
Alfalfa
E. coli Ol 57:111
1.6
Significant
58
a -
EL
reduction
15 min
Alfalfa
Salmonella
2.0
No effect
59
LO
24 h
Alfalfa
E. coliO\51:Hl
>2.0, survivors
present
Slight reduction
60
1— (■
22 h
Alfalfa
Salmonella
2.0
No effect
61
o
— 1->
22 h
Mung bean
Salmonella
5.0
No effect
61
"D
22 h
Alfalfa
E. coli Ol 57 :H7
3.0
No effect
61
O
22 h
Mung bean
E. coli Ol 57 :H7
6.0
No effect
61
10 min
Alfalfa
E. coliO\51:Hl
3.2
No effect
62
10 min
Alfalfa
Salmonella
2.8-3.8
No effect
62, 63
3 min
Alfalfa
E. coliO\51:Hl
>2.3, survivors
present
Reduced rate
64
10 min
Alfalfa
Salmonella
2.0
Slight reduction
63
10 min
Alfalfa
Salmonella
3.9
No effect
65
10 min
Alfalfa
E. coli 0157:H7
4.5
No effect
65
10 min
Mung bean
Salmonella
5.0
No effect
66
10 min
Mung bean
E. coliO\51:Hl
3.9
No effect
66
10 min
Alfalfa
E. coliOl51:Ul
>2.4, survivors
present
Significant
reduction
64
-A
(continued)
TABLE 8.2
Continued
Treatment
Citrex™
Citrex™
Dry heat
Dry heat
Fit™
Fit™
H 2 2
H 2 2
Hydrostatic pressure
Hydrostatic pressure
Lactic acid
Radiation, gamma
Radiation, gamma
Radiation, gamma
Radiation, gamma
Sodium chlorite,
acidified
Sulfuric acid
Ozone, aqueous
Ozone, aqueous
Pulsed UV light
Dielectric heating,
radio frequency
Supercritical C0 2
Water, hot
Water, hot
Water, hot
Conditions
20,000 ppm
20,000 ppm
50°C
70°C
According to label
According to label
8%
8%
300 mPa
300 mPa
5%, 42°C
Various
Various
Various
Various
1200 ppm, 55°C
2N
21 ppm, w/sparging
21.3 ppm, w/sparging
5.6 J/cm , 270 pulses
39 MHz, 1.6kV/cm
4000 psi, 50°C
3-stage: 25 to 50 to 85°C
54° C
80°C
Time
10
mm
10
min
60
min
3h
15
min
15
min
3 min
10
min
15
min
15
min
10
min
3 min
20 min
64 min
20 min
90 sec
26 sec
60 min
30 min,
9 sec, 9 sec
5 min
2 min
Seed type
Alfalfa
Alfalfa
Alfalfa
Alfalfa
Alfalfa
Alfalfa
Alfalfa
Alfalfa
Garden cress
Garden cress
Alfalfa
Alfalfa
Alfalfa
Broccoli
Broccoli
Alfalfa
Alfalfa
Alfalfa
Alfalfa
Alfalfa
Alfalfa
Alfalfa
Alfalfa
Alfalfa
Mung bean
Bacterium
Salmonella
E. coliO\51:Hl
E. coliO\51:Wl
Salmonella
Salmonella
E. coliO\51:Hl
E. coliO\51:Hl
Salmonella
Salmonella
Shigella flexneri
E. coliO\51:W
Salmonella
E. coliO\51:Hl
Salmonella
E. coliO\57:Hl
E. coliO\57:Hl
E. coli Ol 57 :H7
E. coliO\51:Hl
L. monocytogenes
E. coliO\51:Hl
Salmonella
E. coli, generic
E. coli, generic
Salmonella
Salmonella
Log reduction
(CFU/g)
3.6
3.4
1.7
3.0
2.3
>5.4
>2.9, survivors
present
3.2
5.8
4.5
3.0
D-value of 0.97 kGy
D-value of 0.60 kGy
D-value of 1.10 kGy
D-value of 1.11 kGy
> 1.9, survivors
present
5.0
2.2
1.5
4.9
1.7
1.0
>4, no survivors
2.5
>6
Seed germination Ref.
No effect 67
No effect 67
No effect 68
Slight reduction 56
No effect 69
No effect 69
No effect 64
No effect 63
Reduced rate 70
Reduced rate 70
No effect 52
Dosage dependent 71
Dosage dependent 71
Dosage dependent 72
Dosage dependent
Slight reduction
No effect
No effect
No effect
Significant
reduction
No effect 77
No effect 78
No effect 79
No effect 34
No effect 80
72
n
o
64
D"
o
o
73
era
74
o
— k
75
-n
—t
76
=3
Q_
<
era
fD
r+
ex
rt)
Interventions to Ensure the Microbial Safety of Sprouts 195
FIGURE 8.2 Scanning electron micrograph of alfalfa seeds showing extensive cracking
of a seed coat and natural openings: C, crack in the seed coat; H, hilum; M, micropyle.
MN), Tween 80, Vegi-clean™ (Microcide, Inc., Detroit, MI), and Vortex®
(Ecolab). Treating with aqueous chemicals at elevated temperatures can lead
to greater reductions of pathogen populations on seed, but is often detrimental
to seed germination [83]. Addition of high levels of the surfactant Tween 80
(1%, w/v) to 1% Ca(OH)2 led to only an additional 1 logio reduction or less
in the population of salmonella on alfalfa seed [62,63]. Sonication of seed
during treatment with aqueous antimicrobial compounds also did not have a
significant effect, only slightly increasing the log 10 kill obtained [68,83].
Treatment with gaseous acetic acid was reported to eliminate both salmo-
nella and E. coli 0157:H7, but not Listeria monocytogenes, from artificially
inoculated mung bean seed without reducing seed germination [55]. Similar
treatments of inoculated alfalfa seed led to either unacceptable reductions of
seed germination [84] or were not effective [56]. Hot water treatments of alfalfa
seed inoculated with generic E. coli were reported to eliminate the bacterium
[79], but results both with alfalfa seed artificially inoculated with human
pathogens as well as naturally contaminated seed have not been as promising
due to lowered effectiveness and/or detrimental effects on seed germination
[34,85]. Under commercial practice, the ability of hot water treatments to
ensure consistent elimination of bacterial human pathogens from alfalfa seed
was put into question by a recent multistate outbreak of salmonellosis due to
contaminated alfalfa sprouts grown from seed treated with hot water followed
by a soak in low levels (2000 ppm) of chlorine [20]. However, a recent labo-
ratory study indicates that treatment of mung bean seed with hot water may be
an effective seed-sanitizing step. Treatment of seed inoculated with salmonella
at 55°C for 20 minutes, 60°C for 10 minutes, or 70°C for 5 minutes led to
an approximate 5 logio reduction [80]. Treating seed at 80°C for 2 minutes
was even more effective resulting in an over 6 log 10 reduction. None of
196 Microbiology of Fruits and Vegetables
these temperature/time treatments led to a decrease in germination of the
treated seed.
There have been a very limited number of studies on seed sanitization using
naturally contaminated rather than artificially inoculated seed and these studies
have evaluated the efficacy of hot water and chlorine treatments only [44,65,
85]. The use of naturally contaminated seed rather than artificially inoculated
seed may give a more accurate prediction of the efficacy of seed treatments
for eliminating bacterial human pathogens in commercial practice. This may
be due to differences in bacterial populations per gram of seed (normally much
lower on naturally contaminated seed than on artificially contaminated seed
used for laboratory studies), possible differences in the location and physio-
logical status of the pathogens and the potential presence of pathogens in
biofilms. In contrast to studies with artificially inoculated seed treated with
high levels of chlorine, research conducted independently in two laboratories
using alfalfa seed lots naturally contaminated with salmonella indicated that
treatment with chlorine (unbuffered and buffered to neutral pH, from 2,000 to
20,000 ppm) completely eliminated the pathogen [65,85]. However, a third
laboratory published contrasting results using 20,000 ppm of unbuffered active
chlorine also using seed naturally contaminated with salmonella [44]. The
reasons for the differing results between laboratories may include differences in
the degree of mixing during seed treatment as well as differences in the popu-
lation and location of the pathogen on the particular naturally contaminated
seed tested even if originating from the same seed lot.
Several physical treatments have also been tested for sanitizing sprout seed
(Table 8.2). In 2000 the FDA approved exposure of sprout seed to ionizing
radiation at doses up to 8 kGy [86]. Treatment with ionizing radiation can
significantly reduce bacterial pathogens on sprout seed. Exposure of inoculated
alfalfa seed to a 2 kGy dose of gamma irradiation led to a 3.3 and 2.0 log 10
reduction in E. coli 0157:H7 and salmonella populations, respectively, while
still maintaining commercially acceptable yields as well as nutritive values of
sprouts grown from the treated seed [71,87,88]. Higher dosages led to
unacceptable reductions in yields. For alfalfa seed naturally contaminated
with salmonella and treated with gamma radiation, Thayer et al. [89] reported
a Z)-value of 0.81 kGy. An absorbed dose of 4 kGy was required to eliminate
the pathogen, a dosage that results in significant reductions in yield. A required
dosage of 4 kGy for pathogen elimination along with a Z)-value of 0.81 kGy
indicates that individual naturally contaminated seeds may harbor pathogen
populations in excess of 4 log 10 CFU. Electron beam radiation or use of
so-called soft electrons (low-energy electron beam, energies <300 kV) may also
be useful for reducing pathogen populations on the surface of seed [90], but
both have lowered penetration ability compared to gamma radiation.
Various treatment combinations (hurdle concept) for reducing contami-
nants on sprout seed have also been tested. Bari et al. [68] reported that the
combination of dry heat (50°C, 1 hour) followed by treatment with hot acidic
electrolyzed oxidizing (EO) water and sonication was able to reduce popula-
tions of E. coli 0157:H7 on artificially inoculated mung bean seed by 4.6 logio,
Interventions to Ensure the Microbial Safety of Sprouts 197
but the combination treatment was less effective when tested against inoculated
radish and alfalfa seed. Seed germination and subsequent sprout growth were
not adversely affected. In the same study, a dry heat (50°C, 1 hour) seed
treatment in combination with exposure to 2 to 2.5 kGy of gamma radiation
led to the elimination of the pathogen on mung bean, radish, and alfalfa seed,
but resulted in decreases in yield, most significantly for mung bean and radish.
Lang et al. [52] found that successive treatments of alfalfa seed artificially
inoculated with E. coli 0157:H7 with lactic acid and chlorine (2000 ppm) were
slightly more effective than lactic acid treatments alone, but were less effective
than high levels of chlorine (20,000 ppm). Sharma et al. [91] found that treat-
ing alfalfa seed inoculated with E. coli 0157:H7 first with ozone (continuous
sparging in water) followed by a dry heat treatment (60°C, 3 hours) led to a
greater than 4 log 10 reduction of the pathogen population, but survivors were
detected by enrichment. A sequential washing treatment with thyme oil (5 ml/1)
followed by ozonated water (14.3 mg/1) and aqueous C10 2 (25 mg/1) led to a 3.3
log 10 reduction of E. coli 0157:H7 on inoculated alfalfa seed [92].
The large body of research reported subsequent to the release of the
FDA guidance documents [53] indicates that several alternative chemical and
physical treatments may be similar or greater in efficacy to high levels of chlo-
rine for reducing pathogen populations on sprout seed. For sanitizing alfalfa
seed such treatments include seed soaks in 1% Ca(OH) 2 , 1% calcinated cal-
cium, FIT®, 8% H 2 2 , or 2% CITREX™ [62,63,67,69,83,93]. For sanitizing
mung bean seed exposure to gaseous acetic acid or soaking seed in hot water
appear especially promising [55,80]. The efficacy of these alternative chemical
and physical treatments needs to be confirmed by other researchers ideally
using naturally contaminated seed. In contrast to high levels of chlorine,
several of these alternative methods of sanitizing seed may be acceptable for
use by organic growers as well as conventional growers pending any required
regulatory approvals. The cost of some of these alternative methods to the
commercial grower may be prohibitive, however. Cost may not be as much of
an issue for home growers.
8.3.2 Biological
In contrast to the voluminous literature concerning biological control of plant
pathogens [94] as well as numerous studies on the biological control (com-
petitive exclusion) of pathogens in poultry, meat, and dairy products [95,96],
there is little published information on the use of antagonistic microorganisms
to control human pathogens on produce. The ideal biocontrol product for use
on sprout seed and sprouts would contain a nonpathogenic microorganism(s)
that is genetically stable, easily cultured and formulated using low-cost
substrates and materials, has a long shelf life, is easily applied to seeds and/or
sprouts, is highly effective on a variety of sprout types and against several
human pathogens, and is affordable for the grower. For control of pathogens
in poultry, meat, and dairy products, single microbial strains or defined or
undefined consortia of microbes have been tested as antagonists.
198 Microbiology of Fruits and Vegetables
Most of the studies on biological control of bacterial human pathogens on
produce have examined the use of lactic acid bacteria (LAB) as antagonists
[95]. LAB are attractive candidates for commercial biological control agents
due to their common occurrence on sprout surfaces [41,97], their ability to
produce multiple antimicrobial agents including bacteriocins, hydrogen
peroxide, and organic acids in vitro, their extensive use in the food industry
for fermentation, and their lack of known pathogenicity [95]. A strain of
Lactococcus lactis inhibitory in vitro against Listeria monocytogenes due to acid
production was tested for control of the pathogen when the two bacteria were
co-inoculated onto alfalfa seed before sprouting [98]. Results indicated that the
strain was much less inhibitory towards the pathogen in situ than in vitro,
reducing pathogen populations on the sprouts by only 1 logio. In a second
study on LAB, Wilderdyke et al. [99] found that of 58 isolates of LAB isolated
from alfalfa seeds and sprouts, 32 were inhibitory towards the three pathogens
salmonella, E. coli 0157:H7, and Listeria monocytogenes in agar spot tests. One
strain of Lactococcus lactis subsp. lactis was particularly inhibitory towards all
three pathogens on agar media and in broth culture. The same group reported
a significant reduction in populations of Listeria monocytogenes on alfalfa
sprouts after application of a strain of LAB in the seed soak solution [100].
A commercial product containing a lactic acid bacterium is available in Japan
for controlling E. coli 0157:H7 on Daikon radish sprouts [101]. This product
is to be sprayed onto seeds and sprouts several times during the sprouting
process. In our laboratory, we have tested hundreds of plant-associated bac-
teria, primarily isolated from sprout surfaces, for their ability to inhibit growth
of salmonella inoculated to alfalfa seed in small-scale laboratory bioassays
[102]. Of these, a few isolates (none are LAB) have been identified that consis-
tently reduce growth by several log 10 units in small-scale laboratory bioassays.
Currently, the effectiveness of these antagonists is being evaluated in larger
scale experiments and studies on their mode of action are also underway.
More than a single antagonist may be required for controlling pathogens
on germinating seeds of various sprout types due to compositional differences
in the native microflora [103]. Treating artificially contaminated alfalfa seed
with a novel purified bacteriocin, colicin HU194, led to reductions ranging
from 3 logio CFU/g to complete elimination of E. coli 0157:H7. Efficacy was
dependent on the particular strain of E. coli 0157:H7 used for seed inoculation
[104]. Bacteriophages are also being researched as a possible antimicrobial
intervention for application to sprout seed [105]. Biological control agents may
also be useful for reducing spoilage caused by soft-rotting bacteria [106].
8.4 INTERVENTIONS: SPROUTS
A variety of antimicrobial chemicals have been tested as additives to sprout
irrigation water for the purpose of preventing or reducing the growth of
native microflora and bacterial human pathogens. A study in our laboratory
indicated that addition of H 2 2 , Tsunami®, acidified NaC10 2 , Aquatize™
(Bioxy, Raleigh, NC), EDTA, NaP0 4 , and NaOCl at varying concentrations
Interventions to Ensure the Microbial Safety of Sprouts 199
to the irrigation water did not reduce the populations of the native micro-
flora on alfalfa sprouts grown in a commercial-scale tray system by more than
approximately 1 logio without evidence of phytotoxicity [107]. Piernas and
Guiraud [108] reported that spray irrigation of tray-grown rice sprouts
with chlorinated water (100 mg/1) every 6 hours was not effective in reducing
populations of total aerobic bacteria, B. cereus, or L. innocua. Daily spraying
of alfalfa sprouts grown from artificially inoculated seed with chlorine (100 mg/1)
led to reduction of less than 2 logio in the population of salmonella at day
4 of sprouting [51]. Daily irrigation with C10 2 (100 mg/1) did not reduce the
population of total aerobic bacteria on alfalfa sprouts grown in trays, but did
reduce populations of V. cholera up to 2 log 10 when sprouts were grown from
seed inoculated with the pathogen [32]. A reduction of 4 logio f° r total con-
forms was obtained for mung bean sprouts that were subject to irrigation with
0.2 ppm gaseous ozone and 0.3 to 0.5 mg/1 of ozonated water at days 4 to 7 of
sprouting [105]. Rinsing of inoculated alfalfa seed growing in plastic jars with
aqueous C10 2 (25 mg/1) or ozonated water (9.27 mg/1) after 48 or 72 hours of
sprouting was ineffective in reducing populations of E. coll 0157:H7 [92].
However, rinsing with thyme oil (5.0 mg/1) alone or in sequence with C10 2 and
ozonated water led to reductions of up to 2 logio in pathogen populations when
carried out at 24 and 48 hours into the sprouting process. None of the rinsing
treatments were effective at 72 hours, however. Rinsing with water was
ineffective at all time points. Taormina and Beuchat [110] tested a variety of
aqueous antimicrobial chemicals as spray treatments for reducing or elimi-
nating E. coll 0157:H7 from the surface of growing alfalfa sprouts. None of the
chemicals were effective for reducing pathogen populations and only acidified
NaOCl 2 (1200 ppm) controlled the growth of the pathogen. A complication of
addition of antibacterial compounds to the irrigation water is that any patho-
gens present in the spent irrigation water may be killed, but viable pathogen
populations may remain on the sprouts rendering the testing of spent irrigation
water for viable pathogens meaningless [4].
Several postharvest treatments for reducing the populations of native
microbes and pathogens have been examined. Water rinses are not highly
effective in reducing microbes on sprouts with resultant population reductions
of 1 logio or l ess [31,39,110,111]. A 2-minute treatment with aqueous ozone
(23 ppm) did not reduce the population of aerobic microorganisms on alfalfa
sprouts [75]. Dipping in hot water (60°C) for 30 seconds led to a reduction
of 2 logio m the population of total microbes on soybean sprouts [112] and
a similar treatment for 5 minutes led to a reduction of 5 logio in aerobic
plate counts on rice sprouts [113]. Blanching in hot water (90°C, 1 minute) was
reported to reduce microbial counts by 5 logio units for mung bean sprouts
[111]. Rinsing of mung bean sprouts in 1 and 2% lactic or acetic acid reduced
the native microflora by less than 2 logio [HI]- Treatment of rice sprouts with
chlorine (100 mg/1) for up to 10 minutes decreased aerobic plate counts by only
1.5 logio [HI]- Treatment (10 minutes) of inoculated mung bean sprouts
with chlorous acid (HC10 2 ; 268 ppm), NaOCl (200 ppm), or lactic acid (2%)
resulted in a maximum reduction of 1 logio f° r total aerobes [114].
200 Microbiology of Fruits and Vegetables
Blanching in hot water (100°C, 30 seconds) did not eliminate E. coli
0157:H7 from alfalfa sprouts [115]. Treatment (10 minutes) of alfalfa sprouts
with EO water (84 mg/1 of available chlorine) in conjunction with sonication
led to a reduction of 1.5 log 10 in the population of salmonella [116]. Treatment
(64 minutes) with EO water (50 mg/1 of available chlorine) resulted in a
reduction of 3 log 10 of E. coli 0157:H7 on alfalfa sprouts without any reported
changes in appearance [117]. Aqueous ozone treatments (maximum concen-
tration of 20 to 23 ppm, treatment time of 20 to 64 minutes) of alfalfa sprouts
led to a maximum population reduction of approximately 1 to 2 log 10 for
L. monocytogenes and E. coli 0157:H7, respectively [75,118]. The greatest log
reductions reported for a postharvest aqueous chemical treatment were for
HCIO2. Treatment (10 minutes) of inoculated mung bean sprouts with HC10 2
(268 ppm) resulted in a reduction of approximately 5 log 10 of salmonella
and L. monocytogenes. Lactic acid (2%) was also tested in this study, but was
less effective [1 14]. Exposure of inoculated alfalfa sprouts to gaseous acetic acid
or allyl isothiocyanate vapor led to significant reductions in the population
of salmonella, but also led to undesirable changes in sensory quality [56].
Most likely the only postharvest treatment able to inactivate pathogens
that have been internalized into sprouts during the growing process is irradi-
ation. A postharvest treatment with gamma radiation at 2 kGy extended the
shelf life of alfalfa and broccoli sprouts by 10 days due to significant decreases
in the native microflora [72,88]. Doses up to 2.6 kGy did not significantly
change the appearance or nutrient quality of alfalfa sprouts [118]. Salmonella
was eliminated from alfalfa sprouts grown from naturally contaminated seed
when exposed to gamma radiation at a minimum dose of 0.5 kGy [120].
Irradiation of inoculated alfalfa sprouts with 3.3 kGy of beta radiation
(electron beam) eliminated L. monocytogenes without an adverse effect on
quality [121].
8.5 REDUCING THE RISK OF FUTURE OUTBREAKS
Several steps can be taken to minimize the risk of future sprout-related
outbreaks of foodborne illness including the use of good agricultural practices
(GAPs) during the production of sprouting seed as detailed in several recent
government, university, and produce organization publications [122-124].
Sprout seed is obtained from plants grown in the open field and thus subject to
potential contamination by nonpotable irrigation water, manure, domestic and
wild animals, birds, farm machinery, and farm workers. To the author's
knowledge, there are no fields in the U.S. or elsewhere designated solely for the
production of seed destined for use by sprout growers. Settings on harvesting
machinery should be such as to minimize damage to the seed. Cross contami-
nation between clean and contaminated lots of harvested seed can occur in
seed cleaning (conditioning) facilities and also when lots of seed are mixed
before packaging and distribution. Several salmonella serovars were detected
in the waste streams of a seed-cleaning machine in a U.S. alfalfa seed-cleaning
facility indicating the presence of salmonella in the local alfalfa fields where
Interventions to Ensure the Microbial Safety of Sprouts 201
the seed originated [125]. Seed-cleaning machines should be thoroughly
cleaned and sanitized before and between lots of seed destined for sprouting.
Seed scarification has been used historically to increase the germinability of
seed lots that contain a significant amount of hard seed. Scarification involves
the mechanical abrasion of the seed coat to allow for entry of water facilitating
germination. Damage to the seed coat may make elimination of bacterial
pathogens by treatment with chemical sanitizers more difficult [62,81] and
probably should be avoided if possible. There is also the potential for contami-
nation during transit and storage of seed as well as during seed germination,
growth, and harvest.
Commercial sprout growers need to follow good manufacturing practices
(GMPs) and have written standard sanitation operating procedures (SSOPs)
and a hazard analysis and critical control point (HACCP) plan in place [126].
Growers should be thoroughly familiar with the recommendations contained
in the FDA guidance documents which include detailed methods for testing of
spent irrigation water for salmonella and E. coli 0157:H7 [53]. Seed should be
of high quality and all bags of seed should be inspected for evidence of rodent
activity (gnawed holes and presence of urine stains using a blacklight).
Thorough testing of all lots of sprout seed for bacterial pathogens is desirable
and should reduce the risk of sprout-related outbreaks of foodborne disease.
A sampling and testing protocol for use with sprout seed for human pathogens
has been proposed [127]. However, due to the sporadic and low level of
contamination with human pathogens often encountered, a negative sample
test cannot guarantee that the entire lot is pathogen free. Thus, an effective,
approved seed-sanitizing step should be applied by the grower, and the spent
irrigation water or sprouts should be tested for the presence of pathogens.
Irrigation water needs to be of high quality and the use of well water also
requires regular testing for adequate levels of residual chlorine. Postharvest
contamination of sprouts can occur during transit, storage, display, and by
cross contamination in restaurant or home kitchens and adequate precautions
need to be taken.
8.6 RESEARCH NEEDS
Despite considerable research efforts towards the development of sprout seed-
sanitizing methods there is still a need for highly effective, low-cost, easily
implemented, and environmentally benign seed-sanitizing strategies that can be
used by organic and conventional sprout growers. The use of 20,000 ppm
Ca(OCl) 2 presents worker and environmental safety concerns, may not always
be effective in eliminating human pathogens from contaminated seed lots
under commercial practice, and can be highly detrimental to the germination
capacity of some seed types [65]. The potential for internalization of bacterial
human pathogens into sprouts during germination and growth from conta-
minated sprouting seed has been demonstrated, but the location of pathogens
on naturally contaminated seed is still not known. Are the pathogens solely
surfaceborne, sometimes entering into cracks and natural openings such as
202 Microbiology of Fruits and Vegetables
the hilum and micropyle in the seed coat, or are they also present internally
in the seed coat as are some seedborne plant pathogenic bacteria [128]? The
optimization and commercialization of biological control agents for use on
sprouting seed as an alternative to chemical sanitizers is highly desirable.
The ecology of human pathogens on sprouts is not well defined and several
questions remain unanswered. Are pathogens capable of forming biofilms on
sprout surfaces or can they become part of biofilms formed by the native
microflora making their eradication more problematic? What microbial cell
surface components (e.g., curli, fimbriae, flagella, and extracellular poly-
saccharides such as colanic acid and cellulose) are important for the initial
attachment to plant surfaces and subsequent biofilm formation? Does the plant
react in any way to the presence of pathogens on surfaces or in internal tissues?
Could sprout seed cultivars be developed that release high levels of antibac-
terial compounds upon germination that might inhibit growth and survival of
pathogens?
Further research in the areas mentioned above should assist in the
development of improved strategies for reducing the risk of future foodborne
outbreaks allowing for greater consumer confidence in the microbiological
safety of sprouts and ensuring the survival of a strong sprout industry
worldwide. Intervention strategies developed for seeds and sprouts may also be
applicable to ensuring the microbiological safety of other types of produce.
ACKNOWLEDGMENTS
Sincere thanks are due to Drs. Francisco Diez-Gonzalez and Mindy Brashears
for supplying articles in press, to Drs. Jeff Farrar and Glenn Henderson for
supplying information concerning sprout-related outbreaks, and to Drs. Jeff
Farrar, James Smith, and Mary Lou Tortorello for critically reviewing the
manuscript before publication.
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9
Microbiological Safety
of Fresh Citrus and
Apple Juices
Susan ne E. Keller and Arthur J. Miller
CONTENTS
9. 1 Introduction 211
9.2 Juice Production 212
9.3 Physicochemical Properties and Endogenous Juice Microflora 213
9.3.1 Citrus Juice 213
9.3.2 Apple Cider 214
9.4 Pathogens Associated with Fresh Juice and Their
Environmental Sources 216
9.4.1 Enterohemorrhagic Escherichia coli 216
9.4.2 Salmonella Species 217
9.4.3 Cryptosporidium parvum 218
9.4.4 Listeria monocytogenes 218
9.5 Juice HACCP Rule 219
9.6 Importance of SSOPs to HACCP 219
9.7 Application of the 5-Log Standard 221
9.8 Intervention Treatments 222
9.9 Other Juice HACCP Considerations 223
9.10 Labeling 224
9.1 1 Conclusion 224
References 224
9.1 INTRODUCTION
Historically, citrus juices and apple juice or cider were not considered to be
beverages associated with a high risk for causing foodborne illness. These
products were not typically thought of as being exposed to pathogens that were
animal derived, such as salmonella. Secondly, the pH and organic acid content
of these foods was presumed to be too adverse for the survival or growth of
bacterial foodborne pathogens. Nonetheless, incidents of foodborne illness
associated with citrus juice and apple cider occurred as far back as 1922 [1].
211
212 Microbiology of Fruits and Vegetables
Documented evidence of pathogen survival in juice has also existed for some
time, along with proposed mechanisms for acid resistance [2-12]. Therefore,
survival of foodborne pathogens, and the occurrence of serious foodborne
illness outbreaks, including fatalities, have led to new regulation requiring the
implementation of hazard analysis critical control point (HACCP) programs
by juice manufacturers [13]. The regulation requires implementation of a
process capable of reducing the pertinent pathogen by 100,000-fold (5 log
units).
This chapter briefly describes production of citrus and apple juices, their
physical characteristics, and typical microflora. The emphasis is on pathogens
that have been associated with fresh juice and on recent regulations related to
the prevention of foodborne illness outbreaks. Sources of contamination and
intervention methods are also discussed.
9.2 JUICE PRODUCTION
The U.S. Food and Drug Administration (FDA) defines juice as "the aqueous
liquid expressed or extracted from one or more fruits or vegetables, purees of
the edible portions of one or more fruits and vegetables, or any concentrates of
such liquid or puree" [13]. Produce production is beyond the scope of this
chapter, but it is critical to recognize that fruit and vegetables used for juice
manufacture should be produced, harvested, and transported using good
agricultural practices (GAPs). Only high-quality produce should be used. Juice
processing begins with the reception of the raw produce at the processing
facility. Raw produce is inspected and culled according to established good
manufacturing practices (GMPs). Removal of defective raw material is critical
to the production of a quality juice product. This is discussed in greater detail
in Chapter 16. Sound raw produce is then cleaned and sanitized prior to
extraction or maceration. For some products, such as apples and oranges, a
mechanical means of washing may be employed, such as a brusher-washer.
Such methods can efficiently remove soil and extraneous materials, but may be
too harsh for more fragile fruits, such as berries. Sanitizing follows cleaning,
which generally results in some reduction of microbial load at the surface of the
fruit. Both cleaning and sanitizing are described in more detail in Chapter 17.
After appropriate culling, cleaning, and sanitizing, most noncitrus produce
is macerated. Generally, produce is mechanically conveyed to size reduction
equipment such as a hammer mill, crusher, or a grater for processing into a
mash or pulp-like material from which the juice may be extracted. Rice hull
may be added to the mash to improve juice yield during extraction. Any such
added ingredients must also be approved and used according to established
GMPs. Following maceration, some product types, such as tomatoes or grapes,
may be given a mild heat treatment to set color, inactivate enzymes, and/or
improve yield. In general, this treatment is not an effective means to reduce
microbial load. Extraction of the juice follows mild heat treatment, if it is
employed.
Microbiological Safety of Fresh Citrus and Apple Juices 213
The most common juice extraction method from a mash or pulp is batch
hydraulic pressing. The whole or chopped raw fruit or vegetable is placed into
bags that are stacked alternately with plastic separator grid interleaves and
then subjected to hydraulic pressure. Pulpers, with tapered screws or paddles,
that squeeze juice and puree through a cylindrical screen, while carrying the
pomace to one end for discharge, are also common.
Juices may be marketed in both clarified and unclarified styles. For clarified
juices, additional processing aids, such as approved pectinolytic enzymes, are
added to facilitate removal of particles and cloud.
For citrus products an entirely different extraction procedure is commonly
employed by large-scale manufacturers. Citrus fruits are generally not
macerated; rather, the juice is extracted while largely maintaining peel integrity.
This results in limited contact between the juice and the peel. Two types of
equipment are in common use: a mechanical reamer and a pin-point extractor
[14]. Mechanical reamers first cut the fruit in half. The halves are then held
against rotating burrs to extract the juice and pulp. A pin-point extractor
contains a small hollow tube that punctures the peel at one point. Then,
intermeshing mechanical fingers squeeze the fruit surface to force the juice and
pulp out through the hollow extraction tube. Seeds and pulp are separated from
the juice using cylindrical pulpers and finishers.
9.3 PHYSICOCHEMICAL PROPERTIES AND
ENDOGENOUS JUICE MICROFLORA
9.3.1 Citrus Juice
Citrus are nonclimacteric fruits that are allowed to mature on the tree, since
postharvest maturation will not occur. The indices of fruit maturity include
°Brix, acid content, and the Brix/acid ratio. Major U.S. citrus producing
regions, such as California, Florida, and Texas, all have legal maturity stan-
dards. Typically, oranges range from 7 to 14, grapefruit from 10 to 12, and
tangerines from 16 to 17 °Brix [15]. Citric acid is the major acid present in
citrus fruit. At maturity, concentrations of total acid for oranges, grapefruit,
and tangerines are 0.5 to 1.5%, 1.0 to 2.0%, and 0.6 to 2.3%, respectively. The
U.S. Department of Agriculture (USDA) has published standards for grades
of orange juice. Brix standards for pasteurized juice are 11° for grade A and
10.5° for grade B. An acid concentration or pH standard is not established.
However, Brix/acid minimum and maximum ratios are given for grade A and
grade B juice. The typical pH range for most citrus juice is from 3.0 to 4.0 and
cannot be legally altered by added acidulants [16].
Typical aerobic microbial load on citrus fruit is approximately 4.0 log
CFU/cm [17-19]. Yeast and mold populations measured alone seem to show
greater variability than total aerobic populations, but have been reported as
nearly as high as total aerobic microbial load. In citrus juices, acidic condi-
tions, coupled with higher sugar content, result in a microbiological popula-
tion made up primarily of acidolactic bacteria, yeasts, and molds. Lactic acid
214 Microbiology of Fruits and Vegetables
bacteria are reported to be the major spoilage organisms [16,20,21]. Popula-
tions found in fresh citrus juices are reduced compared to populations found
on fruit when appropriate sanitation and extraction methods are used [22].
9.3.2 Apple Cider
Apples are climacteric fruit, with respiration increasing as the fruit matures.
The ripening process will continue postharvest. Indices of maturation are
variety specific; they include hardness, °Brix, and/or color. The composition of
apple juice is dependent on fruit variety. A study by Mattick and Moyer
examined 15 varieties of apple from 8 geographic locations over 3 years [23].
Although 15 varieties were examined, only composite data were reported.
Composite results over this 3-year period showed a mean °Brix of 12.74, with
minimum and maximum values of 9.8 and 16.9, respectively. The mean esti-
mate of composite pH for this same period was 3.69, with a range of 3.23 to
6.54. Total acid calculated as % malic acid was 0.42% with minimum and
maximum values of 0.15 and 0.91%, respectively. Malic acid represents >85%
of the acid present in apples [24]. In an earlier study by Goverd et al., pH values
and acid levels (% malic) were listed for six different apple varieties [25]. Acid
content and pH values varied from 0.17 % with a pH of 4.03 for variety Sweet
Coppin to 1.43 % with a pH of 2.92 for variety Bramley's Seedling. Brix was
not reported.
In a study initiated by the FDA in 1998, seven apple varieties were exam-
ined for pH, % acid, and °Brix prior to fresh juice/cider manufacture [26]. Brix,
pH, and % titratable acidity (TA, calculated as malic acid) averages for fresh
juice/cider made from both fresh and stored apples that were harvested from
trees and from the ground are shown in Table 9.1. All three parameters were
significantly influenced by apple variety (p < 0.0001).
TABLE 9.1
Influence of Apple Variety on pH, °Brix, and % TA Content of Fresh Apple
Juice/Cider
Apple variety
pH a
Brix a
% Acid (malic)
Fuji
3.88 ±0.06
16.8±1.2
0.40 ±0.08
Gala
3.94 ±0.09
14.6±0.4
0.26 ±0.04
Golden Delicious
3.71 ±0.10
13.0±0.4
0.35 ±0.04
Granny Smith
3.46 ±0.05
13.1 ±0.9
0.57 ±0.06
Mcintosh
3.48 ±0.05
12.0±0.7
0.55 ±0.07
Red Delicious
4.06 ±0.09
13.6±0.9
0.20 ±0.03
Red Rome
3.58±0.10
13.4±1.0
0.43 ±0.06
a Values are means and standard deviations of all juices made from fresh and stored, tree- and
ground-harvested apples during the 1999 harvest season in northern California.
Adapted from Keller, S.E., Chirtel, S.J., Merker, R.I., Taylor, K.T., Tan, H.L., and Miller, A.J.,
/. Food Prot., 67, 2240, 2004.
Microbiological Safety of Fresh Citrus and Apple Juices 215
TABLE 9.2
Influence of Apple Variety on the Total Aerobic Microbial Populations Found
in Fresh Apple Juice/Cider
Aerobic microflora in fresh
cider/juice from tree-harvested
unsorted fruit
Apple variety
Total aerobic plate Yeast and mold
count (log CFU/ml) a count (log CFU/ml) a
Fuji 3.93±0.36 3.66±0.13
Gala 2.65±0.04 3.31 ±0.69
Golden Delicious 3.57 ± 0.57 3.27 ± 0.56
Granny Smith 3.32 ± 0.56 2.87 ± 0.66
Mcintosh 2.47 ±0.52 1.97 ±0.83
Red Delicious 3.77±0.60 3.16±0.40
Red Rome 3.85±0.51 3.88±0.13
a Values are means ± standard deviation of 6 replicate composite samples.
Adapted from Keller, S.E., Chirtel, S.J., Merker, R.I., Taylor, K.T., Tan, H.L., and Miller, A.J.,
J. Food Prot., 67, 2240, 2004.
Although cider/juice physiochemical parameters such as °Brix, pH, and %
TA are influenced by variety, there is large overlap in the range of values
obtained for each apple variety. In addition, the range of any given parameter
can be large. For example, the pH of cider/juice from over 32 batches of Fuji
apples averaged 4.02 ±0.30 (FDA, unreported data) [26]. However, individual
batches from Fuji apples ranged from a low pH of 3.80 to a high pH of
4.65. Damaged and dropped fruit also have higher pH values. In a study by
Dingman, fresh, undamaged tree-picked variety Red Delicious apples had a
mean pH value of 3.98 ±0.05, whereas bruised tree-picked, undamaged
dropped fruit, and bruised dropped fruit had mean pH values of 4.57 ±0.11,
4.15 ±0.07, and 4.90 ±0.09, respectively [27]. The range of physiochemical
parameters such as pH of different apple varieties, as well as the influence of
unsound fruit can be significant when compared to minimum levels required
for growth of many foodborne pathogens.
Natural microflora found in fresh apple juice/cider also varies with apple
variety and is significantly influenced by pH, % TA, and Brix (Table 9.2) [26].
Tree-harvested fruit that were culled to remove damaged fruit had total aerobic
microbial populations in juice that ranged from 1.90 to 3.40 log CFU/ml.
Yeast and mold populations in the same juice/cider ranged from 1.99 to 3.32
log CFU/ml. Total aerobic microbial and yeast and mold populations were
also measured in poorer quality, ground harvest fruit. For this group, juice/
cider microbial populations were substantially higher, ranging from 4.19 to
5.43 log CFU/ml for total aerobic populations and from 3.84 to 5.23 log CFU/
ml for yeast and mold populations. Other studies reported similar population
216 Microbiology of Fruits and Vegetables
density ranges, particularly when ground harvested apples were included in
cider production [28-30].
The types of organisms normally associated with fresh apple juice/cider
are typically aciduric microorganisms, due to the pH, acid, and sugar content
normally associated with this product. As with orange juice, human pathogenic
microorganisms such as salmonella and enterohemorrhagic Escherichia coli
are not considered endogenous microflora of the fruit or juice. Rather they are
environmental contaminants originating from animal sources. However, unlike
fresh citrus juice, populations of microorganisms in fresh apple juice/cider are
generally higher than populations found on apples [28,29,31,32].
9.4 PATHOGENS ASSOCIATED WITH FRESH JUICE
AND THEIR ENVIRONMENTAL SOURCES
9.4.1 Enterohemorrhagic Escherichia coli
Escherichia coli is one of the most studied bacteria. It is part of the normal
bacterial flora resident in the intestines of many animals, including humans,
and is commonly used as a nonpathogenic indicator of recent fecal contami-
nation and of fecally associated pathogenic organisms such as salmonella
[33]. However, numerous strains of E. coli exist which are not commensal.
Pathogenic E. coli produces toxins of various types and toxicities that cause
various diseases. These toxins have been described previously [34]. Diar-
rheagenic E. coli are subdivided into six classes based on the symptoms they
produce and virulence factors they possess [35]. Of these groups, the
enterohemorrhagic (EHEC) class is of most concern, due to its low infectious
dose and its association with hemorrhagic colitis (HC), hemolytic uremic
syndrome (HUS), and thrombotic thrombocytopenic purpura (TTP). HUS
occurs primarily in children under 10 years of age and has a mortality of 3
to 5% [35]. Children's susceptibility to HUS led the FDA to issue a warning
in November 2001 to the public concerning the health risk of consuming
untreated juices by children [36].
Although there are several serotypes of EHEC known, the most common
serotype, particularly in the U.S., Canada, Great Britain, and parts of Europe,
is E. coli 0157:H7 [35]. In the years from 1998 to 2000 the Center for Disease
Control (CDC) recorded 86 outbreaks attributed to E. coli. Of these, 68 were
identified as outbreaks caused by E. coli 0157:H7 [37]. The great majority of
these outbreaks were either from meat products or had an unknown source.
Despite the fact that the majority of EHEC outbreaks are not associated with
fresh fruit or juice made from fresh fruit, outbreaks associated with fresh fruit
or fresh juice are of concern, since these products are associated with a healthy
lifestyle and are generally consumed raw.
EHEC strains of E. coli are not normal endogenous microflora of fresh
juice or of the fruit used to produce fresh juice. Their presence on fruit and
in fruit juice is believed to be the consequence of some form of fecal con-
tamination prior to consumption. Cattle have been implicated as a major
Microbiological Safety of Fresh Citrus and Apple Juices 217
reservoir of this organism [38-41]. Wild animals such as deer may be an
additional source of the organism [42]. Wild birds have also been implicated as
vectors for contamination, particularly those living near landfills [43].
Presumably, birds become infected at landfills and then may carry infection
to farm fields and/or cattle. In addition to birds, transfer of E. coli 0157:H7 by
fruit flies has been demonstrated [44].
From epidemiological data it is clear that E. coli 0157:H7 can survive well
enough in low pH juice to result in serious illness. Of particular note was the
Western states outbreak during October 1996 from contaminated apple cider
that resulted in 66 cases of illness and one death [45,46]. Although the pH of
most apple and orange juice is low enough to either significantly slow or inhibit
growth of E. coli, EHEC strains have tolerance to high levels of acid allowing
for extended survival time [3,47]. Tolerance to high acid levels is a complex
induced response involving three distinct mechanisms and is enhanced in
stationary phase cells [3,7,8,47].
9.4.2 Salmonella Species
There are over 2000 serotypes of the genus salmonella that cause human
disease [48]. According to the CDC, there are an estimated 1.4 million cases
annually, with an estimated 500 fatalities [48]. Approximately half of all cases
are caused by serotypes Enteritidis or Typhimurium [48].
Salmonella infections are more commonly associated with animal-derived
foods, such as meat, seafood, dairy, and egg products, rather than juices.
However, outbreaks associated with fresh juice have occurred as far back as
1922 [1]. Early outbreaks resulting in typhoid fever were associated with poor
hygiene by asymptomatic S. Typhi shedding food handlers. As disinfection of
water, sanitation procedures, and hygiene practices have improved, outbreaks
of typhoid fever have become far less common in developed countries. Nonethe-
less, given the dramatic increase of fresh fruit imported from developing
countries, typhoid fever outbreaks associated with these commodities remain a
concern [49]. More recent outbreaks of nontyphoidal salmonellosis in fresh
juice have been attributed to fecal-associated contamination of fruit or poor
processing practices [50-52].
Both E. coli 0157:H7 and salmonella are tolerant to extreme acid environ-
ments. As with E. coli 0157:H7, tolerance in salmonella is inducible and
increases when cells have been adapted either to acid conditions or are in
stationary phase [4,7]. For S. Typhimurium, two major acid tolerance sys-
tems were identified, one associated with log phase and one associated with
stationary phase [2]. Not surprisingly, survival in juice for extended periods has
been observed. Goverd et al. reported survival of S. Typhimurium in apple
cider [25]. Survival in juice above pH 3.6 held at 22°C was reported as greater
than 30 days. Survival was decreased by lower pH and lower temperature.
Survival in orange juice by various salmonella serovars was studied by Parish
et al. [53]. Salmonella serovars Gaminara, Hartford, Rubislaw, and Typhi-
murium were inoculated at log 6CFU/ml into orange juice at pH 3.5, 3.8, 4.1,
218 Microbiology of Fruits and Vegetables
and 4.4. Survival (to below levels of detection) at pH 3.5 ranged from a low
of 14.3 ±0.9 days for S. Typhimurium to a high of 26.7 ±4.0 days for
S. Hartford.
9.4.3 Cryptosporidium parvum
Cryptosporidium parvum is a highly infectious protozoan parasite causing
persistent diarrhea. Common reservoirs are ruminants including cattle, deer,
and sheep [54,55]. Infection with Cryptosporidium does not always result in
severe disease symptoms and the organism is far more dangerous for the
immunocompromised [56]. Cryptosporidium is more commonly associated with
contaminated water. The largest waterborne outbreak in U.S. history occurred
in Milwaukee, WI, in 1993 and affected an estimated 403,000 people [56].
Cryptosporidium cannot replicate in the environment; however, the oocysts are
thick-walled, resistant to chlorine, and persist in the environment. Presumably,
the thick wall also confers some acid resistance, as outbreaks of cryptospor-
idiosis have also occurred from fresh-pressed cider [54,55]. Apple cider-
associated outbreaks were reported in 1993, 1996, and 2003.
9.4.4 Listeria monocytogenes
Although not implicated in a foodborne outbreak associated with fresh juices,
other foodborne pathogens do exist that also exhibit a tolerance/resistance to
high levels of acid. Given an opportunity to contaminate fresh juice, these acid-
resistant organisms could result in foodborne outbreaks. Chief among these
possible pathogens is Listeria monocytogenes. L. monocytogenes is ubiquitous
within the environment, carried by animals, and frequently found on fruits and
vegetables [57-59].
The minimum pH for growth of L. monocytogenes is dependant on the
acidulant. For malic acid, the primary acid found in apple cider/juice, the
lowest pH value for growth of L. monocytogenes is from 4.4 to 4.6 depending
on the strain [60]. Although this pH is somewhat higher than typical fresh
apple cider/juice, some apple cider/juice may fall within a range that will allow
L. monocytogenes growth, particularly if unsound fruit is used in production.
Not all apple cider/juice may have a pH low enough to prohibit growth of
L. monocytogenes. In addition, although L. monocytogenes may not grow at
lower pH values, survival at lower pH similar to E. coli 0157:H7 and
salmonella is possible [60,61]. L. monocytogenes has been isolated from
unpasteurized apple juice [62]. The recently completed L. monocytogenes risk
assessment indicated that consumption of fresh fruit has a low risk for
listeriosis [63]. However, two risk factors need to be considered concerning
juice-associated listeriosis. First, comingling fruit to make juice or cider spreads
the risk over a much larger exposed population, when compared to a single or
limited serving size typically associated with the fruit itself. Second, fresh juice
is frequently consumed by subpopulations at risk for listeriosis, e.g., children
and adults with compromised immune systems. Consequently, it is reasonable
Microbiological Safety of Fresh Citrus and Apple Juices 219
to consider as somewhat likely outbreaks or sporadic cases of listeriosis
associated with fresh juice.
9.5 JUICE HACCP RULE
In response to a series of juice-associated outbreaks, the FDA published its
final rule on January 19, 2001 requiring the application of HACCP to juice pro-
duction [13]. The rule became effective January 22, 2002 for large businesses.
For small and very small businesses effective dates were January 21, 2003 and
January 20, 2004, respectively. As part of the rule, the FDA issued a
performance standard that requires all juice receive a treatment wherein the
pertinent pathogen is reduced in concentration by 100,000-fold (5-log units).
The *'5-log reduction standard" was established based on recommendations
by the National Advisory Committee on Microbiological Criteria for Foods
(NACMCF). NACMCF considered worst-case scenarios, such as might occur
if apples were contaminated directly with bovine feces. The committee included
a 100-fold safety factor in their recommendation for a 5-log reduction pro-
cess to ensure the safety of juice. The FDA also considered regulatory
precedence when setting the 5-log pathogen reduction performance standard.
This same standard is also required for E. coli 0157:H7 reduction in fer-
mented sausage, and FDA has advised that a 5-log process for salmonella
should be used for in-shell pasteurization of eggs [13].
9.6 IMPORTANCE OF SSOPs TO HACCP
HACCP, as applied to juice production, requires that sanitation standard
operating procedures (SSOPs) be developed and consistently applied. For juice
manufacture the SSOP must address eight points: (1) safety of the water that
comes into contact with the product; (2) conditions and cleanliness of food
contact surfaces; (3) prevention of cross contamination; (4) maintenance of
hand washing, sanitizing, and toilet facilities; (5) protection of food, food
contact surfaces, and packaging material from adulteration; (6) proper labeling,
storage, and use of any toxic compounds; (7) control of employee health that
could result in microbial contamination of the food; and (8) exclusion of pests
from the food processing facilities [13].
The juice HACCP rule sparked considerable public comment, especially the
requirement of HACCP for juice processing in lieu of better enforcement of
GMPs and SSOPs. The FDA conducted a survey of cider production facilities
in 1997 [30]. In that survey, 67% of firms had good sanitation, 27% were
marginal, and only 4% were categorized as poor. Microbiological trends
indicated that the cider plant GMPs and SSOPs implemented during processing
did not substantially reduce the level of microbiological contamination between
incoming raw ingredients and finished juice.
Senkel et al. followed the performance of 1 1 Maryland cider producers
before and after HACCP training [29]. Although a significant decrease in
220 Microbiology of Fruits and Vegetables
E. coli positive juice was reported, not all cider processed was negative for
E. coli after the implementation of more stringent processing control. The
efficacy of GMPs and SSOPs in controlling contamination in an experimental
commercial cider plant was examined by Keller et al. [32]. As expected, the lack
of appropriate sanitation controls resulted in significant increases in E. coli
K-12 in juice, when inoculated apples were used for cider processing. However,
re-implementation of appropriate GMPs and SSOPs during cider manufac-
ture failed to yield reduced E. coli levels in juice to below detectable limits,
suggesting that GMPs and SSOPs alone are incapable of ensuring safety of
fresh cider. In addition, considerable cross contamination was observed
between batches of cider when intentionally contaminated and uncontami-
nated apple batches were alternately processed. Cross contamination occurred
even under stringent application of SSOPs. The key findings of this study were
as follows: (1) preharvest prevention of contamination of apples is essential;
(2) if contaminated apples enter the processing environment, juice safety will
be compromised both in the contaminated and subsequent juice batches;
(3) SSOPs and GMPs are critical to the maintenance of a sanitary establish-
ment, but are insufficient controls to reduce hazard levels in juice once a
contaminated batch has been processed; (4) for apple cider production, a
terminal 5-log treatment is essential to ensure product safety. In addition, any
process needs to be validated and verified through a HACCP program.
Processing apple juice/cider is a considerably different process than is used
for most citrus products. During apple juice/cider processing, the whole fruit
including the skin is macerated and juice expressed with pressure applied on
the fruit mash. For citrus processing, juice is commonly extracted using an
automated extractor that separates juice from the fruit peel, seeds, and large
pieces of pulp simultaneously [14]. In theory, only a small hole (approximately
1 inch) is cut into the fruit to extract the juice, and the juice has limited
exposure to peel surface. Pao and Davis studied the transfer of microorganisms
from fruit surface to juice during extraction [64]. Both natural and artificially
inoculated microorganisms were followed during this study. Results indicated
that significant transfer could occur (1.7%) for both naturally occurring
and artificially inoculated organisms. Several different strains of E. coli were
included among the artificially inoculated fruit. Significant cross contamina-
tion was also observed by Martinez-Gonzales et al. during the preparation of
fresh orange juice [65]. Initial counts for inoculated orange surfaces were
2.3 log CFU/cm 2 for S. Typhimurium, 3.6 log CFU/cm 2 for E. coli 0157:H7,
and 4.4 log CFU/cm for L. monocytogenes. Transfer from the fruit to the juice
during mechanical extraction resulted in 1.0 log CFU/ml of S. Typhimurium,
2.3 log CFU/ml E. coli 0157:H7, and 2.7 log CFU/ml L. monocytogenes in
final orange juice. The authors concluded that strict sanitation programs and
decontamination treatments for fruit might be effective control measures to
prevent cross contamination and to reduce risk of foodborne illness.
In summary, studies with both apple and orange juice clearly show the
importance of SSOPs. For both types of product, transfer of both pathogens
and indigenous microflora into product can be reduced by strict adherence to
Microbiological Safety of Fresh Citrus and Apple Juices 221
GMPs and SSOPs. However, despite reductions in microbial levels, results also
clearly indicate that such procedures will be insufficient to ensure elimination
of all pathogens present. Consequently, HACCP, GMPs, and SSOPs are all
required to ensure juice safety.
9.7 APPLICATION OF THE 5-LOG STANDARD
The 5 log pathogen reduction standard must be applied to the most resistant
pertinent pathogen present on the fruit or in the juice. For most juices, this
requires that the standard be applied after the juice is expressed. The surface
skin of many fruits and vegetables is an imperfect barrier to microorganisms
[66]. Internalization is particularly problematic for fruit such as apples.
Temperature differentials between wash water and fruit clearly exacerbate
pathogen internalization [67]. However, even in undamaged apples without the
assistance of a temperature differential, pathogens have been shown to
internalize through the floral tube and other structures or defects. Surface
interventions such as washing or treating with chlorine or hydrogen peroxide
solutions do not typically result in complete destruction of pathogens [68-73].
Ozone (Table 9.3) was similarly unsuccessful in destroying E. coli 0157:H7
inoculated onto apples.
Surface treatments such as with ethanol or heat can result in greater than
5 log reductions in pathogens when the pathogens are spot inoculated onto
the surface of apples (Table 9.3). However, when apples are inoculated by
immersion, without any temperature differential between the fruit and the
inoculum menstruum, surface treatments fail. Surface heat treatment applied
to apples inoculated by immersion into an inoculum menstruum achieved
TABLE 9.3
Reduction in E. co/i 0157:H7 in Apples Using Different Surface Treatments
and Inoculation Methods
Surface treatment
Length of
Reduction
Inoculation method
method
treatment (min)
(log CFU/ml)
Immersion in
Water immersion at 40° C
1.5
1.10
inoculum menstruum
Inoculum spot dried
Water immersion at 40° C
0.5
0.00
on surface
Immersion in
Water immersion at 95°C
0.5
2.40
inoculum menstruum
Inoculum spot dried
Water immersion at 95°C
0.5
6.20
on surface
Inoculum spot dried
Immersion in ozone
10.0
1.16
on surface
saturated water (1.7—1.1 ppm)
Inoculum spot dried
Immersion in 50% ethanol
5.0
6.49
on surface
222 Microbiology of Fruits and Vegetables
no greater than a 3 log reduction [74]. Penetration of heat into the fruit was
measured during the study. Subsurface fruit temperatures remained signifi-
cantly lower than treatment temperatures even after 60 seconds of exposure.
Consequently the lower level of pathogen reduction achieved using an
immersion method of inoculation implies penetration of the pathogen into
the fruit.
With apples, pathogen internalization into the tissue can occur. Typical
processing conditions used ensure a reasonable likelihood of its occurrence.
Consequently, to ensure that all pathogens in noncitrus fruit juice received an
intervention treatment sufficient to result in the prescribed 5 log reduction, the
FDA ruled that the intervention must occur after the juice is expressed. For
fruit such as citrus, internalization potential or likelihood was not as clearly
defined. In December 1999 the FDA asked NACMCF to consider the potential
for internalization of microorganisms into citrus fruit [75]. Data were
considered that demonstrated the internalization and survival potential of
artificially inoculated foodborne pathogens in seemingly intact oranges [76,77].
NACMCF concluded that although laboratory evidence indicated potential
internalization in sound, intact citrus fruit, there was no demonstrated evidence
that such internalization was likely under current industry practices. As a result
of NACMCF conclusions, the FDA determined that the 5 log pathogen
intervention treatment in citrus juice can be applied prior to extraction, pro-
viding only tree-picked, sound, intact fruits are used. The fruit must be cleaned
and culled prior to the 5 log intervention treatment.
9.8 INTERVENTION TREATMENTS
The juice HACCP rule required a 5 log reduction (100,000-fold decrease) in the
pertinent pathogen, but did not specify the means by which the reduction was
to be achieved. This approach facilitates development and implementation of
treatment alternatives to thermal pasteurization. Many of these alternative
treatments are discussed elsewhere in this book. Since most are still under
development, the primary means of juice pathogen reduction remains thermal
pasteurization.
Thermal pasteurization is a well-studied method of pathogen reduction.
D- and z-values for various pathogens are available in the literature [78-81].
To apply thermal pasteurization to juice, processors must first determine the
pertinent pathogen for their product. In most cases the pertinent pathogen will
be either E. coli 0157:H7, salmonella, or C. parvum. Physicochemical factors
influencing thermal death time in juice include pH, viscosity, particulates, and
Brix. Since actual time and temperature parameters may vary depending on
heating menstrua and the equipment used, each processor should verify their
systems once a process has been validated.
An alternative intervention treatment that is currently in use for apple cider
processing is ultraviolet (UV) light irradiation. UV light irradiation was shown
to destroy pathogens in apple cider [82-86]. The efficacy of UV light treatment
Microbiological Safety of Fresh Citrus and Apple Juices 223
on any liquid is strongly and negatively affected by turbidity and the sizes of
any particles present [87-91]. For this reason, using UV treatment requires
turbulent flow to expose all portions of the juice to the light treatment [92].
It is important to note that the majority of studies examining the efficacy of
UV light irradiation in apple cider have been undertaken using systems that do
not achieve turbulent flow. Although limited efficacy with nonturbulent flow
devices has been demonstrated, these units would not conform to current FDA
regulations, which require turbulent flow. In the application of any processing
technology, juice processors have the responsibility of ensuring that all
appropriate government regulations are met.
Juice manufacturers also have the option of combining treatments to
achieve the 5 log pathogen reduction standard. Comes and Beelman
demonstrated 5 log reductions in populations of E. coli 0157:H7 in apple
cider using a combination of fumaric acid, sodium benzoate, and a 25° C
holding time prior to refrigeration [93]. In an earlier study, Uljas and Ingham
achieved 5 log reduction through a combination of freeze-thaw cycles and
preservatives [94]. Ingham and Schoeller went on to test the acceptability to
consumers of a multistep intervention method capable of a 5 log reduction [95].
In this study, despite treatment similar to their first study, some juices did not
show the expected 5 log reduction. The cause of the failure to achieve a 5 log
reduction in these juices was unknown, indicating more research was necessary
before this system could be commercially applied. In addition, consumers
typically rated the multistep-treated juice lower than untreated juice.
Citrus processors may utilize surface treatments to achieve the required 5
log reduction in the pathogen that is most resistant to the intervention
treatment applied. Interventions applied to the surface of citrus fruit must be
applied after the fruit has been cleaned and culled. Interventions aimed at the
surface of citrus fruits are often similar in type to that applied to the extracted
juice. One of the most effective interventions remains thermal treatment. Pao
et at. demonstrated greater than 5 log reductions in pathogen levels when
oranges were submerged at 80° C for 1 or 2 minutes [96].
When surface interventions, such as a thermal treatment, are used in the
production of citrus juice, additional microbiological process verification is
mandated by 21 CFR 120.25. These requirements specify the number and
volume of juice samples that must be tested for generic E. coli. Testing must
be performed according to established standard methods [13]. A "moving
window" scheme is used for the testing protocol. If two of seven samples tested
are positive for E. coli, then the intervention measures in place are considered
inadequate and corrective measure must be taken.
9.9 OTHER JUICE HACCP CONSIDERATIONS
Although the presence of pathogens is a primary concern in the production of
fresh juice, it may not be the only hazard present. HACCP principles require
that an initial hazard analysis be conducted prior to the establishment of
224 Microbiology of Fruits and Vegetables
controls for any identified hazards. Other hazards may include mycotoxins,
such as patulin, and are discussed elsewhere in this book. Depending on
processing and/or packaging methods employed, physical hazards may also
exist. All of these issues should be addressed when developing a process for the
production of juice.
9.10 LABELING
As a last consideration, the juice processor must be aware that interventions
directed at the whole juice, such as thermal pasteurization or UV irradiation,
will prevent the use of the term "fresh" on the juice label. Only citrus juices
produced using a surface intervention, where the juice is expressed after treat-
ment, may be labeled as "fresh" juice products.
9.11 CONCLUSION
Unpasteurized juices have been part of the American culture since colonial
times. Current trends in food and beverage consumption indicate a growing
interest in foods and beverages with a more healthful image. Fresh foods and
beverages, as well as more foods that are minimally processed, are viewed as
part of a healthy diet. Consequently, Americans are demanding more foods
that are minimally processed. To keep such foods and beverages safe and to
meet consumer needs, processors are being challenged to develop multiple
prevention and intervention systems that begin on the farm or orchard and are
carried through to the point of consumption. Research has demonstrated that
GAPs, GMPs, SSOPs, and HACCP are all essential measures to ensure the
food safety of juices.
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10
Microbiological Safety
Issues of Fresh Melons
Dike O. Ukuku and Gerald M. Sapers
CONTENTS
10.1 Introduction 231
10.2 Microflora of Melons 232
10.2.1 Spoilage Organisms 233
10.2.2 Human Bacterial Pathogens 233
10.3 Factors Contributing to Melon Contamination 234
10.3.1 Preharvest and Harvest Conditions 234
10.3.2 Postharvest Conditions 235
10.3.3 Mode of Microbial Attachment to Melons 236
10.4 Efficacy of Conventional Washing 237
10.4.1 Washing in the Packinghouse 237
10.4.2 Laboratory-Scale Washing Studies 237
10.5 Novel Disinfection Treatments 239
10.5.1 Hydrogen Peroxide 239
10.5.2 Hot Water 240
10.5.3 Steam 241
10.5.4 Other 242
10.6 Issues with Fresh-Cut Melons 242
10.6.1 Transfer of Bacteria from Rind to Flesh 243
10.6.2 Outgrowth on Flesh 243
10.6.3 Suppression of Outgrowth 244
10.7 Methodology for Microbiological Evaluation of Melons 244
10.8 Research Needs 246
References 247
10.1 INTRODUCTION
In the U.S., melons are widely available year round and represent an important
dietary component. In 2001 annual per capita consumption was estimated to be
Mention of trade names or commercial products in this chapter is solely for the purpose of
providing specific information and does not imply recommendation or endorsement by the U.S.
Department of Agriculture.
231
232 Microbiology of Fruits and Vegetables
14.9, 11.2, and 2.1 pounds for watermelon, cantaloupe, and honeydew melons
respectively [1]. The value of these commodities in 2003 was reported to be
$346,022,000, $372,965,000, and $93,241,000, respectively [2]. In recent years
fresh-cut melons have become increasingly popular with consumers and now
account for a large and growing proportion of melon consumption.
For most consumers, melons represent a refreshing and healthy dessert
or snack. However, for a small number of consumers, the situation is quite
different; melon consumption has been a source of foodborne illness. At least
17 melon-related outbreaks involving hundreds of cases have been reported
since 1990 [3-5]. Additional outbreaks ascribed to "multiple fruit" or "fresh-
cut fruit" also may have been due to contamination of an unspecified melon
component. While the largest melon-related outbreaks have been attributed to
various salmonella serotypes, other human pathogens including Escherichia
coli 0157:H7, Campylobacter jejuni, and Norwalk-like virus also have been
implicated [4].
Survival and growth of human pathogens including salmonella, E. coli
0157:H7, and Listeria monocytogenes in melon flesh has been demonstrated
[6-8]. Annous et al. [9] reported growth of S. Poona on cantaloupe rind at 20°C.
Salmonella outbreaks in 2000-2002 were traced by the U.S. Food and
Drug Administration (FDA) to melons imported from Mexico [10]. On-farm
investigations in Mexico conducted by the FDA concluded that "measures
were not in place to minimize microbial contamination in growing, harvesting,
packaging, and cooling of cantaloupe." Detection of L. monocytogenes in cut
melons resulted in a recent product recall [11]. FDA surveys of imported and
domestic produce have documented the presence of salmonella and shigella in
cantaloupe [12,13]. The incidence of salmonella on imported cantaloupe (from
Mexico, Costa Rica, and Guatemala) was 5.3% and on domestic cantaloupes
was 2.6%. Shigella also was detected on these samples, an incidence of 2% on
the imports and 0.9% on domestic melons. On October 28, 2002 the FDA
issued an import alert on cantaloupes from Mexico, halting all such shipments.
Subsequently, export of Mexican cantaloupes to the U.S. by a small number of
grower/packers who met FDA safety criteria was resumed [10].
In this chapter some of the production and postharvest handling conditions
that may contribute to microbial contamination of melons are examined.
Studies of the efficacy of conventional washing practices in reducing the micro-
bial load on melons are reviewed. Finally, current research results pointing to
means of improving the efficacy of melon disinfection are examined.
10.2 MICROFLORA OF MELONS
Melons, especially cantaloupe, present a variety of surfaces to which micro-
organisms may bind. In cantaloupe the epidermal cell surface is ruptured with
a meshwork of raised tissue (the net). This net consists of lenticels and phellum
(cork) cells. These cells have hydrophobic suberized walls to reduce water loss
and protect against pathogen ingress. Also imparting a hydrophobic nature to
Microbiological Safety Issues of Fresh Melons 233
the outer surface of cantaloupe is the cuticle composed of waxes and cutin that
cover the epidermal cells [14].
The ability of pathogenic and spoilage-causing bacteria to adhere to
surfaces of melons represents a food safety problem of great concern as well as
a source of economic loss to the produce and fresh-cut industry. The
mechanism of attachment of bacterial cells to plant surfaces has been studied
most extensively for plant pathogens and symbionts [15,16]. The predominant
class of organisms on cantaloupe and honeydew melon were aerobic
mesophilic bacteria followed by lactic acid bacteria, Gram-negative bacteria,
yeasts and molds, and Pseudomonas spp. [17]. The populations of each of the
categories of microorganisms were found to be higher on cantaloupe than on
honeydew, both for whole and fresh-cut melon. Differences in the populations
of the native microflora on honeydew and cantaloupe melons are most likely
due to the rougher surface of the cantaloupe compared to the relatively smooth
surface of honeydew melon. The extensive raised netting on the surface of
cantaloupe melon no doubt provides more microbial attachments sites and
helps to protect attached microbes from being washed from the surface, and
possibly from environmental stresses such as UV radiation and desiccation.
In unwrapped and wrapped sliced watermelon, Pseudomonas spp., E. coli,
Enter obacter spp., and micrococci comprised the predominant microflora [18].
10.2.1 Spoilage Organisms
The primary causative agents for microbial spoilage of melons are mostly
yeasts and molds and, to a lesser extent, bacteria. Several studies have
demonstrated the presence of enteric bacteria, including Enterobacteriaceae
and Pseudomonadaceae, on whole and fresh-cut melons [17]. Microorganisms
responsible for postharvest diseases are not necessarily dominant on the
surface of sound fruits; they are abundant in the environment and can easily
contaminate the melon surfaces. In a study conducted at the Eastern Regional
Research Center, it was found that the spoilage organisms in fresh-cut melon
were mostly yeasts and molds, Pseudomonas spp., and Erwinia spp. [19]. The
level of these organisms in freshly prepared cut melons was very low but
gradually increased during storage at 5 or 20°C.
10.2.2 Human Bacterial Pathogens
The ability of human bacterial pathogens to attach to melon surfaces [20] and
their virulence characteristics must both be considered. Results of a study
examining attachment of bacteria from a mixed cocktail containing multiple
suspensions of individual strains of each genus (salmonella, E. coli, and
L. monocytogenes) on the surface of cantaloupes stored at 4°C for up to 7 days
showed that salmonella has the strongest attachment to the cantaloupe surface
followed by L. monocytogenes and E. coli, either as individual strains or as a
mixed cocktail [20]. The strength of attachment increased slightly for E. coli
over the 7 days of storage, but decreased for L. monocytogenes. Efficacy of
234 Microbiology of Fruits and Vegetables
sanitizer treatments applied to inoculated cantaloupes at 7 days postinocula-
tion was greatly reduced for L. monocytogenes and E. coli but not for
salmonella. Surface irregularities such as roughness, crevices, and pits have
been shown to increase bacterial adherence by increasing cell attachment and
reducing the ability to remove cells [21].
Salmonella is among the most frequently reported causes of foodborne
outbreaks of gastroenteritis in the U.S. [22]. Salmonellosis has been steadily
increasing as a public health problem in the U.S. since reporting began in 1943
[23]. Five multistate outbreaks of salmonellosis have been associated
epidemiologically with cantaloupes. The first in 1990 involved S. Chester,
which affected 245 individuals (two deaths) in 30 states [22]. The second in
1991 involved more than 400 laboratory-confirmed S. Poona infections
and occurred in 23 states and Canada [22]. A 1997 outbreak associated with
S. Saphra was reported (www.cdc.gov/mmwr/preview/mmwrhtml/mm5146a2.
htm). The most recent outbreaks (2000, 2001, and 2002) were due to S. Poona
[5]. Other melons including watermelon have been associated with outbreaks of
foodborne illness [5,24-26]. The implication of these outbreaks is that
improvements are needed at the farm level to limit or minimize contact of
melons with sources of human pathogens, and at the packinghouse level in
sanitizing and processing conditions.
Other human pathogens including E. coli 0157:H7 and shigella are capable
of growth on melon flesh [6,7]. A 1993 outbreak of foodborne illness was
attributed to cantaloupe contaminated with E. coli 0157:H7 (M. Diermayer,
Oregon Health Division, Portland, OR, personal communication).
1 0.3 FACTORS CONTRIBUTING TO MELON
CONTAMINATION
10.3.1 Preharvest and Harvest Conditions
Relatively little definitive information on sources of human pathogen
contamination of melons is available. The FDA suggested that preharvest
contamination of Mexican melons with human pathogens may have resulted
from use of sewage-contaminated irrigation water [10]. Irrigation water,
transported over long distances and distributed to farms through open and
unprotected aqueducts and channels, may become contaminated by animal or
human activity (Table 10.1). Other potential sources may be from feces of
birds [28,29], reptiles [5], or other wildlife in fields, or exposure to airborne
contamination. The latter scenario was demonstrated by Annous et al. [30] in
studies conducted in an apple orchard in close proximity to a pasture. Animal
production activity was observed by one of the authors within several miles of
melon production locations in California and Mexico. However, the limits of
airborne distribution and survival of human pathogens attached to aerosols
has not been reported. Suslow [31] was unable to recover salmonella from more
than 900 individual field-collected melons produced in different regions of
California during 1999-2001. It may be that contamination events in some
Microbiological Safety Issues of Fresh Melons 235
TABLE 10.1
Potential Sources of Melon Contamination
Preharvest
Direct fecal contamination — human, birds, reptiles, insects, other wildlife
Indirect fecal contamination — irrigation water, dust from animal production
During harvest
Poor worker hygiene
Packing plant
Contaminated process water
Poor plant sanitation
Ineffective washing
Cross contamination during washing
Poor worker hygiene
locations are highly sporadic and localized, e.g., to individual melons with
adhering avian feces or insect damage, a melon defect observed by one of the
authors in a California packing shed. Duffy et al. [32] reported that salmonella
isolates obtained from washed cantaloupes in Texas were most closely related
to isolates obtained from equipment and irrigation water, but DNA finger-
printing did not conclusively establish relationships between contamination
sources. Contamination of melons could occur during harvest if worker
hygiene was deficient [10].
Research is needed to identify specific sources of preharvest contamination
of melons and to develop guidelines and good agricultural practices (GAPs)
that reduce the risk of contamination. Appropriate training of farm workers in
personal hygiene and avoidance of behaviors that result in melon contamina-
tion is essential.
10.3.2 POSTHARVEST CONDITIONS
Gagliardi et al. [33] reported in most cases little change or an increase of
indicator microorganisms (total and fecal coliforms and enterococci) on melons
during washing in samples obtained at packing facilities in the Rio Grande
River Valley of Texas. They attributed contamination to the management
of primary wash tanks or hydrocoolers, e.g., use of contaminated river water,
buildup of soil in tanks, and depletion of chlorine. The contamination of
cantaloupes in Mexico may have been due to cooling and washing with
contaminated water [10]. The potential for such contamination also exists in
the U.S. One of the authors has observed melon processing operations in which
cantaloupes were tightly packed in tanks containing chlorinated water, with
minimal opportunities for agitation of the melons or mixing of the water, prior
to fresh-cut processing. Under such conditions, rapid depletion of chlorine at
the melon surface and survival of attached bacteria on contaminated melons
might be expected with the possibility of cross contamination of other melons
in the tank.
236
Microbiology of Fruits and Vegetables
Other potential sources of postharvest sources of contamination include
poor personal hygiene or work practices by workers (one of the authors
observed the failure of packinghouse employees to wear gloves or hairnets
while handling melons; another worker used his foot to move cantaloupes
down a ramp from a receiving platform to a conveyor) and inadequate plant
sanitation. Accumulation of debris from incoming melons was visible on the
aforementioned ramp and conveyors. Conveyors and processing equipment
must be cleaned and sanitized on a regular schedule with sufficient frequency
so as not to allow debris to accumulate and microbial populations to build up
on food contact surfaces.
Such deficiencies can be addressed by development and implementation
of a hazard analysis critical control point (H ACCP) plan and an effective clean-
ing and sanitation program, and adherence to good manufacturing practices
(GMPs). Of equal importance is employee training in food safety. Such
training should be appropriate to the employee's job and in the employee's
native language.
10.3.3 Mode of Microbial Attachment
to Melons
The external surface of cantaloupe melons is characterized by the presence of a
net comprising porous lenticellar tissue on the epidermis [14]. Such tissue
provides numerous attachment sites for microorganisms and also may shield
attached cells from contact with cleaning or antimicrobial agents (Figure 10.1).
Microbial attachment and the possibility of internalization may occur in
the stem scar region. In contrast, honeydew melon and watermelon have a
smooth surface that should be less favorable for attachment and protection of
A
.
K
r
4
fr
&i*
Jp • '
-l
{r
iiJ {
r i
;\t
?"*•
• <
1
MY
*
FIGURE 1 0.1 Scanning electron microscopy image showing bacteria on cantaloupe rind
surface (A) and in lenticel (B).
Microbiological Safety Issues of Fresh Melons 237
microorganisms. Park and Beuchat [34] reported that greater numbers of E. coli
0157:H7 and salmonella cells were inactivated or detached from inoculated
honeydew melon than from cantaloupe when the melons were washed with
sanitizer solutions. Similarly, the population of aerobic microorganisms on
honeydew melon could be reduced to lower levels than the population on
cantaloupes by washing with 200-2000 ppm chlorine solutions [35]. Similar
results were reported by Ukuku and Fett [17].
10.4 EFFICACY OF CONVENTIONAL WASHING
10.4.1 Washing in the Packinghouse
Field-packed melons are not generally washed because of the difficult logistics
of supplying adequate water to mobile washing equipment. Melons trans-
ported to packing plants may be washed by spraying over rollers in flat-bed
brush washers or by immersion in a wash tank [33]. However, these investi-
gators found little or no reduction and in some cases an elevation in microbial
populations on cantaloupes and honeydew melons washed with commercial
equipment in packing plants in the Rio Grande Valley of Texas. This may have
resulted from contamination of the wash water and/or depletion of chlorine by
reaction with organic material. It also may have been due to the limited
efficacy of brush washers in detaching microbial contaminants from melon
surfaces. Annous et al. [36] demonstrated the inability of a flat-bed brush
washer to reduce the population of E. coli on inoculated apples. In contrast,
Materon [37] reported reductions of 3.2 logs in the populations of aerobic
microorganisms on cantaloupes washed by unspecified means in four
commercial packinghouses, also located in the Rio Grande Valley of Texas.
10.4.2 Laboratory-Scale Washing Studies
Laboratory washing studies in which the melons are fully immersed in a
sanitizing solution with scrubbing or agitation have demonstrated that sig-
nificant reductions in microbial populations can be achieved. Ayhan et al. [35]
reported reductions of 1 and 2 logs for the aerobic plate count on whole
honeydew melons and cantaloupes, respectively, after dipping in 200 ppm
chlorine (as sodium hypochlorite) solutions; reductions exceeding 3 logs were
obtained on cantaloupes dipped in 1000 ppm chlorine. Park and Beuchat
[34] compared 200 or 2000 ppm chlorine, 850 or 1200 ppm acidified sodium
chlorite, 0.2 or 1.0% hydrogen peroxide, and 40 or 80 ppm peroxyacetic acid
(Tsunami™) as sanitizers for cantaloupes inoculated with human pathogens.
Population reductions of E. coli 0157:H7 and salmonella cocktails approached
or exceeded 3 logs for all of these treatments except hydrogen peroxide, which
was less effective. Population reductions of total aerobic microorganisms were
substantially smaller than reductions of human pathogen populations. Similar
results were reported for honeydew melons.
238 Microbiology of Fruits and Vegetables
TABLE 10.2
Effect of Postinoculation Storage at 5°C on Efficacy of Chlorine Wash in
Inactivating Salmonella and Listeria and E. coli on Inoculated Cantaloupes
Survivors (log 10 CFU/cm 2 ) a
Bacteria Days postinoculation
Treatment 11
Control
H 2
Cl 2 OOOOppm)
4.6 ±0.2
4.6±0.1
1.5 ±0.2
4.7±0.1
4.6 ±0.2
2.0±0.1
4.6±0.1
4.6±0.1
2.4 ±0.2
3.6±0.2
3.1 =b 0.1
ND
3.5±0.2
3.3 ±0.2
ND
3.5±0.2
3.3 ±0.2
ND
5.0±0.1
4.5±0.1
0.3±0.1
4.5 ±0.2
4.0±0.1
2.0±0.1
2.0±0.1
2.2 ±0.1
2.2 ±0.1
Salmonella
3
5
L. monocytogenes
3
5
E. coli 25922 e
3
5
Note: ND = not detected by plating.
a Values are means ± standard deviation of three experiments with duplicate determinations per
experiment.
b Treatments applied for 3 min.
c Cocktail of Salmonella spp. containing S. Stanley H0558, S. Poona RM2350, and S. Saphra
97A3312. (Data from Ukuku and Fett [74].)
d Cocktail of L. monocytogenes containing strains Scott A., ATCC 15313, LM-4, and H7778.
(Data from Ukuku and Fett [8].)
e Data from Ukuku et al. [41].
Sapers et al. [38] reported reductions in the aerobic plate count on
cantaloupe surface of less than 1 log when rind plugs were washed by
immersion in lOOOppm chlorine, 1% APL KLEEN 246 (an acidic detergent
formulation supplied by Cerexagri; www.cerexagri.com), or 4% trisodium
phosphate. Immersion of whole cantaloupes freshly inoculated with Salmonella
spp. in 1000 ppm chlorine solution for 5 minutes resulted in population
reductions of 3 logs for salmonella [39,40]; however, the reduction was only 2
logs when the treatment was applied 5 days after inoculation (Table 10.2). With
a nonpathogenic E. coli (ATCC 25922), the reduction was greater than 4 logs
with freshly inoculated melons but less than 1.5 logs when the treatment was
applied 72 hours after inoculation [41]. However, with L. monocytogenes, the
time interval between inoculation and treatment had no effect on treatment
efficacy [8].
Barak et al. [42] obtained a 1 log reduction in the population of Pantoea
agglomerans (a surrogate for S. Poona) on inoculated cantaloupe by immersion
in 150 ppm sodium hypochlorite for 20 seconds, followed by a 2-minute cold
water rinse. In studies with cantaloupes inoculated with E. coli 0157:H7,
Materon [37] reported reductions generally exceeding 5 logs from washing by
immersing the melons for 1 or 10 minutes in solutions containing 200 ppm
Microbiological Safety Issues of Fresh Melons 239
chlorine, 1.5% lactic acid, or 1.5% lactic acid + 1.5% hydrogen peroxide at 25
or 35°C. In view of the efficacy data obtained by other investigators, these
extraordinary results are difficult to explain. It is possible that the recovery of
attached bacteria from the melon surface by rubbing with a sponge was
substantially less efficient than predicted by the investigator's validation
procedure. Alternatively, the presence of residual lactic acid or hydrogen
peroxide in the lowest dilutions plated may have been inhibitory to E. coli
0157:H7 on Petrifilm™.
The FDA advises consumers to wash melons with cool tap water with
scrubbing but without use of soap or detergents immediately before eating.
Consumers are also advised to wash cutting boards, utensils, and counter
tops often using hot soapy water followed by diluted bleach as a sanitizer.
Avoidance of cross contamination with meat, poultry, or fish is essential
(www.fda.gov/bbs/topics/ANSWERS/2002/ANS01 167.html/). Fresh-cut pro-
cessing studies conducted by one of the authors clearly demonstrated the need
to develop and rigorously adhere to a strict protocol for sanitizing knives,
cutting boards, and other food contact surfaces and equipment to avoid cross
contamination and achieve an acceptable product shelf life. Attention to detail
was found to be critical [38].
While the literature on efficacy of washing melons is limited and
contradictory, the overall trend suggests that microbial populations attached
to melon surfaces can be reduced by several logs if sanitizers are applied by
immersion of melons in the solution with scrubbing and/or agitation.
Treatment efficacy may be reduced if the time interval between contamination
and washing is greater than one day, a likely situation with preharvest con-
tamination. Since human pathogens transferred from the rind to the flesh are
capable of growth on the flesh surface, the presence of even small numbers of
survivors following a sanitizing wash represents a significant risk to consumers.
Consequently, there is a great need for better methods of disinfecting melons so
that this risk is minimized.
10.5 NOVEL DISINFECTION TREATMENTS
10.5.1 Hydrogen Peroxide
Hydrogen peroxide is classified as generally recognized as safe (GRAS) for use
in food products [43]. It is used as a bleaching agent, oxidizing and reducing
agent, and antimicrobial agent. The FDA specifies approved food uses of
hydrogen peroxide such as treatment of milk used for cheese, preparation of
modified whey, and production of thermophile-free starch. However, the FDA
requires that the residual hydrogen peroxide be removed by physical or
chemical means during processing. Hydrogen peroxide has not yet been
approved by the FDA for washing fruits and vegetables. Antimicrobial activity
of hydrogen peroxide as a preservative for fruits and vegetables [44], salad
vegetables, berries, and fresh-cut melons [45] has been reported. Also it has
been used to control postharvest decay in table grapes [46]. When used as a
240
Microbiology of Fruits and Vegetables
TABLE 10.3
Population of Salmonella spp. on Cantaloupe Rind and Recovered from Fresh-
Cut Pieces Before or After Washing Treatments and Fresh-Cut Preparation
Salmonella population'
Melon
Treatment
Log CFU/cm 2
Log
Log CFU/g
Log
whole melon
reduction
fresh-cut pieces
reduction
Cantaloupe
Control
4.4±0.1
—
2.1 ±0.1
—
Water
4.3 ±0.2
0.1
2.1 ±0.1
0.0
H 2 2 (2.5%)
1.9 ±0.0
2.5
0.4±0.1
1.7
H 2 2 (5%)
2.1 ±0.1
2.3
0.3±0.1
1.8
Honeydew
Control
3.1 =b0.1
1.3 ±0.1
—
Water
2.7 ±0.2
0.4
1.2±0.1
0.1
H 2 2 (2.5%)
ND
-3.0
ND
-1.3
H 2 2 (5%)
ND
-3.0
ND
-1.3
Note: Cocktail of Salmonella spp. containing S. Stanley H0558, S. Poona RM2350, and S.
Newport HI 275 in the inoculum. Melons were completely submerged in bacterial inoculum
(-20°C) for 10 min. ND = not detected by plating.
a Values are mean ± standard deviation of duplicate determinations from three experiments.
From Ukuku D.O., Int. J. Food Microbiol., 95, 137, 2004.
sanitizer for whole melon surfaces at a concentration in the range 2.5 to 5%
H 2 2 , there were significant (p < 0.05) reductions in the populations of
inoculated E. coli and indigenous microflora [41] and approximately 2.3 to 2.6
and 3.0 log CFU/cm reductions of salmonella on cantaloupe and honeydew
melon, respectively (Table 10.3) [40,47]. Treatment of cantaloupes with 5%
hydrogen peroxide at 70°C for 1 minute resulted in a 5.0 log reduction of total
mesophilic aerobes, a 3 log reduction of yeasts and molds, and a 3.8 log
reduction of inoculated salmonella [48]. When the initial level of salmonella on
the melons was 1.9 log CFU/cm , no survivors were detected after treatment
with 5% hydrogen peroxide at 70°C, even with enrichment. However, when the
initial population on melon surfaces was at 3.5 log CFU/cm , the treated
samples were negative for salmonella by plating but were positive upon
enrichment.
10.5.2 Hot Water
Hot water decontamination of whole cantaloupes designated for fresh-cut
processing was found to have major advantages over the use of sanitizers,
including a significant reduction of microbiological populations on melon
surfaces [48]. The major advantage was that it reduced the probability of
potential transfer of pathogenic bacteria from the rind to the interior tissue
during cutting. In experiments carried out in our laboratory, treatment of
cantaloupes, inoculated with S. Poona, with hot water for 1 minute resulted in
a 2.1 log reduction at 70°C and a 3.6 log reduction at 97°C (Table 10.4) [48].
Microbiological Safety Issues of Fresh Melons
241
TABLE 10.4
Inactivation of £. coli ATCC 25922 and S. Poona on Inoculated Cantaloupe
by Surface Pasteurization With Hot Water for 2 min and Reduction of
Transfer to Fresh-Cut Flesh
Experiment
A
B
Target
organism
Treatment
Surviving population
On melons
Time
Temp.
On fresh-cut
(min)
(°C)
(log 10 CFU/cm 2 )
(log 10 CFU/g)
E. coli
2
Control 11
76
86
97
4.0 ±0.6
0.6 ±0.6
ND
ND
—
S. Poona
1
Control
4.7 ±0.1
2.9±0.1
70
2.6±0.1
0.7±0.1
97
1.1 ±0.2
_b
Note: ND = Not detected by plating.
a Untreated.
b
Detectable by enrichment.
Experiment A data from Pilizota, V. and Sapers, G.M., Unpublished data, 2000; Experiment B
data from Ukuku, D.O., Pilizota, V., and Sapers, G.M., /. Food Prot., 67, 432, 2004.
Surviving S. Poona could not be detected by plating on fresh-cut pieces
prepared from cantaloupes treated at 97° C but could be detected after
enrichment, evidence that a small number of survivors were transferred during
fresh-cut preparation. When the initial level of salmonella on the melons was
1.9 log CFU/cm , no survivors were detected after this treatment, even with
enrichment, but with an initial population of 3.5 log CFU/cm", the treated
samples were negative for salmonella by plating but were positive upon
enrichment. Similar reductions in the population of salmonella occurred when
treatments were applied to cantaloupes stored at 5°C for 5 days as for 3 days.
In experiments with E. coli, the efficacy of hot water treatments at lower
temperatures was compared with that at 96°C (Table 10.4). Surviving E. coli
could be detected on inoculated cantaloupe by plating following treatment at
76°C; no survivors were detected at 86 or 97° C. These hot water treatments,
which approach population reductions of 4 log CFU/cm , represent a
substantial improvement over chlorinated water (1000 ppm) or hydrogen
peroxide at ~20°C which yielded reductions of only 2 to 3.0 logs.
Additional information concerning hot water treatment of melons can be
found in Chapter 21.
10.5.3 Steam
The use of steam to treat fruits is somewhat difficult to control due to time
and exact temperature needed to maintain the desired texture. The applica-
tion of steam on whole cantaloupe surface for reduction of microbial
242 Microbiology of Fruits and Vegetables
population would be appropriate since melon has a thick rind that may protect
the interior flesh from deleterious effects of the steam. In a preliminary study
in our laboratory, steam pasteurization of melon surface was not promising
compared to hot water treatment. The inability of the steam to reduce
effectively total microbial populations on whole melon surfaces can be
attributed to the surface roughness where the netting, cracks, and possible
openings due to detached trichome can provide protection to the attached
organisms.
10.5.4 Other
The application of an effective antibacterial agent to the surface of whole
melons may be desirable. There are several reports that nisin, used in
combination with a chelating agent, exhibits a bactericidal effect towards both
Gram-positive and Gram-negative bacteria [49-53]. Treatment of whole and
fresh-cut cantaloupe and honeydew melon with nisin-EDTA significantly
reduced the natural microflora and extended the shelf life [17]. We also found
that sodium lactate was inhibitory to the native microflora on melons [19]. The
antimicrobial activity of lactic acid is due both to a lowering of pH and to
disruption of the outer membrane of Gram-negative bacteria [54]. Application
of lactic acid (2%) as an antimicrobial spray applied to animal carcasses
to reduce surface populations of E. coli 0157:H7 and salmonella has been
reported [55]. Sorbic acid (pK a of 4.76) and its potassium salt are widely used
in foods at a concentration of 0.02 to 0.3% to inhibit yeasts and molds,
but they also have antibacterial activity [56]. However, washing inoculated
whole melons with sodium lactate (2%), potassium sorbate (0.02%), EDTA
(0.2 M), or nisin (50 /xg/ml), when tested individually, did not cause signi-
ficant (p > 0.05) reductions in salmonella populations. Treatment of whole
cantaloupe with nisin-EDTA may lead to both increased shelf life and a
reduced risk of foodborne illness due to contamination with salmonella or
other pathogens [17].
10.6 ISSUES WITH FRESH-CUT MELONS
The visual symptoms of deterioration of fresh-cut produce are flaccidity due to
loss of water, changes in color resulting from oxidative browning at the cut
surfaces, and microbial contamination [57]. Minimally processed fresh fruits
and vegetables provide a good substrate for microbial growth [58,59].
Such substrate may allow proliferation of human pathogenic organisms like
salmonella, L. monocytogenes, and enterotoxigenic E. coli that contaminate
food when proper sanitation is not employed. Microbial spoilage of fresh-cut
melons will depend on storage conditions and the initial microbial population
of the melon. Honeydew melon generally has a lower initial microbial popula-
tion than cantaloupe and also has been found to have a longer refriger-
ated shelf life [17,47]. Similar results were reported for minimally processed
honeydew and cantaloupe melon stored at 4°C, and the authors concluded
Microbiological Safety Issues of Fresh Melons 243
that both the length of shelf life and type of spoilage were related to the type
of fruit [60].
10.6.1 Transfer of Bacteria from Rind to Flesh
Fresh-cut pieces prepared from whole cantaloupe or honeydew melons showed
the presence of mesophilic aerobic bacteria, Gram-negative bacteria, lactic acid
bacteria, Pseudomonas spp., and yeasts and molds [17,47]. The predominant
categories of microorganisms on fresh-cut cantaloupe immediately after fresh-
cut preparation from unwashed whole melons were mesophilic aerobic bacteria
and lactic acid bacteria. For fresh-cut honeydew, mesophilic aerobic bacteria
predominated immediately after fresh-cut preparation. As days of refrigerated
storage increased, other categories of microbes were detected in all samples,
irrespective of initial treatment before fresh-cut preparation. The fact that
the same categories of microorganisms were detected on fresh-cut pieces
during storage as on the whole melon surface indicates that the microbes were
transferred from the rind to the flesh during fresh-cut preparation. Transfer
occurred during cutting and removal of melon rinds.
Salmonella inoculated on whole melon surfaces was recovered in fresh-cut
pieces prepared from inoculated melons [39]. Similarly, Ukuku and Fett [8]
reported survival and transfer of L. monocytogenes population from whole
cantaloupe to fresh-cut pieces. The population on fresh-cut pieces also survived
and increased during storage at an abusive temperature.
Ukuku et al. [48] reported that fresh-cut pieces prepared from cantaloupes
inoculated with initial salmonella populations of 1.9, 3.5, or 4.6 log and treated
with 97° C water or 5% hydrogen peroxide at 70° C were negative for
salmonella by dilution plating, although positive by enrichment (Table 10.4).
However, the populations of salmonella and all classes of native microflora
in fresh-cut pieces prepared from sanitized melons were low compared to
populations in fresh-cut pieces from untreated whole melon.
10.6.2 Outgrowth on Flesh
Populations of all groups of native microorganisms increased in fresh-cut
samples as storage time increased, regardless of the treatment. The population
of salmonella transferred from the untreated melons to the flesh during cutting
averaged 2 log CFU/g for cantaloupe and 1.3 log CFU/g for honeydew. The
population of salmonella on fresh-cut cantaloupe inoculated with 2.56 log
CFU/g increased as storage time increased, especially at an abusive tempera-
ture [19,39] (Figure 10.1). Golden et al. [6] reported growth of salmonella
inoculated directly onto fresh-cut cantaloupe, watermelon, and honeydew
melons during storage at 23°C. Ukuku and Sapers [39] reported growth of
S. Stanley on fresh-cut cantaloupe during storage at 8 and 20° C. Other
investigators have reported that interior watermelon tissues support the growth
of Salmonella spp. [7,61]. All melon-related foodborne outbreaks noted so far
involved melons that were precut and held at unknown temperatures for some
244 Microbiology of Fruits and Vegetables
period of time at restaurants and retail food stores prior to being purchased
and consumed. The inner flesh of melons comprises mainly parenchyma cells
containing sugars, organic acids, and other substances that may be released
upon plant cell injury and support microbial growth. Tamplin [3] suggested
that attention should be directed to cleaning the melons at the time of cutting,
using clean and sanitized utensils and surfaces to minimize contamination of
the edible portion, and immediately consuming or holding cut melon pieces at
cold temperatures.
10.6.3 Suppression of Outgrowth
The application of effective antibacterial agents to the surface of fresh-cut
melons may suppress outgrowth of the native microflora and any human
pathogens. Studies showing antilisterial activity of nisin in TSB or PBS62, and
demonstrating its activity against native microflora on whole and minimally
processed cantaloupe have been reported [17]. However, total elimination of
salmonella on the surface of whole or fresh-cut melon could not be achieved,
probably due to surface irregularities and internalization which reduced the
ability of antimicrobial treatments to contact or remove bacterial cells.
However, treatment with the combinations sodium lactate-potassium sorbate
or nisin-sodium lactate may lead to an increased shelf life and a reduced risk
of foodborne illness from salmonella or other human pathogens; such treat-
ments also appeared acceptable from a quality standpoint [17,63]. The use of
nisin for treating fresh-cut melon may reduce the risk of L. monocytogenes
outgrowth [64].
Bacteriophage was used to control growth and reduce population of
S. Enteritidis on fresh-cut melons [65]. In our most recent study, we found that
the native microflora of cantaloupe and honeydew melon was inhibitory to
L. monocytogenes [66]. Lactic acid bacteria were used to improve microbial
safety of minimally processed fruits and vegetables [67]. Other researchers have
used antagonistic microorganisms isolated from the field to control postharvest
pathogens and colonization of apple surfaces [68].
10.7 METHODOLOGY FOR MICROBIOLOGICAL
EVALUATION OF MELONS
Accurate assessment of the microbiological quality and safety of melons
requires use of suitable sampling, recovery, and detection methods that
take into account the mode of attachment of microorganisms to the melon
surface. This is especially important with cantaloupes because of their complex
surface morphology characterized by netting and the presence of fissures, both
of which are absent on honeydew melons [14,69]. The cantaloupe surface
morphology provides numerous microbial attachment sites and opportunities
for inaccessibility not present on other non-netted melons. However, all melons
Microbiological Safety Issues of Fresh Melons 245
will show variations in surface features that could affect microbial attachment
and growth, especially in the stem scar and ground spot regions.
Beuchat and Scouten [70] conducted a detailed study of survival and
recovery of S. Poona on spot- and dip-inoculated cantaloupes sampled at three
sites: the intact rind, a wound, or the stem scar. Recovery was accomplished
by stomaching excised rind in a wash solution containing 0.1% peptone,
with or without added Tween 80, or by rubbing melons in the same wash
solution within a plastic bag. They demonstrated the equivalence of a number
of combinations of preenrichment broth, enrichment broth, and selective agar
medium in detection of S. Poona recovered from the rind surface. They
reported no difference in recovery of S. Poona from the three sites compared to
when the inoculum was suspended in water or an organic matrix (horse serum);
growth occurred in both spot- and dip-inoculated wounds over 24 hours at 21
and 37°C but not at 4°C. Addition of up to 1.0% Tween 80 to peptone may
have enhanced detachment of S. Poona, recovered by the washing procedure.
The stomaching and wash solution procedures appeared to give equivalent
results.
Annous et al. [71] examined recovery and survival of E. coli NRRL B-766
on spot- and dip-inoculated cantaloupe rind. Less than 1% of the inoculum
applied by spot inoculation to the rind surface could be recovered by excising
plugs containing the inoculation sites and blending. E. coli survival on
inoculated cantaloupe after treatment with 300 ppm chlorine or water at 60°C
was greater if applied by dip inoculation of the melon surface compared to spot
inoculation. The investigators compared two sampling methods for recovering
bacteria from the melon rind surface: (1) excision and blending of 20 replicate
plugs containing inoculation sites for spot inoculation or taken at random
locations for dip inoculation, and (2) removal of the entire spot- or dip-
inoculated rind with an electric peeler. With both methods, the rind samples
were homogenized with peptone water, serially diluted, and plated. The meth-
ods were applied to melons inoculated with E. coli B-766 or S. Poona. A
method was developed for calculating the melon surface area from measure-
ments of the polar and equatorial diameters, based on an assumption that the
cantaloupe was a sphere, oblate spheroid, or prolate spheroid. When expressed
on an area basis, the population estimates for the two methods were the same
with both test organisms (Table 10.5). Expression of the population estimate
on a weight basis would be invalid, however, because of poor correlation
between the rind weight and external surface area. The whole rind method is
less time-consuming and requires less handling than the rind plug method.
Barak et al. [42] compared two elution methods with peeling and blending
for recovery of S. Poona from inoculated cantaloupes. They reported better
recovery with Butterfield's buffer containing Tween 80 as the eluant than with
phosphate-buffered saline, similar recovery when agitation was provided by
shaking or rolling, and better recovery by the elution methods than by peeling
and blending. The last result was attributed to the release of inhibitory
substances during blending.
246 Microbiology of Fruits and Vegetables
TABLE 10.5
Comparison of Rind Plug and Whole Rind Sampling Methods for Recovery
of Salmonella Poona RM 2350 from the Surface of Dip-Inoculated
Cantaloupes
Storage of inoculated 5. Poona population 3 (log 10 CFU/cm 2 )
melon at 20C (h)
Plug method b Whole rind method c
2 4.7 4.3
24 6.3 6.8
48 6.7 7.0
72 6.9 7.0
Note: Inoculum in water; population was 8.7 loglOCFU/ml. XLT-4 agar medium used to
enumerate S. Poona cell densities.
a Mean for 3 melons per trial; no significant difference between plug and whole rind methods.
b Based on total cross-sectional area of 20 rind plugs, each with 20 mm diameter.
c Based on calculated surface area for spheroid or sphere.
Hammack et al. [72] compared methods for the recovery of salmonella
from cantaloupes spot inoculated at levels to provide fractionally positive
results. They obtained better recoveries by soaking in preenrichment broth
as compared to rinsing with the broth, and by detecting the salmonella using
a culture procedure. Such methods would be useful in evaluating melons
subjected to antimicrobial treatments such as surface pasteurization in which
surviving populations are very small or not detectable by ordinary plating.
10.8 RESEARCH NEEDS
While extensive research has been conducted in a number of areas relating to
the microbiological safety and quality of melons, a number of gaps exist that
impede further progress. One deficiency is the relatively small amount of
information concerning melons other than cantaloupe. Another area requiring
more attention is the nature of microbial attachment to melons, especially
conditions favoring biofilm formation and internalization in the netting of
cantaloupes and stem scar of melons. A better understanding of salmonella
adhesion to cantaloupe is needed for the development of more effective
washing treatments to control this organism on melon surfaces and fresh-cut
pieces. With regard to sanitation methods for melons, the promising results
obtained with hot water surface pasteurization should be extended to
additional melons besides cantaloupe, and the possibility of adverse effects
on quality and shelf life should be given further study. As a back-up strategy,
research should be conducted on lower temperature surface treatments used
in combination with other treatments that may be synergistic. Finally, because
of the possibility of low-level survival of pathogens on melon surfaces
Microbiological Safety Issues of Fresh Melons 247
following such treatments and transfer to the flesh during fresh-cut processing,
better means of suppressing outgrowth of survivors by treatment of fresh-cut
melon with preservatives, irradiation, or other means should be investigated.
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Microbiological Safety Issues of Fresh Melons 251
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11
Fresh-Cut Vegetables
Pascal Delaquis
CONTENTS
11.1 Introduction 253
11.2 Fresh-Cut Carrots 254
11.3 Fresh-Cut Cabbage 255
1 1.4 Fresh-Cut Lettuce 256
11.5 Occurrence and Behavior of Human Pathogens in Fresh-Cut
Vegetables 257
1 1.6 Interactions Between Microorganisms and Plant Tissues 259
References 262
11.1 INTRODUCTION
Natural biological processes require that all plants eventually undergo
senescence, death, and decomposition by microorganisms. Physical stresses,
environmental factors, or disease can hasten this end in otherwise healthy
plants, including species that are cultivated by humans for consumption as
fresh vegetables. In this light, the trauma inflicted by harvest may be viewed as
the first in a series of events that ultimately lead to decomposition. Harvest
provokes physiological alterations associated with attempts to maintain
homeostasis, repair injury, and prevent infection by opportunistic micro-
organisms. Postharvest technological interventions are applied in distribution
systems for whole, fresh vegetables to delay quality changes that arise from
these reactions. Unit operations applied in fresh-cut processing invariably
contribute further stresses, particularly where tissues are cut or sliced. The
latter operations are of critical importance, as cutting irrevocably alters meta-
bolic processes and provides ample opportunity for invasion of tissues by
microorganisms. Additional measures are therefore necessary to preserve the
eating quality of fresh-cut vegetables.
Vegetables destined for fresh-cut processing carry complex microbial
populations that may include saprophytic species living in mutually beneficial,
symbiotic relationships with the healthy plant, potential phytopathogens, or
accidental contaminants derived from environmental sources. Microorganisms
derived from the field are described in detail in Chapter 1. Additional species
may be acquired during subsequent handling and processing. Any or all of
253
254 Microbiology of Fruits and Vegetables
these microorganisms can exploit opportunities for growth provided by access
to the rich source of nutrients contained within plant tissues. Selective
pressures derived from agronomic factors (source, field conditions), post-
harvest treatments, the intrinsic properties of raw materials (physical structure,
pH, availability of growth substrates, antimicrobial factors), and processing
(washing, application of antimicrobials, storage atmospheres, temperature)
influence the success of individual species and the composition of microbial
populations in products derived from individual vegetables.
A considerable body of scientific literature indicates that spoilage associa-
tions in fresh-cut vegetables are product-specific, and a complete description
of microbiological phenomena in all such products is beyond the scope of
this work. Instead, three commodities have been selected to illustrate the
influence of inherent and processing variables on the development of spoilage
associations and the fate of undesirable microorganisms in fresh-cut
vegetables.
1 1 .2 FRESH-CUT CARROTS
Fresh-cut carrots are distributed as slices, sticks, shreds (grated carrots), or
bite-size pared products referred to as 'baby" carrots. These are normally
derived from the mature root, although specialty items prepared with imma-
ture plants grown for the purpose are available in the marketplace. The
appearance of "white blush" on cut surfaces is a serious quality problem with
all fresh-cut carrot products. The phenomenon is believed to result mainly
from dehydration of the cut surface but enzymatic reactions leading to
lignification may contribute to the phenomenon. Some processors dip carrot
products in citric acid to delay enzymatic whitening, and most package the
product without drying to avoid rapid dehydration of the surface. The use of
proprietary humectants that prevent dehydration and preserve the appearance
of freshly sliced carrots is also practiced.
Freshly harvested carrots carry microbial populations dominated by
species derived from soil. In-field washing in chlorinated water to remove
excess soil is a common practice, and the harvested crop is often stored prior to
distribution and processing. In some jurisdictions, fungicides such as
thiabendazole and/or iprodione (Rovral) and bactericides such as chlorane
are applied for the control of storage disease [1]. Under ideal conditions storage
temperature is held close to 0°C, and relative humidity is maintained above
90% to reduce respiration rates, limit weight loss, and discourage growth of
microorganisms responsible for postharvest diseases. There is little doubt
that postharvest treatments such as these alter the microflora of raw carrots
destined for processing, although their influence on microorganisms of
significance in fresh-cut products has not been examined in detail. Similarly,
the processing plant is also expected to contribute spoilage microorganisms to
the finished product. Unfortunately, the microbial ecology of the processing
environment is poorly understood.
Fresh-Cut Vegetables 255
Microbiological examination of carrot sticks at various stages of process-
ing has shown that peel is a major source of microbial contaminants on the
finished product. A survey of processing establishments by Garg et al. [2]
revealed that peeling reduced mean total aerobic populations from 6.5 x 10 6 to
3.6 x 10 CFU/g. It should be noted that the samples analyzed for this work
were prepared by blending cut pieces. Hence the actual population density on
the peel of raw carrots may have been several orders of magnitude greater. The
data derived from this study clearly showed that peeling results in the transfer
of microorganisms from the outer epidermis of the raw material to surfaces
exposed by cutting. It is also interesting to note that populations remained
unchanged during slicing or dipping in a chlorinated water ice bath. Mean total
aerobic populations on the finished product were in the range 10 4 to 10 5 CFU/g,
in close agreement with a separate study by Odumeru et al. [3]. Garg et al. [2]
found large populations of Gram-negative psychrotrophic bacteria, particu-
larly Pseudomonas spp., lactic acid bacteria, and fungi on freshly prepared
carrot sticks. The fate of these microorganisms during refrigerated storage
was subject to the influence of preparation method, composition of the atmos-
phere in the package, and storage temperature. Izumi et al. [4] compared
various quality attributes in carrot slices, sticks, and shreds stored in air or
under controlled atmosphere (0.5% 2 , 10% C0 2 ). Although differences were
not always statistically significant, total aerobic microbial populations were
highest in shreds, followed by sticks and slices. Hence there appears to be a
relationship between the degree of damage to plant tissues, available surface
area for colonization, and the extent of microbial growth.
Rapid quality changes in commercially packaged, shredded carrots and the
suspected involvement of microorganisms led to detailed microbiological
examination of these products by Carlin et al. [5-7]. Most are distributed in
impermeable films or rigid containers. Rapid depletion of oxygen to less than
2% and accumulation of carbon dioxide to more than 30% due to accelerated
respiration in plant tissues leads to the establishment of atmospheres condu-
cive to the growth of microaerophilic and facultatively anaerobic species.
Lactic acid bacteria, particularly the heterofermentative species Leuconostoc
mesenteroides, and yeasts quickly become the predominant groups, and both
lactic acid and ethanol accumulate in the product [5]. Controlled atmospheres
containing 15 to 20% C0 2 and 5% 2 can delay these changes [6-7].
Kakiomenou et al. [8] also examined the influence of temperature and various
modified atmospheres on these events. Lactic acid bacteria were always the
dominant group, particularly at higher temperatures (10°C).
1 1 .3 FRESH-CUT CABBAGE
Shredded cabbage is usually distributed in polymeric film bags. Washing in
chlorinated water followed by dewatering by centrifugation are common in
the industry. Citric acid-containing dips are also employed by some manu-
facturers, purportedly to delay both physiological disorders and microbial
256 Microbiology of Fruits and Vegetables
growth, although there is little experimental evidence that such treatments are
effective. Quality defects due to the development of off-flavors, odors, and
changes in color are not uncommon in distribution systems.
Whole cabbage is frequently stored for extended periods of time before
processing. Temperatures are maintained near 0°C and relative humidity above
90%. Stored cabbage is at risk of spoilage by fungi and the use of fungicides
(such as benomyl, thiabendazole) is practiced in some countries [9]. Micro-
biological examination of whole cabbage heads by King et al. [10] showed that
outer leaves carry much higher microbial populations than tissues close to the
core. These ranged from a density of 1.4 x 10 6 CFU/g on the outer leaves to
3.8 x 10 CFU/g in core samples of stored cabbage sampled over a period of
several months. Psychrotrophic bacteria belonging to the genera brevibacter-
ium, chromobacterium, citrobacter, pseudomonas, and xanthomonas were
the largest group of microorganisms present, although yeast and mold were
also recovered in large numbers. Geeson [9] also found that the outer leaves
of stored cabbage carry large populations of fluorescent pseudomonads,
pectolytic bacteria, yeasts, and molds. In the latter study, postharvest applica-
tion of benomyl and thiabendazole by drenching was correlated with higher
bacterial populations in cabbage stored for a period of several months at
various relative humidities.
Unfortunately, the impact of unit operations on the microbiology of
shredded cabbage has not been examined in detail. However, commercial
experience has shown that removal of outer leaves prior to slicing improves the
microbiological stability of shredded products. Shredding likely distributes
remaining contaminants throughout the product and provides extensive oppor-
tunities for growth on cut surfaces. Hao et al. [1 1] examined the microbiology
of shredded cabbage stored in both low- and high-oxygen transmission films
at 4, 13, and 21°C. Carbon dioxide levels rose and oxygen levels declined faster
in the headspace above product packaged in less permeable films. Surprisingly,
the observed spoilage patterns were consistent for all treatments, and
populations of aerobic bacteria tended to dominate the spoilage association.
However, lactic acid bacteria populations increased to high densities regardless
of packaging treatment or storage temperature. The development of sourness
and blowing of packages can occur in these products and has been associated
with extensive growth of these bacteria. Growth of this relatively minor
component of the raw cabbage microflora can be stimulated by the addition of
salt for the manufacture of fermented products. Reasons for their rapid growth
in shredded cabbage despite the presence of active, large populations of Gram-
negative, psychrotrophic bacteria remain unclear.
1 1 .4 FRESH-CUT LETTUCE
Iceberg, leaf, and mixed lettuces remain the most common fresh-cut products
in the marketplace. Unlike carrot or cabbage products, raw materials for their
manufacture are not stored for extensive periods of time prior to processing.
Fresh-Cut Vegetables 257
Field lettuce carries populations of microorganisms derived from soil [12].
Gram-negative bacteria, particularly fluorescent pseudomonads, and smaller
numbers of yeast make up the bulk of the initial microbial flora, which ranges
in density between 10 4 and 10 6 CFU/g [2,13-15]. Densities of microorganisms
also vary with location in the plant, and outer leaves tend to carry higher
microbial populations.
The need to control browning reactions catalyzed by oxygen in packaged
cut lettuce has led to extensive use of technologies designed to maintain anoxic
atmospheres during storage. Control over browning can be achieved by
vacuum packaging, flushing with nitrogen, or the incorporation of atmos-
pheres containing various levels of oxygen, nitrogen, and carbon dioxide.
Selective pressures exerted by gas composition of the atmosphere are expected
to influence the spoilage association. However, observations drawn from
numerous studies on the microbiology of lettuce packed under oxygen-reduced
atmospheres suggest that storage temperature exerts more influence on the
development of the spoilage association than gaseous composition. Mesophilic
and psychrotrophic populations tend to be similar in stored shredded lettuce,
while yeast and mold and lactic acid bacteria populations generally remain low
[2,13,14]. Spoilage microflora are always dominated by species of psychro-
trophic Pseudomonas spp. [14-16]. Evidence of microbial spoilage appears
earlier in products with high initial microbial loads and at higher storage
temperatures.
The effects of unit operations on the microbiology of fresh-cut lettuce are
not well characterized. Bolin et al. [17] examined the influence of cutting
method, washing, centrifugation, initial microbial load, packaging, and tempera-
ture on the storage stability of iceberg lettuce. Unfortunately, microbiological
assessments were not carried out for all experimental treatments. Deleterious
quality effects were associated with tissue damage induced by cutting with dull
blades, inadequate water removal, and storage at high temperatures. There is
little doubt that some of these factors could influence the development of
microbial populations in the package. High initial microbial loads were also
correlated with reduced keeping quality.
11.5 OCCURRENCE AND BEHAVIOR OF HUMAN
PATHOGENS IN FRESH-CUT VEGETABLES
Any food product delivered to consumers without the application of
treatments that completely eliminate microbial contaminants may serve as a
vehicle for the transmission of microorganisms capable of causing disease.
Hence, concerns about the potential for transmission of human pathogens
through fresh-cut vegetables are warranted because raw horticultural products
are naturally contaminated with large numbers of microorganisms, and they
are not subjected to lethal processing treatments prior to distribution.
Microbial populations on field vegetables may occasionally include potential
pathogens acquired in the production environment, either from natural sources
258 Microbiology of Fruits and Vegetables
TABLE 11.1
Some Outbreaks of Foodborne Illness Linked to Fresh-Cut Cabbage and
Lettuce Products
Etiological agent Commodity Ref.
Listeria monocytogenes Cabbage coleslaw 44
Clostridium botulinum (A) Cabbage salad 45
Shigella sonnei Iceberg lettuce 46
E.coliOlSTJn Mesclun lettuce 47
Hepatitis A Iceberg lettuce 48
E. coli 0157:H7 Shredded carrots 49
or as a result of human activity (see Chapter 1). Foodborne pathogens have
been isolated from market fresh-cut vegetable products in various parts of the
world [18-20]. In addition, processing plants frequently receive raw materials
from a variety of sources and the potential exists for cross contamination of
product. Despite these apparent vulnerabilities, relatively few documented
outbreaks of foodborne illness have been conclusively linked to fresh-cut
vegetables. Table 11.1 lists some outbreaks that have implicated fresh-cut
carrots, cabbage, and lettuce, and these examples serve to illustrate the poten-
tial for transmission of various infectious agents through fresh-cut vegetables.
It should be stressed that epidemiological investigations of outbreaks involving
such products are fraught with difficulties. Considerable time delays between
definitive association and sampling of suspected products may reduce the
likelihood of detection. In addition, microbiological analyses often lack the
sensitivity required to detect small populations of target microorganisms in
environmental samples containing complex background microflora in high
populations. This problem is acute for some pathogens, particularly species of
shigella, due to a lack of effective selective enrichment protocols. Hence,
currently held assumptions about the behavior of human pathogens in fresh-
cut vegetable products are largely derived from research.
The behavior of pathogenic microorganisms has been examined in fresh-
cut cabbage under laboratory conditions. For example, Kallander et al. [21]
... 9
were unable to detect Listeria monocytogenes (detection limit 10 CFU/g) in
shredded cabbage stored under air or under 70% carbon dioxide and 30%
nitrogen after 6 days at 25°C. A reduction in pH related to the growth of lactic
acid bacteria was evident at this temperature. In contrast, the species was
capable of growth in cabbage stored at 5°C, irrespective of atmosphere
composition, and little or no growth of lactic acid bacteria was detected.
Omary et al. [23] inoculated shredded cabbage with Listeria innocua, a
surrogate species for L. monocytogenes. Packaging films with oxygen trans-
mission rates ranging from 5.6 to 6000 cm 2 /m /24 hours were employed for
storage of samples at 11°C. Listeria innocua populations initially declined but
eventually increased in samples subjected to all treatments. Hence, the results
of studies carried out in the laboratory may lead to different individual
Fresh-Cut Vegetables 259
conclusions. Nevertheless, the sum of this research suggests that Listeria spp.
can grow in packaged fresh-cut cabbage and that temperature is a critical
determinant for the fate of this species.
The behavior of L. monocytogenes in cut lettuce has also been examined
and variable results are reported from individual studies. A gradual decline in
populations was reported by Francis and O'Beirne [22] at 3°C and by
Kakiomenou et al. [8] at 4°C. Beuchat and Brackett [24] found there was no
change in cut lettuce stored at 5°C, but Steinbruegge et al. [25] observed
exponential growth at higher temperatures. The effect of modified atmospheres
on the fate of L. monocytogenes is also unpredictable. Francis and O'Beirne
[22] found that growth was enhanced in shredded lettuce stored under 100%
N2 instead of an aerobic packaging system. Jacxsens et al. [26] recorded similar
observations for shredded iceberg lettuce stored at 7°C under a 2 to 3% 2 ,
2 to 3% C0 2 , 94 to 96% N 2 atmosphere. However, contradictory conclusions
were drawn from the work of Beuchat and Brackett [24] where no difference
was found between samples stored at 10°C in air or in a 3% 2 /97% N 2
gas mixture. Hence, the effects of modified atmospheres on the fate of
L. monocytogenes in cut lettuce remain uncertain.
Comparatively little is known about the behavior of human pathogens in
fresh-cut carrots, likely due to the limited number of foodborne infections
conclusively associated with this commodity. Nevertheless, some interesting
observations have been derived from research with inoculated products.
Bagamboula et al. [27] found that Shigella spp. populations gradually fell in
grated carrots stored at either 7 or 12°C. The spoilage association in the
product was dominated by lactic acid bacteria, and it was concluded that a
gradual decline in pH was largely responsible for a gradual die-off of the
inoculum. Viable cells were recovered from all products after 7 days.
Analysis of published research indicates that temperature has a major
influence on the fate of pathogens in fresh-cut vegetable products. The main-
tenance of low temperatures evidently provides the best means to prevent
growth, particularly for members of the family Enterobacteriaceae. Listeria
monocytogenes poses a unique challenge given the ability of the species to grow
at refrigeration temperatures. However, the plurality of experimental outcomes
at higher temperatures or where modified atmospheres are applied to packag-
ing systems suggests that additional factors influence the behavior of this
pathogen in fresh-cut vegetable products.
1 1 .6 INTERACTIONS BETWEEN MICROORGANISMS
AND PLANT TISSUES
The shelf life of fresh-cut vegetables including lettuce, cabbage, and carrots is
limited mainly by the appearance of enzymatically induced discolorations.
Control over these quality defects currently relies on the maintenance of low-
oxygen atmospheres or the application of dips containing enzyme inhibitors,
but additional means to alleviate the problem are under investigation.
260
Microbiology of Fruits and Vegetables
The application of mild heat treatments or heat shocks before packaging shows
promise for this purpose [28-31]. Heat shocks applied between 47 and 50°C for
90 to 180 seconds delay browning in iceberg lettuce by several days in
refrigerated product. In addition, these treatments improve disinfection of
the product, and reductions of up to 3 log CFU/g are readily achieved.
Unfortunately, the advantages of heat treatments are negated by accelerated
microbial growth during subsequent refrigerated storage. Faster growth of
spoilage bacteria has been observed, and inoculation with L. monocytogenes
and E. coli 0157:H7 confirmed that growth is enhanced by prior heat
treatment of the lettuce [28,32]. A similar effect occurs in shredded cabbage
subjected to heat treatments. The example provided in Figure 11.1 shows total
aerobic microbial populations in shredded cabbage stored under refrigeration
in low oxygen permeability films after treatment in 100 jig/ml NaOCl solutions
heated to various temperatures (Delaquis, unpublished data). Evidently,
washing in a cold chlorinated water solution had little effect on the develop-
ment of the spoilage association. Application of heated chlorinated water
reduced the initial population by 2 log CFU/g or more. However, the rate of
population growth and the ultimate size of the spoilage population were
greater in cabbage subjected to the heat treatments.
Removal of a substantial part of the native microflora could provide a
competitive advantage to survivors of heat treatments or to contaminants
acquired postprocess. There is evidence that additional factors are responsible
for these effects, however. The classic plant pathology literature describes
numerous constitutive and induced antimicrobial systems in plants [33]. The
economic impact of species that are pathogenic to crops in the field or those
responsible for losses during prolonged storage has stimulated considerable
=>
LL
O
O
9
8
7
6
5
4
3
2
1
Unwashed
Cold wash (5° C)
| J 49° C, 90 seconds
51° C, 90 seconds
Days in storage, 1° C
FIGURE 11.1 Total aerobic populations in shredded cabbage stored at 1°C in oxygen-
permeable film after treatment in a lOOug/m; NaOCl solution heated to various
temperatures. The cabbage was dried in a centrifuge prior to packaging. (P.J. Delaquis,
2004. Unpublished data.)
Fresh-Cut Vegetables
261
♦ Fresh lettuce
□ Stored 1 day
a Stored 2 days
o Stored 3 days
12 16
Time (h)
28
FIGURE 11.2 Fate of Listeria monocytogenes in aqueous extracts prepared from
packaged, shredded cut iceberg lettuce stored aerobically for 0, 1,2, and 3 days at 15°C.
(P.J. Delaquis, 2004. Unpublished data.)
research in this area. In contrast, comparatively little is known about the
influence of intrinsic plant defense mechanisms on the fate of significant
spoilage microorganisms or foodborne pathogens in fresh-cut vegetable or fruit
products. Reports of antimicrobial activity in vegetable extracts provide
evidence that some plant constituents may have a role in the microbial ecology
of these products. Conner et al. [34] found that an unidentified cabbage juice
extract inhibits the growth of Listeria monocytogenes. Carrot extracts have also
been widely reported to inhibit fungi [35], foodborne bacteria and yeast [36],
and Listeria monocytogenes [36-40].
Accelerated development of microbial populations in products subjected
to heat shocks hints that intrinsic barriers to growth normally present in
physiologically intact plant tissues may be disrupted by processing. This hypo-
thesis was tested in our laboratory by inoculation of Listeria monocytogenes in
iceberg lettuce tissue extracts prepared from tissues stored aerobically for up to
three days, as shown in Figure 11.2. The results of these experiments provided
evidence that an antilisterial factor or factors is elaborated by cut lettuce tissues
stored under aerobic conditions. Application of heat treatments before storage
and preparation of the extracts reduces this effect, however, as shown in
Figure 11.3. The chemical nature of the inhibitor(s) responsible for this effect
in iceberg lettuce is not yet known. There is little doubt that heat shocks and
unit operations applied in processing and preservation have a major impact on
the physiology of plant tissues. For example, heat shocks [41] and modified
atmospheres [42] inhibit the activity of phenylalanine ammonia lyase (PAL), a
key enzyme in the development of discolorations in cut lettuce tissues. The
enzyme catalyzes the first step in a series of complex reactions that leads to
the accumulation of phenylpropanoid intermediates, including phenolic
compounds such as caffeic, ferulic, and chlorogenic acids. Several of these
262
Microbiology of Fruits and Vegetables
8
12 16
Time (h)
♦ Untreated, day
a Hot wash, day
■ Cold wash, day
O Untreated, day 3
a Hot wash, day 3
□ Cold wash, day 3
28
FIGURE 11.3 Fate of Listeria monocytogenes in aqueous extracts prepared from
packaged, shredded iceberg lettuce stored aerobically for and 3 days at 15°C following
a three-minute wash in cold (4°C) or warm (47°C) water. (P.J. Delaquis, 2004.
Unpublished data.)
compounds exhibit antimicrobial activity in vitro [43], but it remains unknown
whether they influence the fate of microorganisms in packaged cut lettuce.
Clearly, much remains to be learned about the interaction between
microorganisms and plant tissues. Improved characterization of intrinsic
factors, which affect the fate of microorganisms in fresh-cut vegetables, will
undoubtedly enhance our understanding of fundamental interactions in these
complex microbial ecosystems. Furthermore, a more complete understanding
of these interactions could lead to significant practical outcomes. In the future,
it may be possible to exploit intrinsic barriers to restrict microbial growth for
the development of novel preservation processes for fresh-cut vegetables.
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12
Outbreaks Associated
with Cyclospora and
Cryptosporidium
Ynes R. Ortega and Charles R. Sterling
CONTENTS
12.1 Introduction 267
12.2 Overview of the Parasites 267
12.3 Sources of Contamination 269
12.4 Description of the Foodborne Outbreaks for Both Parasites 270
12.5 Detection and Enumeration Methodologies 271
12.6 Interventions for Decontamination 273
12.7 Conclusions 274
References 275
12.1 INTRODUCTION
Parasites have frequently been implicated in food and waterborne outbreaks in
the U.S. and elsewhere. This may be related to the development of more
sensitive and specific assays for parasite identification and changes in diet and
food consumption habits. To satisfy the demands of consumers, more fresh
produce is being imported. Food management and production practices in
developing countries are not necessarily similar to those in the developed
world. This chapter covers the parasites Cryptosporidium and cyclospora, as
they have been implicated in several food and waterborne outbreaks.
12.2 OVERVIEW OF THE PARASITES
Cryptosporidium is a protozoan parasite that belongs to the subphylum
Apicomplexa. Because of large and significant waterborne outbreaks
associated with it, much has been learned about the epidemiology, immu-
nology, and biology of this parasite. Cryptosporidium parvum was considered
to be the only species of public health significance; however, studies involv-
ing pediatric populations, the immunocompromised, travelers, and endemic
populations have demonstrated that humans can also be infected with
C. meleagridis (of turkeys), C. canis (of dogs), C. felis (of cats), and C. muris
267
268 Microbiology of Fruits and Vegetables
(of cattle and rodents). Molecular studies have also demonstrated that the
previously described C. parvum is actually at least two different species:
C. parvum (zoonotic) and C. hominis (considered anthroponotic, i.e., human-
to-human). Both are morphologically similar [1,2].
The infective stage of Cryptosporidium is the oocyst, which is excreted
in the feces of infected hosts. These oocysts are immediately infectious to
a susceptible host and contain four fully differentiated and infectious
sporozoites. Once ingested, oocysts excyst in the gastrointestinal tract,
releasing infective sporozoites. Sporozoites infect the epithelial cells of the
ileum preferentially and may continue colonization of all the small intes-
tine and bile ducts if the host is immunocompromised, thus making eradi-
cation of the parasite more difficult. Once parasites have entered the epithelial
cells they are compartmentalized in a parasitic vacuole which is extracyto-
plasmatic, but intracellular. This site localization is unique, and the parasite
depends upon an elaboration of membrane surface at the cytoplasmic inter-
face called the feeder organelle which allows for the transport of select
nutrients to the parasite. Once established, the parasite multiplies asexually,
producing type I and II meronts containing 8 and 4 merozoites, respectively.
The first asexual meront generation can multiply indefinitely in the absence
of immunity while those that have progressed to the second generation
eventually differentiate and produce gamonts (macro- and microgametocytes).
A microgametocyte fertilizes the macrogamont and leads to the formation
of a zygote. When mature, the zygote will become either a thin- or thick-walled
oocyst. The former constitutes approximately 20% of oocysts produced and
is autoinfective, while the latter is environmentally resistant and fully infectious
when excreted to the environment [3].
Cryptosporidiosis is characterized by abundant and persistent diarrhea,
fever, and abdominal pain. There is no effective therapy but new drugs
are being evaluated with promising results [4-6]. The immune system of
the infected individual plays a crucial role in eradicating the parasite.
In immunocompromised patients cryptosporidiosis can be fatal [7].
Cyclospora cayetanensis is also a parasite of the subphylum Apicomplexa.
Fourteen different species of cyclospora have been described in rodents
and insectivores [8]. Cyclospora cayetanensis is considered to be exclusively
anthroponotic [9]. In 1999 three other species infecting nonhuman primates
were described. These cyclospora species are morphologically similar to
C. cayetanensis, but, based on their host specificity and 18S DNA sequence
homology, are different [10,11].
The life cycle of cyclospora begins when oocysts are excreted in the feces
of an infected individual. These oocysts are unsporulated and take approxi-
mately two weeks under optimal environmental conditions to sporulate
fully and become infectious. Differentiated oocysts contain two sporocysts,
each containing two sporozoites. When ingested and upon passage through the
gastrointestinal tract, oocysts rupture and sporocysts are released. Enzymes
and bile salts induce the release (excystation) of the sporozoites, which in
turn invade the epithelial cells of the small intestine, forming an intracellular
Outbreaks Associated with Cyclospora and Cryptosporidium 269
parasitic vacuole. Based on histological observations of biopsies of infected
individuals, type I and II meronts are produced. Gametocytes have also been
observed, suggesting that cyclospora has a life cycle similar to that of
Cryptosporidium and other coccidia. After zygote formation, oocysts are
formed and excreted to the environment [12].
There are nonspecific fingerprinting tools for traceback studies; however,
characterization of the internal transcribed spacers 1 (ITS1) sequences may be
used for these purposes. The ITS1 sequences of clones of all five raspberry-
associated isolates were identical, consistent with their origin from a single
source. One of the two Guatemala isolates and two Peruvian isolates contained
multiple ITS1 sequences [13,14].
1 2.3 SOURCES OF CONTAMINATION
Cryptosporidium can be acquired by ingestion of contaminated water and
foods. The oocysts are already infectious when excreted; therefore, cryptos-
poridiosis can also be transmitted via the fecal-oral route involving person-to-
person or animal-to-person transmission. Recreational [15-18] and drinking
waters [19,20] have been responsible for various Cryptosporidium waterborne
outbreaks. Chlorine concentrations suitable for drinking water have proven
insufficient to inactivate Cryptosporidium oocysts. Prepared foods and apple
cider have also been described as sources for Cryptosporidium contamination
[21-24]. Inappropriate manipulations of foods by food handlers [25] or fruits
contaminated with cattle feces have also been sources of Cryptosporidium
contamination. Zoonotic transmission has also been important, particularly
for hikers who drink river or lake water without disinfecting it. Cryptospori-
dium can infect a diverse variety of animals, particularly cattle. Zoonotic
contaminations have been described in veterinary students and animal
caretakers [26,27]. Outbreaks in day care centers have also been reported.
Children are highly susceptible to infection, and transmission of this parasite
can be high if proper hygiene practices are not in place, either at home or day
care centers [28-33]. The same conditions are favorable for Cryptosporidium
outbreaks in hospitals [34, 35].
Investigations of cyclospora outbreaks suggest that it can be acquired
when ingesting water [36-38] and food [39-44] that contain the parasite's
oocysts. Cyclospora has been isolated from fresh produce in the U.S. and
elsewhere [36,45,46], suggesting that foods play an important role in cyclos-
pora transmission. The mechanisms and dynamics of transmission of
cyclospora are more complicated than those of Cryptosporidium since oocysts
are not fully sporulated and therefore infective when passed from an infected
human.
Contaminated water used for irrigation [47] or pesticide spraying support
oocyst survival [48]. Aerosols, insects [49-51], and contaminated water courses
and streams used in crop irrigation may factor in the introduction of viable
oocysts into fresh produce.
270 Microbiology of Fruits and Vegetables
12.4 DESCRIPTION OF THE FOODBORNE
OUTBREAKS FOR BOTH PARASITES
Cryptosporidium can be found worldwide and infects a variety of hosts,
including humans. The incidence of Cryptosporidium can vary depending upon
the population and location from 0.6 to 20%. In the U.S., cryptosporidiosis is
associated with 0.4 to 1% of cases of diarrhea [52]. The Cryptosporidium oocyst
is resistant to normal environmental conditions, but desiccation can render
oocysts noninfectious. Foodborne outbreaks of Cryptosporidium have been
associated with foods prepared in homes, suggesting direct contamination by
food handlers. In Maine, in 1993, an outbreak was linked to unpasteurized apple
cider. A farm with livestock used dropped apples for the preparation of the
apple cider [24]. In 1995, in Minnesota, chicken salad was implicated in 15 cases
of cryptosporidiosis. Another outbreak involving apple cider occurred in 1996
in New York [53]. Sixty-six persons developed cryptosporidiosis and one died.
In 1997, 54 cases of cryptosporidiosis were probably connected to the con-
sumption of green onions. Two of the 14 food preparers were positive for
Cryptosporidium. In 1 998 an outbreak was associated with consumption of meals
in one of two cafeterias of a university in Washington D.C. Epidemiological
investigation concluded that the outbreak was caused by C. hominis and the
most probable source was an ill food handler who prepared raw produce [25].
Milk, salad, sausage, and tripe have also been suspect foods in travelers
with cryptosporidiosis entering the U.S. from Mexico, the U.K., and Australia.
Although no cases of cryptosporidiosis associated with shellfish have been
reported, the presence of human strain of Cryptosporidium has been reported
in mussels and oysters retailed for human consumption [54-57].
Cyclospora is endemic in certain countries of tropical regions. Much of
what we know about cyclospora has resulted from studies performed in those
settings. A disease of the tropics and developing countries found its way to
the developed countries when the latter started importing produce that is
in demand throughout the year. If cyclospora is endemic in such export-
ing countries, it is possible that if good agricultural practices (GAPs) are
not implemented in those particular fields, human feces can be carried to the
products, either by crop manipulation with contaminated hands, or
contaminated irrigation water. Cyclospora is highly resistant to environmental
conditions and will attach to the surface of the produce and remain viable
for longer periods of time. Most fecal contaminants may not remain viable for
long periods of time, thus explaining why other foodborne outbreaks have
not been reported in parallel with cyclospora outbreaks.
Four commodities have been implicated with cyclospora foodborne
outbreaks: raspberries, basil, lettuce, and snow peas. Since the early 1990s
sporadic cases of cyclosporiasis were reported in the U.S., but no source of
contamination was identified. The first large cyclospora outbreak occurred
in 1995 in Florida. Strawberries were initially implicated in the outbreak,
but later epidemiological investigations suggested that raspberries were
responsible. In 1996, 1465 cases of cyclosporiasis were reported in 20 states
Outbreaks Associated with Cyclospora and Cryptosporidium 271
in the U.S. In 1997, 41 clusters comprising 762 cases were reported during
the months of April and May in 9 states. Raspberries, basil, and lettuce were
implicated in this outbreak. Also during April to June of the same year, 250
laboratory-confirmed sporadic cases were reported. In all instances, imported
raspberries were associated with the outbreak. As a result of this, Guatemala
voluntarily suspended the exportation of raspberries. In 1998 a few sporadic
cases of cyclosporiasis were reported in the U.S., but Canada continued to
import berries from Guatemala and experienced a large outbreak [58,59].
Surveys of fresh produce have described the presence of cyclospora
[40,41,46]. In 2004 the U.S. Food and Drug Administration (FDA) issued
an alert to consumers that two outbreak clusters of cyclosporiasis may be
associated with raw basil and mesclun/spring salads served in Texas and
Illinois. In February 2004 approximately 54 individuals in Wheaton, Illinois,
and 38 people in Irvin, Texas, were stricken with cyclosporiasis. During June
and July 2004 approximately 50 potential cases of cyclosporiasis were asso-
ciated with a residential facility. Epidemiological and traceback studies linked
the cases to consumption of raw Guatemalan snow peas [60]. Throughout
the years, sporadic cases of cyclospora continue to occur in the U.S., suggest-
ing that cyclospora is either being introduced to the U.S. by imported produce,
or by food handlers who are carriers of this parasite. More studies are needed
to determine the actual distribution of cyclospora in the U.S., both in human
populations and environmental samples.
12.5 DETECTION AND ENUMERATION
METHODOLOGIES
Methodologies for the identification and isolation of Cryptosporidium in water
have been thoroughly studied. The EPA Method 1623 [61,62] is based on the
recovery of Cryptosporidium oocysts and giardia cysts by filtration/IMS
(immunomagnetic separation)/FA (fluorescent antibody) (EPA-821-R-99-006)
of up to 101 of water. Filtration can be performed using either the Pall Gelman
HV Envirochek® capsule or the IDEXX Filta-Max™ filter. Cysts or oocysts
are then captured by IMS using Dynabeads® GC-Combo (Dynal, Inc.) or
Aureon CG (Aureon Biosystems) kits. Once the oocysts are recovered, they are
identified using immunofluorescent assays from Merifluor® G/C (Meridian
Diagnostics, Inc.), Aqua-Glo™ G/C Direct (Waterborne, Inc.), or Crypto-
Glo™ (BioTechFrontier, Inc.). Recovery efficiency for Cryptosporidium
parvum is 60 to 80%. Heat incubation of IMS-tagged oocysts resulted in
recoveries of 71 and 51% and DAPI confirmation rates in reagent and river
water of 93 and 73%, respectively [63]. Method 1623 has several limitations
and interferences. IMS can be affected by water turbidity and the presence of
silica, clay, humic acids, other organisms, etc. The presence of iron and pH will
also affect oocyst recovery.
Electrochemiluminescence (ECL) technology has been used to identify
Cryptosporidium in environmental water samples of up to 10,000 nephelo-
metric turbidity units [64].
272 Microbiology of Fruits and Vegetables
It is also important to determine whether the parasites are viable and of
public health relevance. Molecular assays will aid in speciation and sub-
typing of the parasites. These include polymerase chain reaction (PCR), reverse
transcription (RT)-PCR, nested PCR, and an isothermal amplification nucleic
acid sequence-based amplification (NASBA) method [65-70]. After isolation
of the parasites, extraction of the oocyst DNA is of critical importance.
Oocysts can be broken by boiling, mechanical disruption with glass beads,
digestive enzymes (proteinase-K, lysozyme) with 10% SDS, freeze/thaw, micro-
wave, sonication, and commercial kits (DNA and RNA) or automated systems
(contamination free) [71-73].
In water samples, various Cryptosporidium parvum mRNAs have been
used as molecular targets for detection [65]. The mRNA coding of C. parvum
for hsp70 was amplified using NASBA methodology with a detection limit of
80fmol amplicon/test. [74].
Because mRNA denatures quickly, oocyst viability can be determined
using RT-PCR for Cryptosporidium using the hsplO and the (3-tubulin genes.
An electrochemical enzyme-linked immobilized DNA-hybridization assay
using the C. parvum hsp70mRNA could distinguish dead from live oocysts.
No cross-reactivity was observed with other bacterial and parasitic organisms,
including Cryptosporidium muris [75].
In vitro cultivation recognizes parasites that are both viable and have the
ability to penetrate and replicate within host cells. Infectivity can be deter-
mined using animal models, but C. hominis, which is the anthroponotic species,
is host specific and is not infectious in neonatal mouse models.
Some of these methods have been used in food matrices. An IMS-PCR
assay was able to detect <10 C. parvum oocysts in milk [76].
A laser scanning cytometry method (ChemScanRDI), coupled with
immunofluorescence detection with differential interference contrast (DIC)
confirmation, has also been evaluated and compared with manual micro-
scopic enumeration of Cryptosporidium oocysts. The recovery rate was 50% at
seeding levels from 30 to 230 oocysts. Laser scanning cytometry does eliminate
the low sample throughput, operator subjectivity, and operator fatigue using
conventional microscopy [77].
Although these methodologies have been described for environmental
waters, they have not been fully validated in foods. The wide variety of produce
and foods that could potentially be involved in parasite transmission makes
selecting a unique method for isolation difficult. Detection using molecular
diagnostic assays is also challenging because of the presence of inhibitors that
could mask the presence of these parasites in foods.
Immunoassays have been developed for the use of Cryptosporidium identi-
fication in water samples. An indirect immunoffuorescent assay has also
proven to be useful in food matrices [23,78].
Most of the purification techniques that work for Cryptosporidium have
proven to be effective in purifying cyclospora oocysts (Ortega, personal com-
munication). Sucrose and cesium chloride gradients used for Cryptospori-
dium can be used for cyclospora. Water filtration systems have also proven to
Outbreaks Associated with Cyclospora and Cryptosporidium 273
concentrate cyclospora from water sources. To date, monoclonal antibodies
to cyclospora have not been produced. This is not only because of the limi-
ted sources of oocysts, but also because the cell wall has poor antigenic
properties. One useful approach has been to use magnetic beads coated with
the lectin WGA [79].
Various methodologies have been described to identify cyclospora using
conventional clinical assays. When environmental samples are examined, auto-
fluorescence can prove useful, although this is not a specific assay. A PCR
method, initially designed for clinical samples, has worked well with food
matrices; however, other Eimeria spp. also had the same amplification product
as cyclospora [80]. A restriction fragment length polymorphism using the Mnl
1 enzyme could differentiate between cyclospora and eimeria. The biggest
challenges when using PCR for cyclospora are the methodologies used to
extract DNA from the low number of oocysts likely to be encountered, and
how to control for the presence of PCR inhibitors. Various methodologies,
including chelating matrices and freeze/thaw cycles, FTA membranes, and
DNA extraction kits, have been described [72,80]. The use of an extraction-
free, filter-based protocol (FTA) to prepare DNA templates for use in PCR to
identify C. cayetanensis and C. parvum oocysts and microsporidia spores has
been described. As few as 10 to 30 C. cayetanensis oocysts per 100 g of fresh
raspberries could be detected [72].
To control for PCR inhibitors, addition of BSA or milk has improved the
sensitivity of the assay. The PCR assay in raspberries, basil, and mesclun
lettuce could detect 40 or fewer oocysts per 100 g of raspberries or basil, but
had a detection limit of around 1000 per 100 g in mesclun lettuce [72,81].
Real-time PCR can also detect DNA specifically from as few as 1 oocyst of
C. cayetanensis per 5ul reaction volume [82].
Determining the viability of cyclospora oocysts has proven to be very
difficult. To date, there are no susceptible animal models or in vitro cultiva-
tion methods. Sporulation rates have been used to determine if a particular
treatment has affected the oocyst viability. This, however, may not have any
bearing on oocyst infectivity. Electrorotation has been used as a method to
determine oocyst viability [83]. This method needs to be validated when an
in vivo or in vitro system for cyclospora becomes available.
12.6 INTERVENTIONS FOR DECONTAMINATION
Various sanitizers and disinfectants have been evaluated for Cryptosporidium.
Oocysts will remain viable if kept in moist environments, but are very sensitive
to desiccation. Moist heat treatments or pasteurization of Cryptosporidium
oocysts at 45° C for 5 to 20 minutes inactivate the parasite [84].
Chemical agents commonly used for disinfection of contaminated environ-
mental surfaces and medical devices such as endoscopes have been evaluated
for their effect on Cryptosporidium viability. Exposure of C. parvum to steam,
ethylene oxide, and Sterrad 100 and hydrogen peroxide at concentrations of 6
274 Microbiology of Fruits and Vegetables
and 7.5% for 20 minutes resulted in population reductions of 3 logs or greater.
Peracetic acid (0.2% for 20 minutes), sodium hypochlorite (5.25% for 10
minutes), a phenolic, a quaternary ammonium compound (10 minutes), 2%
glutaraldehyde (45 minutes), and or/Zzo-phthalaldehyde (20 minutes) did not
completely inactivate oocysts [85].
The effect of ultraviolet radiation from low- and medium-pressure mercury
arc lamps on Cryptosporidium parvum oocysts has been evaluated. Two and
three log units inactivation have been achieved at approximately 10 and
o • • • 4 • • •
25 mJ/cm /sec, respectively [86]. Use of static mixers for dissolution of ozone in
drinking water treatment plants may contribute to C. parvum inactivation [87].
Flash pasteurization of cider inoculated with Cryptosporidium oocysts at 70 or
71.7°C, both for 10 or 20 seconds, reduced viability by at least 4.9 logs (or
99.999%) when determined using a tissue culture assay. A 3.0 log (99.9%) and
4.8 log (99.9985) inactivation were achieved when oocysts were treated for
5 minutes at 70 or 71.7°C, respectively. Current practices of flash pasteuriza-
tion in the juice industry are sufficient to inactivate contaminant oocysts [88].
An electrochemically produced mixed-oxidant solution (MIOX; LATA Inc.)
was considerably more effective in inactivating Cryptosporidium parvum
oocysts than free chlorine. A 5 mg/1 dose of mixed oxidants produced a > 3 log
(>99.9%) inactivation of Cryptosporidium parvum oocysts in 4 hours [89].
12.7 CONCLUSIONS
There is still limited knowledge of parasite transmission dynamics with respect
to both Cryptosporidium and cyclospora, and much research is required in the
arena of inactivation strategies. Because of changes in population diets, food
production, and management, and improved diagnostic assays, more cases of
parasitic infections are being reported. It is also important to note that there is
a change in population demographics, with more susceptible groups increasing
in numbers (the elderly, children, and the immunocompromised).
Epidemiological features of Cryptosporidium lead to the almost over-
whelming conclusion that the incidence of foodborne cryptosporidiosis is
underestimated. The low numbers of oocysts in suspected samples and the lack
of more sensitive detection methods adapted for oocyst detection in food
undoubtedly contribute to this under-reporting. Control of foodborne out-
breaks caused by parasites such as Cryptosporidium and cyclospora is directly
related to methods that prevent food contamination in the first place. Removal
or inactivation of oocysts of both parasites is a formidable task, since these
organisms strongly attach themselves to produce surfaces. Oocysts have proven
highly resistant to sanitizers and disinfectants, particularly at concentrations
that would not affect the organoleptic characteristics of the fresh produce.
Possible vehicles of transmission have been suspected to be contaminated
soils, fertilizers, pesticide solutions, and irrigation water containing human or
animal waste. Washing hands, appropriate hygiene, and GAPs may contribute
to the prevention of pathogens in ready-to-eat foods.
Outbreaks Associated with Cyclospora and Cryptosporidium 275
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13
Patulin
Lauren Jackson and
Mary Ann Dombrink-Kurtzman
CONTENTS
13.1 Introduction 282
13.2 Methods of Analysis 283
13.2.1 Introduction 283
13.2.2 Thin-Layer Chromatography (TLC) 283
13.2.3 Gas Chromatography (GC) 284
13.2.4 Liquid Chromatography (LC) 284
13.2.5 Micellar Electrokinetic Capillary Chromatography
(MECC) 285
13.2.6 Other Methods 285
13.3 Toxicological Effects of Patulin 286
13.3.1 Introduction 286
13.3.2 Acute Toxicity Studies 286
13.3.3 Immunotoxicity Studies 286
13.3.4 Reproductive Toxicity and Teratogenicity Studies 287
13.3.5 Genotoxicity Studies 287
13.3.6 Carcinogenicity Studies 287
13.3.7 Mechanism(s) of Toxicity 288
13.4 Regulatory Aspects 288
13.5 Fungal Species Producing Patulin in Foods 288
13.6 Natural Occurrence of P. expansum and Patulin in
Fruits and Vegetables 289
13.7 Factors Affecting Patulin Production 290
13.7.1 Introduction 290
13.7.2 Physical, Chemical, and Microbial Properties of Apples . . 291
13.7.3 Environmental Factors 292
13.8 Approaches for Controlling Patulin Levels 293
13.8.1 Introduction 293
13.8.2 Preharvest 294
13.8.3 Harvest 295
13.8.4 Postharvest 295
13.8.4.1 Introduction 295
13.8.4.2 Washing Treatments 296
13.8.4.3 Culling, Sorting, and Trimming 297
281
282 Microbiology of Fruits and Vegetables
13.8.4.4 Chemical, Heat, and Biological Control, and
Irradiation Treatments 297
13.8.4.5 Storage 299
13.8.4.6 Controls for Processed Apple Products 300
13.9 Conclusions 300
References 301
13.1 INTRODUCTION
Mycotoxins are a chemically diverse group of toxic secondary metabolites
produced by filamentous fungi. They are responsible for significant financial
losses for the food industry, particularly any aspect of the industry that har-
vests, stores, processes, or uses commodities or ingredients. Mycotoxins elicit a
variety of acute and chronic toxic effects in domestic animals and humans
including reduced growth efficiency, vomiting, reproductive problems, cancer,
and immunosuppression [1,2]. Worldwide, mycotoxins pose a threat to public
health, agriculture, and economics [3].
Patulin is a mycotoxin produced by fungi belonging to several genera
including penicillium, aspergillus, and byssochlamys. Although patulin can
occur in many molding fruits, grain, and other foods, the major source of
patulin contamination is apples with blue mold rot, and in apple cider or apple
juice pressed from moldy fruit. Penicillium expansum is believed to be the major
fungal species contributing to patulin in apple products. Mold growth occurs
when the surface tissue of fruit has been damaged by improper handling, insect
or storm damage, and is often followed by production of patulin. P. expansum
and patulin contamination of fruit can occur before harvest, but they are more
commonly found as contaminants of apples postharvest and during storage.
Thermal processing is effective in destroying microorganisms such as bacteria,
yeast, and most fungi. However, patulin is fairly heat resistant, especially in
acidic environments. The stability of patulin is illustrated by the presence of the
toxin in shelf-stable apple products (juices, concentrates, jellies, baby foods,
etc.) [4-7]. Since the compound persists in heated juices, it has been suggested
that the presence of patulin in processed apple products may be a good
indicator of the quality of the fruit used in production.
Patulin has been demonstrated to be acutely toxic [8], genotoxic [9],
teratogenic [8,10,11], and possibly immunotoxic [12,13] to animals. Although
the toxicity of patulin in humans has not been demonstrated conclusively, there
is a desire to limit its concentration in apple juice since young children and
infants are major consumers of this product, and the effects of long-term
exposure to patulin are not known. Many countries, including the U.S., have
set regulatory limits for patulin in apple products of 50 ug/1 or less.
This chapter reviews the literature on the chemical properties of patulin,
methods for monitoring the occurrence and levels of patulin in food, regulation
of patulin levels, factors affecting growth of P. expansum and patulin
Patulin 283
FIGURE 13.1 Chemical structure of patulin (4-hydroxy-4//-furo[3,2-c]pyran-
2(6//)-one).
formation, and methods for controlling the levels of this toxin in apple
products.
1 3.2 METHODS OF ANALYSIS
13.2.1 Introduction
Patulin, 4-hydroxy-4//-furo[3,2-c]pyran-2(6//)-one (Figure 13.1), is a low-
molecular-weight (MW 154) of, ^-unsaturated y-lactone with a melting point
of 110°C. Patulin is stable under acidic conditions and resistant to thermal
treatments, but it is unstable at alkaline pH [14]. The toxin is soluble in water,
ethyl acetate, methanol, acetonitrile, and acetone, and less soluble in diethyl
ether and benzene. It reacts with sulfhydryl groups such as those in cysteine
and glutathione, free amino groups, sulfur dioxide, and ascorbic acid [15-17].
Patulin is metabolized by yeast {Saccharomyces cerevicae) in fermenting cider
into a variety of compounds including E-ascladiol and Z-ascladiol [18].
As more countries have passed regulatory limits for patulin in apple
products, there have been increasing efforts to develop sensitive, selective, and
rapid procedures for measuring patulin levels in food. Monitoring of patulin in
apple juice, apple juice concentrates, and apple cider is performed to comply
with regulatory limits set by the U.S. Food and Drug Administration (FDA)
and regulatory agencies throughout the world [19]. The majority of the
methods currently used are based on the Association of Official Analytical
Chemists (AOAC) official methods, involving liquid-liquid extraction of
patulin with ethyl acetate, followed by use of high-performance liquid chroma-
tography (HPLC) for detection and quantification. If there is need for
confirmation of the amount of patulin in a product, gas chromatography/mass
spectrometry (GC/MS) is performed. Shephard and Leggott [20] published an
excellent review of the chromatographic methods used to determine patulin
levels in fruit products. The following is an overview of the analytical methods
used for quantifying patulin in food.
13.2.2 Thin-Layer Chromatography (TLC)
The first method developed for detection of patulin and adopted as an AOAC
official method (AOAC Official Method 974.18) involved the use of normal
phase TLC [21]. Apple juice is extracted with ethyl acetate, and the extract
284 Microbiology of Fruits and Vegetables
partially purified on a silica gel column. Patulin is eluted, concentrated, and
detected by TLC using normal phase silica gel plates which are typically
developed in toluene/ethyl acetate/formic acid and then sprayed with 3-methyl-
2-benzothiazolinone hydrochloride (MBTH). Patulin appears as a yellow-
brown fluorescent spot under UV light at 366 nm. The method has a limit of
detection of ~20 jig patulin/1 apple juice. More recently, Prieta et al. [22]
described an analytical method for patulin using diphasic dialysis for
extraction of patulin from juice, followed by separation on TLC silica gel
plates, detection with MBTH, and quantification by densitometry. The authors
reported a detection limit of 50 jig patulin/1 juice and extraction recovery of
65% [20]. TLC remains the method of choice for detection of patulin in many
parts of the world, especially in developing countries.
13.2.3 Gas Chromatography (GC)
Although not the method of choice of researchers and regulatory laboratories,
GC methods have been developed for analysis of patulin. These methods
generally involve the formation of trimethylsilyl ether derivatives and detection
by electron capture or mass spectrometry (MS) [20]. Tarter and Scott [23]
described the use of heptafluorobutyrate (HFB) derivatives of patulin with
chromatographic separation on a nonpolar fused silica capillary column and
electron capture detection. Application of the method to naturally contami-
nated apple juice gave a detection limit of 10 \ig patulin/1. A recent publication
by Llovera et al. [24] described the detection of underivatized patulin by MS at
a detection limit of 4 \xg patulin/1 apple juice.
13.2.4 Liquid Chromatography (LC)
HPLC methods are the most commonly employed methods for the quanti-
tation of patulin in fruit juices. HPLC requires a large initial cash investment,
but provides good sensitivity, precision, and ease of use. In addition, a skilled
and experienced staff is required to operate and maintain the HPLC
equipment.
Almost all published methods involve liquid-liquid extraction of patulin
into ethyl acetate, a cleanup step using a sodium carbonate solution to remove
interfering phenolic compounds, and HPLC with UV detection to separate and
detect patulin [25,26]. HPLC columns typically are reversed phase (CI 8), and
the mobile phases tend to be predominately mixtures of water and acetonitrile
(up to 10%v/v) or tetrahydrofuran (up to 5%v/v). Although patulin can be
detected with single-wavelength UV detectors (276 nm), many laboratories
use photodiode array (PDA) detectors to detect patulin and spectrally dis-
tinguish the compound from coextracted compounds such as polyphenols and
hydroxymethyl furfural (HMF).
The HPLC procedure described by Brause et al. [26] was subjected to an
interlaboratory study on method reproducibility and accuracy. In this
collaborative study, 22 laboratories analyzed apple juice spiked with 20 to
Patulin 285
200 ug patulin/1 as well as naturally contaminated juice containing 3 1 ug
patulin/1 [25,26]. Mean recovery of patulin spiked into juice was 96%. Based on
the results of this collaborative study, the method was adopted as a first action
method by AOAC International (AOAC Official Method 995.10) [27].
A second AOAC official method for determination of patulin by HPLC
describes the analysis of clear and cloudy apple juices and apple purees. The
method (AOAC Official Method 2000.02) [28] is based on a publication by
MacDonald et at. [29]. This method differs from that of Brause et al. [26] in the
use of pectinase prior to extraction to remove the cloudiness present in some
juice samples.
Solid phase extraction (SPE) methods recently have been developed for
extracting and purifying patulin from apple juice. In the method developed by
Trucksess and Tang [30], patulin was extracted from undiluted apple juice with
a reversed-phase SPE (Oasis, Waters, Milford, MA) column. The column was
washed to remove interfering compounds, and patulin was eluted and then
detected by HPLC with a recovery of 93 to 104% [30]. Recently, Eisle and
Gibson [31] modified the method of Trucksess and Tang [30] and reduced
analysis time to approximately 1 hour including extraction and HPLC analysis
steps.
13.2.5 Micellar Electrokinetic Capillary
Chromatography (MECC)
Neutral compounds, such as patulin, or mixtures of neutral and charged
compounds can be analyzed by MECC [32]. There are various advantages for
using MECC. Only a small amount (2 ml) of sample is used and less organic
solvent is consumed compared to HPLC methods. The method is rapid, with
total run time of 10 minutes, and has a low limit of detection (3.8 ug patulin/1).
For samples having patulin levels < 75 ug/1, 2 ml of sample is extracted with
ethyl acetate. The extract is passed through anhydrous sodium sulfate,
evaporated under nitrogen, reconstituted in 0.1 ml acidic water solution (pH 4),
and analyzed immediately by MECC.
13.2.6 Other Methods
Although the previously described HPLC methods give accurate and precise
measurements of patulin levels in fruit products, they can be laborious, and
their results are not attainable for at least several hours. Efforts are being made
to develop immunochemical techniques for rapidly ( < 30 minutes) quantifying
patulin in juice products. As opposed to other mycotoxins, no commercial
enzyme-linked immunosorbent assay (ELISA) kits are available for patulin.
Production of suitable antibodies for use in ELISA kits is needed. Many
research groups have attempted to produce antibodies capable of detecting
patulin. Unfortunately, these efforts have not met with success.
An alternative approach for detection of patulin may be to develop
methods for the synthesis of molecularly imprinted polymers (MIPs), highly
286 Microbiology of Fruits and Vegetables
crosslinked polymers, capable of binding specifically to patulin. During the
polymerization process the template (patulin) interacts with one or more of
the functional monomers present. When the template or structurally related
compound is removed from the polymer, a cavity capable of binding patulin
remains. Because MIPs have the advantage of high chemical and physical
stability, they have been described as "plastic antibodies" and have the poten-
tial for use in place of antibodies in applications such as affinity separation
assay systems and biosensors [33]. A paper describing the synthesis of MIPs
with selective binding properties for the mycotoxin ochratoxin A was published
by Jodlbauer et al. [34]. A critical component for success in MIP synthesis is the
availability of compounds that can serve as mimics of the template of interest
(patulin).
13.3 TOXICOLOGICAL EFFECTS OF PATULIN
13.3.1 Introduction
Study of patulin's toxicity began over 60 years ago when the compound was
first isolated from Penicillium patulum (now called P. griseofulvum) and found
to possess antimicrobial properties. Patulin was later isolated from other fungal
species and given the names clavacin, claviformin, expansin, mycoin, and
penicidin [35]. During the 1940s research was aimed at finding pharmaceutical
uses for patulin. For example, patulin was tested as a treatment for the com-
mon cold as well as an ointment for treating fungal infections [36]. However,
animal studies revealed that, in addition to antibiotic properties, patulin also
possessed toxic effects [37,38].
Research on the toxicological properties of patulin has shown the
compound to be acutely toxic in animals and to have possible genotoxic,
immunotoxic, and teratogenic effects. Patulin toxicity data have been reviewed
in detail [38-40]. In addition, the FDA [19] independently reviewed the
available information on patulin toxicity.
13.3.2 Acute Toxicity Studies
In acute toxicity studies with a variety of experimental animals, the LD 50
values for patulin, as well as the lesions observed, varied [41]. Overall, patulin
produced lesions to the gastrointestinal tract, including epithelial degeneration,
hemorrhaging, and ulceration of the gastric mucosa [41-43]. Other lesions
included edema of the lungs and brain, visceral organ congestion, and hepatic
and renal necrosis [44]. The oral LD 50 in rats, mice, and hamsters has been
reported in the range 30 to 48mg/kg body weight [41].
13.3.3 Immunotoxicity Studies
Studies on the effects of patulin on the immune system have shown con-
flicting results. At relatively high doses, patulin has been shown to have
Patulin 287
immunosuppressive properties ranging from cytotoxicity in rat alveolar
macrophages to increases in neutrophils [12,13]. However, Llewellyn et al.
[37] found that 28 days of oral exposure to patulin at levels comparable to
human exposure from apple juice did not have toxic effects to the immune
system of female B6C3F! mice.
13.3.4 Reproductive Toxicity and Teratogenicity
Studies
Reproductive toxicity and teratogenicity studies in mice, rats, and chicken
embryos indicate that patulin is a possible teratogen. Dailey et al. [8] reported
no reproductive or teratogenic effects in mice or rats dosed with patulin at
levels of up to 1.5mg/kg body weight/day. However, maternal toxicity and
an increase in the frequency of fetal resorptions were observed at higher
levels, which indicate that patulin was embryotoxic. When injected into the air
cell of chick eggs, patulin was reported to be embryotoxic at levels of 2.35 to
68.7|ig/egg depending on the age of the embryos, and teratogenic at levels of
1 to 2|ig/egg [45]. Roll et al. [10] found that patulin, when administered
intraperitoneally to female mice on day 12 and 13 of pregnancy, caused an
increase in the incidence of cleft palates and malformation of the kidneys of the
developing fetuses.
13.3.5 Genotoxicity Studies
Although patulin failed to show mutagenicity in the Ames test and other
bacteria-based assays, it has been shown to produce chromosomal damage in
mammalian systems [9,10,40]. Patulin was shown to be potent inducer of
chromatid-type aberrations to Chinese hamster V79E cells, but did not increase
sister-chromatic exchange (SCE) frequency. In contrast, Liu et al. [46] reported
that patulin caused a significant dose-dependent increase in SCE frequency
in both Chinese hamster ovary cells and human lymphocytes. Induction of
chromosome damage and micronuclei formation in mammalian cells suggest a
possible clastogenic property of patulin [46]. Nucleic acid synthesis and protein
synthesis have also been reported to be inhibited by patulin [47,48].
13.3.6 Carcinogenicity Studies
From the available toxicological data, it is unclear whether patulin is a
carcinogen. In a study by Dickens and Jones [49], patulin, when administered
subcutaneously twice a week to rats for 15 months, induced sarcomas at the
injection sites. However, in two long-term studies, patulin administered orally
by gavage was not carcinogenic in rats or mice [44,50]. In their review of
these studies, IARC39 concluded that no evaluation could be made of the
carcinogenicity of patulin to humans and that there was inadequate evidence in
experimental animals [39].
288 Microbiology of Fruits and Vegetables
13.3.7 Mechanism(s) of Toxicity
While patulin has been found to exhibit cellular toxicity in in vivo and in vitro
tests, the mechanisms of cellular toxicity are not clear. Patulin alters the plasma
membrane functions in cultured LLC-PK1 renal cells through an inhibition of
Na + -K + ATPase [51,52]. The compound also inhibits several key biosynthetic
enzymes including RNA polymerase and aminoacyl-tRNA synthetases [53].
Due to its electrophilic nature, patulin reacts readily with cellular nucleophiles
such as the sulfhydryl-containing compounds cysteine and glutathione [15].
The mode of action of patulin may be through oxidation of critical sulfhydryl
groups in cell membranes or in enzymes [52]. Patulin adducts formed with
cysteine or glutathione were less toxic than the unmodified compound in acute
toxicity, teratogenicity, and mutagenicity studies [40,54]
1 3.4 REGULATORY ASPECTS
At present, there are no published toxicological or epidemiological data to
indicate whether consumption of patulin is harmful to humans. Products con-
taining patulin have probably been consumed for long periods of time, yet
accounts of human toxicity caused by patulin exposure from food do not exist.
However, there is a desire to limit patulin levels in apple products since infants
and young children are major consumers of these foods and the effects of long-
term exposure to patulin are not yet known.
Based on the results of reproductive toxicity and long-term toxicity studies
involving animals, the Joint Food and Agriculture Organization/World Health
Organization Expert Committee on Food Additives (JECFA) established a
provisional maximum daily intake for patulin of 0.4|ig/kg body weight [55].
At least ten countries have established action levels of 50 (ig/1 for patulin in
apple juice, and several have established lower limits (25 to 35 jig/1) [3,56]. The
FDA has established a 50 |ig/l action level for patulin in single strength and
reconstituted apple juice [19].
13.5 FUNGAL SPECIES PRODUCING PATULIN IN
FOODS
A variety of fungi are reported to be capable of producing patulin in defined
media including Aspergillus clavatus, A. giganteus, A. terreus, Byssochlamys
fulva, B. nivea, Paecilomyces variotii, Penicillium carneum, P. clavigerum,
P. concentricum, P. coprobium, P. dipodomyicola, P. expansum, P. glandicola,
P. griseofulvum (formerly known as P. patulum, P. urticae), P. roqueforti, P.
sclerotigenum, and P. vulpinum [61-66]. However, P. expansum is considered
the major producer of patulin in food, and, in particular, pome fruits such as
apples. In pure culture, P. expansum is reported to produce over 59 secondary
metabolites including patulin, citrinin, cyclopiazonic acid, chaetoglobosins A
and C, roquefortine C, penicillic acid, and ochratoxin. However, only some of
Patulin 289
these metabolites (patulin, citrinin, chaetoglobosins, roquefortine) were
actually detected in apples and apple products [61-66].
Reviews on the physiology and growth characteristics of P. expansum and
other fungi that produce patulin have been published [67,68]. In addition,
Doores [69] wrote a comprehensive review of the microbiology of apples and
apple products. Penicillium expansum is a psychrophile [67]. The optimum
growth temperature for this species is near 25°C, but there are reports of
growth of the organism at — 3°C [68]. Minimum water activities for spore
germination are 0.82 to 0.83 [70]. Penicillium expansum has a very low require-
ment for oxygen; the organism was found to grow at atmospheric oxygen levels
of less than 2%. Carbon dioxide concentrations of up to 15% have been found
to stimulate growth of the organism [68]. The growth characteristics of the
fungus help explain the finding of P. expansum and patulin in apples stored
under modified atmosphere conditions [71].
Although patulin is typically not destroyed during pasteurization of juices,
P. expansum and its spores typically do not survive this thermal treatment.
However, other species of patulin-producing fungi (B. fulva and B. nivea)
produce spores that are resistant to processing at temperatures of 90°C [72].
Consequently, there is a possibility of patulin production in stored juices
if spores of these fungi germinate. At present, it is unclear if heat-resistant
ascospores contribute significantly to the patulin content of apple juice
products.
13.6 NATURAL OCCURRENCE OF P. expansum AND
PATULIN IN FRUITS AND VEGETABLES
Penicillium expansum is one of the most pervasive and destructive postharvest
pathogens of pome fruits such as apples and pears, but it can affect other fruits
including tomatoes, strawberries, avocados, bananas, mangoes, grapes,
peaches, and apricots [3,68]. The primary habitat of P. expansum is in fruit
storage and packinghouse facilities, but it can also be found in orchard soil,
seeds of various plants, and on the surface and in the core of unblemished fruit.
The fungus is primarily a wound pathogen, gaining entrance through fresh
mechanical injuries such as stem puncture, bruises and insect injuries, hail or
weather-related damage, and fingernail scratches caused by fruit pickers [73].
There are also reports of the fungi entering apple fruits through open calyx
canals, at the point of attachment of stem to fruit, and through skin lenticels
[73]. The infection often occurs while apples are still on the tree, but it remains
latent until the fruit is harvested and stored [66]. The appearance of the decay
caused by P. expansum is characterized by rotten areas that are soft, watery,
and light brown in color. The surface of older lesions may be covered by
bluish-green spots that initially are white in color [73].
Although P. expansum can be isolated from the surface of a wide variety of
fruits, patulin has only been detected in apples, pears, blueberries, cherries,
peaches, plums, strawberries, raspberries, and mulberries [3]. The organism is
290 Microbiology of Fruits and Vegetables
rarely isolated from vegetables [67]. The mere presence of P. expansum does not
necessarily imply that patulin will be present since mycotoxin production is
influenced by many factors including environmental conditions, cultivar and
nutritional status of the fruit, the microbial load on the fruit, and strain of the
fungus [3,74].
Patulin is found with greater incidence and concentration in apples than in
other fruit, and they contribute the vast majority of patulin in the human diet
[19]. The toxin has been detected in intact fruit, juice, cider, applesauce,
and apple puree. Whole apples (table fruit) are not believed to contribute
significantly to human exposure since contaminated fruit is often discarded or
trimmed to remove moldy areas before it is eaten. The greatest exposure to
patulin comes from consumption of apple juice and cider pressed from moldy
fruit [19].
Numerous surveys have been published on the incidence and concentration
of patulin in apples and apple products [5-7,75-82]. Harwig et al. [83] surveyed
61 samples of whole apples from different orchards in Canada. Penicillium
expansum was isolated from 42 of the samples, while patulin was found in 28
samples of expressed juice at levels up to 240 u.g/1. Wilson and Nuovo [76]
analyzed 100 samples of freshly pressed apple cider and detected high levels of
patulin (up to 45,000 u.g/1) in several samples of cider produced from
organically grown fruit. The authors concluded that cider samples with the
highest patulin concentrations were made from ground-harvested and rotten
fruit. In contrast to these results, Malmauret et al. [84] and Riteni [82] reported
no significant difference in patulin levels from fruit grown organically
compared to conventionally grown fruit. In surveys of apple products obtained
in Turkey and New South Wales, Australia, Yurdun et al. [79] and Burda [6]
reported that >25% of juice samples contained > 50u.g patulin/1, and several
samples contained 500 to 1000 ug patulin/1. Watkins et al. [78] analyzed apple
juice purchased in Victoria, Australia, and found that >65% of samples were
contaminated with patulin, and >33% had levels over 50u.g patulin/1. In
contrast, Ritieni [82], Leggott and Shephard [80], and Lai et al. [85] reported
that patulin levels in almost all tested apple products purchased in Italy, South
Africa, and Taiwan, respectively, were < 50 u.g/1. A survey conducted of apple
juices purchased between 1994 and 2000 in the U.S. revealed that 12.6% of
juices had patulin levels over 50ug/l, and approximately 6% had levels
> 100u,g/l [81]. Overall, surveys of apple products indicate that, although the
incidence of patulin contamination is fairly high, levels of contamination are
typically less than 50 u.g patulin/1.
13.7 FACTORS AFFECTING PATULIN PRODUCTION
13.7.1 Introduction
At present, there are a number of factors known to affect production of patulin
in apple products. Within a species, the mycotoxigenic potential of a fungus
depends mainly on the strain of fungus. Although genetic variation may be the
Patulin 291
ultimate cause of the differences between strains with regard to fungal growth
and mycotoxin production, physical and chemical properties of the food and
environmental factors such as incubation temperature and time are also
important factors [86]. Patulin production in fruit is believed to be affected by
many factors including apple cultivar, geographical location where the fruit is
grown and harvested, climate, preharvest treatments, method of harvest,
surface defects on the fruit, postharvest treatments, and storage conditions.
At present, it is not clear which of these factors plays the greatest role in
mycotoxin production or how they can be manipulated to prevent or reduce
patulin contamination of apple products. A better understanding of the aspects
influencing patulin production may aid in developing effective means for
controlling mycotoxin formation in food. The following is a description of
some factors known to affect patulin production in apples.
13.7.2 Physical, Chemical, and Microbial
Properties of Apples
Several investigators have shown that fruit cultivars differ in their suscept-
ibilities to P. expansum rot and to patulin formation in the apple tissue.
Jackson et al. [87] reported that cider pressed from ground-harvested Red
Delicious apples had significantly higher levels of patulin than cider prepared
from other apple cultivars (Golden Delicious, Granny Smith, Fuji, Gala,
Macintosh, Red Rome). Of the four cultivars studied by Spotts and Mielke
[88], Royal Gala apples were found to be most resistant to decay from
P. expansum, while Fuji apples were least resistant. These results, along with
those published by others [65,70,76,86,89,90], indicate that apple cultivars
differ substantially in susceptibility to blue mold rot. The differences in
cultivars may be due to unique physical and chemical characteristics of each
cultivar such as skin thickness and strength, flesh firmness, pH of flesh, sugar
levels, levels of antimicrobial compounds, and other apple constituents
[86,89,91]. Apple cultivars with an open calyx are at greater risk for patulin
development within the apple core [92]. Since core rot is often not detected,
juice or cider pressed from affected fruits may have high patulin levels.
Penicillium expansum can be isolated from the surface of unblemished fruit,
although the fungus typically does not grow until it is able to make contact
with the flesh of the fruit. The pathogen enters the fruit through skin breaks
caused by bird, insect or weather-related damage, improper handling, and
through damaged lenticels near bruised areas [73]. Several investigators
reported greater patulin levels in apple juice pressed from damaged than from
sound fruit [76,87,93,94]. Susceptibility to surface wounds and bruising is
influenced by apple cultivar, but it also may be a function of degree of maturity
of the fruit, since skin layers soften during the ripening process [3,67]. Other
factors such as mineral imbalances (e.g., high nitrogen, low calcium) are
believed to increase susceptibility of fruit to infection. Mineral imbalances are
caused by improper fertilization, excessive or too little rain, and poor soil
conditions [95].
292 Microbiology of Fruits and Vegetables
At present, the effects of the chemical composition of fruit on patulin
production are not well understood. Apples are composed of a complex
mixture of sugars (primarily fructose, glucose, and sucrose), oligosaccharides,
and polysaccharides, together with malic, quinic, and citric acids, polyphenols,
amides and other nitrogenous compounds, soluble pectin, vitamins, minerals,
water, and a variety of esters. The relative proportions of these components
depend on the apple cultivar, the conditions under which the apples were
grown, the state of maturity of fruit at the time of pressing, and extent of
damage to the fruit. Patulin is produced over the range of pH values found in
apple juice (3.2 to 3.8) and is stable at these pH values, but degrades at
higher pH values [32,91,96]. McCallum et al. [36] found that the concen-
tration of patulin formed in juice was correlated negatively with the pH value.
Prusky et al. [97] reported that Penicillium spp. colonization and growth are
enhanced by low pH in the host tissue. They also found that P. expansum
actively reduced the pH during decay development by causing accumulation
of fumaric and gluconic acid in the fruit tissue.
The mineral content of apples may influence degree of decay by postharvest
pathogens. Calcium is believed to be the major mineral nutrient affecting apple
quality and storage life [98,99]. The effect is thought to be partly due to the
role of the mineral in preventing physiological disorders in the developing
fruit [99-102]. Calcium is also believed to improve fruit firmness by forming
complexes with pectic substances in the cell wall [101,103]. The influence of
other minerals and antimicrobial compounds in apples (e.g., phenolic
compounds) on their susceptibility to fungal rot and mycotoxin production
is not known at this time. Research is needed to determine how the apple
constituents affect patulin formation.
Microbiological factors can influence patulin formation. The micro-
biological flora on apples and other fruit differs according to geographic area,
climatic conditions, pesticide or fungicide treatments, cultivar, presence of
competitive microorganisms, harvest method, and postharvest treatments [69].
Spores of P. expansum are found in soil, on plant surfaces, and in air and are
transferred to dump tank and flume water in packinghouses by contaminated
wooden picking bins and fruit [104]. Early findings by Sommer et al. [74]
indicate that the presence of a patulin-producing species does not necessarily
imply patulin production in apples. Factors like incubation temperature, lesion
size, and substrate, also play important roles. Substantial differences have been
noted among P. expansum strains in terms of growth kinetics and patulin
production [86]. McCallum et al. [86] found that highest patulin levels were
those from isolated strains displaying aggressive growth and profuse mycelial
development.
13.7.3 Environmental Factors
Understanding the environmental conditions influencing mycotoxin produc-
tion is important so that storage environments can be made unfavorable for
fungal growth and toxin production. Temperature is one of the major factors
Patulin 293
that affect the shelf life of apple fruits and their rate of deterioration by fungi
[60]. The optimum temperature for patulin production has been reported in
the range 23 to 25°C [86,90]. Although patulin production tends to decrease
as temperature is decreased, patulin can be produced at low temperatures
(0 to 4°C). Consequently, refrigerated storage is not practical to inhibit totally
patulin production [60,105]. Storage time affects the degree of decay since
apples lose their natural resistance to infection with time [95].
Modified atmospheres can suppress both fungal growth and patulin
formation in apples. Modified atmosphere storage has been used for over 30
years as a means for extending the storage life of fresh produce. A modified
atmosphere of high carbon dioxide and low oxygen has been found to inhibit
the growth and sporulation of some fungi and the production of such
mycotoxins as aflatoxin, penicillic acid, and patulin [105-107]. Paster et al. [90]
found that an atmosphere of 3% C0 2 and 2% 2 completely inhibited patulin
production by P. expansum at 25°C, but production occurred in atmospheres
of 2% C0 2 and 10 or 20% 2 . Use of subatmospheric pressure, a type of
modified atmosphere, to extend storage life of fresh produce was studied by
Adams et al. [108] as a method for reducing growth and patulin production by
P. expansum and P. patulum. This work showed that pressures as low as
160mmHg are needed to control fungal growth and patulin production.
Moodley et al. [109] monitored patulin formation in whole apples stored
(14 days, 25°C) in polyethylene bags with different gas combinations. They
found that polyethylene, the most widely used material for retail packages of
apples, inhibited toxin production in apples by 99.5% and fungal growth by
68%, even in the absence of a modified atmosphere, when compared to
unpackaged apples.
13.8 APPROACHES FOR CONTROLLING PATULIN
LEVELS
13.8.1 Introduction
Apples and other pome fruit are major food crops, with over 40 million tons
being produced worldwide [110]. Fungal diseases, and in particular blue mold
rot from P. expansum, cause significant economic losses in the fruit growing
and processing industries. The losses from this disease can be significant (up to
10% of stored fruit) but can be substantially reduced by following proper
sanitation and control measures. An integrated approach, including careful
handing of fruit and strict hygiene in orchard, packinghouse, and in storage,
must be used for controlling P. expansum and hence patulin formation in fruit.
Reducing patulin levels in fruit juice and other processed apple products can be
achieved through the use of sound, healthy fruit, modified atmosphere storage,
culling of damaged and rotted fruit, trimming of rotted tissue, filtering juice
through activated carbon, and fermentation of cider with added yeast [80].
Guidelines for reducing postharvest decay of apples and other fruits have been
294 Microbiology of Fruits and Vegetables
published [73,95,111,112]. Codex Alimentarius Commission [92] published
recommendations for preventing patulin contamination of apple juice.
The next sections outline preharvest, harvest, and postharvest methods for
controlling patulin levels in apple products.
13.8.2 Preharvest
Although patulin production in fruit is believed to occur mainly postharvest,
several factors pertaining to the growing conditions of fruit trees may influence
fungal infection and mycotoxin production in apples. Codex Alimentarius
Commission [92] outlined good agricultural practices (GAPs) that may reduce
the likelihood of infection of fruit trees. Trees should be trimmed of dead
and diseased wood and mummified fruits and pruned to allow proper air flow
and light penetration [92]. It has been demonstrated that fruit with mineral
imbalances are more susceptible to infection by P. expansum and other fungal
pathogens. Supplementing fruit trees with foliar calcium sprays during the
growing season and use of minimal amounts of nitrogen fertilizer are some
methods for reducing preharvest infection of apple fruit by fungi [95,98].
Calcium is believed to reduce decay by maintaining the firmness of cell walls
during ripening [98,1 13]. Ammonium molybdate tetrahydrate has been studied
as foliar and soil treatment of several crops. When ammonium molybdate was
applied as a preharvest treatment to apple trees, a significant reduction in blue
mold decay was observed in the treated apples after three months' cold storage
[114]. Tests in vitro showed that the mode of action of the chemical is by
inhibiting germination of P. expansum spores [114].
Postharvest decay can be reduced by preharvest applications of fungicides.
Studies on the effectiveness of applications of ziram fungicide showed an
average reduction in decay of 25 to 50% [112,115]. Synthetic fungicides are
being developed to protect produce from a number of postharvest diseases.
However, problems associated with use of synthetic fungicides, such as pro-
liferation of fungicide-resistant pathogen strains, as well as concerns about
public health and environmental contamination, have increased the need for
development of alternative treatments [116].
During the past five years, biological control of postharvest fungal diseases
with naturally occurring antagonists (yeasts and bacteria) has become an
alternative to synthetic fungicide control [116]. The commercial products
Aspire {Candida oleophila strain 182, Ecogen Inc., Langhorne, PA) and
Biosave 10 and BioSave 11 (Pseudomonas syringae strains ESC10 and
ESC11, EcoScience Corp., Worcester, MA) are examples of commercial
biocontrol products available in the U.S. Biocontrol agents act by colonizing
the wounds of apples where decay proliferates. The organisms are believed to
inhibit growth of fungal pathogens by utilizing all of the available nutrients in
the wound. Although most success with biological control has been with
application of the antagonists to fruit postharvest, but before storage, there has
been some degree of success at preharvest treatment of apples with antagonists.
Nunes et at. [117] reported that although preharvest treatments with Candida
Patulin 295
sake were less effective than postharvest treatments against P. expansum, about
54% control was achieved by spraying the organism on Golden Delicious
apples while still on the tree. More work is needed to determine the efficacy of
preharvest biocontrol of P. expansum and to determine if biocontrol affects
postharvest formation of patulin in fruit.
13.8.3 Harvest
The condition of produce at harvest determines the length of time the crop can
be stored [112]. Stage of maturity at harvest is believed to be one of the main
factors determining the susceptibility of fruit to mechanical damage and to blue
mold rot during postharvest storage. Fruits become increasingly susceptible to
fungal invasion during ripening as the pH of the tissue increases, soluble sugars
build up, skin layers soften, and defense barriers weaken [118,119]. To reduce
undesirable biochemical changes, apples should be picked when mature but not
fully ripe to ensure that they can be stored for several months [112].
Studies indicate that bruising and skin punctures substantially increase
the susceptibility of fruit to decay. Gentle handling of fruit by pickers during
harvest and care during transport of the fruit from the orchard to the packing-
house, juice processing plant, or storage may prevent injury to the fruit [73].
Rain during harvest allows for increased fungal contamination and
infection [95]. Consequently, fruit should be harvested in dry weather con-
ditions and quickly transferred to cold storage. Fallen fruit in the orchard
should be discarded and not sold for the fresh market or used in processed
apple products. Jackson et al. [87] reported significantly higher patulin levels in
cider produced from ground-harvested apples than from tree-picked fruit. The
process of falling from the tree may result in cuts or cracks in the apple peel
that become infected from fungal spores from the soil.
One of the major methods for controlling P. expansum infection of apples
is improved sanitary practices during harvest [95,120]. This includes reducing
contamination of packing/storage bins with orchard soil by cleaning and
sanitizing bins before use. Studies by Spotts and Cervantes [120] found that
while steam was the most effective treatment on wood and plastic bins, chlorine
compounds, sodium o-phenylphenate (SOPP), and quaternary ammonia
compounds were also effective sanitizing agents. Sodium hypochlorite was
more effective on P. expansum spores on plastic than on wood bins [120].
Benomyl, iprodione, and captan were generally not effective disinfectants.
13.8.4 Postharvest
13.8.4.1 Introduction
Approximately 75% of the world apple crop is marketed as fresh whole apples,
with the remaining 25% finding its way into processing, primarily into apple
juice and cider [121]. After harvest, a portion of the apple crop is transported
to packinghouses where it is packaged for the fresh (table) fruit market.
296 Microbiology of Fruits and Vegetables
Fruit not sold for the fresh market is processed into juice and other products,
or is stored at cold (0 to 4°C) temperatures with or without modified
atmosphere to extend the shelf life and to provide a constant supply of raw
material for the fresh market and for the processed apple industry [122]. Since
the majority of patulin forms in fruit postharvest, considerable efforts have
been devoted to developing strategies for reducing proliferation of fungal
pathogens and contamination with patulin during storage. This section
outlines some of the major postharvest controls of patulin in apple products.
13.8.4.2 Washing Treatments
Organic matter (soil, plant material, decayed apples) can act as a reservoir of
fungal spores that contaminate fruit. It is important to maintain sanitary
conditions in all areas where fruit is packaged, stored, and processed. Proper
sanitation includes washing and sanitizing packing machinery, the walls and
floors of storage rooms, and the surfaces of all processing equipment [112].
Water systems (water flumes), used to float apples from field bins to bulk
tanks, minimize mechanical damage to fruit. Flume water typically contains
chlorine (sodium hypochlorite) or SOPP to reduce fungal spore load [112,123].
Active chlorine levels in flume water must be maintained periodically to ensure
spores are destroyed. Other chemicals that can reduce spore levels include
chlorine dioxide [124] and ozone [125], although both are not commonly used
disinfectants for flume water. Physical removal of fungal spores by filtration
has been reported to remove >92 to 99% of P. expansum conidia from flume
water [126].
According to recent surveys of industry practices, the majority of apple
packagers and processors wash apples upon receipt or immediately before
chopping and pressing to remove soil, rot, pesticide residues, insects, micro-
organisms, and other extraneous material [127-129]. Apples are typically
washed in dump tanks containing water or chlorinated water, with brusher-
scrubbers, and/or with high-pressure water sprayers [122,128]. Since
P. expansum and patulin are associated with the soft rot of apples, washing
may result in the removal of rotten areas of the apple and the partitioning of
patulin into the cleaning water [87,93]. Jackson et al. [87] found that washing
ground-harvested apples in a dump tank before pressing reduced patulin levels
in the resulting cider by 10 to 100%, depending on the initial patulin levels and
type of wash solution (water vs. chlorine) used. Sydenham et al. [94] found that
patulin levels in cider decreased from 920 to 190 (ig/1 after Granny Smith apples
were washed with water. Acar et al. [130] reported that patulin levels were
reduced by up to 54% when apples were washed with a high-pressure water
spray. Total removal of patulin during the wash treatments is unlikely since
patulin can diffuse up to 1 cm into healthy tissue [131]. Wash solutions other
than chlorine that have had efficacy in reducing mold counts in apples include
electrolyzed oxidizing water [132] and ozone [125], although their effects on the
patulin content of apples are not known.
Patulin 297
13.8.4.3 Culling, Sorting, and Trimming
Removal of decayed or damaged fruit or trimming moldy portions of apples
prior to packaging or processing have been reported to reduce patulin levels in
apple juice [4,93,94,131,133]. Wilson and Nuovo [76] surveyed 100 samples of
fresh cider and found that samples having the highest patulin levels were
produced by cider mills that did not remove decayed apples before pressing.
Similarly, Sydenham et al. [93,94] reported that removal of rotten fruit prior to
pressing significantly reduced patulin levels in cider produced from apples
stored at ambient temperatures for 7 to 35 days. Jackson et al. [87] reported
that patulin was not detected when apples were culled prior to pressing, but
was found in five out of seven varieties when cider was pressed from unculled
fruit. Although removing visibly decayed fruit before processing is a proven
method for reducing patulin levels in apple products, there is no guarantee that
culling alone can totally eliminate patulin. Apples with "invisible" sources of
fungal rot (core rot) can contaminate apple juice, cider, or puree with patulin if
they are not removed before processing. Apple cultivars susceptible to core rot
should be cut in half and fruit with signs of decay removed before processing.
In large-scale operations where this culling procedure is not practical, other
methods for detecting apples with core rot are needed.
13.8.4.4 Chemical, Heat, and Biological Control, and
Irradiation Treatments
Prior to storage, apples are often drenched with diphenylamine along with a
fungicide (thiabendazole) to prevent superficial scald [104]. Since some strains
of pathogens are developing resistance to fungicides, there has been a push to
use alternative postharvest control methods [113,134]. Treatments that have
shown some promise include the use of essential oils [135], organic acid fumi-
gants [136], calcium salts [98], carbonate and bicarbonate [137,138], chitosan
[139-141], 2-deoxy-D-glucose [142,143], heat, biological control, irradiation
[118], and combinations of these treatments.
Spraying cinnamon oil, cinnamaldehyde, or a potassium sorbate solution
on the surface of apples extended shelf life with respect to decay by P. expansum
[135]. Complete inhibition of patulin formation in liquid culture was found
with 0.2% lemon oil, and >90% inhibition was observed using 0.05% lemon
oil and 0.2% orange oil [60]. It is unclear if these treatments can be used
commercially to reduce fungal decay in apples or if they affect the shelf life
of fruit.
Preliminary studies by Sholberg et al. [136] indicate that fumigation of fruit
with short-chain organic acids prevents decay and could become an important
alternative to liquid sterilants such as sodium hypochlorite. The number of
lesions on apples caused by P. expansum decreased exponentially with
increasing time of fumigation with vinegar or acetic acid vapors [136,144].
Drawbacks to the use of acid fumigants include the need for an airtight
enclosure and the corrosiveness of the vapors to steel [136].
298 Microbiology of Fruits and Vegetables
Fungal decay in apples was reduced by postharvest application of calcium
solutions to fruit [98,113]. Direct application of calcium to fruit can be
accomplished by dipping or spraying fruit with calcium solutions or with
vacuum or pressure infiltration [99]. Calcium helps to maintain firmness of the
apple and to decrease the incidence of physiological disorders that enable
fungal pathogens to infiltrate the fruit tissue.
There has been an increased interest in the use of prestorage heat
treatments to prevent fungal decay of fruit. Heat can be applied to fruit as a
hot water dip, as steam, as hot dry air, and by short hot water rinses [145-148].
Leverentz et al. [134] reported that holding Golden Delicious apples at 38°C
for four days reduced decay after three months of storage at 0°C without
reducing fruit quality. Fallik et al. [148] reported that Golden Delicious apples
treated with a 15-second hot water (55°C) rinse followed by a brushing
treatment had less P. expansum decay than untreated apples or apples given a
dry heat treatment (96 hours at 38°C). One explanation for the enhanced
stability of the heat-treated fruit is that heated apples softened more slowly
than nonheated fruit. In addition, heat treatments may recrystallize the wax
layer on the surface of the apple peel or increase synthesis of wax in the peel
[148,149].
A promising alternative to chemical treatments is biological control of
postharvest pathogens [116,117,134,150,151]. Decay caused by P. expansum
has been controlled in pome fruits by bacterial and yeast antagonists in several
laboratory and pilot storage tests [134]. At least one yeast-based product and
two bacteria-based products are now commercially available for treating
apples after harvest. Several more are being developed for commercialization
[151-153]. Although biological control agents have exhibited excellent control
of fungal rot in fruit, their efficiency is sometimes lower than chemical control,
and they do not always give consistent results [112,153]. Microbial antagonists
have a poor ability to eradicate preexisting infections, while chemical
treatments are frequently more effective at controlling established infections
[153]. Use of a combination of microorganisms could improve the spectrum
of activity and reduce the required concentration of biocontrol agents [116].
El Ghaouth et al. [139,140] reported enhancing the biological efficacy of the
yeast Candida saitoana by combining it with either glycochitosan or with the
sugar 2-deoxy-D-glucose. Both approaches increased the protective and
curative activity of the yeast in controlling postharvest diseases. Droby et al.
[153] found that application of 2% sodium bicarbonate in combination with
Aspire consistently enhanced its biocontrol performance against penicillium
rot in apples. Similarly, McLaughlin et al. [154] demonstrated that the addi-
tion of calcium salts to yeast cell suspensions enhanced the ability of Pichia
guilliermondii to control postharvest diseases of apple. Pichia guilliermondii is
found as an occasional clinical isolate and therefore is of questionable safety
as a biocontrol organism [155]. In another study, a combination of a heat
treatment and the use of a yeast antagonist was more effective than either
treatment alone [134]. Clearly more work is needed to identify a combination
of treatments to control penicillium rots in apples. Additional research is also
Patulin 299
needed to determine if these treatments are able to inhibit patulin formation in
fruit [153].
Aziz and Moussa [118] studied the effect of gamma irradiation on
mycotoxin production in fruits stored under refrigeration conditions. After 28
days of storage, nonirradiated fruits were contaminated with higher levels of
mycotoxins (including patulin) than irradiated (3.5 kGy) samples. Mycotoxin
production was reported to decrease with increasing irradiation dose.
Although UV light has a lethal effect on bacteria and fungi, little has been
done to study the effects of UV irradiation on mold levels on apples. However,
Stevens et al. [156] reported that applying a yeast antagonist to fruit after UV
irradiation was the most effective treatment in reducing storage rot in peaches.
Use of gamma and UV irradiation to control fungal rot in fruit deserves
further study.
13.8.4.5 Storage
After harvest, apples are generally kept in cold storage at —1 to 3°C with or
without modified atmospheres. These treatments can extend the shelf life of
apples from 9 to 28 weeks, depending on apple cultivar [109]. Since apple
cultivars differ in their susceptibility to postharvest diseases, cultivars with
resistance to mechanical damage and infection should be chosen, especially if
they will be kept in long-term storage.
Although fungal growth is dramatically reduced at temperatures < 10°C,
the growth of P. expansum and production of toxin were not prevented during
cold storage [89,90,157]. Paster et al. [90] found that patulin levels in apples and
pears inoculated with different strains of P. expansum generally increased with
increasing storage temperature from to 25°C. Similarly, Beer and Amand [89]
reported that Macintosh apples stored at 4°C had substantially lower patulin
levels than fruit stored at 15 or 24°C. Apples are typically kept in cold storage;
however, when suitable refrigerated storage is not available, apples are stored
in the open in ambient conditions (i.e. deck storage). Sydenham et al. [94]
reported that patulin levels in deck-stored apples were 2445 ug/1 as opposed to
90 |ig/l in comparable refrigerated stored fruit. Fungal growth and patulin
formation increased with the length of storage [90,94]. Overall, the research
presented here indicates that apples should be kept in refrigerated storage when
possible to slow mold growth and reduce mycotoxin production.
Several researchers studied the effects of modified atmosphere con-
ditions and found that gas composition affected P. expansum growth and
patulin formation in fruit [90,158-160]. Lovett et al. [71] reported that juice
from modified atmosphere-stored apples (3% 2 , 1 to 3% C0 2 , to 3.3°C, and
>90% relative humidity; 14 weeks) had 500 |ig patulin/1 while juice made from
air-stored apples had 2000 to 3000 jag patulin/1. Stitton and Patterson [160]
reported that use of high (>3%) C0 2 atmospheres (greater than used for
commercially stored apples) was an effective fungistatic treatment for stored
apples. However, excessively high levels of C0 2 (>8%) negatively affected the
quality of some apple cultivars. Johnson et al. [159] reported a lower incidence
300 Microbiology of Fruits and Vegetables
of penicillium rots in apples stored at lower 2 conditions (0.75% 2 ) than
under higher 2 levels (1.0 to 1.25%). While in modified atmosphere storage,
apples should be examined periodically for fungal decay [92].
1 3.8.4.6 Controls for Processed Apple Products
Treatments that have shown promise at reducing patulin levels in apple juice
include filtration, centrifugation, use of charcoal, addition of ascorbic acid,
and fermentation [16,109,161-164]. Bissessur et al. [164] evaluated the effec-
tiveness of several clarification processes for the reduction of patulin in apple
juice. Pressing followed by centrifugation resulted in 89% reduction in levels of
the toxin. Patulin reductions using paper filtration, enzyme treatment, and
fining with bentonite were 70, 73, and 77%, respectively. These data suggest
that patulin tends to bind to the apple solids, which are removed from the juice
during treatment. Activated carbon treatment has also shown promise as a
method for reducing patulin levels in apple juice [163,165,166].
Several compounds have the ability to modify chemically patulin,
rendering the toxin undetectable in some analyses. Yazici and Velioglu [167]
found that adding vitamins (thiamine hydrochloride, pyridoxine hydrochlor-
ide, and calcium-d-pantothentate) to apple juice before storage at 4°C for
6 months reduced patulin levels by 55.5 to 67.7% versus controls (no vitamin
addition) that had 35.8% reduction in levels of the toxin. It is unlikely that
the use of these vitamins to reduce patulin levels in juice has any practical
value. Adding ascorbic acid (0 to 3%w/v) to apple juice has been reported
to reduce patulin levels by up to 80%, as measured by HPLC [16]. The
mechanism by which patulin interacts with ascorbic acid needs to be studied
in more detail.
Patulin is known to become analytically undetectable during the
production of cider from contaminated apple juice [18,168]. Analysis of
patulin-spiked fermentations by HPLC showed the appearance of two major
metabolites of patulin, one of which appeared to be E-ascladiol [18]. More
work is needed to determine the toxicity of these metabolites of patulin.
13.9 CONCLUSIONS
Contamination of apples and apple products with P. expansum and patulin
causes considerable financial losses for apple growers and processors.
Considerable efforts have been made to understand the conditions by which
fungal pathogens such as P. expansum infect fruit and produce patulin. Fungal
growth and mycotoxin production are known to result from an interaction of
many factors, including the chemical and physical properties of the affected
fruit crop, genetics of the fungus, environmental conditions, and preharvest,
harvest, and postharvest conditions. In order to devise strategies for prevent-
ing patulin formation, more research is needed to understand how these
factors separately and together can be used to prevent fungal and mycotoxin
Patulin 301
contamination. The research to date indicates that an integrated approach,
including careful handling of fruit to prevent structural damage and strict
hygiene in the orchard, packinghouse, storage, and processing facility, is
essential for reducing P. expansum decay and patulin formation. Research also
indicates that only sound fruit should be used for processed apple products.
Fallen fruit should be discarded and not sold for the fresh market or used in the
manufacture of processed apple products. Culling or removing damaged and
moldy fruit before processing is an effective method for reducing patulin
contamination of juice and cider. In addition, washing whole fruit before
pressing and filtering juice have been successful at reducing patulin levels in
juice products.
More research is needed to identify apple cultivars that are resistant to
fungal decay, especially those cultivars that are stored for extended lengths of
time. Although chemical treatments with synthetic fungicides traditionally
have been used to control fungal pathogens in fruit, biological control has
shown promise in preventing decay. As described in this chapter, postharvest
treatment of whole apples with biological antagonists, heat, and calcium and
other chemical treatments have been demonstrated as effective at reducing
fungal rot. However, information is lacking on how these and other treatments
affect patulin formation in fruit. As more countries have passed regulatory
limits for patulin in juices and other apple products, there is an increasing need
to develop analytical methods that can rapidly (< 30 minutes) quantify patulin
in food.
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14
Safety of Minimally
Processed, Acidified,
and Fermented
Vegetable Products
F. Breidt, Jr.
CONTENTS
14.1 Introduction 314
14.2 Vegetable Microflora 314
14.2. 1 Washing Procedures 316
14.2.2 Biocontrol in Minimally Processed
Vegetable Products 316
14.3 Fermented Vegetables 318
14.3.1 Fermentation Chemistry 319
14.4 Acidified Vegetables 319
14.4.1 Definitions and Regulations for Acid
and Acidified Foods 320
14.4.2 Pathogenic Bacteria 321
14.5 Organic Acids and Destruction of Pathogens 322
14.5.1 Specific Effects of Acids 323
14.5.2 Genetic Regulation of Acid Resistance 325
14.6 Conclusions 327
Acknowledgments 327
References 327
Paper no. FSR04-21 of the Journal Series of the Department of Food Science, North Carolina
State University, Raleigh, NC 27695-7624. Mention of a trademark or proprietary product does
not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or
North Carolina Agricultural Research Service, nor does it imply approval to the exclusion of other
products that may be suitable.
313
314 Microbiology of Fruits and Vegetables
14.1 INTRODUCTION
Food fermentation technology likely originated sometime between 8,000 to
12,000 years ago as plants and animals were being domesticated in the Middle
East, Africa, and Asia [1-3]. The development of primitive pottery technology
likely led to early fermentation experiments, either planned or unplanned.
Cheese, bread, and alcoholic beverages may have resulted from the
fermentation of milk, grains, fruits, and vegetables stored in ceramic jars or
pots. If these "spoiled" or fermented products were found to have desirable
sensory properties, they may have been developed as the first processed or
fermented foods [2]. An important characteristic of fermentation was the
increase in the storage lifetime during which foods could be safely eaten. The
microbial nature of food fermentation or foodborne illnesses was not
understood, however, until the advent of the science of microbiology in the
late 19th century. The fermentation of vegetables by lactic acid bacteria (LAB)
is now well understood as an effective means of preserving and ensuring the
safety of foods [4,5]. LAB are being considered for use in nonfermented
vegetable products as a means of ensuring safety and preventing spoilage [6-8].
Fermented and acidified vegetable products, such as sauerkraut, kimchi, olives,
and cucumber pickles, not only have desirable sensory qualities, but also have
an excellent safety record with no known reported cases of foodborne illness.
1 4.2 VEGETABLE MICROFLORA
The microflora on fresh fruits, grains, and vegetables can range from as low as
10 to 10 colony forming units (CFU) per gram [9,10]. On pickling cucumbers,
for example, the aerobic microflora is typically between 10 to 10 6 CFU/ml for
fresh fruit, with LAB less than 10 CFU/g [11]. In the absence of processing,
degradative aerobic spoilage of plant material by mesophylic microorganisms
occurs, with Pseudomonas spp., Enterobacter spp., and Erwinia spp. initiating
the process [10]. A variety of pathogens, including Salmonella spp., Shigella
spp., Aeromonas hydrophylia, Yersinia enter ocolitica, Staphylococcus aureus,
Campylobacter, Listeria monocytogenes, Escherichia coli, and others, may be
present on fresh vegetable products [12-15]. Pathogens on fruits and vegetables
may also include enteric, hepatitis, or polio viruses [16]. A variety of sources
may contribute to the occurrence of pathogenic bacteria on fruit and vege-
table crops, including exposure of plants to untreated manure or contaminated
water, the presence of insects or birds, personal hygiene practices of farm
workers, postharvest washing or hydrocooling water, and conditions of storage
during distribution [12,14]. A study comparing the use of organic fertilizer
(composted manure) and inorganic fertilizer from farms in Minnesota showed
significantly higher coliform counts on the organically grown vegetables
[17]. However, in this and related studies [18,19], pathogens, including E. coli
0157:H7, were not detected.
Removal of pathogenic and spoilage bacteria from fruits and vegetables
has proved difficult. Surface adherence of bacteria (Figure 14.1) may serve
Safety of Minimally Processed, Acidified, and Fermented Vegetable Products
315
10um150kV250E3 0001/00 SE
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. s ■-■ ■ -
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-
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00 SE
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FIGURE 14.1 Attachment of pathogenic bacteria to cucumber fruit. Adhesion
of bacteria to the surfaces of pickling cucumbers (Calypso variety) with wax: (A)
Staphylococcus aureus; (B) Lactobacillus plantarum; (C) Listeria monocytogenes; (D)
Salmonella typhimurium; (E) Enterobacter aerogenes ATCC 13048. Bar 5, 10mm.
(From Reina, L.D., Fleming, H.P., and Breidt, F., /. Food Prot., 65, 1881-1887, 2002.)
to enhance survival of bacteria during washing or sanitizing treatments.
Bacterial cell surface charge and hydrophobicity measurements have been
found to correlate with the attachment of cells to surfaces of cantaloupes and
cucumbers [20,21]. Dewaxing cucumber fruit led to increased adhesion of
L. monocytogenes and decreased adhesion for other bacteria with higher
relative surface hydrophobicity, including salmonella, lactobacilli, and
316 Microbiology of Fruits and Vegetables
staphylococci [20]. Biofilms of bacteria may be more resistant to sanitizing
agents and organic acid treatments than free or planktonic cells [22-24]. It is
likely that the vast majority of microorganisms in food processing environ-
ments occur in multispecies or multistrain biofilms on food or equipment
surfaces [25,26].
14.2.1 Washing Procedures
Washing procedures with water or chemical sanitizers typically result in only
a 1 to 2 log 10 decrease in bacterial cell numbers [24]. Hydrocooling proce-
dures used for some fruits immediately after harvest may even serve to increase
internalization of bacteria due to the vacuum created as internal gases in
fruits and vegetables contract with the reduction in temperature [27,28].
Bacteria may be protected in inaccessible locations on fruits and vegetables,
such as the cores and calyx of apples [29]. Attachment to wounded regions
or entry into the interior of fruits and vegetables through wounded regions or
stomata, pores, or channels may occur [20,30-32].
The packaging and storage conditions for minimally processed vegetable
products, including the use of modified atmosphere packaging, may
significantly alter microbial ecology. The extended shelf life of some minimally
processed vegetable products may result in an undesirable "safety index,"
a concept developed to define the risks associated with modified atmosphere
packaged foods [33]. This safety index is defined as the ratio of spoilage
to pathogenic bacteria in foods, measured as the relative cell concentrations
of these organisms. It has been argued, however, that the primary effect of
modified atmosphere packaging in extending the sensory quality of vegetable
products may be to decrease the metabolic activity of the vegetable material
[34]. In a model system, it was found that growth rates for L. monocytogenes,
A. hydrophilia, and Bacillus cereus may be reduced by modified atmosphere
conditions, but final cell density was not affected [35]. One major source of
concern is that Clostridium botulinum spores have been isolated from a variety
of vegetables, and this organism may, under the right conditions of tempe-
rature, pH, and atmosphere, grow and produce toxin in minimally processed
vegetable products if the O2 concentrations drop to 1% or lower [10].
14.2.2 Biocontrol in Minimally Processed
Vegetable Products
The survival and growth of bacteria on vegetable products can depend on the
competitive microflora present and the environmental conditions and
processing treatments [15,36]. The use of competitive microflora to enhance
the safety of minimally processed foods, including vegetable products, has been
proposed by a number of authors [5,37-39]. LAB have been nominated for this
role, partly because of their GRAS (generally regarded as safe) status and their
common usage in food fermentations. Application of this approach for mini-
mally processed fruit and vegetable products has led to mixed results. Vescovo
Safety of Minimally Processed, Acidified, and Fermented Vegetable Products 317
and co-workers isolated LAB from salad vegetables and, subsequently,
re-inoculated the vegetables with both the biocontrol cultures and selected
food pathogens, including aeromonas, salmonella, staphylococcus, and listeria
species [6,40]. The added LAB cultures were found to reduce or prevent the
growth of microbial pathogens. Conversely, a Lactobacillus delbruckii lactis
strain, known to inhibit E. coli on chicken skin due to the production of
hydrogen peroxide, did not alter the survival of E. coli 0157:H7 on fresh-cut
vegetables, possibly due to the presence of catalase on the plant surfaces [8].
Competition from aerobic microflora isolated from fresh vegetables,
other than LAB, including yeasts, Bacillus spp. and Pseudomonas spp., can
influence the survival and growth of microbial food pathogens. Pseudomonas
spp. have been shown to enhance [41], inhibit [42-44], or have no effect [45] on
the growth of L. monocytogenes in fruits and vegetables. A variety of pseudo-
monas and aeromonas isolates from fresh vegetables were found to confer
inhibitory activity against E. coli, salmonella, listeria, and staphylococcus
strains using an agar diffusion assay [46]. Competition studies have shown iron
sequestration by siderophores may influence the competition between
pseudomonads and L. monocytogenes [42,47], although some Listeria spp.
may be able to use exogenous siderophores as an iron source [48]. Buchanan
and Bagi [49] demonstrated that the effects of salt and temperature can control
the outcome of competitive growth of a L. monocytogenes Scott A and a
Pseudomonas fluorescens culture that was screened for the inability to produce
siderophores or bacteriocins. In a study by Del Campo et al. [45], competition
for nutrients between a Scott A strain of L. monocytogenes and saprophytic
bacteria from green endive was investigated. Enterobacteriaceae and pseudo-
monas were grown in competition with L. monocytogenes in minimal media
and media supplemented with yeast extract. In this case, enterobacteriaceae
but not pseudomonads species were effective in reducing the growth of the
L. monocytogenes culture. Because culture filtrates from enterobacteriaceae
were found to have no inhibitory effects in broth supplemented with yeast
extract, the data indicated that competition for nutrients (not end product
inhibition) was responsible for the inhibitory effect [45].
These studies illustrate the complexity of microbial interactions in and on
fruit and vegetable products. Varying environmental conditions may include
changes in the availability of nutrients, salt concentration, temperature, atmos-
phere, pH, and others. While further research is clearly needed, the use of
protective cultures should only be considered as a supplement to good manu-
facturing practice, not as a substitute for the proper handling and packaging
of vegetable products [5]. The use of biocontrol cultures may, therefore, be
considered to enhance existing hurdle technology to prevent the growth of
pathogens in foods. The hurdle concept [50] advocates the use of multiple
preservative factors to prevent the growth of pathogens. In fresh fruit and
vegetable products, the main factors affecting the growth of the indigenous
bacterial populations are sanitation, modified atmosphere packaging, and
refrigeration, as well as the competitive interactions of bacteria.
318 Microbiology of Fruits and Vegetables
Bacteria cultures selected for use in biocontrol applications should ideally
be isolated from the products for which they are intended to be used [39].
Development of successful biocontrol strategies for fresh fruit and vegetable
products may include the following steps: (1) isolation of potential biocon-
trol LAB from the product for which they are intended to be used; (2) reduction
of the total microflora in and on the vegetable product by one of a variety
of procedures, including heat, washing using chemical sanitizers, irradiation, or
others; (3) addition of the biocontrol culture to achieve an appropriate
initial population, as determined experimentally; (4) storage of the product
under refrigeration temperatures [39]. The shelf life of the product would then
be dictated by the growth of the biocontrol culture, but, to be successful,
the growth rates of a biocontrol culture presumably should be faster than
that of the target pathogens. While rapid growth and production of inhibitory
metabolites may be desirable from a safety standpoint, this may be a liability
as far as the quality of the product is concerned. Breidt and Fleming [7]
investigated the kinetics of acid production and inhibition of L. monocytogenes
by L. lactis using a mathematical modeling approach [7]. It was observed that
the growth and death of the L. monocytogenes culture could only be accu-
rately predicted by the model if pH was assumed to be the limiting variable,
rather than acid concentration, with cessation of growth around pH 4.6.
Further studies to characterize the kinetics of bacterial competition are needed
to aid in the development of biocontrol strategies.
1 4.3 FERMENTED VEGETABLES
Under the anaerobic conditions found with brined vegetables, rapid ferm-
entation by LAB and yeasts occurs, resulting in the destruction of most other
microflora, usually within a few days of the onset of fermentation [51].
In the U.S., cucumber pickles and sauerkraut represent the majority of ferm-
ented vegetable products. For pickles, fermentation was the primary means of
preservation until the 1940s, when direct acidification and pasteurization of
cucumber pickles was introduced (reviewed by Fleming et al. [51]). Currently,
fermented cucumbers represent roughly 30% of commercial production of
pickles, mostly for institutional markets (hamburger dill slices), with the
majority of the retail market being nonfermented acidified pickles which are
pasteurized to destroy vegetative microflora.
Vegetable fermentations typically begin with heterofermentative LAB, such
as Leuconostoc mesenteroides and end with the most acid-resistant homo-
fermentative LAB, usually Lactobacillus plantarum [1,52,53]. Lactobacillus
plantarum is able to tolerate a lower internal pH than other LAB, and this
feature may allow it to predominate in the terminal stages of most vege-
table fermentations [54]. During the fermentation of cucumbers and cabbage,
hexose sugars, including glucose and fructose, are typically converted to
lactic acid by homofermentative LAB via the Embden-Myerhof-Parnas
pathway, while the heterofermentative LAB will produce a combination of
Safety of Minimally Processed, Acidified, and Fermented Vegetable Products 319
lactic acid and acetic acid or ethanol, along with C0 2 via the phospho-
ketolase pathway [55]. When fructose is present, LAB can use this sugar as an
electron acceptor, producing mannitol, which subsequently can be converted
anaerobically to lactic acid with an appropriate electron acceptor [56].
In cucumber fermentation where malate is present, L. plantarum and other LAB
have been found to carry out a decarboxylation of malate to produce lactic
acid and C0 2 [57]. This one-step reaction occurs via malolactic enzyme, and is
analogous to the amino acid decarboxylation reactions described below [119].
During the reaction, a proton is taken up from the surrounding medium,
which helps to buffer cellular pH and causes the pH in the surrounding
medium to rise.
14.3.1 Fermentation Chemistry
In the U.S., commercial cucumber fermentations are typically carried out
with 5 to 6% NaCl, while cabbage fermentations are carried out with 2 to
3% NaCl [51]. During the growth of LAB in vegetable fermentations, a
variety of antimicrobial metabolic end products are produced, including org-
anic acids, peroxides, amines, thiols, bacteriocins, and other enzymes and
compounds [1,4,5,58-61]. These inhibitory compounds begin to accumulate
in the initial stages of fermentation. A combination of several factors,
including organic acids from the fermentation (up to 2 to 3% organic acids
may be produced), complete fermentation of available sugar, terminal pH
values around 3 to 3.5, and salt, can serve to destroy most vegetative bac-
terial cells, including human pathogens. Desirable textural and nutritional
properties of the fermented vegetables may be maintained during storage in the
fermentation brine for extended periods of time (a year or more) without
refrigeration.
14.4 ACIDIFIED VEGETABLES
For nonfermented, acidified vegetable products, acetic acid is commonly used
as an acidulant. At a concentration of 3.6% or greater, acetic acid-acidified
foods can be preserved without the addition of other antimicrobial agents or
use of heat treatments [62,63]. For pickled pepper products, acidification with
2% acetic acid to pH values around 3.2 was found to prevent microbial growth
for 6 months or more [64]. In general, preservation by organic acids alone
results in products that can only be consumed in small amounts, as condi-
ments, or as ingredients in other foods. Many acidified vegetable products
contain between 0.5 and 2% acetic acid and are pasteurized to prevent
spoilage, as well as to ensure safety. For nonfermented pickled vegetables,
the combination of heat treatments, acid, and sugar concentration (for sweet
pickles) serves to prevent microbial growth. Fresh-pack cucumber pickle
products typically contain between 0.5 and 1% acetic acid. A recommended
pasteurization procedure consists of heating to an internal temperature to 74°C
for 15 minutes [65].
320 Microbiology of Fruits and Vegetables
Both acidified and fermented vegetable products have enjoyed an excellent
safety record with few or no reported cases of foodborne disease resulting from
consumption of these products. Recently, however, there have been reports of
disease outbreaks in juice products with pH values below 4.0, in the same range
as many fermented and acidified vegetable products. Escherichia coli 0157:H7
and salmonella serotypes have caused serious illness and death from the con-
sumption of apple cider and orange juice [66,67]. These disease outbreaks
have raised questions about the safety of acidified and fermented vegetable
products. While pathogenic microorganisms have not been found to grow in
these products due to the low pH (typically below 4.0), these microorganisms
may adapt to acid conditions and survive for extended periods [68]. Acid types
and concentrations vary considerably for acidified foods. Factors affecting
acid inhibition of microbial pathogens include the pH of the product, as well
as specific effects of the acid or acid anion on cellular enzymes or membranes,
and the ability of bacteria to transport protons and organic acids out of the
cell interior [69-72].
1 4.4.1 Definitions and Regulations for Acid and
Acidified Foods
Acid foods are defined in the U.S. Code of Federal Regulations (21 CFR part
1 14) as foods that have a natural pH value at or below 4.6. These foods include
fermented vegetables; vegetable fermentation is considered a "field process"
and typically results in a product with a final pH below 4.6. A pH value
of 4.6 is used in the definition of acid foods because this is a limiting pH at
or below which C. botulinum spore outgrowth and neurotoxin toxin production
is prevented [73]. Foods with pH values above 4.6 are defined as low-acid
foods, and, when packaged in hermetically sealed containers, must be made
commercially sterile as defined in 21 CFR part 113. Acidified foods are defined
in 21 CFR part 114 as foods to which acid or acid food ingredients have been
added that have a water activity (a w ) greater than 0.85 and have a finished
equilibrium pH value at or below 4.6. The regulation requires producers of
acidified foods to verify that the final equilibrium pH is maintained at or below
4.6 to ensure safety. This regulation governing acidified foods in the U.S.
was promulgated by the U.S. Food and Drug Administration (FDA) in 1979.
At that time, vegetative pathogenic microorganisms were not considered to
be a significant risk for acidified or fermented food products. Included in
the regulation, however, is the requirement for a heat process "to the extent
that is sufficient" to destroy vegetative cells of microorganisms of public
health significance or those of nonhealth significance capable of reproducing
in the product. The regulations governing acidified foods are, therefore,
based primarily on the pH needed to prevent botulism, and do not include any
specification about the type or concentration of acid needed to meet the pH
requirement.
In a study of beef carcass wash water, a treatment with 0.2% (33.3 mM)
acetic acid and a pH of approximately 3.7 showed that an E. coli 0157:H7
Safety of Minimally Processed, Acidified, and Fermented Vegetable Products 321
strain survived for up to 14 days at 15°C, while cell numbers dropped about
4 log cycles [74]. In that study, competitive microflora were also present and
could have influenced the survival of the E. coli strains. A statistical analysis
of several published studies showed that, under typical storage conditions for
apple cider (which typically has a pH value less than 4.0 and contains malic
acid), the acid conditions alone were not sufficient to ensure a 5 log reduction
in the cell numbers of E. coli [75]. From these and other studies [68,75-79], it is
clear that the potential for E. coli to survive for extended periods in acidified
vegetable products with a pH below 4 clearly exists, and pasteurization for
some acidified food products may be needed to ensure safety.
14.4.2 Pathogenic Bacteria
After recent outbreaks of E. coli 0157:H7 in apple cider and salmonella in
orange juice [66,67], the FDA in 2001 proposed that all new process filings
(which are required for the production of acidified foods) should include
a heating or pasteurization step. Of primary concern was E. coli 0157:H7
because of its low infectious dose and lethal sequelae which can result from
infection [80,81]. Escherichia coli and other food pathogens have been
shown to have inducible acid resistance mechanisms [76,82-85]. If only pH is
considered, acid-resistant pathogens might, therefore, pose a potential threat
to acidified foods. It is likely that the organic acids present in these products
have contributed to their excellent safety record because some acidified
products have been produced safely for many years without heat treatments
[84], although quantitative measurements of the independent effects of
organic acids and pH on the killing of pathogens in these products are
lacking. In response to the pathogen outbreaks in juice products, 21 CFR part
120 was promulgated in 2001. This regulation mandated a HACCP (hazard
analysis critical control point) system with a processing step designed to deliver
the equivalent of a 5 log reduction in target pathogen populations in juices.
Typically, a heat pasteurization process is used, based on thermal destruction
time data for inactivation of E. coli 0157, which was found to be the most
heat- and acid-resistant pathogen in fruit juices [86]. In recent experiments
(Breidt, unpublished data), the thermal resistance of E. coli 0157:H7 and
L. monocytogenes was found to be identical under the conditions typical of
acidified pickle products, and salmonella strains were significantly less
heat-resistant. Similarly, salmonella was found to be less heat resistant than
L. monocytogenes or E. coli 0157:H7 in fruit juices [86]. For the variety of
acidified vegetable products currently available, the time and temperature
needed to ensure a 5 log or greater reduction (although a 5 log reduction is not
currently mandated by existing federal regulations) in numbers of microbial
pathogens will depend on the type and concentration of organic acid present,
the composition of the brine or suspending medium during heating, heat
resistance of the microorganisms, and other factors.
Some pickled pepper products with high concentrations of acetic acid
(greater than 2% acetic acid) and pH values around 3.1 to 3.3 may not need
322 Microbiology of Fruits and Vegetables
a heat treatment to ensure the destruction of acid-resistant pathogens
because sufficient acid is present. In a study of firmness retention with
unpasteurized pickled peppers, which typically have pH values around
3.1 to 3.3, and cucumbers, using 2 to 5% acetic acid, microbiological stability
was achieved for a 6-month period [64] for all products tested. A heat
process is typically not used for these pickled peppers because sliced
peppers are susceptible to softening during pasteurization. Historically,
pasteurization treatments were designed to prevent spoilage by LAB in brined
vegetables and inactivate softening enzymes. Currently, most commercial
acidified vegetable products with pH values between 3.3 and 4.1 are produced
using a pasteurization process to prevent spoilage. In addition, low water
activity and preservatives can reduce the amount of acetic acid needed
for preservation. A preservation prediction chart showing the effects of
acid and sugar in preventing the growth of spoilage yeasts in sweet pickles
was developed in the 1950s [62]. The acid concentrations that will ensure the
death of microbial pathogens for many acidified foods remain to be
determined.
14.5 ORGANIC ACIDS AND DESTRUCTION
OF PATHOGENS
Organic acid preservatives have widespread application for preventing
food spoilage and contribute to the manufacture of safe food products
[87-89]. The survival or death of pathogenic bacteria in acid and acidified
foods has been investigated in a variety of products, including apple cider
[68,90-93], mayonnaise, dressings and condiments [76,84,94,95], and fermented
meats [96-98]. The mechanism of action of organic acids is commonly
attributed to acidification of the cytoplasm of target cells, but also to
intracellular accumulation of anions [99]. The protonated form of weak acid
preservatives may diffuse across microbial cell membranes and then dissociate
in the cell cytoplasm, releasing protons and anions because the intracellular pH
must be maintained at a higher value than the external environment. Internal
acid anion concentrations may correlate with the cessation of growth.
Goncalves et al. [100] found that the specific growth rate of L. rhamnosus
approached zero at approximately 4 molar lactate (anion), with pH values
between 5.0 and 6.8. In vegetable fermentations, L. plantarum was found to
tolerate a lower internal pH than other LAB and, therefore, would have lower
acid anion concentrations.
Data on the relative effects of various organic acids and preservatives
on the inhibition of microbial pathogens are often conflicting in the scientific
literature. For example, Young and Foegeding [101] showed that with equal
initial pH values in brain-heart infusion broth ranging from 4.7 to 6.0 and
on an equimolar basis, the order of effectiveness in inhibiting the growth of
L. monocytogenes for three weak organic acids was acetic > lactic > citric.
However, when based on initial undissociated acid concentrations, the order
Safety of Minimally Processed, Acidified, and Fermented Vegetable Products 323
was reversed. Ostling and Lindgren [102] determined MIC values for the
inhibition of L. monocytogenes by lactic, acetic, and formic acids. They found
lactic acid was the most inhibitory over a range of pH values from 4.2 to 5.4,
with an MIC value of less than 4mM (protonated acid) for aerobic growth and
less than 1 mM for anaerobic growth. They used cells grown in glucose-
containing nutrient broth and reported MIC values for the protonated acid as
no growth for 5 days. Similar MIC values for the inhibition of growth of
Listeria innocua were reported as 217 mM sodium lactate at pH 5.5,
corresponding to about 5mM protonated lactic acid [103], and 4.7 mM
protonated lactic acid in another study [7]. Buchanan and Edelson [104] looked
at the effects of a variety of organic acids on E. coli 0157:H7 at a fixed
concentration of 0.5% and pH 3.0. They examined the effects of citric, malic,
lactic, and acetic acids on the viability of this organism; variables included
growth phase and the presence or absence of glucose in the growth medium.
The ability of the cells to survive when held in an acid solution varied in a
strain-dependent manner. For nine strains, lactic acid was the most effective
at reducing the viable cell population, and HC1 was the least effective [104].
This study clearly demonstrated that strain-to-strain variability, as well as
growth conditions (induction of acid resistance by growth in the presence
of glucose), must be considered in studies of the effects of weak acids and low
pH on E. coli.
The effect of acetate on E. coli 0157:H7 was investigated by Diez-Gonzalez
and Russell [70,105]. They investigated intracellular pH, acetate anion
accumulation, glucose consumption rates, and intracellular potassium
concentrations. They showed that E. coli 0157:H7 cells could divide in the
presence of about twice as much intracellular acetate anion (80 vs. 160 mM)
as E. coli K12. In cells grown at a constant pH of 5.9, E. coli 0157:H7 lowered
its internal pH to close to 6.0 and accumulated significantly less anion when
compared to E. coli K12, which kept a constant internal pH of 7. To test
the theory that acetate acted as an uncoupler (i.e., ferrying protons across
the E. coli cell membrane), Diez-Gonzales and Russell [105] compared the effects
of acetate and the uncoupler carbonylcyanide-m-chlorophenylhydrazone
(CCCP). They found that the effects of acetate and CCCP differed, specifically
in reference to intracellular ATP concentrations of E. coli 0157:H7. Acetate
had very little or no effect on intracellular ATP, even at concentrations greater
than 200 mM, while about 10 mM CCCP reduced intracellular ATP
concentrations by about 50%. These and similar experiments showed that
acetate was having effects other than simply acting as an uncoupler on E. coli
0157:H7. It was also apparent from these studies that E. coli 0175:H7 and
E. coli K12 regulate internal pH differently.
14.5.1 Specific Effects of Acids
A complicating factor in the study of acid inhibition of microorganisms is
that protonated acids and pH (which are interdependent variables linked
by the Henderson-Hasselbalch equation for common conditions) may both
324
Microbiology of Fruits and Vegetables
independently inhibit growth [106], or they may interact. Tienungoon et al.
[107] modeled the probability of growth of L. monocytogenes using a logis-
tic regression procedure with a function relating specific growth rate to
temperature, water activity, pH, lactic acid, and lactate ion concentrations.
They found that their equation accurately predicted conditions allowing
growth using their own laboratory data, as well as examples from the literature
[107]. They presented no data, however, on the growth/no growth interface
for L. monocytogenes, based on protonated acid and pH; they cited a lack of
independent data sets available in the literature.
To address the safety concerns of the FDA and the acidified foods
industry, Breidt et al. [79] investigated the specific effects of organic acids
independent of pH. This study was made possible by using gluconic acid as
a noninhibitory low pH buffer. While gluconic acid has been investigated
for use as an antimicrobial agent in meats [108,109], it has not proven to be
as effective as acetic or lactic acid. The antimicrobial effects of gluconic acid
solutions were found to be primarily due to pH rather than to specific effects
of the acid itself [79]. No change in the log reduction time (D value) was
observed over a 100-fold range of gluconic acid concentrations (Figure 14.2).
By using gluconic acid as a noninhibitory buffer, the inhibitory effects of
pH alone were compared with the combined effects of pH and acetic acid,
while holding ionic strength, temperature, and other variables constant [79].
As expected, survival of E. coli 0157:H7 was reduced with the addition
of acetic acid at concentrations typically found in acidified foods, and with
3.5 n
3.0-
CD
_^
CO
>
2.5
2.0
1.5
1.0
0.5
0.0
T
X
fi
i
0.002 M 0.02 M 0.2 M
Acid concentration
FIGURE 14.2 Effects of acetic and gluconic acid on the destruction of Escherichia coli
0157:H7 (cocktail of strains). The log reduction times (D values) for acetic (black bars)
and gluconic (gray bars) acids at 0.002, 0.02, and 0.2 M concentrations in water at 25°C
and pH 3.1. The error bars indicate the upper 95% confidence intervals. No statistically
significant difference was detected for the gluconic acid D values (p > 0.05). (From
Breidt, F., Hayes, J.S., and McFeeters, R.F., /. Food Prot., 67, 12-18, 2004.)
Safety of Minimally Processed, Acidified, and Fermented Vegetable Products 325
increasing temperature for a given pH and ionic strength. Gluconic acid may
have wider application as a noninhibitory buffer for similar experiments with
other organic acids.
In addition to the proposed mechanisms for the effects of weak acids
on microorganisms mentioned above (acidification of the cytoplasm and
intracellular accumulation of anion), the effectiveness of these compounds
may be modulated by additional factors. Examples of these include: specific
effects of the acid or acid anion on cellular enzymes or membranes, the internal
buffering capacity of cells, proton pumping at the expense of cellular ATP, and
facilitated transport of acid molecules, among others. To investigate the
relative importance of these effects for the inhibition of yeasts with sorbic
acid, Stratford and Anslow [71] compared the effects of acids with similar
pK values (acetic acid, pK = 4.76; sorbic acid, pK = 4.74) and used structural
analogs of sorbic acid that have similar lipophilic properties. Interestingly,
a variety of structural analogs, including aldehydes and alcohols, had
similar MIC values to sorbic acid (which was 3 mM) for the inhibition of a
Saccharomyces cerevisiae strain, and a survey of yeast strains showed sorbate
resistance correlated with ethanol tolerance [71]; they proposed that sorbic
acid acted specifically on yeast membranes. Krebs et al. [69] examined
glycolysis intermediates in yeast cells treated with benzoate and showed
an increase in the intracellular concentrations of glucose 6-phosphate and
fructose 6-phosphate, while frucotose 1,6-bisphosphate and triose phosphate
concentrations were reduced. The specific inhibition of phosphofructokinase,
however, could be attributed to a lack of ATP required for the function
of this enzyme [69]. Alakomi et al. [72] showed that lactic acid had a
specific membrane effect on Gram-negative bacteria. They found that lactic
acid could sensitize E. coli 0157:H7, pseudomonas, and salmonella to lytic
agents such as detergents and lysozyme, presumably by disrupting the outer
membrane. Lactic acid (5mM, pH 3.5) was found to have a greater ability to
liberate lipopolysaccharides from the outer membrane of Salmonella serovar
Typhimurium than a 1 mM EDTA solution under similar conditions [72]. The
effect of sorbate on the germination of C. botulinum spores was investigated
[110]. This study indicated that sorbate inhibited spore outgrowth by disrupt-
ing the cell membrane after the start of germination. In addition to membrane
effects, organic acids may have a variety of other possibly minor effects on
the inhibition of microorganisms. A review by Shelef [88] cites additional
effects of lactate salts on the inhibition of microorganisms. These effects
include lowering water activity, chelating iron, and the inhibition of lactate
dehydrogenase.
14.5.2 Genetic Regulation of Acid Resistance
Induction of acid resistance genes in E. coli can be accomplished by growing
cells statically to stationary phase in media containing an excess of glucose,
resulting in a pH of about 5.5 [104]. The acid resistance systems in E. coli
and other pathogenic bacteria are also subject to crosstalk, or regulation by
326
Microbiology of Fruits and Vegetables
Acid stress
GadE
Catabolite
repression
RpoS
J\
GadB
GadC
V
h!Lzh
GadA
GadX
V
GadX
GadW
GadW
FIGURE 14.3 Regulatory network governing gadA/BC expression and glutamate-
dependent acid resistance. (Adapted from Ma, Z., Gong, S., Richard, H., Tucker, D.L.,
Conway, T., and Foster, J.W., Moi Microbiol., 49, 1309-1320, 2003. With permission.)
stresses other than acid [1 1 1-113]. A clear example of this crosstalk is exhibited
by E. coli 0157:H7 in which acid resistance is induced in response to heat stress
[114], and heat tolerance is induced in response to acid stress [115]. Crosstalk
can be mediated by two-component (sensor-effector) regulatory systems used
by bacteria, where sensor kinases phosphorylate noncognate regulatory
proteins [116,117]. The precise nature of the signal(s) recognized by the cells
for controlling acid resistance remains unclear, although considerable research
has been carried out investigating genes induced by exposure to acid and
other stresses.
Escherichia coli has several known inducible acid resistance systems
that allow the organism to respond to the presence of organic acids and
low pH in the environment [118,119]. The most well studied system uses
decarboxylation of glutamic acid as a means for modulating internal pH [120].
The system consists of two inducible proteins, glutamate decarboxylase (GadA
and an isozyme GadB), and an antiport transporter (GadC) for glutamate and
the decarboxylated product of glutamate, gamma-aminobutyric acid. The
genetic regulation of this system has been found to be quite complex
(Figure 14.3). RpoS, a sigma factor produced in response to stress, mediates
expression of two regulatory proteins, GadW and GadX, that control expres-
sion of the decarboxylase and transport proteins [121]. In addition, there
is a two-component regulatory system that responds to (unidentified) exter-
nal acid signals and can cause expression of the proteins of the glutamate
decarboxylase system through the action of another regulatory protein,
GadE [118,122]. The other acid resistance systems include arginine and lysine
decarboxylase systems [119] similar to the glutamic acid system and a glucose-
repressed, acid-induced system also controlled by RpoS which does not
require external amino acids [83]. Inducible acid resistance mechanisms
have been observed in a variety of other food pathogens, including Salmonella
Safety of Minimally Processed, Acidified, and Fermented Vegetable Products 327
spp., L. monocytogenes, Shigella flexneri, B. cereus, and others [111,123-126].
As the details of gene regulation of acid resistance of microbial food pathogens
become clearer, strategies may be devised to help prevent the survival of these
pathogens in acidified foods.
14.6 CONCLUSIONS
Preservation of vegetables by fermentation is one of the earliest and most
widespread technologies developed by humans. Fermented and acidified
vegetable products are produced and consumed in every culture and society
around the world, usually based on traditional processing methods. This is
because the products produced are safe even in the absence of refrigerated
storage, due to the inhibitory metabolites, primarily organic acids produced
by lactic acid bacteria. The lactic acid bacteria may also be used to control
spoilage of fresh vegetable products. The factors influencing microbial
competition during fermentation or spoilage of fresh vegetable products
have proved to be difficult to understand, but biocontrol strategies have
the potential to ensure the safety and control the microbial ecology of food
spoilage for many types of nonfermented foods. Significant challenges remain,
however, in understanding the mode of action of organic acids in killing
bacterial pathogens, and how those pathogens respond and adapt to acid
challenge.
ACKNOWLEDGMENTS
This work was supported in part by a research grant from Pickle Packers
International, Inc., St. Charles, IL. The author thanks Mr. Jim Cook of the
M.A. Gedney Company, Mr. Mike Woller of Dalton's Best Maid Products,
Inc., Dr. Roger McFeeters of USDA/ARS and NC State University, and
Dr. Henry Fleming, formerly of USDA/ARS and Professor Emeritus NC
State University, for helpful advice and contributions to this chapter, and
Ms. Dora Toler for excellent secretarial assistance.
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Section IV
Interventions to Reduce
Spoilage and Risk of
Foodbome Illness
15
HACCP: A Process Control
Approach for Fruit and
Vegetable Safety
William C. Hurst
CONTENTS
15.1 Introduction 339
15.2 What Is HACCP? 340
15.3 Applying the HACCP Concept 342
15.4 Prerequisites for HACCP 343
15.5 Planning and Conducting an HACCP Study 344
15.6 Conducting a Hazard Analysis/Risk Assessment Study
(HACCP Principle 1) 345
15.7 Using SPC to Ensure HACCP Control 347
15.8 Identifying and Stabilizing Variability at CCPs
(HACCP Principle 2) 348
15.9 Conducting Process Capability Analyses to Verify
Critical Limits (HACCP Principle 3) 351
15.10 Establishing SPC Monitoring Procedures
(HACCP Principle 4) 354
15.11 Determining Corrective Action Procedures
(HACCP Principle 5) 356
15.12 Confirming HACCP Implementation Through
Verification Activities (HACCP Principle 6) 357
15.13 Establishing Documentation and Record Keeping
(HACCP Principle 7) 359
15.14 Summary 361
References 362
15.1 INTRODUCTION
Hazard Analysis Critical Control Point (HACCP) has been called a logical,
cost-effective, systematic approach toward food safety management. It
involves the identification, evaluation, and control of potential hazards that
339
340 Microbiology of Fruits and Vegetables
might contaminate fruits and vegetables during handling between the farm
and consumer. The basic objective of HACCP is to ensure consistent and safe
food production. By identifying in advance potential problems in a fruit or
vegetable operation and establishing control measures at those stages critical
for food safety, potential microbiological, chemical, or physical hazards can
be reduced, prevented, or eliminated. HACCP is based on two important
concepts in safe food production: prevention and documentation [1]. HACCP
is a tool that can determine how and where safety hazards may exist in
a food operation and how to prevent their occurrence. Once located, these
hazards may then be confirmed and controlled through documentation
(record-keeping) procedures.
During HACCP implementation and maintenance, an understanding of the
production system in relation to process control is of paramount import-
ance. There are many sources of variation that must be recognized and reduced
to achieve consistent control over product quality and safety. Properly
defining and documenting a process is an important first step toward understand-
ing and gaining control over the process. Summers has identified a process
as simply taking inputs and performing value-added activities on those inputs
to create an output [2]. Take, for example, the fresh-cut (minimally processed)
produce process for making a salad product. Inputs such as raw materials
(lettuce, red cabbage, carrots), people (plant workforce), machines (processing
equipment), and methods (machine settings, quality control, etc.) perform
value-added activities (grading, cutting, washing, drying, and packaging) to
transform whole produce into a 3/8 inch chopped salad product contained
in a 14-ounce bag. Complementary to this production process is the need to
ensure consistent product safety. Thus HACCP serves as that portion of the
establishment's overall process control system that focuses on safety [3].
The need and value of process control technology and its integration
into the HACCP process is addressed in this chapter. Although HACCP
is often referred to as a preventive system, from a statistical standpoint
HACCP would be more appropriately described as a means of minimizing
the variability of safety parameters in a processing system [3]. However, in
order to achieve consistency of operation, process control techniques must be
coupled with HACCP principles to keep those parameters being monitored
under control and within safety limits. This chapter also focuses on how
a statistical approach to safety, such as statistical process control (SPC), can be
effectively applied to the HACCP process.
15.2 WHAT IS HACCP?
HACCP is a science-based, objective, and proactive method of assuring food
safety by focusing on hazard identification and control at its source. Origi-
nally developed in the early 1960s by the Pillsbury Company, the U.S. Army
Natick Laboratories, and the National Aeronautics and Space Administration
(NASA) to develop safe food for astronauts, HACCP is derived from failure
HACCP: A Process Control Approach for Fruit and Vegetable Safety 341
mode and effect analysis (FMEA). This is an engineering system that looks
at a product, all of its components and manufacturing stages and asks what
can go wrong within the total system [4].
HACCP-like controls for thermally canned foods were first mandated
by the U.S. Food and Drug Administration (FDA) in 1973, followed by
regulations concerning acidified foods (marinated vegetables) that likewise
mandated HACCP tools [5,6]. The National Advisory Committee on Micro-
biological Criteria (NACMCF) prepared the first official HACCP document in
1989, which was subsequently issued by the FDA as a proposed regulation
for recommending HACCP as the food safety program of choice for the
entire U.S. food industry [7,8]. In 2001 the FDA issued its mandatory
juice HACCP regulations that apply to the production of all fresh fruit and
vegetable juices [9].
Although not yet mandated by the FDA, HACCP for the fresh-cut
produce industry presents some unique challenges because there is no defini-
tive kill step for pathogens (e.g., retorting, pasteurization, acidification) in the
processing operation. Instead, HACCP must incorporate a series of interven-
tion steps or hurdles, such as using antimicrobial agents in flume wash water,
applying modified atmosphere packaging techniques, and following consis-
tent good sanitation and low-temperature management practices to retard
pathogen growth [10-12]. Today, HACCP implementation is recommended
or required by FDA/U.S. Department of Agriculture (USDA) throughout the
food industry in the U.S. [13]. HACCP has achieved international acceptance
with the recognition by the World Health Organization (WHO) as the most
effective means of controlling foodborne disease [14].
Traditionally, the fruit and vegetable industry has used the concept of
end-line product testing to attempt to ensure a high level of safety and quality
of products. Today the defect detection approach has given way to defect
prevention whereby a process is monitored as a product is being manufac-
tured to determine when adjustments are required to maintain stability and
where a change is needed to reduce inherent variability [15]. This preven-
tion approach is in harmony with HACCP, which also focuses on prevention,
whereby potential hazards are identified and controlled within the food
processing environment to prevent unsafe products from being made [16].
While prevention is obviously superior to detection, it is not always easy
to accomplish. This is because all processes are inherently unpredictable in
nature and gravitate toward the natural force of entropy or disorder [17]. The
only way to overcome the effects of entropy is to utilize statistical tools to find
and eliminate the causes of disorder in the production system so the process
can be brought back under control.
The National Academy of Sciences (NAS) drafted a report in 2003 that
included science-based tools for food safety regulatory use in ensuring safe
food production for the American consumer. It recommended the establish-
ment of performance standards that would guide fruit and vegetable produ-
cers and processors to an appropriate means of ensuring food safety when
operating under a HACCP system [18]. The committee also recommended the
342 Microbiology of Fruits and Vegetables
use of SPC as the most appropriate scientific tool for monitoring performance
standards in fruit and vegetable operations and for developing the science-
based food safety criteria that processors will need to verify and validate their
HACCP programs.
15.3 APPLYING THE HACCP CONCEPT
The HACCP concept is implemented through a logical sequence of activities,
known as the HACCP study, that utilizes the seven principles of HACCP
(Table 15.1) to produce a HACCP plan.
By definition, a HACCP plan is a document prepared in accordance with
the principles of HACCP to ensure control of hazards which are significant
for food safety in the segment of the food chain under consideration [19].
A HACCP system is a system that identifies, evaluates, and controls hazards
that are significant for food safety. Thus, the HACCP study should yield a
HACCP plan which is then implemented as the HACCP system [20].
Design, implementation, and maintenance of a HACCP plan are not an
easy job. When Pillsbury first decided to implement HACCP, the CEO publicly
stated that all raises, promotions, and evaluations would be based on deve-
loping and implementing HACCP to ensure safe food production [21]. Now,
that was a strong statement of support! Employees ultimately determine the
success or failure of HACCP. Therefore, training programs are essential to
develop a positive attitude about food safety and to help empower personnel
to maintain the HACCP program. Implementing HACCP takes time;
TABLE 15.1
Seven Principles of HACCP
Principle 1 Conduct a hazard analysis. Construct a flow diagram of the steps of a process to
determine where significant hazards exist and what control measures should be
instituted
Principle 2 Determine critical control points (CCPs) required to control the identified hazards.
CCPs are any steps where hazards can be prevented, eliminated or reduced to
acceptable levels
Principle 3 Establish critical limits (CLs). These are specifications (target values and tolerances)
that must be met to ensure that CCPs are under control
Principle 4 Establish procedures to monitor CCPs. These are used to assess when a process must
be adjusted to maintain CCP control
Principle 5 Establish corrective actions to be taken when monitoring indicates that a particular
CCP is not under control
Principle 6 Establish verification procedures for determining whether the HACCP program is
working correctly
Principle 7 Establish documentation procedures concerning all activities with records appropriate
to these principles and their application
From USDA-FSIS, Hazard Analysis and Critical Control Point Principles and Application
Guidelines, 1997 (http://www.fsis.usda.gov/OPHS/nacmcf/past/JFP0998.pdf).
HACCP: A Process Control Approach for Fruit and Vegetable Safety 343
experience has shown that installation and implementation can take from
six months to two years.
Today, there are numerous manufacturers promoting computer soft-
ware programs, manuals, and services that can aid in the development of
generic HACCP systems. While these models are useful tools to demonstrate
how to create a HACCP plan, it must be emphasized that they should not
be used "out of the box," as the generic HACCP plan may not be appli-
cable for every facility, processing line, or product. Since each HACCP plan
is process-specific, the plan must be tailored to address the unique aspects of
the production, including processing and preparation operations, equip-
ment being used, the foods being prepared, and the training of personnel [22].
15.4 PREREQUISITES FOR HACCP
It has been pointed out by Sperber et al. that HACCP cannot exist as a stand-
alone food safety program [23]. Rather, it must be supported by a strong
foundation of prerequisite programs. While not a formal part of HACCP,
the prerequisite programs are written, implemented procedures that address
operational conditions and provide the documentation to help an operation
run more smoothly to maintain comprehensive safety assurance. Standard
operating procedures (SOPs), good agricultural practices (GAPs), good manu-
facturing practices (GMPs), and sanitation standard operating procedures
(SSOPs) are the basis for establishing a comprehensive sanitation program in
a fruit and vegetable processing plant or packinghouse facility.
SOPs are step-by-step instructions that outline how an operation is to
be carried out in such a way that ensures that all processing steps critical
for product safety are accomplished in an orderly fashion. SOPs provide the
detailed framework and safety continuum between agricultural production
and commercial processing of fruit and vegetable products. GAPs are a
collection of HACCP-like principles that have been extended to the on-farm
production and postharvest handling activities in order to minimize micro-
bial contamination of fresh fruits and vegetables [24]. They ensure that the
fresh produce industry, for which the traditional HACCP methods do not
necessarily fit, will have a systematic and proactive method to reduce
potential product contamination and thereby ensure safety [25]. GMPs,
sanctioned by the FDA, are the minimal sanitary requirements that must be
met by a food packer or processor to ensure safe and wholesome food
for interstate commerce [26]. GMPs are broadly written, based on federal
regulations, general in nature, are not intended to be plant-specific, do not
establish deviation limits, and do not describe corrective action require-
ments [27]. SSOPs focus more narrowly on specific procedures that allow
a fruit or vegetable processing plant to achieve sanitary process control in
its daily operation. SSOPs can be categorized into two types: preoperational
and operational SSOPs. Preoperational SSOPs are the sanitary procedures
carried out prior to the start of production each day. Operational SSOPs refer
344 Microbiology of Fruits and Vegetables
to sanitary actions taken during production to prevent product contami-
nation or adulteration [19]. More extensive information on these programs
as applied to fresh produce have been reviewed by Gorny and Hurst [27,28].
15.5 PLANNING AND CONDUCTING
AN HACCP STUDY
Before the actual HACCP plan design begins, several preliminary tasks
must be accomplished: (1) identifying the scope of the study, (2) assembling
the HACCP team and providing adequate training, (3) describing the
product for which the HACCP plan is being developed and its intended use,
and (4) developing and verifying a flow chart of the operation required to
produce the product. It is important to remember that a separate HACCP
plan must be developed for each product or processing line in the plant
operation. This means that each HACCP plan will be plant-specific and
must be uniquely tailored for each packer or processor.
Task 1: setting limits for the HACCP study. First, the terms of reference
for the HACCP study should be established at the outset to define where
the HACCP plan begins and ends. The model for discussion in this chapter
considers only the preparation of shredded iceberg lettuce at a fresh-cut
processing plant. Where complex production operations of a product are
concerned, it can be simpler (and safer) to break down the handling
chain into smaller segments (e.g., lettuce production, distribution to the
consumer) and link the operations together later to form the overall HACCP
system [20].
Task 2: the HACCP team. A HACCP coordinator with good commu-
nication skills and knowledge of HACCP techniques should be appointed
to lead, coordinate, and build the HACCP team. Selection of team members
should be based on their working knowledge of the entire process and
their ability to contribute unique aspects of the operation toward ensuring
the safety of the product. Team members should be multidisciplinary and
multifunctional. The HACCP team may include a microbiologist, quality
assurance and sanitation personnel, production operations, engineering
and maintenance, purchasing or procurement, marketing and sales, and
on-line personnel [4].
Task 3: product description. The HACCP team must first develop a
complete description of the product under study. Information on key
parameters that determine safety must be included (e.g., specific refrigera-
tion temperature for fresh-cut produce, pasteurization temperature for
processed juices, pH value for acidified vegetables). A complete description
detailing its form, size, packaging and storage requirements, shelf life,
instructions for use, and intended consumer must be defined, as demonstrated
for fresh-cut lettuce in Table 15.2.
Task 4: flow chart of process. A flow chart can be a picture worth a
thousand words when used to describe in clear and simple terms the steps
HACCP: A Process Control Approach for Fruit and Vegetable Safety 345
TABLE 15.2
Product Description, Use, and Distribution
Common name Shredded lettuce; prepared from refrigerated iceberg lettuce; trimmed,
cored, and cut; washed in a solution of potable water and chlorine
Type of package Packed in food-grade plastic bags, 8oz to 10 lb units
Length of shelf life, at Optimum shelf life of 14 days if refrigerated at 34 to 38° F (1.1 to 3.3° C)
what temperature?
Labeling instructions Bag and/or box contains "processed on" or "use by" date
Where will it be sold? Foodservice operations and retail markets
Intended use and For use in salads and sandwiches by foodservice customers; prepackaged
consumer units for in-house use by consumer
Special distribution Product distributed under refrigeration, stored in refrigeration at 34 to
control 38°F(1.1 to 3.3°C)
From IFPA Technical Committee, HACCP for the Fresh-Cut Produce Industry, IFPA, Alexandria,
VA, 2000. With permission.
involved in a process. A block-type flow diagram is typically used for simplicity
of understanding by the HACCP team. Its purpose is to facilitate hazard
analysis and assist in the identification of critical control points (CCPs). Also,
the flow diagram serves as a record of the operation and a future guide for
employees, regulators, and customers who must understand the process [1].
Once the flow diagram has been completed, it needs to be verified for
accuracy and completeness. Ideally, confirmation should be made by having
HACCP team members "walk the process" whereby the flow diagram is
compared with what actually happens, as it happens [20]. This also should
include confirmation of activities during the night shift or weekend running
of the operation.
15.6 CONDUCTING A HAZARD ANALYSIS/RISK
ASSESSMENT STUDY (HACCP PRINCIPLE 1)
Using the flow diagram and description of the product as a guide, the HACCP
team must conduct a hazard analysis of the process as the first step in the
formal HACCP plan design. By definition, a hazard is a biological, chemical,
or physical agent in, or condition of, food with the potential to cause an
adverse health effect [19]. The purpose of hazard analysis is to develop a list
of hazards at each step of the process that are of such significance that they
are reasonably likely to cause illness or injury if not effectively controlled.
For fruit and vegetable component operations to which HACCP may be
applied, these hazards may be introduced as inputs during the crop produc-
tion process, during the plant process, or as outputs from the process and
the final product. Consideration of the myriad of conceivable hazards that
might contaminate fresh and processed fruit and vegetable products is not
within the scope of this chapter. However, extensive listings of hazards have
346 Microbiology of Fruits and Vegetables
been reviewed [19,20,29]. Based on epidemiological data and industry exp-
erience, the primary safety issue in fresh-cut processing is microbiological
contamination.
Hazard analysis consists of a two-part process: hazard identification and
hazard evaluation. During hazard identification, the HACCP team gene-
rates a list of all potential hazards associated with each step in the process,
by following the flow diagram. Brainstorming and Pareto diagrams are
two problem-solving tools that can aid in this process. Brainstorming stimu-
lates creative, exhaustive thinking that can help team members by identifying
every conceivable hazard so that none are missed. Pareto diagrams allow
the team to prioritize identified hazards based on their relative importance
to safety [30].
Step two of hazard analysis is hazard evaluation. Each identified hazard
must be carefully considered based on its severity in the extent of exposure and
likely occurrence (risk) in a product. For example, Listeria monocytogenes
and Clostridium botulinum are both potential microbiological hazards in the
fresh-cut produce industry. However, L. monocytogenes, due to its ubiquity,
is a greater health risk to consumers of fresh-cut produce, even though
C. botulinum would be considered a more severe hazard based on mortality
rates [27]. Potential listeria contamination within the processing and/or storage
environment of fruit and vegetable facilities is a constant threat to products
since there is a zero tolerance regulation for this hazard in ready-to-eat
(RTE) foods.
Another problem-solving tool, the cause-and-effect diagram, can be
utilized to find ways to minimize environmental hazards such as listeria.
Constructing a cause-and-effect diagram of a process requires the HACCP
team to itemize all possible locations where a problem or hazard could occur,
to look for all causes of a problem, and to collect data to evaluate the possible
risk. A cause-and-effect diagram highlighting the processing environment
as the possible source of listeria contamination to fruit and vegetable
products would show the effect or problem (e.g., listeria contamination in
a fresh-cut plant) at the end of a horizontal arrow or spine. Primary causes
(e.g., poor sanitation in the processing room, produce cooler) are represen-
ted by oblique arrows entering the spine. Secondary causes (e.g., condensate
on overhead pipes, electrical conduits, floors, in drains, and on refrigera-
tion units) are represented by perpendicular arrows off the primary cause
arrow [27].
An important component of hazard evaluation is risk assessment, which
is defined as a scientifically based process of four activities: (1) hazard
identification, (2) exposure assessment, (3) dose-response assessment, and
(4) risk characterization. A comprehensive discussion of risk assessment with
relationship to HACCP cannot be covered in this chapter, but excellent
resources to consult include Tapia et al. [31] and Forsythe [32].
Once all potential hazards have been identified and their risks to the
final consumer of the product evaluated, the HACCP team must consider
what preventive measures are to be used to control the hazard. Preventive
HACCP: A Process Control Approach for Fruit and Vegetable Safety 347
measures are those actions or activities that are required to control hazards,
eliminate hazards, or reduce their effect to an acceptable level. More than one
control may be required for a specific hazard that occurs at different parts
of the production process. For example, if the hazard is Listeria monocyto-
genes on raw fruit or vegetables, which are consistently heat-treated in the in
the manufacture of a processed juice, then pasteurization could be the
appropriate control measure for this hazard. If the same hazard arises from
environmental contamination during ingredient assembly of a refrigerated
product which is given no heat treatment, such as fresh-cut produce, other
control measures (environmental sanitation, personnel sanitation, refrigeration
management) would be required.
It cannot be overemphasized that a thorough hazard analysis is essen-
tial to the design of an effective HACCP plan. If this is not done correctly,
and the significant hazards requiring control within the plan are not properly
identified and evaluated, the HACCP program will not be effective, regardless
of how well the plan is followed [33].
15.7 USING SPC TO ENSURE HACCP CONTROL
SPC emphasizes building product safety and quality into the manufacturing
process by focusing on the process rather than the product. SPC tools are used
to measure and interpret different kinds of variation that affect the behavior of
a process. An important part of dealing with variation in a process control
system is to have knowledge of the extent of variability. The critical point in
process control is not to eliminate all variation, but to have any variation that
is present in the process to be stable and predictable.
The key to controlling safety and quality in manufacturing is to under-
stand how to recognize the different types of variation present in a process.
Common cause variation is the inherent, random chance events expected in
every process which fluctuate in a normal, predictable manner. Variation
is small in magnitude and therefore difficult to locate and eliminate from
a process. Special cause variation is sporadically induced variation that impacts
a process, causing large fluctuations that are easily discernable and there-
fore can be effectively eliminated from a process [34] (see Figure 15.1).
The basic objective of SPC is to use valid statistical methods to identify the
existence of special causes of variation and to eliminate them from a process.
This will produce a stable, constant-cause system which can be measured
and controlled [35].
Although SPC is the most effective tool to achieve process control in
an operation, most processes do not naturally operate in a state of control.
They tend to deteriorate over time. Process control is defined as the function-
ing of an operation within predetermined statistical limits, such that only
common cause (controlled) variation is occurring among its manufactured
products [36]. While a process may be in control initially, it will not remain
there. The only way to determine whether a process is in or out of control is
348
Microbiology of Fruits and Vegetables
Special cause variation
• Incorrect setting
• Faulty materials
• Machine breakdown
• Operator error
• Incorrect specification
• Change in temperature
i
Common cause variation
• Limits of measurement
• Limits of control
• Variation in materials
• Environmental fluctuation
• Vibration of machines
• Skill of workers
I
Eliminate
Measure and control
FIGURE 15.1 Distinguishing between special and common cause variation. (Adapted
from Cullen, J. and Hollingum, J., Implementing Total Quality, IFS Publications,
Bedford, U.K., 1987.)
to interpret the data gathered from the process with the appropriate statistical
tool. That tool is the statistical control chart.
Control chart methods provide an objective and statistically valid means
to assess the nature of ongoing processes, and, as such, are particularly
applicable to HACCP monitoring [3]. Control chart theory is based upon the
notion that the parameter being measured, when in statistical control, will vary
normally (e.g., only common cause variation) about a central value [37].
Control chart methodology is the only SPC tool that can distinguish between
common cause (inherent) and special cause (unnatural) variation in a process.
The control chart allows the highlighting of special cause variation, if present,
when monitoring a process. If the special cause variation source can be found
and eliminated in the process, then the process will exhibit only common
cause variation. Only when common cause variation is the only source of
variation present is the process in a state of statistical control. What makes
statistical control so important? The essence of statistical control is predic-
tability. A process is predictable when it is in a state of statistical control, and
it is unpredictable when it is not in a state of statistical control [38].
So how can HACCP be made a more effective prediction tool for
safety hazards in the production process? The key lies in integrating SPC
methodology into the HACCP plan. Achieving process control in a HACCP
monitoring system will assist the processor in systematically and predictably
demonstrating control of identified safety parameters [39]. It will also provide
warning signs signifying out-of-control status so that corrective action can
be taken to return the system to the established safety limits [27].
15.8 IDENTIFYING AND STABILIZING VARIABILITY
AT CCPs (HACCP PRINCIPLE 2)
Once all significant hazards have been identified through hazard analysis,
they must be controlled at some point in the process. Determining the identity
HACCP: A Process Control Approach for Fruit and Vegetable Safety 349
and location of CCPs is the second principle in HACCP design and is really
the heart of the HACCP plan. A CCP is a step at which control can be applied
and is essential to prevent or eliminate a food safety hazard, or to reduce it
to an acceptable level [7]. For every significant hazard identified, one or more
CCPs must be designated to control or eliminate the hazard. For example,
the thermal process given to canned vegetables at the retort step would be
designated as a CCP in the low-acid foods industry, while proper acidification
or pH adjustment in the brine kettle step would be the CCP in the acidified
foods industry. Sometimes it is not possible to eliminate a potential micro-
biological hazard, only to minimize it to an acceptable level. In the fresh-cut
produce industry, proper water chlorination at levels > 1 ppm free available
chlorine in flumes and dip tanks becomes the CCP [27].
Unfortunately, there is no simple, clear-cut answer to the question of
how many CCPs a HACCP plan may need and where should they be located.
It depends on plant layout and design, the product being produced, the
ingredients used, equipment age and condition, processing methods emplo-
yed, and, especially, the effectiveness of the prerequisite programs imple-
mented. Often an SSOP or SOP can be incorporated to control a hazard
rather than a CCP. To keep HACCP programs plant-friendly and sustain-
able, Bernard recommends that the number of CCPs be kept to a minimum,
and none should be redundant [33]. Redundancy will also add to the cost
of record keeping. Experience has shown that HACCP plans that are
unnecessarily cumbersome will likely be the ones that fail.
However, many points in a flow diagram not identified as CCPs may
be considered control points. A control point (CP) is any step at which bio-
logical, physical, or chemical factors can be controlled [7]. Many types
of control points can exist in fruit and vegetable operations, including those
that address quality control (color, flavor, texture), sanitary control (SSOPs,
GMPs), maintenance (calibration of equipment), and process control (fill
weights, seal closures).
Pinpointing the right CCPs is the most crucial and problematic aspect of
an effective HACCP program [40]. Therefore, a common strategy to facilitate
the proper identification of CCPs is to use the CCP decision tree (Figure 15.2)
[7,41]. The decision tree consists of four questions that are asked for each
process step for which hazards have been identified during hazard analysis.
The answer to each question will direct the process of elimination and
ultimately lead to a decision as to whether a CCP or CP is required at that step.
A benefit of the CCP decision tree is that it forces and facilitates HACCP team
discussion and teamwork and ensures a consistent approach to every hazard at
each step [42]. However, as pointed out by Wedding, this is not a perfect tool
and is not a substitute for common sense and process knowledge, because
complete reliance on the decision tree may lead to false conclusions [41].
To determine the kind of variability that might exist at a specific CCP, data
of the parameter used to maintain control at this location must be collected
and statistically analyzed. For example, the conditions that influence
variability at a CCP, like pasteurization temperature for fresh juices, must be
350
Microbiology of Fruits and Vegetables
Yes
I
Q1 : Do preventive
measures exist for the
identified hazard?
Modify step,
process or
product
Q2: Does this step eliminate
or reduce the likely
occurrence of a hazard to an
acceptable level?
I
No
CCP
Yes
"}No
Q3: Could contamination with
identified hazard(s) occur in
excess of acceptable level (s)
or could these increase to
unacceptable levels?
f
I
Is control of this step
necessary for safety?
Not CCP
Stop!
Q4: Will a subsequent step, prior
to consuming the food, eliminate
the identified hazard(s), or reduce
the likely occurrence to an
acceptable level?
I
Not CCP
Stop!
Not CCP
Stop!
CCP
FIGURE 15.2 Critical control point decision tree.
understood and controlled within an acceptable range to ensure that a safe
product is manufactured. Processes with a high degree of variability, especially
when that variability is not recognized or understood, are more likely to
produce unacceptable and possibly hazardous food [3].
Before data collection begins, the appropriate control chart must be
determined for the parameter to be evaluated at the CCP. Two principal
categories of control charts are employed in SPC work: variable and attribute.
Variable control charts use actual measurements (e.g., temperatures, chlorine
concentration, oxidation-reduction potential (ORP), pH values) for charting.
Attribute control charts use pass-fail information (metal inclusion, cull fruit
presence, foreign objects) for charting. Smith presents a good description of
the different types of charts in each category [43].
To create the chart, individual data, normally arranged into subgroups,
are sampled from the process. The average value of the data is then calcu-
lated and becomes the centerline of the chart. Using statistical formulae
specific for each chart type, upper and lower trial control limits are calculated.
HACCP: A Process Control Approach for Fruit and Vegetable Safety 351
They describe the spread of the process. Finally, the individual (or averaged)
measured values are plotted on the control chart. Once the chart is con-
structed, it presents a picture of the types of variation occurring in the process
over the time at which the samples were taken. If one or more plotted
points exceed either trial control limit, special cause variation has become a part
of the process, forcing it out of statistical control. If a cause can be assigned
to each value exceeding the control limit, then it can be discarded from the
data and new control limits can be computed from the remaining data.
However, if no cause can be found and corrected, then the points cannot be
removed from the chart [2]. Once the assignable causes have been eliminated,
the revised control charts should be in control. If a process shows only
common cause variation present, it is stable, and the process of improvement
can begin.
When first implemented, SPC will do a good job of finding areas of
high variability (special cause variation) in a process. This results in readily
demonstrated points exceeding the control limits. However, as more problems
are solved, those remaining will be more subtle in their variation [44].
When an unusual number of nonrandom points produces a pattern on
a control chart, none being beyond the control limits, this signifies that the
process is unstable and on the verge of going out of control. While a dozen
or more of these patterns may occur in a process, Evans has characterized
the five most common ones, as shown in Figure 15.3 [45]: (a) shift — seven
or more consecutive points on one side of the center line of a control chart;
(b) run — a pattern of seven points consecutively climbing or falling in a
control chart; (c) cycling — short repeated patterns of points having alternate
high peaks and low valleys on a control chart; (d) instability — unnatural and
erratic swings on both sides of the chart over time with points often lying
near or on the control limits; and (e) stratification — 14 or more consecutive
points hugging the center line on the control chart. When these patterns occur,
it is a warning signal that something has gone wrong in the process and
immediate action is needed to avoid loss of control.
15.9 CONDUCTING PROCESS CAPABILITY
ANALYSES TO VERIFY CRITICAL LIMITS
(HACCP PRINCIPLE 3)
The third step in HACCP plan development is to set safety boundaries,
or critical limits, for each CCP identified in the hazard analysis. A critical
limit (CL) is defined as a maximum or minimum value to which a biological,
chemical, or physical parameter must be controlled at a CCP to prevent,
eliminate, or reduce to an acceptable level the occurrence of a food safety
hazard [7]. CLs are individual values that signify whether the variation of the
control measure implemented at the CCP is capable of remaining within
its safety boundaries. It is important to note that CLs cannot be arbitrarily
set based on the variation in a process. They are not control limits; they must
352
Microbiology of Fruits and Vegetables
R chart-limits based on 50 samples
7.742 i ^ - ^ UCL
3.663
000 * ' ' ' ' ' ' ' * * ' ' 1 ' ' ' ' L ' ' ' * ' ' ' ' ' ' * ' ' ' ' ' * ' ' ' ' ' ' * ' * * ' * ' * ' LCL
10 20 30 40 50
(a) Shift
X-bar chart-limits based on 50 samples
6.117
5.588
UCL
■ * i ■ 1 1 1 ■ ■ 1 1 ' 1 1 1 i ■■ 1 1 ■■■ 1 1 j ■■ 1 1 1 .,.,.., i [_r_/|_
5.051
10 20 30 40 50
(c) Cycling
X-bar chart-limits based on 50 samples
4.350i UCL
3.238
\fwj^A/V^
CL
p 1 P5 ""''"''' mln i ■ ■ i i ..!»... i in. I |_Q|_
10 20 30 40 50
(e) Stratification
R chart-limits based on 50 samples
2.874 i UCL
1.380 I
Q QQQ I 1 1 i l > 1 1 1 1 I i 1 1 1 1 1 1 1 1 1 1 lit l 1 1 1 1 1 1 1 1 I i 1 1 1 1 fSy_i i lIiTI |_Q|_
10 20 30 40 50
(b) Run
X-bar chart-limits based on 50 samples
5.478 r
5.289
UCL
CL
Li.Lll.lllllLJMlllLillllllll LCL
10 20 30 40 50
(d) Instability
FIGURE 15.3 Interpreting control chart patterns. (Adapted from Evans, J.R.,
Statistical Process Control for Quality Improvement: A Training Guide to Learning
SPC, 1st ed. Copyright 1991. Reprinted by permission of Pearson Education, Inc.,
Upper Saddle River, NJ.)
be scientifically determined. In many cases, the appropriate CLs may not be
readily apparent or available to HACCP team members. Wedding has listed
some sources to consult for this information, including scientific research
articles, government documents, trade association guidelines, in-plant studies,
university extension publications, and industry experts [46]. If outside sources
are used to establish CLs, they should be documented and become part of
the HACCP plan.
Once CLs based on scientific data have been determined for each CCP,
a capability study must be conducted on the HACCP process to ensure it
can be realistically and consistently maintained within these defined limits.
As noted by Evans, the process must first be in a state of statistical control
before performing a process capability study [45]. The major function of a
capability analysis is to determine by measurement how well the control
measure used at that CCP is functioning when compared to the specifications
HACCP: A Process Control Approach for Fruit and Vegetable Safety 353
set at the CL [43]. Establishing CLs that may be outside the capability of
the process will ultimately jeopardize the integrity of the entire HACCP
plan [47].
Several texts reference how to conduct a process capability study [2,43].
Assessment of process capability is required to determine the relationship
between the natural process variation and specified tolerances. Thus, indivi-
dual temperature readings for producing safe juices should always operate
within the CLs for safe pasteurization temperature. Evans has expressed
process capability as the ratio of the tolerance width to the natural process
variation. In the context of HACCP, this would be defined as shown in
Figure 15.4 [45]. As noted by the CL definition, only one-sided limits are
necessary for most HACCP capability studies. When this is the case, the
formulae in Figure 15.5 apply. Examples of when this might be appropriate
include the maximum pH allowed for an acidified vegetable product where
no heat treatment is applied during the process and the product is stored
in ambient temperature, or the minimum scheduled process temperature
allowed for a low-acid canned vegetable to ensure safety [3]. When a CL
is violated, it signals that an unsafe product may have been manufactured at
this CCP. Immediate action must be taken to bring the CCP back into its CL
range. Also, any product manufactured at the time the CL was violated must
be held for evaluation and/or reprocessing.
When CLs have been set for all CCPs, the task is to keep the para-
meter being measured in control within the established tolerances. This may or
may not be an easy job depending on the kind of variation in the process.
Cp =
tolerance width of CL CL y - CL L
natural variation 6a
Where:
C P = quotient of tolerated variation
CLy = upper critical limit of CCP
CL L = lower critical limit of CCP
6a = actual process variation, assuming a normal distribution
FIGURE 15.4 Calculating upper and lower critical limits at a CCP.
Cp„=%^ or C Pi = CL l
u 3a rL 3a
Where:
C P = process capability in C P = process capability in
relationship to the relationship to the
upper Critical Limit lower Critical Limit
CL y = upper Critical Limit CL L = lower Critical Limit
x = process average x = process average
3a = only right side of 3a = only left side of
normal distribution normal distribution
FIGURE 15.5 Calculation formulae for a process requiring only one critical limit.
354 Microbiology of Fruits and Vegetables
Establishing operating limits is a practical means to help prevent routine
violation of the CLs [46]. Operating limits are criteria that are more
stringent than CLs and are established at a level that would be reached before
the critical limit is violated [48]. Process adjustment should be taken when
the operating limit is exceeded to avoid loss of control and the need to take
corrective action at the CL.
15.10 ESTABLISHING SPC MONITORING
PROCEDURES (HACCP PRINCIPLE 4)
Selection of the correct monitoring system is an essential part of any HACCP
study because it is what the HACCP team relies upon to maintain control at
the CCPs. By definition, monitoring is a planned sequence of observations
or measurements to assess whether a CCP is under control and produces
an accurate record for future use in verification [7]. Monitoring serves to
(1) track the operation of a process and enable the identification of trends
toward a CL that may trigger process adjustment; (2) identify when and
where there was a loss of control (a deviation occurred at the CCP) such that
corrective action is needed; and (3) provide written documentation of the
process control system [48].
The HACCP team will be responsible for designing the monitoring acti-
vities at each CCP, as well as training the individual(s) who will carry out the
monitoring. These individuals should have the authority to stop the opera-
tion and to take corrective action if the CL is violated. All records and
documents associated with monitoring CCPs should be recorded, signed,
and dated by the person doing the monitoring, and, where necessary, assessed
by a designated manager with overall responsibility for the food product [4].
Procedures must identify what control measures will be monitored, how
frequently monitoring should be performed, what procedures will be used, and
who will perform the monitoring [27].
Physical (e.g., temperature, ORP millivolts, metal detection) and chemical
(e.g., chlorine, pH, acidity) monitoring systems are always the preferred
methods of monitoring because of their ease and real-time data feedback.
Monitoring systems may be continuous (e.g., recording on a continuous
circular chart the pasteurization temperatures of juice filled into bottles) or
discontinuous (e.g., measuring residual free chlorine in fresh-cut vegetable
flume water at specific intervals). Monitoring equipment systems may be
online (e.g., temperature and ORP probes, metal detectors) or offline
(e.g., chlorine test kits, determination of titratable acidity, water activity
measurements). The equipment chosen for CCP monitoring must have the
degree of sensitivity to control hazards accurately. Daily calibration or
standardization is necessary, and records should be maintained on these
procedures, to become a part of the support documentation for the HACCP
plan [27]. While microbiological testing is not suitable for controlling CCPs,
HACCP: A Process Control Approach for Fruit and Vegetable Safety 355
the above measurements (excluding metal detection) can serve as an indirect
measure of microbiological control at the CCPs.
Statistical control charting is ideally suited for HACCP monitoring
of designated CCPs, because it provides an early warning signal of the need
for corrective action before a CCP is violated. In terms of process con-
trol, however, all statistical control charts are not created equal. Variable
control charts are much better than attribute charts in detecting an impend-
ing change in a process. This is because variable charts use quantitative data
measurements while attribute charts work with qualitative data. Variable
charts can pinpoint the relative position of plotted points within a CL such that
if there is a move toward the boundary, or if an unusual pattern of points
signals there is trouble in the process, corrective action can be taken
immediately before the CL is compromised. In contrast, attribute charts
utilize a pass/fail system of data gathering, and cannot signal a change until
the problem has already occurred. Therefore attribute charts are poor tools
for anticipating process change [49].
It is important to note, however, that process control may not be
HACCP control. If the common cause variation of the parameter monitor-
ing a CCP is too great, the process may exceed the CL. Thus, a process in
statistical control may not be capable of producing a safe product. Likewise,
the parameter monitoring a CCP (ORP in millivolts) may be within the
CL, but not in statistical control. In fact, any one of four scenarios may
exist, as demonstrated in Figure 15.6 [27]. Thus, to ensure product safety, the
important point is that SPC limits must have less variability than HACCP
limits [50].
It must be remembered that any statistical chart that relies on plotted
data averages may obscure extreme values that could pose a health hazard [37].
While plotted averages for a CCP may be within critical limits, individual
values may be above or below the CL for safety. For this reason, it is recom-
mended to first monitor CCPs using individual values plotted on individual/
moving range charts to be certain they can remain within their predeter-
mined CLs [49]. Once process stability has been achieved, then one can proceed
to construct average/range charts. These are better indicators of any process
shift that may occur for a CCP within the CL.
The availability of numerous SPC software packages has increased
significantly in the last 1 5 years, because of listings and reviews in publications
such as Quality Progress, Journal of Quality Technology from the American
Society for Quality (ASQ), as well as from vendors on the Internet. Much
of the fruit and vegetable industry has moved from charts on paper and
clipboards to computer-controlled processing. It is now possible to load raw
data into software that creates control charts and performs capability studies
with great speed and accuracy. However, even though this technology is time
and cost saving, there is the danger that operators do not understand the
theoretical background on which these programs are based and therefore
can draw inappropriate conclusions based on computer results. As Cullen
and Hollingum point out, a computer will carry out complicated calculations
356
Microbiology of Fruits and Vegetables
>
CL
Chlorine monitoring
730
690
UCL
-LCL
O 650 -.-• CL
-I — I— I — I — I — I — I — \—\
Time (0.5 hr)
(a) Process in statistical control
and within critical limits (CL)
730
Chlorine monitoring
UCL
I— I — I — I — I — r
Time (0.5 hr)
(b) Process in statistical control,
but outside of critical limits (CL)
>
c_
CL
O
730
690
650
Chlorine monitoring
UCL
Time (0.5 hr)
(c) Process out of statistical control,
but within critical limits (CL)
>
E
DC
o
730
690
Chlorine monitoring
UCL
650 - -
Time (0.5 hr)
(d) Process out of statistical control,
and outside of critical limits (CL)
FIGURE 15.6 Monitoring chlorine concentration in ORP (mV) using SPC and HACCP
methodology. (Adapted from Hurst, W.C., Safety aspects of fresh-cut fruits and
vegetables, in Fresh-Cut Fruits and Vegetables: Science, Technology and Market,
Lamikanra, O., Ed., CRC Press, Boca Raton, FL, 2002. With permission.)
very quickly, but unless the user fully understands the significance of the
figures and graphs generated, the user can easily move to some extremely
misleading conclusions [35]. Computers are no substitute for a thorough
training in the fundamentals of SPC.
15.11 DETERMINING CORRECTIVE ACTION
PROCEDURES (HACCP PRINCIPLE 5)
Since the natural forces of entropy cause all HACCP processes to deteriorate
toward a state of disorder, deviations from CLs will occur, and corrective
actions will be needed [38]. By definition, a corrective action must be taken
when a CL deviation is identified by monitoring a CCP [7]. Tompkin pointed
out that corrective action involves four activities: (1) bringing the process
back into its CL through process adjustment; (2) determining and correct-
ing the cause of the deviation; (3) determining the disposition of the non-
compliant product; and (4) recording the corrective action taken and the
disposition of the noncompliant product [51].
When CLs are violated at a CCP, predetermined (developed in advance
for each CCP and included in the HACCP plan) corrective action proce-
dures must be initiated. Slade noted that a corrective action should take care
HACCP: A Process Control Approach for Fruit and Vegetable Safety 357
TABLE 15.3
Steps for Determining Product Disposition
1 . Determine if the product presents a safety hazard, based on:
(a) Expert evaluation
(b) Biological, chemical, or physical testing
2. If no hazard exists, the product may be released.
3. If a potential hazard exists, determine if the product can be:
(a) Reworked or reprocessed
(b) Diverted for an alternative use
4. If potentially hazardous product cannot be handled as described in step 3, the product must be
destroyed
of the immediate (short-term) problem as well as provide a long-term solu-
tion [9]. It may be necessary to determine the root cause of the deviation
to prevent future recurrence. A CL failure that was not anticipated or one
that reoccurs should result in the adjustment of the process or product and
a re-evaluation of the HACCP plan. Because of the great diversity in fruit
and vegetable products, and variation in the equipment used, type of proces-
sing, raw materials, etc., specific corrective actions must be developed for
each CCP, according to the parameters of processing. When a deviation
occurs and nonconforming product is produced, there are four steps for deter-
mining product disposition, as outlined in Table 15.3 [9].
Individuals who have a thorough understanding of the product,
process, and HACCP plan should be assigned responsibility for writing the
corrective action procedures and overseeing that the corrective actions are
implemented [33]. Likewise, a log entry for each corrective action procedure
should identify the person responsible for taking action to control product
safety. HACCP plan records should contain a separate file in which all
deviations and corresponding actions are maintained in an organized fashion.
15.12 CONFIRMING HACCP IMPLEMENTATION
THROUGH VERIFICATION ACTIVITIES
(HACCP PRINCIPLE 6)
Verification and validation comprise principle 6 of the HACCP study and
must be carried out for each identified CCP in the HACCP plan. Verification
is defined as the application of tests, procedures, and evaluations, in addi-
tion to monitoring, that determine the validity of the HACCP plan and that
the system is operating as written [7]. Verification deals with implementa-
tion of the HACCP plan. It must be a routine part of the daily production
process. The purpose of verification is to confirm through documentation
that food safety has been achieved at the CCPs according to the implemented
HACCP plan [52].
358 Microbiology of Fruits and Vegetables
Major verification activities include plant audits, calibration of instru-
ments and equipment, CCP records review, targeted sampling, and micro-
biological testing [48]. The objective of audit verification is to compare
actual practices with what is contained in the HACCP plan. Audits can be
performed by a member of the HACCP team, plant management, outside
experts or consultants, regulatory agencies, and customers. If the calibration
of instruments and equipment is not done on a scheduled and frequent basis,
significant deviations at a CCP might go unnoticed, thus creating a potential
health hazard. If this happens, the CCP would be considered out of control
since the last documented acceptable calibration. The CCP record review
involves examining two types of records generated at each CCP: monitor-
ing and corrective action. These records are valuable management tools,
providing documentation that CCPs are operating within established safety
parameters and that deviations are being handled in a safe and appropriate
manner. Verification also includes targeted sampling and microbiological
testing. Vendor compliance can be checked by targeted sampling when receipt
of material is a CCP, and purchase specifications are relied on as control
limits. Microbiological testing can be used as a verification tool to determine
if the overall operation is under control.
In 1986 the International Commission on Microbiological Specifications
for Food (ICMSF) published a statistically based acceptance sampling plan
for testing microbiological hazards in foods [53]. Although acceptance
sampling utilizes random sample collection and has good statistical validity,
the nonrandom distribution of pathogens and low probability for detection
gives no guarantee that their number is below a safe level or that they are
absent from fruit and vegetable products [54]. While acknowledging this
inherent flaw, acceptance sampling is based on sound science and therefore
can serve as an important verification tool in the design and validation of
process control through monitoring [3]. Microbiological results obtained for
a food product can tell about the process: whether the process at the time
of sampling was good (under control) or bad (out-of-control) in its abatement
of microorganisms and/or human pathogens. Also, to improve verification,
the stringency of acceptance sampling plans can be increased by modifying
the operating characteristic (OC) curves associated with these plans to
increase sample size draw or lower acceptance numbers. These more rigorous
criteria for acceptance will ensure a higher probability for making the correct
decision to pass or fail inspection by these plans [37]. As noted by Leaper,
some end product testing, particularly for verification purposes, will always
be required by customers to document product safety to consumers [4].
Validation of the HACCP plan should be performed whenever there are
indications that a process is unstable or out of control, or whenever there is a
change in product, formulation, or processing equipment. HACCP revalida-
tion should be performed on a periodic basis, even if no changes have occurred
in the process, so the plan will retain its support base [33].
HACCP: A Process Control Approach for Fruit and Vegetable Safety 359
15.13 ESTABLISHING DOCUMENTATION AND
RECORD KEEPING (HACCP PRINCIPLE 7)
Establishing accurate documentation and efficient record keeping is essential
to the successful application of HACCP. Documentation demonstrates that
the principles of HACCP have been correctly applied. Records provide the
written evidence that all HACCP activities were carried out as specified.
Record keeping, admittedly, can be a boring, tedious task; yet, in the words
of a USDA inspector, if it was not written down, as far as he was concerned,
it was not done! Experience has demonstrated that inadequate or inefficient
record keeping is a major reason for HACCP audit failures.
All HACCP records should be kept in a separate master file so that only
product/process safety is reviewed during a HACCP audit. Software systems
are available to assist in the documentation of HACCP plans and keeping of
records. The FDA requires that HACCP records be kept on file for at least one
year from the date of production for refrigerated foods (e.g., fresh-cut
produce). Although record keeping may appear to be a burden, there are sound
reasons for this activity which will benefit the processor, including the
following [27]:
1. Documents that all CCPs are within CLs to ensure product safety.
2. Provides the only reference available for produce traceability once
the product leaves the plant.
3. Documents corrective actions taken when CLs were exceeded.
4. Provides a monitoring tool so process adjustments can be made to
prevent loss of control.
5. Provides data for review during regulatory, customer, and third-party
auditing.
6. Provides demonstrable evidence that procedures and processes were
followed in strict accordance with the written HACCP plan.
Record keeping includes records that go beyond those that are tabulated
on a day-to-day basis. NACMCF endorses the maintenance of four types of
records [7]:
1 . Summary of the hazard analysis — includes records on the HACCP
team's deliberations on the rationale for determining hazards and
control measures.
2. The HACCP plan for each product, including records of the
product description, distribution and end use, verified flow diagram,
and all HACCP plan summaries addressing the seven required
components.
3. Support documentation — CCP records, CL records monitoring and
corrective action records, verification and validation records.
TABLE 15.4
Excerpt of HACCP Plan Summary Page for Fresh-Cut Lettuce
©
Process
Biological/chemical/
Critical limits
Monitoring procedures/
Corrective actions/
Verification procedures/
step;
physical
frequency /person
person responsible
person responsible
CCP
tin
hazard description
responsible
Water
Biological: L.
Potable water
Prior to start of processing
QC personnel
QC personnel will
flume
monocytogenes;
containing
and each 30 minutes there-
will adjust water
maintain chlorine,
wash;
E. <%>//0157:H7;
> 1 ppm free
after, QC personnel will
chemistry to
pH, temperature
CCP IB
Salmonella
residual chlorine
monitor free chlorine using
maintain pH and
monitoring logs; CCP
spp., Shigella spp.,
for 30 seconds;
standardized test kit, and
chlorine added to
deviations/corrective
other microbial
at pH < 7.0
a calibrated pH meter will
maintain CL;
action logs, calibration
pathogens
be used to monitor pH
held product will
logs for thermometer,
three times per shift
be rewashed and
CL deviations
noted in log
pH test and chlorine test
kit used; microbiological
tests will be run on
finished product at least
once per year to validate
pathogen absence.
HACCP plan will be
revalidated at least one
per year
HACCP records
From IFPA Technical Committee, HACCP for the Fresh-Cut Produce Industry, IFPA, Alexandria, VA, 2000. With permission.
HACCP coordinator
reviews all HACCP
records weekly, HACCP
coordinator will conduct
calibration tests; plant
manager reviews records
daily, state food
inspector/FDA audits,
customer audits/
internal/consultant
audit once per year, all
records kept at least one
year; random sampling/
testing product to ensure
process verification
n
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o
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HACCP: A Process Control Approach for Fruit and Vegetable Safety 361
4. Daily operational records — includes records generated daily
detailing control of the HACCP process for each CCP (specifically,
monitoring, corrective action, and verification).
HACCP records can be quite diverse and may include procedures
for monitoring, calibrating, corrective action, and verifying and validating a
CCP. An example of the records necessary to maintain and document control
at the water flume wash step CCP of a fresh-cut lettuce operation is given in
Table 15.4.
15.14 SUMMARY
To meet the challenges of today's food safety issues, the FDA has increased its
emphasis toward programs that are proactive and prevention oriented. The
most comprehensive, science-based program to date for reducing pathogen
contamination in fruit and vegetable products is HACCP. It has become the
industry standard for ensuring food safety. Unlike past traditional approaches
which relied on end-product testing, HACCP focuses on continuous control
and monitoring of CCPs to ensure safety all along the production and
processing continuum. Because all product and processing operations tend to
vary over time, however, it becomes important to be able to identify and
quantify the type of variation present in them. Unfortunately, an inherent
weakness of HACCP is that it can neither identify variation within a process
nor provide any advanced warning as to when a CCP has a high probability of
exceeding its CL, causing loss of control within the safety zone. If HACCP is to
be a truly effective prevention tool, it must be linked to appropriate procedures
that both monitor and verify that a process can remain in control and safe.
The reliability and effectiveness of HACCP as a safety tool can be
greatly strengthened by the incorporation of statistical quality control (SQC)
methods, namely SPC and acceptance sampling, into its structure. SPC is an
objective, quantitative, and statistically valid means of predicting CCP control
during monitoring. In SPC, the data generated can be used on a continuous
basis to assess whether any unacceptable trends are developing over a period
of time at a CCP and whether the process is in statistical control. In similar
fashion, acceptance sampling brings the scientific method to HACCP
verification activities. It can validate and verify that a process is not only
operating in a safe zone of control but is producing a safe product.
Integration of SPC into a HACCP program will provide several benefits.
First, it will bring about a culmination to any processor's/packer's HACCP
plan in that statistically valid control charts will demonstrate to customers
evidence of product safety. Second, it will provide an on-going and conti-
nuous improvement of all processes which will have a positive impact on
the company's "'bottom line." Third, it will satisfy future government regu-
lations that are moving toward the requirement that a fresh produce processor/
packer or fresh-cut processor be able to document compliance with product
performance standards.
362 Microbiology of Fruits and Vegetables
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16
Effect of Quality Sortin
and Culling on the
Microbiological Quality
of Fresh Produce
Susanne E. Keller
CONTENTS
16.1 Introduction 365
16.2 Grade Standards 366
16.3 Effectiveness of Good Agricultural Practices (GAPs) 366
16.4 Effectiveness of Sorting and Sorting Methods 367
16.5 Impact on Food Safety 370
References 371
16.1 INTRODUCTION
Quality sorting and culling of fresh produce is performed to separate damaged
or decayed produce from undamaged sound produce. The principal motivation
for such sorting is financial. Consumers are not likely to purchase fresh fruit
or vegetables that are noticeably damaged or decayed. Produce destined for
further processing, rather than fresh consumption, will also demand a better
price if quality is higher. Poor-quality produce will result in higher processing
cost, greater losses, and shorter shelf life of the final product.
Although the principal motivation for quality sorting may be financial,
another more important motivation should be the desire to provide a safe and
nutritious product. Damaged and decayed produce can have substantially
higher levels of microorganisms than undamaged sound produce. Storing or
processing such damaged and decayed produce with sound produce may result
in the spread of spoilage organisms, resulting in further losses and lower quality
of finished products. In addition, foodborne pathogens can find greater ingress
in damaged and decayed produce, resulting in a significant increase in the risk
of foodborne illnesses. Consequently, the prompt removal of unsound produce
will impact not only on costs, but the safety of the product as well, be that fresh
or processed produce.
365
366 Microbiology of Fruits and Vegetables
16.2 GRADE STANDARDS
In general, produce is sorted according to established standards. Standards for
many types of produce are established by the U.S. Department of Agriculture
(USDA) and can be obtained at http://www.ams.usda.gov/standards. Stan-
dards can vary for produce from different areas. Oranges produced in Florida
have somewhat different standards than oranges produced in California.
Differences in grade standards are related to different growing conditions
and climate which influences characteristics such as sugar and acid levels. The
highest grades are generally represented by produce that is free of blemishes,
cuts and bruises, and any decay.
Numerous environmental and mechanical factors can influence product
grade. Growing conditions and weather can dramatically affect produce
quality. The consequences of environmental and mechanical factors for apples
were examined by Baugher et al. [1]. Economic losses due to various defects
resulting from both environmental and mechanical factors were measured for
apples at nine packinghouses. Severe drought and high temperatures caused
a significant increase in losses due to undersized fruit, cork spot, and spray
injury. Some types of defects, particularly bacterial soft rot of fresh fruits
and vegetables, have been associated with an increased incidence of pathogen
contamination. Wells and Butterfield found higher salmonella contamination
(59%) in wash water from fruit and vegetables that were affected by bacterial
soft rot [2]. Wash water used for healthy produce had a lower incidence of
salmonella contamination (33%). Other defects, such as undersized fruit,
would appear to be primarily cosmetic and therefore of less importance in
relation to spoilage or food safety. However, research suggests that lower
quality produce, regardless of defect type, is more prone to spoilage. In a study
on the effect of tomato grade on subsequent spoilage, it was determined that
lower grade tomatoes inoculated with Erwinia carotovora subsp. carotova had
higher levels of spoilage and infection after 14 days in storage than higher
grade tomatoes [3]. Although the tomatoes were inoculated with E. carotovora,
82.4% of the infection was due to Alternaria alternate, and only 17.6% was due
to the bacterium. The bacterium did, however, increase decay in lower grade
tomatoes to a greater extent than higher grade. This study suggested surface
blemishes of any type promoted postharvest decay.
16.3 EFFECTIVENESS OF GOOD AGRICULTURAL
PRACTICES (GAPs)
Good agricultural practices (GAPs) fundamentally influence the level of
microflora on produce and products made from produce. In 1998 the U.S.
Food and Drug Administration (FDA) published a document in conjunction
with the USDA entitled Guide to Minimize Microbial Food Safety Hazards
for Fresh Fruits and Vegetables. This document was published as a draft
on April 13 and as a final version on October 26, 1998 [4,5]. The document
Quality Sorting and Culling of Fresh Produce 367
outlines hazards associated with common agricultural practices such as
irrigation water, application of manure, field and facility sanitation, packing,
and transportation. Although this document does not specifically address
sorting and culling, inclusion of damaged and decayed produce amplifies the
risks described in it.
The risk of subsequent spoilage and invasion of foodborne pathogens can
be reduced through careful handling and appropriate phytosanitary measures
including appropriate sorting and culling. The majority of spoilage organisms
found on fruits and vegetables at harvest are essentially opportunistic organ-
isms that require physical injury or excessive softening to gain entry to host
tissue [6]. Many plant pathogens will infect adjacent areas of healthy produce
once they have become established in damaged produce. In addition, such
damaged and rotted areas particularly in ordinarily acidic tissue allow growth
and survival of foodborne pathogens that might otherwise not survive.
Escherichia coli 0157:H7 survival was enhanced in bruised apples [7]. The
bruised tissue was found to have significantly higher pH values that allowed
growth of the pathogen. Survival of E. coli 0157:H7 was also enhanced when
apples were wounded and inoculated with the plant pathogen Glomerella
cingulata [8]. This enhanced survival and growth was again attributed to
increases in pH that accompanied infection by G. cingulata.
In addition to the increased risk of infection with foodborne pathogenic
organisms, decayed produce may be contaminated with toxins produced by the
invading microorganisms. Patulin is one mycotoxin that is produced primarily
by Penicillium expansium, responsible for blue mold rot on apples, pears, and
other fruit. Levels of patulin in apple juice have been correlated to the level
of decay found on the apples [9]. Clearly, improper sanitation and handling
of damaged produce where pathogens have greater ingress to internal tissue
would represent a greater risk of foodborne illness than the use of sound
produce. Consequently, proper sorting and removal of any damaged and
decayed produce is an essential prerequisite in the prevention of foodborne
illnesses.
16.4 EFFECTIVENESS OF SORTING AND SORTING
METHODS
That removal of damaged and decayed fruit and vegetables prior to storage
or subsequent processing should reduce microbial loads and thus reduce any
risk of foodborne illness would seem intuitive. However, data to document
the actual level of reductions achieved by sorting and culling are not always
available. In data provided by the Florida Department of Citrus, juice
microflora was 4.5 ±0.71ogCFU/ml with no grading, 3.7 ± l.OlogCFU/ml
with light grading, and 2.2 ± 0.6 log CFU/ml with moderate grading [10]. Light
grading was defined as splits removed, 0.5 to 1% of fruit stream; and moderate
grading was splits, peel plugs, and significant blemishes removed, 2 to 3% of
incoming fruit stream.
368
Microbiology of Fruits and Vegetables
Sorting and removing poor-quality apples during cider production results
in a reduction in aerobic counts in the final cider. In one recent study,
cider was produced from seven different apple varieties. Production variables
included method of harvest, quality sorting, and storage. Cider from fresh
ground-harvested fruit, considered to be lowest quality and greatest risk, had
significantly greater numbers of aerobic microflora (4.89 log CFU/ml) than any
cider from tree-harvested fruit (Figure 16.1) [11]. Yeast and mold populations
were also elevated in ground-harvested fruit (Figure 16.2). Cider from unsorted
and unculled fruit had an average of 3.45 log CFU/ml, whereas cider from
sorted and culled apples had an average of 2.88 log CFU/ml (p < 0.05) [11].
As with ground-harvested fruit, yeast and mold populations were elevated in
cider from apples that were not sorted and culled prior to cider production.
Along with the examination of microflora levels in sorted or unsorted
apples, the cider in the same study was also tested for changes in the level of
patulin [12]. Patulin levels varied depending on storage and variety. No patulin
was detected in cider produced from any fresh tree-harvested fruit. However,
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FIGURE 16.1 Differences in Tukey box plots of aerobic plate counts (APC) of pooled
apple varieties due to harvest conditions, storage, and culling. Means sharing a letter
were not statistically different at the p < 0.05 level. FTC = fresh, tree-harvested, culled;
FTU = fresh, tree-harvested, unculled; FGU = fresh, ground-harvested, unculled;
STC = stored, tree-harvested, culled; STU = stored, tree-harvested, unculled. (Modified
from Keller, S.E. et al., J. Food Prot., 67, 2240, 2004. With permission.)
Quality Sorting and Culling of Fresh Produce
369
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Cider
FIGURE 16.2 Differences in Tukey box plots of yeast and mold populations in pooled
apple varieties due to harvest conditions, storage, and culling. Means sharing a
letter were not statistically different at the p < 0.05 level. FTC = fresh, tree-harvested,
culled; FTU = fresh, tree-harvested, unculled; FGU = fresh, ground-harvested, unculled;
STC = stored, tree-harvested, culled; STU = stored, tree-harvested, unculled. (Modified
from Keller, S.E. et ah, J. Food Prot., 67, 2240, 2004. With permission.)
cider pressed from controlled atmosphere (CA) stored apples did contain
significant levels of patulin. These patulin levels were significantly reduced in
most varieties when the apples were culled prior to cider production. Chapter
13 contains a more in-depth review of patulin in apple cider.
Sorting and culling are included in a 1999 survey of industry practices
published by the USD A [13]. This report covers 30 fruit and vegetable com-
modities in 14 states. The report focuses on fruit and vegetable commodities
that are predominately consumed raw, as these represent a substantially greater
risk than produce that undergoes further processing. Of all the produce in the
survey, the vast majority was harvested by hand. For fruit acres, 94% was
harvested by hand only, whereas for vegetable acres, 87% was harvested by
hand. The majority of packers in this survey manually sorted both fruit
and vegetables. It should be noted, however, that produce in this survey was
intended for the fresh (consumed raw) market, and for many specific types of
produce in the survey, data were unavailable. Nonetheless, the survey reported
that 100% of apples, table grapes, broccoli, celery, cucumbers, cantaloupes,
and honeydew melons were manually sorted.
That manual sorting and culling are used for the majority of fresh produce
is not surprising. Mechanical handling and sorting can result in substantial
damage, increased losses, and may incur considerable expense to implement.
370 Microbiology of Fruits and Vegetables
In a study on grade-lowering defects in grapefruit, mechanical injury levels
were found to increase from pregrading areas (5.6%) to final grading (8.6%)
and to final packaging (12.3%) [14]. Increased injury was also related to
increased levels of bacterial soft rot in Bell peppers [15].
Despite such reported problems, mechanical handling and sorting have an
advantage in speed. For manual sorting, fruit or vegetables are sorted either
in the field or at the packinghouse. Most frequently, a sorting table and/or
conveyor are used. Care must be taken that the speed of the conveyor is slow
enough to allow individual examination by personnel of each piece. Most
mechanical systems consist of two basic elements, a conveyor and some means
of separation, based on specific fruit or vegetable characteristics such as size,
weight, color, and/or the presence of defects. The simplest of such instruments
sorts using uniformity of size and shape by means of appropriate holes or
sizers. Such simple sorting machines still require considerable worker atten-
tion, but can significantly increase sorting speed, particularly for less fragile
produce. Such instruments would not, however, detect other defects.
More elaborate and modern instruments that sort by color and defects
using camera-based systems and computer technology are available commer-
cially. Produce is conveyed in traditional fashion to a scanning area where it is
scanned and analyzed using computer programs to determine appropriate
grade. Use of such a method was described by Leemans et al. for apples [16].
Leemans et al. reported correct classification up to 78% for Golden Delicious
apples and 72% for Jonagold. Classification was based on external quality
parameters such as color and texture.
Both manual sorting and machine sorting based on camera methods detect
defects and characteristics external to the fruit or vegetable. However, some
defects may be small and difficult to detect. Other defects may be internal and
not detectable using any surface examination methods. More recently, research
has been directed at the development of methods that may allow the detection
of such internal defects in a nondestructive manner. These new methods are
based primarily on X-ray imaging, magnetic resonance imaging (MRI), or
near-infrared spectroscopy (NIR) spectroscopy [17-24].
Some produce types are not particularly suitable for machine sorting,
frequently because of their fragile nature. For such produce, researchers look
for novel means of assessing quality that may require less manual manipulation
of the produce. One such method that has potential is the use of an electronic
system that measures aromatic volatile gas emissions. Such a method was
employed by Simon et al. to assess the quality of blueberries [25]. The method
successfully detected differences in maturation levels and detected damaged
fruit in closed containers of blueberries.
16.5 IMPACT ON FOOD SAFETY
The Guide to Minimize Microbial Food Safety Hazards for Fresh Fruits and
Vegetables, jointly published by the FDA and the USD A, clearly describes
Quality Sorting and Culling of Fresh Produce 371
where the greatest risks of foodborne pathogen contamination are likely to
occur. It makes the assumption that damaged and decayed produce will not be
included during any further processing or packaging. The consequence of such
inclusion would be significantly higher microbial loads, shorter shelf life, and
a substantially increased risk of infiltration of pathogens internally in the
produce.
There is no intervention currently available that, if applied solely to the
surface of the fruit or vegetable, will destroy any internal foodborne pathogen.
Consequently, produce with internal foodborne pathogens that is minimally
processed or consumed raw will present a substantial risk of foodborne illness.
It is therefore critical to prevent the initial contamination of fresh produce so
that internal contamination does not occur and that internally contaminated
produce is not used for the fresh market. Appropriate quality sorting and
culling of damaged and decayed produce are essential to reducing the risk that
such potentially contaminated fruit will enter the fresh market.
The critical nature of appropriate sorting and culling was recognized by the
FDA in the final HACCP (hazard analysis critical control points) juice
regulation [26]. Because of the risk of pathogen infiltration into fresh produce,
all juice must receive a treatment that will give a 5-log reduction in the pertinent
pathogen. The treatment must be applied post-extraction, so all juice is treated,
with the exception of citrus products. For citrus products, the 5-log treatment
may be applied to the surface of the fruit. The risk of infiltration by patho-
gens in sound and intact citrus fruit was believed to be too small to merit a
mandated treatment to the extracted juice. However, it is important to note
that the legislation clearly states that such surface treatment must be applied
only to sound, intact fruit and after the fruit has been sorted and culled.
Although the incidence of foodborne illness caused by the consumption of
fruits and vegetables is low, there is some evidence that this risk is increasing
[27]. Part of the increase may be due to the increased consumption of fresh
produce, perceived by many consumers to be a healthy food choice. With an
increase in consumption of fresh produce comes increased risk particularly if
that produce is not handled in a safe and sanitary manner. Appropriate quality
sorting and culling to ensure damaged and potentially contaminated produce
does not enter the fresh market is not just an economically sound approach,
it is an important component in the prevention of foodborne illness.
REFERENCES
1. Baugher, T.A., Hogmire, H.W.J., and Lightner, G.W., Determining apple
packout losses and impact on profitability, Appl. Agric. Res., 5, 343, 1990.
2. Wells, J.M. and Butterfield, J.E., Salmonella contamination associated with
bacterial soft rot of fresh fruits and vegetables in the marketplace, Plant Dis.,
81, 867, 1997.
3. Blender, R.J., Sargent, S.A., Brecht, J.K., and Bartz, J.A., Effect of tomato
grade on incidence of decay during simulated shipping, Proc. Fla. State Hortic.
Soc, 105, 119, 1992.
372 Microbiology of Fruits and Vegetables
4. U.S. Food and Drug Administration, Draft guidance for industry: guide to
minimize microbial food safety hazards for fresh fruits and vegetables, Fed.
Regist., 63, 18029, 1998.
5. FDA, Guide to Minimize Microbial Food Safety Hazards for Fresh Fruits
and Vegetables, U.S. Food and Drug Administration, 1998.
6. Chesson, A., Maceration in relation to post-harvest handling and processing
of plant material, J. Appl. Bacteriol., 48, 1, 1980.
7. Dingman, D.W., Growth of Escherichia coli 0157:H7 in bruised apple
(Malus domestica) tissue as influences by cultivar, date of harvest, and source,
Appl. Environ. Microbiol., 66, 1077, 2000.
8. Riordan, D.C.R., Sapers, G.M., and Annous, B.A., The survival of Echerichia
coli 0157:H7 in the presence of Penicillium expansium and Glomerella cingulata
in wounds on apple surfaces, J. Food Prot., 63, 1637, 2000.
9. Kadakal, C. and Nas, S., Effect of apple decay proportion on the
patulin, fumaric acid, HMF and other apple juice properties, /. Food Saf., 22,
17, 2002.
10. Kelsey, F. and Pao, S., Factsheet 2, Fresh Citrus Juice. Key Factors in
Determining Fresh Citrus Juice Quality and Safety, Vol. 2003, Florida
Department of Citrus, 1999.
11. Keller, S.E., Chirtel, S.J., Merker, R.I., Taylor, K.T., Tan, H.L., and Miller,
A. J., Influence of fruit variety, harvest technique, quality sorting, and storage
on the native microflora of unpasteurized apple cider, J. Food Prot., 67,
2240, 2004.
12. Jackson, L.S., Beacham-Bowden, T., Keller, S.E., Adhikari, C, Taylor, K.T.,
Chirtel, S.J., and Merker, R.I., Apple quality, storage, and washing treatments
affect patulin levels in apple cider, /. Food Prot., 66, 618, 2003.
13. U.S. Department of Agriculture, Fruit and Vegetable Practices: 1999,
Agricultural Statistics Board NASS, USDA, 2001, p. 1.
14. Miller, W.M. and Burns, J.K., Grade lowering defects and grading practices for
Indian River grapefruit, Proc. Fla. State Hortic. Soc, 105, 129, 1992.
15. Carballo, S.J., Blankenship, S.M., Ritchie, D.F., and Boyette, M.D., Com-
parison of packing systems for injury and bacterial soft rot on Bell pepper fruit,
HortScience, 4, 269, 1994.
16. Leemans, V., Magein, H., and Destain, M.F., On-line fruit grading according to
their external quality using machine vision, Biosyst. Eng., 83, 397, 2002.
17. Bull, C.R. and McFarlane, N.J.B., X-ray detection of subsurface damage
in fruits and vegetables, Postharvest News and Information, 9, 29N, 1998.
18. Clark, C.J. and Burmeister, D.M., Magnetic resonance imaging of browning
development in "Braeburn" apple during controlled-atmosphere storage under
high C0 2 , HortScience, 34, 915, 1999.
19. Clark, C.J., McGlone, V.A., and Jordan, R.B., Detection of brownheart
in "Braeburn" apple by transmission NIR spectroscopy, Postharvest Biol.
Technoi, 28, 87, 2003.
20. Fraser, D.G., McGlone, V.A., Kunnemeyer, R. and Jordan, R.B., NIR (Near
Infra- Red) light penetration into an apple, Postharvest Biol. Technoi, 22,
191,2001.
21. Gonzalez, J.J., Valle, R.C., Bobroff, S., Biasi, W.V., Mitcham, E.J., and
McCarthy, M.J., Detection and monitoring of internal browning developments
in "Fuji" apples using MRI, Postharvest Biol. Technoi, 22, 179, 2001.
Quality Sorting and Culling of Fresh Produce 373
22. Keener, K.M., Stroshine, R.L., and Nyenhuis, J.A., Evaluation of low field
(5.40 MHz) proton magnetic resonance measurements of Dw and T2 as methods
of non-destructive quality evaluation of apples, /. Am. Soc. Horticult. Sci., 124,
289, 1999.
23. Schatzki, T.F., Haff, R.P., Young, R., Can, I., Le, L.C., and Toyofuku, N.,
Defect detection in apples by means of X-ray imaging, Trans. Am. Soc. Agric.
Eng., 40, 1407, 1997.
24. Upchurch, B.L., Throop, J.A., and Aneshansley, D.J., Detecting internal
breakdown in apples using interactance measurements, Postharvest Biol.
Technol., 10, 15, 1997.
25. Simon, J.E., Hetzroni, A., Bordelon, B., Miles, G.E., and Charles, D.J.,
Electronic sensing of aromatic volatiles for quality sorting of blueberries, J. Food
Sci., 61, 967, 1996.
26. U.S. Food and Drug Administration, 21 CFR Part 120, Hazard analysis
and critical control point (HACCP); procedures for the safe and sanitary
processing and importing of juice, Final rule, Fed. Regist., 66, 6137, 2001.
27. Anon., Analysis and Evaluation of Preventive Control Measures for the Control
and Reduction/Elimination of Microbial Hazards on Fresh and Fresh-Cut
Produce, Vol. 2003, U.S. Food and Drug Administration, 2001.
17
Washing and Sanitizin
Treatments for Fruits
and Vegetables
Gerald M. Sapers
CONTENTS
17.1 Introduction 376
17.2 Conventional Washing Technology 376
17.2.1 Washing Agents 376
17.2.1.1 Chlorine 376
17.2.1.2 Alternatives to Chlorine 378
17.2.2 Washing Equipment 383
17.2.2. 1 Types of Washers 383
17.2.2.2 Efficacy of Washers 385
17.2.3 Factors Limiting the Efficacy of Washing 387
17.3 Novel Washing Technology 387
17.3.1 Hydrogen Peroxide 387
17.3.2 Trisodium Phosphate and Other Alkaline
Washing Agents 388
17.3.3 Organic Acids 389
17.3.4 Other Experimental Antimicrobial Washing Agents 390
17.3.5 Synergistic Treatment Combinations 390
17.4 Foodservice and Home Applications 391
17.4.1 FDA Recommendations 391
17.4.2 Other Options 392
17.4.3 Commercial Equipment and Wash Formulations
for Home or Foodservice Use 393
17.5 Conclusions 394
Acknowledgments 394
References 394
Mention of trade names or commercial products is solely for the purpose of providing specific
information and does not imply recommendation or endorsement by the U.S. Department of
Agriculture.
375
376 Microbiology of Fruits and Vegetables
17.1 INTRODUCTION
The detection of human pathogens in fresh produce and occurrence of
outbreaks of foodborne illness associated with contaminated produce, as
documented in previous chapters, represent serious public health problems.
Contamination of fruits and vegetables with human pathogens or organisms
causing spoilage also has important economic consequences. Consequently,
it is in the interests of the produce industry to develop interventions to reduce
the risk of microbial contamination. If contamination is likely during
crop production or harvest, it is usually better to reduce this risk by avoid-
ance of contamination sources through implementation of good agricultural
practices (GAPs). However, this is not always possible, and in such situa-
tions the grower/shipper or processor must depend on washing and sanitizing
treatments as a second line of defense. If produce contamination occurs post-
harvest and contamination sources cannot be eliminated through improve-
ments in plant layout, implementation of good manufacturing practices
(GMPs), and improvements in plant sanitation, then washing and sanitizing
of produce and equipment become the first line of defense. The subject of
washing and sanitizing technology for fresh produce has been reviewed
previously [1-3].
In this chapter we review the efficacy, advantages, and disadvantages
of conventional washing and sanitizing agents for fresh fruits and vegetables.
We also examine the regulatory status of interventions for decontamination
of produce and equipment. We examine the types of equipment available for
treatment application and their performance. We briefly consider some of the
factors that limit the efficacy of cleaning and sanitizing agents and methods
of treatment. We examine the potential of new treatments for produce decon-
tamination. We also consider the problem of decontamination of fresh fruits
and vegetables in foodservice situations or in the home. This chapter does not
examine vapor-phase treatments, surface pasteurization, nonthermal physical
treatments, or biological control methods, all of which are covered elsewhere
in the book.
1 7.2 CONVENTIONAL WASHING TECHNOLOGY
17.2.1 Washing Agents
17.2.1.1 Chlorine
Most freshly harvested fruits and vegetables are washed by the grower,
packer, or processor to remove soil, plant debris, pesticide residues, and
microorganisms from the commodity surface. This may be accomplished
by spraying or immersion in water or solutions containing one of a number of
cleaning or sanitizing agents, using equipment designed for each particular
commodity type, e.g., leafy vegetables, root vegetables, fruit vegetables, tree
Washing and Sanitizing Treatments for Fruits and Vegetables 377
fruits, or melons. Chlorine is the most widely used sanitizing agent for fresh
produce. It may be added to wash water as Cl 2 gas or, more commonly, as
sodium or calcium hypochlorite. In water, at pH levels and concentrations
used on produce, these chlorine sources are converted to hypochlorous
acid and hypochlorite ion in a ratio determined by the solution pH [4,5].
At pH 6.0, roughly 97% of the unreacted chlorine is hypochlorous acid,
whereas, at pH 9.0, 97% is hypochlorite ion. The antimicrobial activity of these
solutions is due largely to hypochlorous acid rather than to hypochlorite.
The concentration of chlorine in a wash solution is sometimes expressed
as total available chlorine (or total residual chlorine = combined residual
chlorine + free residual chlorine), based on the calculated amount present
in the added hypochlorite or chlorine, or determined by oxidation of KI to
I 2 , which may not be indicative of the actual potency as a sanitizer because
of the inclusion of reaction products such as monochloramine which are
not very effective as sanitizers. Preferably, the chlorine concentration can be
expressed as free available (or residual) chlorine, the sum of hypochlorous
acid and hypochlorite ion concentrations [5]. The total or free chlorine
concentration can be monitored by means of test kits, based on colorimetry
(www.chemetrics.com; www.emscience.com, www.hach.com), or by measure-
ment of the oxidation-reduction potential (ORP). Chlorine is highly reactive
with certain types of compounds in organic materials and soils that are
leached or washed from fruits and vegetables. If this chlorine sink is excessive,
the free chlorine concentration will be depleted rapidly. Computerized
ORP systems that monitor the pH and chlorine concentration can be used to
control the level of chlorine in a wash tank in such situations (www.pulsein
struments.net; numerous other suppliers listed on www.globalspec.com).
Use levels of chlorine will depend on allowable levels, the commodity, and
the anticipated microbial load. The U.S. Food and Drug Administration (FDA)
specifies a use level for washing fruits and vegetables not to exceed 0.2% when
followed by a potable water rinse [6]. The U.S. Environmental Protection
Agency (EPA) exempts calcium hypochlorite "from the requirement of a
tolerance when used preharvest or postharvest in solution on all raw agricultural
commodities" [7]. The concentration range of 50 to 200 ppm is commonly
used for most commodities. However, as much as 20,000 ppm calcium hypo-
chlorite may be used to sanitize alfalfa seeds intended for sprout production
because of the failure of other treatments to disinfect adequately seeds
and sprouts, and the high risk that sprouts grown from contaminated seeds
may be a source of salmonella or Escherichia coli 0157:H7 outbreaks [8-10].
Chlorine is highly effective for inactivating planktonic cells of bacteria,
yeasts, molds, and viruses, although bacterial and fungal spores are consider-
ably more resistant [5]. However, chlorine is less effective for inactivating
bacteria attached to produce surfaces or embedded within the product [11-18].
Typically, population reductions of native microflora on produce surfaces
or of human pathogens on inoculated produce are no greater than 2 logs
(99%). While such reductions can greatly reduce spoilage, they are insufficient
to ensure safety in the event of contamination with human pathogens.
378 Microbiology of Fruits and Vegetables
The activity of chlorinated water may be increased by the addition of
an acidulant or buffer so that the pH is shifted from an alkaline value (about
pH 9) to the neutral to slightly acidic range (pH 6 to 7), thereby increasing the
proportion of hypochlorous acid in the equilibrium mixture. Organic acids
such as citric acid or mineral acids such as phosphoric or hydrochloric acid can
be used for this purpose. If the solution pH is too low (e.g., below pH 4),
hypochlorous acid may be converted to free chlorine which is subject to off-
gassing. This will result in a loss of activity and may be potentially hazardous.
Additionally, equipment corrosion is enhanced as pH levels drop below as well
as rise above neutrality. Unpublished data obtained at the Eastern Regional
Research Center indicated that hypochlorite solutions acidified with a mineral
acid were more stable than solutions acidified with citric acid [19]. Buffers
for hypochlorite solutions are available commercially (www.cerexagri.com).
The effectiveness of chlorine in inactivating microorganisms on produce
may be enhanced by adding a surfactant to the solution so that it can penetrate
into the irregular crevices and pores on produce surfaces where microorganisms
may lodge and escape contact with a sanitizer. Several commercial surfactant
formulations have been developed for this purpose (www.cerexagri.com/usa/
Markets/Cleaners). Addition of a nonionic surfactant improved the efficacy of
chlorine against decay fungi in pears [20,21]. Washing formulations contain-
ing sodium hypochlorite, buffers, and surfactants have been described by
Park et al. [22] and marketed by Bonagra Technologies under the name Safe-
T-Washed (www.bonagra.com). The efficacy of chlorine in reducing the
microbial flora of shredded iceberg lettuce was increased by elevating the
solution temperature to 47°C [23]. However, no greater reduction of non-
pathogenic E. coli (ATCC 25922) populations on inoculated apples was obtained
when apples were washed at 50 or 60°C compared to 20°C using 200 ppm Cl 2
(added as sodium hypochlorite), adjusted to pH 6.5 with citric acid [19].
Chlorine's major advantages are its broad spectrum of antimicrobial acti-
vity, ease of application, and low cost. However, chlorine is highly corrosive
and may damage stainless steel equipment after prolonged exposure. Its other
major disadvantages are rapid depletion in the presence of a high organic load
[24], and the potential carcinogenicity and mutagenicity of its reaction products
with organic constituents of foods [25-27]. This is a matter of concern to
processors, regulators, and consumers [28]. For these reasons, and the desirabi-
lity of obtaining greater population reductions, the development of alternative
sanitizing agents has been an active area of research, and a limited number
of agents suitable for use on fresh produce have been commercialized.
Electrolyzed water, a technology developed largely in Japan [29,30], is
really a special case of chlorination. This technology is discussed in detail in
Chapter 22.
17.2.1.2 Alternatives to Chlorine
A number of commercial detergent formulations have been developed for
washing fruits and vegetables. In addition, three approved sanitizing agents
Washing and Sanitizing Treatments for Fruits and Vegetables
379
Sanitizing
Use level
agent
(ppm)
Advantages
Chlorine
50-200
Easy to apply, inexpensive,
effective against all microbial
forms, not affected by hard
water, easy to monitor, FDA
approved
Ozone
0.1-2.5
TABLE 17.1
Advantages and Disadvantages of Commercially Available Sanitizing Agents
for Washing Fresh Fruits and Vegetables
Disadvantages
Decomposed by organic
matter, reaction products may
be hazardous, corrosive to
metals, irritating to skin, activity
pH-dependent, population
reductions limited to < 1-2 logs
Requires on-site generation,
requires good ventilation,
phytotoxic at high concentra-
tions, corrosive to metals, diffi-
cult to monitor, higher capital
cost than chlorine, no residual
effect, population reductions
limited to < 1-2 logs
Must be generated on-site,
explosive at high concentrations,
not permitted for cut fruits
and vegetables, population
reductions limited to < 1-2 logs,
generating systems expensive
Population reduction limited
to < 1-2 logs, strong oxidant,
concentrated solutions may
be hazardous
Chlorine
dioxide
1-5
Peroxyacetic
acid
<80
More potent antimicrobial
than chlorine, no chlorinated
reaction products formed,
economical to operate,
self-affirmed GRAS, but FDA
review possible, activity not
pH-dependent
More potent than chlorine,
activity not pH-dependent,
fewer chlorinated reaction
products formed than with
Cl 2 , effective against biofilms,
FDA approved, residual
antimicrobial action, less
corrosive than CI2 or O3
Broad spectrum antimicrobial
action, no pH control required,
low reactivity with soil, effective
against biofilms, FDA
approved, no hazardous
breakdown products, no on-site
generation required,
monitoring not difficult,
available at safe concentration
are available as alternatives to chlorine: chlorine dioxide (or acidified sodium
chlorite), ozone, and peroxyacetic acid. The advantages and disadvantages of
the agents described in the following sections are compared in Table 17.1.
17.2.1 .2.1 Detergent Formulations
Among the detergents approved by the FDA for washing produce are
sodium ft-alkylbenzenesulfonate, sodium dodecylbenzenesulfonate, sodium
mono- and dimethyl naphthalenesulfonates, sodium 2-ethylhexyl sulfate,
and others [6]. These formulations may be neutral in pH, acidic due to the
presence of citric or phosphoric acid, or alkaline because of the addition
380 Microbiology of Fruits and Vegetables
of sodium or potassium hydroxide. Major suppliers of detergent formulations
for produce cleaning include Cerexagri (formerly Elf Atochem N.A., Inc.,
source of Decco products) (800-221-0925; www.cerexagri.com), Microcide,
Inc. (www.microcideinc.com), and Alex C. Fergusson, Inc. (800-345-1329;
www.afcocare.com).
These products are designed to remove soil and pesticide residues
from produce and do not contain antimicrobial agents per se. Relatively little
information is available concerning the ability of these products to remove
or inactivate microorganisms attached to produce surfaces. However, their use
can result in significant population reductions. Sapers et al. reported that
some commercial washing formulations could achieve population reductions
as great as 1 to 2 logs in decontaminating apples inoculated with a non-
pathogenic E. coli, comparable to reductions obtained with hypochlorite [16].
When these products were applied at 50°C instead of at ambient temperature,
a 2.5 log reduction was obtained. Wright et al. [31] reported similar efficacy
with a commercial phosphoric acid fruit wash and with a 200 ppm hypo-
chlorite wash, each applied to apples inoculated with E. coli 0157:H7. Kenney
and Beuchat [32] compared the efficacy of representative commercial clean-
ing agents in removing or inactivating E. coli 0157:H7 and S. muenchen on
spot-inoculated apples. They obtained reductions as great as 3.1 logs with an
alkaline product and as great as 2.7 logs with an acidic product, reductions
generally being greater with salmonella. Raiden et al. [33] compared the
efficacy of water, sodium lauryl sulfate, and Tween 80 in removing Salmonella
spp. and Shigella spp. from the surface of inoculated strawberries,
tomatoes, and leaf lettuce. They obtained high removal rates but concluded
that the detergents were no more effective than water. However, this result
may have been a reflection of the brief time interval (1 hour) between
inoculation and treatment, which may have been insufficient for strong
bacterial attachment. In nature, the interval between preharvest contamina-
tion and postharvest application of a wash may be days or weeks, sufficient
time for strong attachment and even biofilm formation.
In a study of cantaloupe rind decontamination, Sapers et al. [34] repor-
ted reductions in the total aerobic plate count of about 1.3 logs when the rind
was washed with a 1% solution of a commercial produce wash containing
dodecylbenzene sulfonic acid and phosphoric acid (pH 2) at 50° C. Sequential
washing with this product followed by treatment with 1% hydrogen pero-
xide, both at 50°C, resulted in a 3.1 log reduction. Both washes extended
the shelf life of fresh-cut cantaloupe prepared from the treated melons. No
significant population reductions were obtained when the cantaloupe rind
was washed with aqueous solutions of sodium dioctyl sulfosuccinate or sodium
2-ethylhexyl sulfate.
17.2.1.2.2 Chlorine Dioxide
Solutions of chlorine dioxide and acidified sodium chlorite have been
used commercially as alternatives to chlorine for sanitizing fresh produce.
Washing and Sanitizing Treatments for Fruits and Vegetables 381
Chlorine dioxide is considered to be efficacious against many classes of
microorganisms [5]. Chlorine dioxide and acidified sodium chlorite are
approved by the FDA for use on fresh produce [35,36], but chlorine dio-
xide is not permitted for use on fresh-cut products. Chlorine dioxide must
be generated on-site, usually by reaction of sodium chlorite with an acid
or chlorine gas. Information concerning various proprietary generating and
stabilizing systems are available from suppliers such as Vulcan Chemical
(800-873-4898), Alcide Corp. (Sanova®; www.alcide.com/sanova), CH20 Inc.
(Fresh-Pak™; www.ch2o.com), Rio Linda Chemical Co., Inc. (916-443-4939),
Bio-Cide International, Inc. (Oxine®; www.biocide.com), International
Dioxcide (www.idiclo2.com), Alex C. Fergusson (800-345-1329; www.afco
care.com), CDG Technology, Inc. (www.cdgtechnology.com), and others.
Unlike chlorine, chlorine dioxide is claimed to be effective over a broad
range of pH levels, more resistant to neutralization by the organic load, and
unlikely to produce trihalomethanes (see Oxine Technical Data Sheet;
www.bio-cide.com). Chlorine dioxide also is claimed to be less corrosive than
chlorine and to be effective against bacteria in biofilms. However, generation
of chlorine dioxide by reaction of sodium chlorite with acid or Cl 2 must be
carefully controlled to avoid production of high concentrations of CIO2 gas
which can be toxic and explosive (MSDS for IVR-San 15 sodium chlorite;
www.ch2o.com). Additionally, unlike chlorine, chlorine dioxide dissolves
in water as a gas and is subject to off-gassing if the water is moving or used
in washers. In that situation, special venting would be required to prevent
worker discomfort.
The efficacy of chlorine dioxide in disinfecting produce is comparable to
that of chlorine. Published reports indicate that chlorine dioxide and related
products were potentially effective in preventing potato spoilage by Erwinia
carotovora [37], reducing populations of E. coli 0157:H7, S. Montevideo,
and poliovirus on inoculated strawberries [38], reducing the population of
E. coli 0157:H7 on inoculated apples (but at a treatment level 16 times the
recommended concentration) [39], and suppressing decay in pears [40].
Treatments were less effective in suppressing microbial growth on the surface
of cucumbers [41]. Fett obtained only a 1 log reduction in alfalfa sprouts
irrigated with acidified sodium chlorite [42]. Population reductions of
L. monocytogenes on uninjured surfaces of inoculated green bell peppers,
washed with C10 2 solution (3 mg/1), were about 2 logs greater than could
be achieved with a water wash, but reductions were negligible on injured
surfaces [43]. In contrast, these investigators obtained population reductions
of 7.4 and 3.6 logs on uninjured and injured surfaces of peppers, respectively,
using a C10 2 gas treatment (see Chapter 18).
17.2. 1.2. 3 Ozone
The efficacy of ozone in killing human pathogens and other microorganisms in
water is well established [44], and it is widely used as an alternative to chlorine
in municipal water treatment systems and for production of bottled water
382 Microbiology of Fruits and Vegetables
[45]. Ozone is effective in killing food-related microorganisms [46] and has
been approved for use on foods by the FDA [47]. Potential applica-
tions of ozone in disinfecting foods have been reviewed [48,49]. Ozone is
effective in reducing bacterial populations in flume and wash water and
may have some applications as a chlorine replacement in reducing
microbial populations on produce [50,51]. Ozone treatment was effective in
suppressing decay of table grapes by Rhizopus stolonifer [52]. Use levels of
0.5 to 4.0 |ig/ml are recommended for wash water and 0.1|ig/ml for flume
water [53,54].
However, not all ozone treatments show high efficacy. Ozone treatment of
fresh-cut lettuce, inoculated with a mixture of natural microflora, yielded
reductions of only 1.1 logs [18]. Treatment of lettuce, inoculated with
Pseudomonas fluorescens, with 10u.g/ml of ozone for 1 minute achieved less
than a 1 log population reduction [50]. While ozone treatment of apples
inoculated with E. coli 0157:H7 was effective in reducing populations on the
surface (3.7 log reduction), reductions were < 1 log in the stem and calyx
regions [55]. Ozone treatment of pears (5.5u.g/ml water for 5 minutes) was
ineffective in reducing postharvest fungal decay [56]. Population reduc-
tions obtained by ozone treatment of alfalfa seeds inoculated with E. coli
0157:H7 were only marginally better than those for water-treated controls
[57]. In another study, ozone treatment of alfalfa seeds, inoculated with
L. monocytogenes, was ineffective in reducing the population of this pathogen,
while treatment of inoculated alfalfa sprouts reduced the L. monocytogenes
population by < 1 log and was phytotoxic to the sprouts [58]. These results are
probably a reflection of the difficulty in contacting and inactivating bacteria
attached to produce surfaces in inaccessible sites (see Chapters 2 and 3).
One of the major advantages claimed for ozone is the absence of poten-
tially toxic reaction products. However, ozone must be adequately vented
to avoid worker exposure [48]. Ozone has to be generated on-site by passing
air or oxygen through a corona discharge or UV light [48]. A number of
commercial systems for generating ozonated water for produce washing are
available. Information about commercial ozone generators is available on-line
from Air Liquide (www.airliquide.com), Praxair, Inc. (www.praxair.com),
Novazone (www.novazone.net), Pure Ox (www.pureox.com), Osmonics, Inc.
(www.osmonics.com/food), Ozonia North America, Inc. (www.ozonia.com),
Lynntech, Inc. (www.lynntech.com), Clean Air & Water Systems, Inc.
(360-394-1525), Electric Power Research Institute (EPRI; www.epri.com),
and others. For information about ozone gas disinfection treatments, see
Chapter 18.
17.2.1 .2.4 Peroxya cetic A cid
Peroxyacetic acid (peracetic acid) is an equilibrium mixture of the peroxy
compound, hydrogen peroxide, and acetic acid [59-61]. The superior
antimicrobial properties of peroxyacetic acid are well known [59]. Peroxyacetic
acid is approved by the FDA for addition to wash water at concentrations
Washing and Sanitizing Treatments for Fruits and Vegetables 383
not to exceed 80ppm [6]. Under EPA regulations, an exemption from the
requirements of a tolerance was established for peroxyacetic acid as an
antimicrobial treatment for fruits and vegetables at concentrations up to
lOOppm [62]. Much higher concentrations are permitted for sanitizing food
contact surfaces [63]. Peroxyacetic acid decomposes into acetic acid, water, and
oxygen, all harmless residuals.
Peroxyacetic acid is recommended for use in treating process water, but
Ecolab, one of the major suppliers, is also claiming substantial reductions in
microbial populations on fruit and vegetable surfaces [64]. However, company
literature provides insufficient information on methodology to assess treatment
efficacy (www.ecolab.com/initiatives/foodsafety). Population reductions for
aerobic bacteria, coliform bacteria, and yeasts and molds on fresh-cut celery,
cabbage, and potatoes treated with 80ppm peroxyacetic acid were less than
1.5 logs [65]. Addition of 40ppm Tsunami 100 (the Ecolab peroxyacetic acid
product) to the irrigation water used during sprout propagation did not
suppress the outgrowth of the native microflora [42]. Treatment with 100 ppm
Tsunami reduced the population of E. coli 0157:H7 and S. Montevideo
on inoculated strawberries by about 97% [38]. Several published studies
have looked at the efficacy of peroxyacetic acid against E. coli 0157:H7
on inoculated apples. Attempts to disinfect apples, inoculated with E. coli
0157:H7, by washing with 80 ppm peroxyacetic acid 30 minutes after inocu-
lation resulted in a 2 log reduction compared to a water wash [31]. However, in
another study where inoculated apples were held for 24 hours before washing
(allowing more time for attachment), an 80 ppm peroxyacetic acid treatment
reduced the E. coli 0157:H7 population by less than 1 log; at 16 times the
recommended concentration, a 3 log reduction was obtained [39]. Sapers et al.
[16] reported similar results with apples inoculated with a nonpathogenic
E. coli. Like ozone and chlorine dioxide, low concentrations of peroxyacetic
acid are effective in killing pathogenic bacteria in aqueous suspension [59].
Addition of octanoic acid to peroxyacetic acid solutions increased efficacy
in killing yeasts and molds in fresh-cut vegetable process waters but had
little effect on population reductions on fresh-cut vegetables [65].
Peroxyacetic acid is a strong oxidizing agent and can be hazardous to
handle at high concentrations, but not at strengths marketed to the produce
industry. Peroxyacetic acid is available at various strengths from Ecolab, Inc.
(www.ecolab.com), FMC Corp. (www.fmcchemicals.com), and Solvay Interox
(www.solvayinterox.com).
17.2.2 Washing Equipment
17.2.2.1 Types of Washers
Washing equipment for produce is designed primarily for removal of soil,
debris, and any pesticide residues from the harvested commodity. The design of
most commercial equipment has not taken into account requirements for the
reduction of microbial populations on produce surfaces although this is a
desirable goal of washing.
384
Microbiology of Fruits and Vegetables
(b)
(e)
(f)
FIGURE 17.1 Commercial washing equipment for fruits and vegetables: (a) flat-bed
brush washer; (b) U-bed brush washer; (c) rotary washer; (d) pressure washer; (e, f)
flume washers; (g) helical washer.
Washing and Sanitizing Treatments for Fruits and Vegetables
385
Numerous types of washers have been developed for cleaning fresh fruits
and vegetables, varying in complexity from a garden hose used for cleaning
apples prior to farm-scale cider production (an unsatisfactory procedure due
to lack of control) to sophisticated systems employing rotating brushes
and applying heated water under pressure with agitation. The more common
types of commercial washers for produce include dump tanks, brush washers,
reel washers, pressure washers, hydro air agitation wash tanks, and immersion
pipeline washers (Figure 17.1). Major suppliers of such equipment are listed
on the Postharvest Resources website of the University of Florida (http://
postharvest.ifas.ufl.edu). The choice of washer for a particular commodity will
depend on such characteristics of the commodity as shape, size, and fragility.
It is obvious that equipment requirements are quite different for cut lettuce
than for tomatoes or potatoes.
1 7.2.2.2 Efficacy of Washers
The efficacy of commercial flat-bed and U-bed brush washers in removing
or inactivating a nonpathogenic E. coli on artificially contaminated apples was
investigated by Annous et al. [66] and Sapers [3]. These studies demonstrated
that the E. coli population could be reduced by about 1 log (90%) by passage of
the apples through a dump tank with minimal agitation (Table 17.2). However,
further cleaning of the apples in a flat-bed brush washer had little further
effect on the E. coli population, irrespective of the cleaning or sanitizing agent
used (water, 200 ppm Cl 2 , 1% acidic detergent, 8% trisodium phosphate, 5%
H2O2). Similar results were obtained with a U-bed brush washer. Subsequent
studies by the investigators showed that the bacteria that had attached in the
TABLE 17.2
Decontamination of Apples Inoculated with f. coli (Strain K12) with
Sanitizing Washes Applied in a Flat-Bed Brush Washer
f. co/i (log 10 CFU/gr
Wash treatment
Temp. (°C)
Before dump tank
After dump tank
After brush washer
Water
20
5.49 ±0.09
4.92 ±0.37
4.81 ±0.26
50
5.49 ±0.09
5.03 ±0.1 5
4.59 ±0.08
200ppmCl 2
20
5.87 ±0.07
5.45 ±0.05
5.64 ±0.23
8% Na 3 P0 4
20
5.49 ±0.09
5.02 ±0.43
4.98 ±0.02
50
5.49 ±0.09
5.02 ±0.08
4.75 ±0.45
1% acidic detergent
50
5.87 ±0.07
5.49 ±0.03
5.42 ±0.50
5% H 2 2
20
5.87 ±0.07
5.46 ±0.40
5.27 ±0.09
50
5.87 ±0.07
5.54±0.31
5.49±0.10
a Mean of four determinations ± standard deviation.
From Annous, B.A. et al., J. Food Prot., 64, 159, 2001. Reprinted with permission. Copyright
International Association for Food Protection, Des Moines, IA.
386 Microbiology of Fruits and Vegetables
TABLE 17.3
Distribution of £. coli (ATCC 25922) on Surfaces of Inoculated Apples Before
and After Washing with 5% H 2 2 at 50°C
Log 10 (CFU/cm 2 )
24 h after inoculation 72 h after inoculation
Location Inoculated
Washed
Inoculated
Washed
Skin except at calyx and stem ends 4.77
2.05
4.37
1.63
Skin at calyx end of core 7.26
5.20
6.79
4.46
Skin on stem end of core 6.63
5.06
5.61
4.89
From Sapers, G.M. et al, J. Food Set, 65, 529, 2000.
Reprinted
with
permission.
relatively inaccessible stem and blossom ends of the apples, or were internalized
within the latter region, survived washing while E. coli attached elsewhere
on the apple surface were readily inactivated (Table 17.3). Greater efficacy
was obtained when the apples were washed by full immersion in a sanitizing
solution with vigorous agitation [67].
Gagliardi et al. [68] examined commercial practices for washing melons
produced in the Rio Grande River Valley of Texas. They reported little or no
reduction in the population of coliforms, fecal coliforms, enterococci, and
fecal enterococci in cantaloupes and honeydew melons that were washed with
water in a tank and then spray rinsed on a conveyor line. Use of chlorinated
water in the secondary rinse appeared to reduce the populations of fecal
coliforms and fecal enterococci but not total coliforms and enterococci.
Laboratory-scale washing studies with cantaloupes that had been dip-
inoculated with Salmonella Stanley or a nonpathogenic E. coli (ATCC
25922) demonstrated that the population reductions obtained by immersion
of the melons in 200 ppm Cl 2 or 5% H 2 2 decreased as the time interval
between inoculation and washing increased from 24 hours to 5 days [69,70].
However, the efficacy of these treatments in inactivating L. monocytogenes on
inoculated cantaloupes was not dependent on the length of storage between
inoculation and treatment [71]. Sapers et al. obtained minimal inactivation of
E. coli B-766 (a surrogate for S. Poona) when dip-inoculated cantaloupes
were immersed in 300 ppm Cl 2 for 3 minutes [72]. Apparently, cantaloupes
are especially difficult to disinfect, even if fully immersed in the sanitizing
solution. This may be due to the movement, attachment, and possible biofilm
formation by the targeted bacteria within inaccessible pores in the netting so
that contact between the sanitizing solution and the attached bacteria is
minimal. This is borne out by the success of treatments with 5% H 2 2 at
70° C or near boiling water where heat penetration contributes to the efficacy
of the antimicrobial treatment [73] (see Chapter 10). Such treatments can
Washing and Sanitizing Treatments for Fruits and Vegetables 387
greatly reduce the risk of transfer of human pathogens from the rind surface
to the flesh during fresh-cut processing.
17.2.3 Factors Limiting the Efficacy of Washing
The action of commercial washing agents and equipment in removing or
inactivating microorganisms on fresh produce is not well understood. In
general, microbial populations on produce surfaces are not easily detached or
inactivated for a number of reasons discussed in Chapters 2 and 3. Briefly,
the microbial contaminants may become strongly attached to the produce
surface by physical forces within a short time of contamination or incorporated
within a biofilm over a longer time period. Microbial contaminants may
be located in a protected attachment site, e.g., a cut surface, puncture, or pore,
where a wash solution cannot reach. Microorganisms also may become
internalized within the commodity either during crop production or when
submerged in water in a packing plant dump tank or flume as a consequence
of infiltration driven by a negative temperature differential or by hydrostatic
pressure. Consequently, the inaccessible population will escape direct contact
with a cleaning or sanitizing agent in a commercial washer. These conditions
are discussed in greater detail in an earlier review article [3] and in Chapter 3.
1 7.3 NOVEL WASHING TECHNOLOGY
Because the commercially available alternatives to chlorine discussed above
generally cannot achieve population reductions of human pathogens on
contaminated produce much in excess of 2 logs, which is insufficient to ensure
safety, a number of experimental treatments have been examined to obtain
greater efficacy. The efficacy and regulatory status of some of these
experimental treatments are described in the following.
17.3.1 Hydrogen Peroxide
Hydrogen peroxide is a highly effective antimicrobial agent against bacteria
but is less active against yeasts, fungi, and viruses [59]. Characteristics and
potential food applications of hydrogen peroxide as a sanitizer for produce
were recently reviewed by the author [74]. Hydrogen peroxide may be
considered as a potential alternative to chlorine. Numerous studies have
demonstrated the efficacy of dilute hydrogen peroxide in sanitizing fresh
produce including mushrooms [75-77], apples [16,67,78], melons [34,69,70,73],
eggplant, and sweet red pepper [80]. In side-by-side comparisons, dilute
(1 to 5%) hydrogen peroxide washes were at least as effective as 200 ppm
chlorine [16,79]. When applied to apples with vigorous agitation at an elevated
temperature (50 to 60°C), population reductions approaching 3 logs were
obtained [67]. However, temperatures exceeding 60°C could not be used
without inducing browning of the apple skin. Hydrogen peroxide treatments
388 Microbiology of Fruits and Vegetables
were ineffective in decontaminating sprouts [42] or the seeds used to produce
sprouts [81].
While treatment with hydrogen peroxide vapor can reduce microbial
populations on grapes [82], melons [83], and prunes [84], required treatment
times are long compared to the application of a dilute hydrogen peroxide
dip [85]. The vapor treatments proved to be ineffective with apples [86] and
produced discolorations with mechanically damaged berries [85].
The regulatory status of hydrogen peroxide as a washing agent for produce
is unclear. The FDA has jurisdiction if the washing treatment is applied as part
of a processing operation, while the EPA has jurisdiction if the treatment is
applied to a raw commodity. While fresh produce clearly falls within the EPA
regulations, fresh-cut produce is under FDA regulations. However, if the wash
treatment is applied to the raw produce before cutting, and if this operation
is carried out in a receiving area, separate from the processing room, it
would appear that EPA regulations apply. Under FDA regulations, hydrogen
peroxide is GRAS (generally recognized as safe) for some specified food
applications, provided that residual H 2 2 is removed "by appropriate physical
and chemical means during processing,'' but the regulation does not cover
hydrogen peroxide as a washing or sanitizing agent for produce [87]. According
to an Agency Response Letter (GRAS notice no. GRN 000014, May 26, 1999)
a petition to the FDA to amend the regulation would be required to seek
approval for a new application (in this case, reduction of the microbial load
on onions prior to dehydration; http://vm.cfsan.fda.gov/~rdb). Peroxyacetic
acid formulations, which contain low levels of hydrogen peroxide (59 ppm),
are approved by the FDA for use in washing fruits and vegetables [6]. A
higher concentration is permitted if the formulation is used to sanitize food
contact surfaces [63] Under EPA regulations, postharvest hydrogen peroxide
applications to produce as an antimicrobial treatment are exempt from the
requirements of a tolerance if the concentration is <1% per application [88].
The presence of residual hydrogen peroxide should not represent an
obstacle to use of this agent as a produce sanitizer. Most fruits and vegetables
contain sufficient catalase to permit rapid breakdown of residual peroxide to
water and oxygen. Peroxide residues could not be detected in mushrooms,
apples, or cantaloupes following hydrogen peroxide wash treatments [16,34,77].
Information on hydrogen peroxide applications can be obtained from
FMC Corp. (www.fmcchemicals.com), Solvay Interox (www.solvayinterox.
com), US Peroxide (h2o2.com), and Degussa Corp. (www.degussa.com).
BiosSafe Systems (www.biosafesystems.com) is marketing a formulation
containing hydrogen peroxide and peroxyacetic acids (Storox®) for sanitizing
fruits and vegetables; the recommended maximum use level is 0.27%.
17.3.2 Trisodium Phosphate and Other Alkaline
Washing Agents
Trisodium phosphate (TSP) has been marketed by Rhodia Specialty Phos-
phates (www.rhodia-phosphates.com) as an antimicrobial rinse (AvGard®,
Washing and Sanitizing Treatments for Fruits and Vegetables 389
Assur-Rinse®) to reduce human pathogen populations on processed beef
and poultry. TSP is classified as GRAS by the FDA [89].
The antimicrobial activity of TSP probably is due to its high pH (pH 12)
which disrupts the cytoplasmic membrane [90,91]. Highly alkaline washes
based on sodium and potassium hydroxide (pH 11 to 12) resulted in 3 log
reductions in the population of a nonpathogenic E. coli on surface-inoculated
oranges [92]. A 30-minute dip in 0.25% calcinated calcium suspension, another
highly alkaline product derived from oyster shells (pH 10), reduced the
native bacterial population on cucumbers by about 2 logs [93]. In a more recent
study, Bari et al. [94] reported population reductions exceeding 5 logs on
tomatoes that had been surface inoculated with E. coli 0157:H7, salmonella
strains, or L. monocytogenes and treated with 0.5% calcinated calcium. These
exceptionally high population reductions (for a wash) may be a reflection
of the brief interval (30 minutes) between inoculation and treatment used
by these investigators. Sapers et al. [67] obtained population reductions
approaching 3 logs when apples that had been dip-inoculated with E. coli
(ATCC 25922) were washed with 5% hydrogen peroxide, followed by brushing
the calyx and stem areas with a paste of calcinated calcium; the population
reduction was < 2 logs with only the peroxide wash. TSP solutions (12 to 15%)
were highly effective in reducing S. Montevideo populations on inoculated
tomato surface but failed to inactivate completely this organism in the tomato
core tissue [95]. Survival in the latter tissue probably resulted from bacterial
infiltration. Sapers et al. [78] reported a 2 log reduction in a nonpathogenic
E. coli strain on inoculated apples washed with 4% TSP at 50°C. A 1% TSP
wash reduced the population of E. coli 0157:H7 and S. Montevideo on
strawberries by 93 and 96%, respectively [38]. Treatment of lettuce with
2% TSP was ineffective in killing L. monocytogenes [14]. Addition of 0.3% TSP
to the irrigation water was ineffective in reducing the native microflora on
alfalfa sprouts [42]. TSP was reported to be highly effective in inactivating
E. coli 0157:H7 in biofilms but less effective against S. Typhimurium and
L. monocytogenes in biofilms [96].
1 7.3.3 Organic Acids
Organic acids such as lactic and acetic acids are effective antibacterial
agents [97] and are classified by the FDA as GRAS [98,99] (21CFR184.1005;
21CFR184.1061). Lactic acid dips and sprays are used commercially to decon-
taminate animal carcasses containing E. coli 0157:H7, L. monocytogenes,
and salmonella [100] (see additional information from Purac America,
Inc., www.purac.com). Lactic acid rinses might have applications for the
decontamination of fruits and vegetables. A 5% acetic acid wash was reported
to reduce the population of E. coli 0157:H7 on inoculated apples by
about 3 logs [31]. In another study, apples that had been inoculated with
E. coli 0157:H7 were treated with 5% acetic acid at 55°C for as long as
25 minutes. While the E. coli population was greatly reduced in the apple
390 Microbiology of Fruits and Vegetables
skin and stem areas, as many as 3 to 4 logs survived in the calyx tissue [101].
In a more recent study, application of 2.4% acetic acid to apple disks that had
been inoculated with S. mbandaka or S. Typhimurium resulted in population
reductions of 1.1 and 1.4, respectively [102]. However, the combination of
5% acetic acid with 5% hydrogen peroxide yielded a population reduction
approaching 4 logs. It is not clear whether organic acid treatments would
produce off-flavors or discoloration in treated produce.
17.3.4 Other Experimental Antimicrobial
Washing Agents
Cetylpyridinium chloride (CPC) is being marketed as Cecure® for use in oral
hygiene products and may have application as an antimicrobial rinse for fresh
produce and other foods. Yang et al. [103] reported population reductions in
the range 1 to 2 logs for S. Typhimurium and E. coli 0157:H7 on inoculated
fresh-cut lettuce, treated by spraying with 0.3% CPC. Similar reductions were
obtained with strawberries inoculated with E. coli 0157:H7 or S. Montevideo
and immersed in 0.1% CPC at 43°C [38]. However, regulatory approval for
this agent has not yet been obtained (www.safefoods.net/cecure.htm).
Activated lactoferrin, which prevents attachment of bacteria to meat, is
approved by the FDA and USDA for application to beef as a carcass rinse
[104] (also see www.activinlf.com). However, there are no reports of its
applicability to fruits and vegetables. Silver and copper ions are known to exert
antimicrobial activity against bacteria in water [105], and ion generators have
been marketed for disinfection of water in swimming pools, irrigation systems,
and various other commercial applications (Tew Manufacturing Corp., 800-
380-5839; T.P. Technology pic, www.tarn-pure.com). Application of this
technology to produce packing lines and dump tanks at recommended levels
of 0.50 ppm copper and 0.035 to 0.05 ppm silver has been proposed (Tew
Manufacturing Corp.), but published efficacy data are lacking, and the
regulatory status of such applications is unclear.
17.3.5 Synergistic Treatment Combinations
Certain combinations or sequences of treatments may show synergism in
inactivating or detaching microbial contaminants on produce. Such behavior
might be anticipated if the individual treatments have different modes of
action, e.g., cell membrane disruption and oxidation. Several examples of
promising combination treatments have been reported: the sequential washing
of cantaloupes with detergents and hydrogen peroxide [34] and the appli-
cation of an acetic acid-hydrogen peroxide combination to inoculated apple
disks [102]. Lin et al. [106] investigated the inactivation of E. coli 0157:H7,
S. enterica serotype Enteritidis, and L. monocytogenes by combinations of
hydrogen peroxide and lactic acid and hydrogen peroxide with mild heat.
Further research in this area may yield treatment combinations that show
Washing and Sanitizing Treatments for Fruits and Vegetables 391
greater efficacy towards bacteria located in punctures or pores or incorporated
in biofilms on produce surfaces.
17.4 FOODSERVICE AND HOME APPLICATIONS
While conventional sanitizing agents, applied to produce with commercial-
scale washing equipment, have the capability of achieving 1 to 2 log population
reductions in contaminated produce, this option is not generally available for
foodservice and consumer applications. Consumers and operators of
delicatessens, restaurants, and other foodservice establishments do not have
the technical skills or knowledge to prepare the more potent sanitizer solutions
used commercially nor do they have access to commercial washing equipment.
Duff et al. [107] developed an economic model to evaluate the potential cost-
effectiveness of a disinfection program that targets high-risk food preparation
activities in household kitchens. They concluded that such a program would be
cost-effective. What options are available to consumers and foodservice
managers so that they can provide some meaningful level of protection to their
families or customers?
17.4.1 FDA Recommendations
The FDA advises consumers to: "Wash all fresh fruits and vegetables with
cool tap water immediately before eating. Don't use soap or detergents. Scrub
firm produce, such as melons and cucumbers, with a clean produce brush.
Cut away any bruised or damaged areas before eating." Consumers are also
advised to:
Wash surfaces often. Cutting boards, dishes, utensils, and counter tops
should be washed with hot soapy water and sanitized after coming in contact
with fresh produce, or raw meat, poultry, or seafood. Sanitize after use with
a solution of 1 teaspoon of chlorine bleach in 1 quart of water. Don't cross
contaminate. Use clean cutting boards and utensils when handling fresh produce.
If possible, use one clean cutting board for fresh produce and a separate one for
raw meat, poultry, and seafood. During food preparation, wash cutting boards,
utensils, or dishes that have come into contact with fresh produce, raw meat,
poultry, or seafood. Do not consume ice that has come in contact with fresh
produce or other raw products (www.fda.gov/bbs/topics/ANSWERS/2002/
ANS01167.html/).
In the situation where a particular fruit or vegetable is suspect, more
specific advice is provided. For example, in response to an outbreak of
hepatitis A in green onions, the FDA recommended: "Cook green onions
thoroughly. This minimizes the risk of illness by reducing or eliminating the
virus. Cook in a casserole or saute in a skillet" and iC Cook sprouts. This
significantly reduces the risk of illness" [108]. While a kill step is undoubtedly
effective, it would not be applicable to many fruits and vegetables that would
392 Microbiology of Fruits and Vegetables
no longer be considered "fresh" if subjected to a cook or full blanch and would
lose their appeal to consumers. Washing produce without a sanitizer is not
likely to achieve the population reductions that can be obtained with
commercial sanitizing agents and equipment.
1 7.4.2 Other Options
Alternative methods of surface sanitizing cantaloupes were examined by Barak
et al. [109]. They reported reductions in the bacterial load of 70, 80, and 90%
by scrubbing the melons with a vegetable brush in tap water, washing with
soap, and dipping in 150ppm sodium hypochlorite, respectively. However, a
three-compartment sanitation method comprising washing with an antimicro-
bial soap, scrubbing with a brush in tap water, and immersion in a
hypochlorite solution resulted in a 99.8% reduction. Population reductions
exceeding 5 logs were obtained on cut iceberg lettuce, inoculated with E. coli
CDC1932, by washing with diluted vinegar (1.9% acetic acid); in contrast,
washing with diluted bleach solution (180 ppm available chlorine) and lemon
juice (0.6% citric acid) yielded 1.6 and 2.1 log reductions, respectively [110].
However, the vinegar treatment resulted in some product damage. Application
of a solution containing 1.5% lactic acid and 1.5% hydrogen peroxide as a 15-
minute soak at 40°C was reported to yield greater than 5 log reductions in the
population of E. coli 0157:H7, Salmonella enteritidis, and Listeria mono-
cytogenes on spot-inoculated apples, oranges, and tomatoes [111]. However, in
both studies, the surviving bacteria were recovered by a rinsing procedure such
that only unattached, exposed cells were being recovered and not bacteria that
were embedded in fruit tissues or biofilms or attached to fruit surfaces. This
may have yielded unrealistically high population reductions. Smith et al. [112]
evaluated a commercial peroxyacetic acid formulation intended for food-
service applications (Victory produce wash; Ecolab, St. Paul, MN; www.eco-
lab.com) for reducing the bacterial load on lettuce; small reductions (~llog)
were obtained. Lukasik et al. [38] compared various washing treatments,
including consumer-oriented products (detergents, Fit® and Healthy Harvest)
on inoculated strawberries; population reductions for E. coli 0157:H7, S.
montevideo, and several viruses were between 1 and 2 logs. Parnell and Harris
[113] compared water, sodium hypochlorite, and vinegar as consumer washes
for reducing salmonella on spot-inoculated apples. Population reductions
obtained with vinegar and chlorine washes were 2 to 3 logs greater than
reductions obtained with water. Treatment with sodium hypochlorite and
vinegar yielded comparable reductions in the population of natural microbiota
of lettuce [114]. A study of consumer acceptance of a home use antibacterial
solution for sanitizing apples indicated that consumers would be unwilling
to use a procedure requiring the 15-minute heat and soak step [115].
Venkitanarayanan et al. [116] reported that an electrolyzed water treatment
was effective in inactivating foodborne pathogens on smooth plastic kitchen
cutting boards. They did not investigate scarred cutting boards which might be
expected in a kitchen or foodservice situation.
Washing and Sanitizing Treatments for Fruits and Vegetables 393
17.4.3 Commercial Equipment and Wash
Formulations for Home or
Foodservice Use
Some manufacturers of commercial equipment for sanitizing produce have
developed small-scale units suitable for consumer and foodservice use.
Systems based on use of electrolyzed water are being marketed by Sterilox
Technologies, Inc. (www.steriloxtechnologies.com) and Hoshizaki America,
Inc. (www.hoshizakiamerica.com). Small-scale systems based on ozone are
being marketed by Sterilion Ltd (www.performancesystems.com/medical.htm)
and UltrOzone (UC Davis Postharvest Technology Center; 1-866-21-
OZONE).
A number of commercial fruit and vegetable wash formulations intended
for consumer use are being marketed, but little information is available about
their performance in reducing microbial populations. Fit®, a produce wash
produced by Procter & Gamble Co. and marketed for a number of years, did
show some antimicrobial activity in addition to removing dirt, wax, and other
residues [117,118], although no claims were made by the company that the
consumer product had antimicrobial activity. They did make such a claim for a
"Pro Line Fit" intended for commercial rather than consumer use. Fit is now
marketed by HealthPro Brands, Inc. (www.healthprobrands.com). JohnsonDi-
versey markets a Hard Surface Sanitizer/Fruit & Vegetable Wash (Product
4444) claimed to have antimicrobial activity (www.jwp.com/jwp/ProdInfo.nsf/;
click on foodservice, then sanitizers). Another product with documented
antimicrobial activity is Pro-San®, previously marketed as Vegi-Clean®
(www.microcideinc.com/prosan.htm). A product derived from oranges and
other GRAS ingredients and claimed to have antibacterial properties is
marketed under the name CitroBio for postharvest processing, use in retail
misting systems, or as a produce wash for consumers (www.citrobio.com).
Grapefruit seed extract (Citricidal®) is reputed to have antimicrobial properties
(www.biochemresearch.com) and is being marketed as a consumer-use cleaner
and disinfectant for fruits and vegetables (www.pureliquidgold.com). Other
produce washes include: Veggie Wash® marketed by Beaumont Products
(www.citrusmagic.com), Nature Clean Fruit & Veggie Wash (claimed to
remove bacteria) (www.smallplanetinc.com, www.healthyhomeservices.ca,
www.frankross.com), CleanGreens! (www.cleangreensinc.com), and Organi-
clean (www.organiclean.com).
In addition to these commercial products, recipes for fruit and vegetable
washes can be found on the internet. Typical examples include diluted
3% hydrogen peroxide (www.wellnesstoday.com), and vinegar and 3% hydro-
gen peroxide sprays applied individually to produce (http://myexecpc.com/
~mjstouff/articles/vinegar.html). One source suggests use of 35% hydrogen
peroxide around the house, a potentially dangerous recommendation;
specific uses for produce treatment call for use of 3 or 5% solutions (http:
h2o2hydrogenperoxide.com/additrion.html).
394 Microbiology of Fruits and Vegetables
17.5 CONCLUSIONS
The efficacy of conventional washing technology in reducing populations of
human pathogens and other microorganisms on fresh produce surfaces is
limited to 1 to 2 logs, a significant improvement compared to the unwashed
produce but insufficient to ensure food safety. Incremental improvements in
washing efficacy can be obtained through buffering, addition of surfactants,
temperature elevation, full immersion, and washing with vigorous agitation.
However, greater population reductions cannot be obtained because of the
strength of microbial attachment to produce and location of attached
microorganisms in inaccessible sites. Approved alternatives to chlorine may
provide certain technical advantages and avoid disadvantages such as
formation of toxic reaction products, but differences in antimicrobial efficacy
are small. Washing agents developed for foodservice or home use may
exhibit antimicrobial activity, but safe and uniform application may be
problematic without the controls available for large-scale produce packing
and processing applications. Microbial reduction benefits claimed by many
purveyors of home-use formulations, especially those marketed via the
internet, are unsubstantiated. Experimental washing agents, if found to be
technically and economically feasible, or synergistic sequences or combi-
nations of treatments may provide addition gains in efficacy over current
technology, but attainment of high levels of safety such as afforded by a 5 log
reduction in pathogen populations is unrealistic. Use of other technologies
such as surface pasteurization or irradiation may be required to reach this
level of safety.
ACKNOWLEDGMENTS
The author thanks Prof. Jerry A. Bartz at the University of Florida in
Gainesville and Prof. William C. Hurst at the University of Georgia for their
thorough and constructive review of this chapter.
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of a Tolerance, Code of Federal Regulations Title 40, Part 180, Section
180.1197.
89. 21CFR182.1778, Sodium Phosphate, Code of Federal Regulations Title 21, Part
182, Section 182.1778.
90. Mendonca, A.F., Amoroso, T.L., and Knabel, S.J., Destruction of Gram-
negative food-borne pathogens by high pH involves disruption of the
cytoplasmic membrane, Appl. Environ. Microbiol., 60, 4009, 1994.
91. Sampathkumar, B., Khachatourians, G.G., and Korber, D.R, High pH
during trisodium phosphate treatment causes membrane damage and destruc-
tion of Salmonella enterica Serovar Enteritidis, Appl. Environ. Microbiol., 69,
122, 2003.
92. Pao, S., Davis, C.L., and Kelsey, D.F., Efficacy of alkaline washing for the
decontamination of orange fruit surfaces inoculated with Escherichia coli,
J. Food Prot., 63, 961, 2000.
93. Isshiki, K. and Azuma, K., Microbial growth suppression in food using
calcinated calcium, JARQ, 29, 269, 1995.
94. Bari, M.L. et al., Calcinated calcium killing of Escherichia coli 0157:H7,
Salmonella, and Listeria monocytogenes on the surface of tomatoes, J. Food
Prot., 65, 1706, 2002.
95. Zhuang, R.-Y. and Beuchat, L.R., Effectiveness of trisodium phosphate for
killing Salmonella montevideo on tomatoes, Lett. Appl. Microbiol., 232, 97, 1996.
96. Somers, E.B., Schoeni, J.L., and Wong, A.C.L., Effect of trisodium phosphate
on biofilm and planktonic cells of Campylobacter jejuni, Escherichia coli
0157:H7, Listeria monocytogenes and Salmonella typhimurium, Int. J. Food
Microbiol., 22, 269, 1994.
97. Foegeding, P.M. and Busta, F.F., Chemical food preservatives, in Disinfection,
Sterilization, and Preservation, 4th ed., Block, S.S., Ed., Lea & Febiger,
Philadelphia, 1991, chap. 47.
98. 21CFR184.1005, Direct Food Substances Affirmed as Generally Recognized as
Safe, Listing Of Specific Substances Affirmed as GRAS, Acetic Acid, Code of
Federal Regulations, Title 21, Part 184, Subpart B, Section 184.1005.
99. 21CFR.184.1061, Direct Food Substances Affirmed as Generally Recognized as
Safe, Listing of Specific Substances Affirmed as GRAS, Lactic Acid, Code of
Federal Regulations, Title 21, Part 184, Subpart B, Section 184.1061.
100. Castillo, A. et al, Lactic acid sprays reduce bacterial pathogens on cold beef
carcass surfaces and in subsequently produced ground beef, /. Food Prot., 64,
58, 2001.
101. Delaquis, P.J., Ward, S.M., and Stanich, K., Evaluation of pre-pressing sanitary
treatments for the destruction of Escherichia coli 0157:H7 on apples destined
for production of unpasteurized apple juice, Technical Report No. 9901,
Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre,
Summerland, BC V0H 1Z0, 2000.
102. Liao, C.-H., Shollenberger, L.M., and Phillips, J.G., Lethal and sublethal action
of acetic acid on Salmonella in vitro and on cut surfaces of apple slices, /. Food
Sci., 68, 2793, 2003.
400 Microbiology of Fruits and Vegetables
103. Yang, H. et al., Efficacy of cetylpyridinium chloride on Salmonella Typhimur-
ium and Escherichia coli 0157:H7 in immersion spray treatment of fresh-cut
lettuce, /. Food ScL, 68, 1008, 2003.
104. Naidu, A.S., Activated lactoferrin: a new approach to meat safety, Food
Technol., 56, 40, 2002.
105. Yahya, M.T. et al., Disinfection of bacteria in water systems by using
electrolytically generated copper: silver and reduced levels of free chlorine,
Can. J. Microbiol., 36, 109, 1990.
106. Lin, C.-M. et al., Inactivation of Escherichia coli 0157:H7, Salmonella enterica
Serotype Enteritidis, and Listeria monocytogenes by hydrogen peroxide and
lactic acid and by hydrogen peroxide with mild heat, /. Food Prot., 65, 1215,
2002.
107. Duff, S.B. et al., Cost-effectiveness of a targeted disinfection program in
household kitchens to prevent foodborne illnesses in the United States, Canada,
and the United Kingdom, /. Food Prot., 66, 2103, 2003.
108. FDA Talk Paper, Consumers Advised That Recent Hepatitis A Outbreaks Have
Been Associated With Green Onions, U.S. Food and Drug Administration,
Nov. 15, 2003 (www.fda.gov/bbs/topics/ANSWERS/2003/ASNS01262.html;
accessed Aug. 14, 2004).
109. Barak, J.D., Chue, B., and Mills, D.C., Recovery of surface bacteria from and
surface sanitization of cantaloupes, /. Food Prot., 66, 1805, 2003.
110. Vijayakumar, C. and Wolf-Hall, C.E., Evaluation of household sanitizers for
reducing levels of Escherichia coli on iceberg lettuce, /. Food Prot., 65, 1646,
2002.
111. Venkitanarayanan, K.S. et al., Inactivation of E. coli 0157:H7, Salmonella
Enteritidis and Listeria monocytogenes on apples, oranges, and tomatoes by
lactic acid with hydrogen peroxide, /. Food Prot., 65, 100, 2002.
112. Smith, S. et al., Efficacy of a commercial produce wash on bacterial
contamination of lettuce in a food service setting, J. Food Prot., 66, 2359, 2003.
113. Parnell, T.L. and Harris, L.J., Reducing Salmonella on apples with wash
practices commonly used by consumers, J. Food Prot., 66, 741, 2003.
114. Nascimento, M.S. et al., Effects of different disinfection treatments on the
natural microbiota of lettuce, J. Food Prot., 66, 1697, 2003.
115. McWatters, K.H. et al., Consumer acceptance of raw apples treated with an
antibacterial solution designed for home use, /. Food Prot., 65, 106, 2002.
116. Venkitanarayanan, K.S. et al., Inactivation of Escherichia coli 0157:H7 and
Listeria monocytogenes on plastic kitchen cutting boards by electrolyzed
oxidizing water, J. Food Prot., 62, 857, 1999.
117. Beuchat, L.R. et al., Development of a proposed standard method for assessing
the efficacy of fresh produce sanitizers, /. Food Prot., 64, 1103, 2001.
118. Harris, L.J., Efficacy and reproducibility of a produce wash in killing Salmonella
on the surface of tomatoes assessed with a proposed standard method for
produce sanitizers, /. Food Prot., 64, 1477, 2001.
18
Gas-/Vapor-Phase
Sanitation
(Decontamination)
Treatments
Richard H. Linton, Yingchang Han,
Travis L. Selby, and Philip E. Nelson
CONTENTS
18.1 Introduction 402
18.2 Chlorine Dioxide Gas 402
18.2.1 Physical, Chemical, and Safety Properties of C10 2 Gas . . . 402
18.2.2 Antimicrobial Properties of Aqueous and Gaseous C10 2 . 404
18.2.3 C10 2 Gas Generation 404
18.2.4 General Gas/Vapor Treatment Systems 407
18.2.5 Mechanisms for Microbial Inactivation 409
18.2.6 Factors Influencing C10 2 Gas Treatment 409
18.2.7 Efficacy in Reducing Microorganisms on Different
Produce Surfaces 412
18.2.8 Effects of C10 2 Gas Treatment on Quality of Produce ... 414
18.3 Ozone Gas 414
18.3.1 Properties of Ozone 414
18.3.2 Potential Applications of Ozone in the Food Industry. ... 415
18.3.3 Generation of Ozone 415
18.3.4 Treatment Systems 416
18.3.5 Mechanisms of Inactivation of Microbes 417
18.3.6 Factors Influencing Sanitation Treatment by Ozone Gas 417
18.3.7 Efficacy in Reducing Foodborne Microorganisms on
Produce Surfaces 418
18.3.8 Effects of Ozone Gas Treatments on Quality of Fruits
and Vegetables 42 1
18.4 Allyl Isothiocyanate Gas 422
18.4.1 Properties 422
18.4.2 Mechanisms and Factors Influencing Sanitation
Treatment 422
401
402 Microbiology of Fruits and Vegetables
18.4.3 Efficacy in Reducing Foodborne Pathogens on Produce
Surfaces 422
18.4.4 Effects of AITC Vapor Treatment on Quality of Fruits
and Vegetables 422
18.5 Other Gases/Vapors 423
18.5.1 Hydrogen Peroxide Vapor 423
18.5.2 Acetic Acid Vapor 424
18.5.3 Other Natural Plant Volatiles 425
18.6 Present and Future Applications of Gaseous/Vapor-Phase
Antimicrobials for Decontamination of Fresh Produce 426
18.7 Regulatory Considerations 427
References 428
18.1 INTRODUCTION
Since the 1950s chemical gases, such as ethylene oxide, propylene oxide,
formaldehyde, and (3-propiolactone, have been used to sterilize medical
products and biological preparations that are not compatible with heat or
radiation sterilization. In the 1980s sterilization of these products using
chemical gases, such as chlorine dioxide (CIO2), ozone, and hydrogen peroxide
vapor or plasma, emerged as a new technology [1-4]. For the purpose of this
chapter, the gaseous form is considered as the direct application of the gas
phase. The vapor form is considered as the application of a vaporized chemical
from a liquid starting material.
In more recent years applications of such gaseous chemical disinfectants
are gaining interest in the food industry for reducing microorganisms. Research-
ers have successfully used C10 2 gas to sterilize bulk orange juice storage tanks
[5] and have also found that ozone gas can eliminate insects in grain storage
facilities without harming food quality or the environment [6,7]. Other studies
have focused on potential applications of gaseous or vapor-phase antimicro-
bials for decontamination of fruits and vegetables. Such antimicrobials being
studied include CIO2 gas, ozone, allyl isothiocyanate vapor, hydrogen peroxide
vapor, acetic acid vapor, and natural volatile compounds (methyl jasmonate,
tr arts -anethole, carvacrol, cinnamic aldehyde, eugenol, linalool, and thymol).
C10 2 and ozone gases are examples of promising technologies which have been
shown to lead to high microbial reductions. This chapter reviews the efficacy of
these antimicrobials in inactivation of microorganisms on fruits and vegetables.
18.2 CHLORINE DIOXIDE GAS
18.2.1 Physical, Chemical, and Safety Properties
of Cl0 2 Gas
C10 2 is a neutral compound of chlorine in the +IV oxidation state.
It disinfects by oxidation; however, it does not chlorinate. The major
Gas-/Vapor-Phase Sanitation (Decontamination) Treatments
403
oxidation/reduction is:
C10 2( aq)+e-= ClO^ (E = -0.954 V)
(1)
CIO2 gas is usually mixed with nitrogen or air and has a yellowish green color.
It has an odor similar to chlorine and sodium hypochlorite and can be easily
detected at levels as low as about 0.1 ppm in air. C10 2 gas has a density
2.4 times that of air. It is unstable as a gas and can decompose to chlorine
and oxygen with noise, heat, flame, and a minor pressure wave at low
concentrations. Selected physical, chemical, and safety properties of CIO2 gas
are summarized in Table 18.1. C10 2 is also highly water soluble and does not
TABLE 18.1
Physical, Chemical, and Safety Properties of Chlorine Dioxide Gas and Ozone
Property
Molecular weight
Oxidation potential
Melting point
Boiling point
Density, 25°C, 760mmHg
Vapor pressure
Solubility limit
Heat of formation at 298.15 K
Entropy at 298.15 K
Heat capacity at 298.15 K
Heat of vaporization at
boiling point
Heat of solution
Explosion velocity, pure gas
Explosion velocity, in air
Explosion concentration, in air
or >76mmHg
Permissible exposure limit (PEL)
specified by the Occupational
Safety and Health Administration
(OSHA), in air
Short-term exposure limit specified
by OSHA, in air, for 15 min
Chlorine dioxide gas
67.45 g/mol
-0.954V
-59°C
11°C
2.757 g/1
10 kPa at -34.3°C;
100 kPa at 10.5°C
~3 g/1, aqueous, 25°C,
34.5 mmHg;~20 g/1,
aqueous, 0-5°C,
70-100mmHg
89.1kJ/mol
263.7 J/mol/K
46.0J/mol/K
30 kJ/mol
6.6kcal/mol
1250m/s
50 m/s
>10% v/v
0.1 ppm
0.3 ppm
Ozone
48.00 g/mol
-2.07 V
-192.7°C
-111.9°C
1.962 g/1
lOkPaat -139.7°C;
lOOkPaat -111.5°C
-0.57 g/1, 100% ozone,
20°C, 760mmHg
142.7 kJ/mol
238.9 J/mol/K
39.2 J/mol/K
Unknown
Unknown
Unknown
Unknown
Unknown
0.1 ppm
Unknown
Modified from Lide, D.R., CRC Handbook of Chemistry and Physics, 82nd ed., CRC Press, Boca
Raton, FL, 2001; Occupational Safety and Health Administration, Air Contaminant Exposure
Standards, 29 CFR 1910.1000, chap. XVII, 6, July 1, 1991; Gates, D., The Chlorine Dioxide
Handbook, American Water Works Association, Denver, CO, 1998; EPA, Ozone, in Guidance
Manual Alternative Disinfectants and Oxidants, Environmental Protection Agency, 1999, chap. 3;
Kim, J., Yousef, A.E., and Dave, S., /. Food Prot., 62, 1071, 1999.
404 Microbiology of Fruits and Vegetables
hydrolyze readily but remains in solution as a dissolved gas [13]. C10 2 cannot
be compressed or stored commercially as a gas because it decomposes with
time and is highly explosive at high concentration (>10% in air) or under
pressure. Therefore, CIO2 is generated on-site. The permissible exposure limit
(PEL) or time-weighted average (TWA) of C10 2 gas in air is 0.1 ppm, specified
by the Occupational Safety and Health Administration [9].
18.2.2 Antimicrobial Properties of Aqueous and
Gaseous Cl0 2
C10 2 is strong oxidizing agent that has broad and high biocidal effectiveness.
Aqueous C10 2 effectively inactivates bacteria [14-18] including human
pathogens [19-21] and bacterial spores [14,24], viruses [22,23], and algae [25].
CIO2 has approximately 2.5 times the oxidation capacity of chlorine [15] and
has been shown to produce a bactericidal effect equivalent to seven times that
of chlorine at the same concentration in poultry processing water [26].
Advantages of C10 2 over chlorine also include effectiveness at low concentra-
tion, nonconversion to chlorophenols that result in residual odors and off-
flavors, ability to remove chlorophenols already present from other sources,
effectiveness at high and low pH values, and inability to react with ammonia or
humic acid to produce harmful chloramines and trihalomethanes [27].
Therefore, the use of C10 2 as an alternative disinfectant to chlorine is
attractive not only in the drinking water industry but also in the food industry.
Gaseous C10 2 has been used successfully for sterilization of medical
implements in the medical science area for years [2,3,28]. More recently gaseous
C10 2 was used to decontaminate B. anthracis contaminated areas of the Hart
senate office building and the Brentwood postal sorting facility in Washington
D.C. [29] (Table 18.2). In recent years additional applications of gaseous C10 2
in the food industry have been studied. Research has demonstrated that C10 2
gas is highly effective in reducing foodborne pathogens on fruit and vegetable
surfaces [31-34], spoiled orange juice isolates from epoxy-coated storage tank
surfaces [5], and bacillus spores on paper, plastic, epoxy-coated stainless steel,
and wood surfaces [30] (Table 18.2). These results have demonstrated that
C10 2 gas treatments are a promising surface decontamination technology
which could be applicable to the food industry. The efficacy and potential
applications of C10 2 gas treatment for decontamination of fruit and vegetables
are reviewed later in this chapter.
18.2.3 Cl0 2 Gas Generation
C10 2 gas is most often generated based on the reaction between chlorine gas
and sodium chlorite. The principal reaction can be described as:
2NaC10 2 +Cl 2 -► 2C10 2 +2NaCl (2)
Figure 18.1 shows an example of a C10 2 gas generation system from CDG
Technology, Inc. (Bethlehem, PA; http://www.cdgtechnology.com). Chlorine
o
CD
O
TABLE 18.2
Efficacy of CI0 2 Gas Treatments in Inactivation of Microorganisms on Different Surfaces
Surface
Paper strips and aluminum
foil cups
Analytical paper disk
Paper, plastic, wood
Paper in Tyvek/film pouch
Epoxy-coated stainless steel
Pathogenic microorganism
Bacillus sub til is spores
Bacillus sub til is niger spores
Bacillus thuringiensis spores
Bacillus sub t His spores
Lactobacillus spp.,
Penicillium spp., S. cerevisiae
CI0 2 gas treatment conditions
Continuous, >40mg/l, 1 h,
27°C, 60% RH
Continuous, 6-7mg/l, 1 h, 23°C,
70-75% RH
Batch, 15mg/l, 30min, 22° C,
>90% RH
Continuous, 5mg/l, 30min,
room temperature, 70% RH
Batch, 10mg/l, 30min, 22° C,
>90% RH
Total population
reduction/surface
Ref.
>61ogCFU
2
>61ogCFU
3
> 5 log CFU
30
>61ogCFU
28
> 6 log CFU
5
CD
O)
LO
CD
r-t"
CD
r-t-
o'
o
fD
n
o
ST
3
CD
i— h
o
H
—,
fD
CD
i— h
3
=3
o
406
Microbiology of Fruits and Vegetables
Pressure
regulators
CD
en
o
CD
O
On/off
Pressure gauge
c
CD
D)
O
Control
valve
Flow
meter
l
o
Lf)
T
Chlorine dioxide
+ nitrogen
►
To use point
Gas cylinders
FIGURE 18.1 Schematic of a CDG GasiSolid™ C10 2 gas generation system. (From
CDG Technology, Inc. With permission.)
I Chlorine dioxide gas generator and control system
pn
j
i
Photometer
Real time
chlorine dioxide
measurement
1
2
3
Target
chamber
Reagent
gas
Relative
humidity
probe
tef^-1-
t
*l/\ *
j Gas injec
tion
FIGURE 18.2 Schematic of a ClorDiSys C10 2 gas generation and control system. (From
ClorDiSys Solutions, Inc. With permission.)
gas (4%) in nitrogen flows into a Saf-T-Chlor™ reactor cartridge containing
thermally stable sodium chlorite; approximately 140 g and 1300g C10 2 gas are
produced by a bench- and a pilot-scale generator, respectively. Another
example of a C10 2 gas generator is manufactured by ClorDiSys Solutions, Inc.
(Lebanon, NJ, http://www.clordisys.com) which uses a similar approach, but
produces approximately 900 g C10 2 gas by flowing 2% chlorine gas in nitrogen
through three sodium chlorite cartridges (Figure 18.2).
ICA TriNova, LLC (Forest Park, GA) has developed a Z-Series C10 2
technology that involves generating C10 2 gas by mixing two dry solids: a C10 2
precursor and an activator (Figure 18.3). Sodium chlorite or sodium chlorate is
used as the C10 2 precursor in either crystalline or impregnated forms. The
Gas-/Vapor-Phase Sanitation (Decontamination) Treatments 407
FIGURE 18.3 Z-series products for C10 2 generation. (From ICA TriNova, LLC. With
permission.)
activator is a granular porous solid impregnated with an acid or with an acid
precursor. C10 2 gas is produced by a disproportionation reaction as the two
dry solids are mixed:
4H + +5NaC10 2 -» 4C10 2 +NaCl + 4Na + +2H 2 (3)
The Z-series products can produce approximately 0.1 mg to 50 g of C10 2 .
This product comes packaged in sachets, small tubs, and buckets based on
different applications.
18.2.4 General Gas/Vapor Treatment Systems
Batch and continuous C10 2 gas treatment systems have been developed and
used for decontamination of fruits and vegetables [31-34,36-38,40-42].
Typically, the batch system (Figure 18.4) includes a C10 2 gas generator (such
as a bench-scale CDG Technology or ClorDiSys Solutions generator), a
treatment chamber, a diaphragm vacuum pump, and a thermo-hydro recorder.
Produce samples are placed on expanded stainless steel shelves inside the
chamber. C10 2 gas is generated from the generator and is stored in a Teflon
storage bag before being injected into the chamber using a gas-sampling
syringe. The injected volume of C10 2 gas (known concentration) is deter-
mined based on required C10 2 gas concentration for the treatment chamber
volume. The injected C10 2 gas is distributed and circulated in the chamber by a
diaphragm vacuum pump. Relative humidity (RH) and temperature inside the
chamber are monitored using a thermo-hydro recorder. To measure C10 2
gas concentration, a certain amount (5 to 60 ml) of C10 2 gas-air mixture is
taken out of the chamber using a gas-sampling syringe and dissolved in
408
Microbiology of Fruits and Vegetables
Thermo-hygro
1 1
1
P
meter
o n n
Treatment
. .1 ! _ 1 — .
o o o
cylinder
n n n
T
CI0 2 gas
generator
o o o—
Fruits or
vegetables
<Z) -ww
>+
^Injection
L
v
CI0 2 gas
storage
Pump
FIGURE 18.4 Schematic of a batch laboratory C10 2 gas treatment system for fruits and
vegetables. (Patent pending, Purdue University.)
deionized water, followed by analysis using a DPD (7V,7V-diethyl-/?-phenylene-
diamine) colorimetric analysis kit and a VVR photometer (CHEMetrics, Inc.,
Calverton, VA).
A continuous laboratory-scale C10 2 gas treatment system mainly consists
of a bench-scale generator, a treatment chamber, a C10 2 gas dilution panel,
a diaphragm vacuum pump, an ultrasonic humidifier, a wireless thermo-
hygrometer, and a continuous CIO2 gas monitor. CIO2 gas from the generator
is diluted with filtered air and passes through the treatment chamber. Relative
humidity in the chamber is controlled using an ultrasonic humidifier. During
the treatment, C10 2 gas concentration is continuously monitored using the
continuous C10 2 monitor (InterScan Corp., Chatsworth, CA; http://www.
gasdetection.com). An automated continuous pilot-scale C10 2 gas treatment
system has also been developed (Figure 18.5). C10 2 gas is generated using a
Mindox-M generator from ClorDiSys Solutions, Inc. Fruit and vegetables are
placed on two movable shelves in a 4001 stainless steel chamber. After
preconditioning (humidifying) the chamber using a humidifier (50 to 95%),
the C10 2 gas is fed in and circulated by a gas blower. The gas concentration,
RH, and pressure relief are continuously monitored and controlled by the
Mindox-M generator. Each treatment cycle is programmed and run auto-
matically. After the treatment, the products may be washed by spraying
filtrated water inside the chamber for 5 to 10 minutes to reduce residual C10 2 ,
if any residuals remain on the product and/or in the chamber.
Concentrations of C10 2 are reported either as ppm in volume or mg/1.
When using C10 2 as a solution, 1 ppm is equivalent to approximately 1 mg/1.
However, when C10 2 is used as a gas, 1 mg/1 C10 2 is equivalent to appro-
ximately 332 ppm in volume under standard conditions (0°C, 1 atm) and
362 ppm under normal conditions (25°C, 1 atm). These conversions are based
on the ideal gas law. In this chapter, the conversion factor of 1 mg/1 = 362 ppm
is used.
Gas-/Vapor-Phase Sanitation (Decontamination) Treatments
409
Treatment
chamber
InterScan CI0 2
gas monitor
Ultrasonic
humidifier
ClorDisys CI0 2 gas
generator and monitor
FIGURE 18.5 Continuous pilot-scale C10 2 gas treatment system for fruits and
vegetables. (Patent pending Purdue University.)
18.2.5 Mechanisms for Microbial Inactivation
The mechanism of microbial inactivation by aqueous CIO2 has been explored,
but it is not fully understood. C10 2 has been shown to react readily with amino
acids (cysteine, tryptophan, and tyrosine), but not with viral ribonucleic acid
(RNA) [44,45]. Bernarde et al. [16] suggested that the primary inactivation
mechanism was the disruption of protein synthesis. However, Roller et al. [46]
indicated that the inhibition of protein might not be the primary inactivation
mechanism. The increase of the permeability of the outer membrane was
considered as another mechanism due to reactions of the outer membrane
protein and lipids with C10 2 [13,45,47]. Berg et al. [48] reported that gross
cellular damage involving significant leakage of intracellular macromolecules
did not occur for C10 2 -treated E. coli, but the cells lost control of potassium
efflux, which may be the primary lethal physiological event. Because C10 2
gas is highly water soluble, it may inactivate microorganisms in a similar way
as the aqueous form of C10 2 . However, C10 2 gas and/or its radicals may
directly diffuse or penetrate into microbial cells to cause damage. Therefore, to
understand completely the inactivation mechanism of aqueous and/or gaseous
C10 2 , extensive research is needed.
18.2.6 Factors Influencing Cl0 2 Gas Treatment
The efficacy of C10 2 gas treatment for decontamination of produce is affected
by gas concentration, exposure time, RH, temperature, cut or intact surfaces,
and microbial inoculation sites. The effects of C10 2 gas concentration (0.1 to
0.5mg/l), RH (55 to 95%), treatment time (7 to 135 minutes) and temperature
(5 to 25°C) on inactivation of E. coli 0157:H7 on green peppers have been
410
Microbiology of Fruits and Vegetables
Relative humidity (%)
75
E,
c
o
'■+— I
c
<D
o
c
o
o
CM
o
o
1.2 1.4 1.6 1.8 2.0
1.2 1.4 1.6 1.8 2.0 "1.2 1.4 1.6 1.8 2.0
1.8 2.0
1.2 1.4 1.6 1.8 2.0
Log time (min)
1.8 2.0
FIGURE 18.6 Effects of C10 2 gas concentration (0.1 to 0.5mg/l), RH (55 to 95%), time
(15 to 135 minutes), and temperature (10 to 20°C) on log reductions of E. coli 0157:H7
on inoculated green peppers. Shaded areas represent reductions greater than 5 logs. Log
time at 1.2, 1.4, 1.6, 1.8, and 2.0 correspond to 15.8, 25.1, 39.8, 63.1, and 100 minutes,
respectively. (From Han, Y. et al., J. Food Prot., 64, 1128, 2001. With permission.)
studied using response surface methodology [34]. A predictive model developed
in this study suggests that increasing C10 2 gas concentration, treatment time,
RH, and temperature all significantly (P<0.01) increased the inactivation
of E. coli 0157:H7. Contour plots (Figure 18.6) for E. coli 0157:H7 log
reduction, generated using a predictive model, showed a good comprehensive
picture of the model, in which the shaded areas indicate treatment conditions
that give greater than a 5 log reduction. C10 2 gas concentration was the most
important factor in the predictive model followed by time, RH, and then
temperature. The interaction between C10 2 gas concentration and RH
indicated a synergistic effect. Other research also indicates that a higher RH
results in a higher bacterial inactivation rate by C10 2 gas [1-3,5]. High RH can
moisturize treatment surfaces possibly leading to a thin layer of water droplets.
These water droplets can further absorb and dissolve large amounts of C10 2
gas due to high water solubility. Thus, the localized and concentrated C10 2
contributes to microbial inactivation on the surfaces of fruits and vegetables.
This may explain why C10 2 gas is unique and more effective compared to other
gaseous disinfectants.
Similar to aqueous sanitation treatments, C10 2 gas treatment may also be
less effective in reducing microorganisms on cut produce surface compared
Gas-/Vapor-Phase Sanitation (Decontamination) Treatments 411
TABLE 18.3
Log Reduction of E. co/i 0157:H7 Inoculated on Uninjured and Injured Green
Pepper Surfaces After CI0 2 Gas Treatments for 30 min at 20°C Under 90 to
95% Relative Humidity
Log reduction after different CI0 2 gas treatments
Sample 3 0.15mg/l 0.30 mg/l 0.60 mg/l 1.2mg/l
c
Uninjured surface 2.90 ± 0.09 A <j 3.99±0.07 Ac 7.27±0.68 Ab 8.04±0 Aa c
Injured surface 1.67 ±0.08*1 1.87 ±0.03,*. 3.03±0.02 Bb 6.45±0.02 Ba
a The initial populations of E. coli 0157:H7 on surface-uninjured and surface-injured green peppers
were 7.9 ±0.29 log CFU/5g.
b Values in the same column with different uppercase subscript letters are significantly different
(P < 0.05). Values in the same row with different lower subscript letters are significantly different
(P < 0.05).
c No viable E. coli 0157:H7 was detected using the end-point method after 1.2 mg/l C10 2 gas
treatments.
From Han, Y. et al., Food Microbiol, 17, 643, 2000. With permission.
to an uncut or intact surface. Studies [31] have shown that injuries to the
wax layer, the cuticle, and underlying tissue layers of green pepper surfaces
increased bacterial adhesion, growth, and resistance to washing and C10 2 gas
treatments. Han et al. [32] reported that CIO2 gas treatments (0.15 to 1.2 mg/l
CIO2) had significantly higher reductions for inoculated E. coli 0157:H7
on uninjured green pepper surfaces than on injured surfaces (jP<0.05)
(Table 18.3). Microphotographs obtained using confocal laser scanning
microscopy (CLSM) illustrated that bacteria preferentially attached to injured
surfaces, where the bacteria could be protected from the treatment. Similar
results have been reported for inactivation of Listeria monocytogenes on
uninjured and injured green pepper surfaces by aqueous and gaseous C10 2
treatments [33].
Microbial inoculation sites also influenced the efficacy of C10 2 gas treat-
ments. Du et al. [40,41] inoculated L. monocytogenes and E. coli 0157:H7 on
three sites of an apple: stem cavity, calyx cavity, and pulp skin. After CIO2
gas treatments, bacteria on the pulp skin were less resistant to the treatment
compared to the other two sites. At each inoculation site, however, bacterial
inoculation levels (at 6, 7, and 8 log CFU/site) did not affect log reductions
after treatment [40].
To determine accurately the efficacy of CIO2 gas treatments, appropriate
bacterial recovery and enumeration methods should be used. In evaluating the
effectiveness of a sanitation treatment on these pathogens in fruits and
vegetables, one of the common problems is overestimation of the treatment
effectiveness, because sublethally injured cells have not been taken into account
since direct selective plating enumeration methods are normally used. Han
et al. [35] reported that a membrane-transferring surface-plating method
412
Microbiology of Fruits and Vegetables
Polycarbonate membrane
Polycarbonate membrane
Non-selective medium
(2-4 hr at 37°C)
Selective medium
(24-48 hr at 37°C)
FIGURE 18.7 Schematic of a membrane-transferring surface-plating method for
enumeration of C10 2 -treated bacteria.
was better for recovering uninjured and C10 2 -injured E. coli 0157:H7 and
L. monocytogenes on green peppers after C10 2 gas treatments compared to
traditional methods using direct surface-plating and overlay surface-plating.
In this method (Figure 18.7), a 100 ul bacterial sample is first spread over a
polycarbonate filter membrane (0.4 urn pore size, 90 mm diameter) (Osmonics
Co., Westboro, MA) previously placed on the surface of a tryptic soy agar
plate. Plates are incubated at 37°C for 2-4 hours to repair injured cells. Then
the membranes are gently transferred onto appropriate selective media using
sterile tweezers, followed by further incubation for 20 to 40 hours at 37°C. The
membrane-transferring surface-plating method is also able to quantify low
levels (<21ogCFU/ml) of surviving bacteria by using a filtration procedure
to concentrate bacterial populations in test samples [41]. An end-point deter-
mination method has been successfully used to detect a complete inactivation
of inoculated bacteria on apple surfaces after C10 2 gas treatments [40,41]. This
method is useful to validate the efficacy of a C10 2 gas treatment when known
levels of bacteria are applied to produce surfaces.
18.2.7 Efficacy in Reducing Microorganisms on
Different Produce Surfaces
The efficacy of batch and continuous C10 2 gas treatments in reducing patho-
genic and spoilage microorganisms on several fruits and vegetables, including
green peppers, apples, potatoes, strawberries, cantaloupes, and lettuce, has
been evaluated (Table 18.4). Results show that more than a 5 log reduction of
selected pathogens such as E. coli 0157:H7, L. monocytogenes, and Salmonella
spp. can be achieved on these produce surfaces while maintaining acceptable
quality, except for lettuce leaves where leaf discoloration was noted. Gaseous
C10 2 has been shown to be a more effective sanitizer for fruits and vegetables
than aqueous C10 2 and chlorinated water wash. More than 61ogCFU of
L. monocytogenes on uninjured green pepper surfaces and 3.51ogCFU on
injured surfaces were inactivated using a batch C10 2 treatment system with
3mg/l C10 2 gas treatment for 10 minutes at 20°C and 90 to 95% RH.
However, a 3mg/l aqueous C10 2 treatment for 10 minutes at 20° C achieved
Gas-/Vapor-Phase Sanitation (Decontamination) Treatments
413
TABLE 18.4
Efficacy of CI0 2 Gas Treatments in Inactivation of Microorganisms on
Different Produce Surfaces
Surface
Pathogenic
microorganism
Green pepper E. coli 0157:H7
L. monocytogenes
Salmonella spp.
Apple E. coli 0\51:H7
E. coli
L. monocytogenes
Strawberry E. coli 0157:H7
L. monocytogenes
E. coli Q15T.H7
Cantaloupe
Lettuce
Potato
L. monocytogenes
Salmonella spp.
L. monocytogenes
Erwinia carotvora,
natural flora (aerobic
bacteria, yeast, and
mold)
CI0 2 gas treatment
conditions
Batch, 0.6mg/l, 30min,
22°C, >90% RH
Continuous, 0.6mg/l,
lOmin, 22° C, >90% RH
Batch, 4.0mg/l, 10 min,
22°C, >90% RH
Batch, 0.3mg/l, 3h, 4°C
Batch, 4.8mg/l, 30 min,
22°C, >90% RH
Batch, 4.0 mg/1, 30 min,
22°C, >90% RH
Continuous, 3.0 mg/1,
10 min, 22°C,
>90% RH
Continuous, 6.0 mg/1,
10 min, 22° C, >90% RH
Batch, 0.2 mg/1, 30 min,
22°C, >90% RH
Vaporized C10 2 by
purging air through
500 ppm acidified Oxine
at61/hfor lh
Log
reduction
Ref.
7.31ogCFU/site 31, 32, 34
6.31ogCFU/site
33
5.51ogCFU/site
37
5.51ogCFU/site
41
4.4 log CFU/g
43
4.81ogCFU/site
40
5.11ogCFU/site
36
5.31ogCFU/site
>61ogCFU/site
38
>61ogCFU/site
4.51ogCFU/site
42
2.01ogCFU/25g
49
3-4 log CFU/g
39
only 0.4 and 3.7 log reductions on injured and uninjured green pepper surfaces,
respectively, while water washing for 10 minutes showed 0.4 and 1.4 log
reductions, respectively [33]. Costilow et al. [50] also found that up to 105 mg/1
aqueous C10 2 failed to reduce the population of microorganisms present on
fresh cucumbers. Zhang and Farber [51] reported that C10 2 solution treat-
ment (5 mg/1, 10 minutes) at 22°C resulted in only a 0.8 log reduction of
L. monocytogenes on both cut lettuce and cut cabbage. The mechanism of why
aqueous CIO2 is not as effective as the gaseous form needs further study.
A continuous CIO2 gas treatment showed a higher efficacy compared to
that of a batch treatment. Using a batch system (Figure 18.4), a 4.0 mg/1
C10 2 gas treatment for 30 minutes at 22°C and under 90% RH achieved an
approximately 5 log reduction of E. coli 0157:H7 and L. monocytogenes on
strawberry surfaces [36]. With a continuous treatment system, more than a 6
log reduction of E. coli 0157:H7 and L. monocytogenes on strawberries was
achieved using a 3.0 mg/1 C10 2 gas treatment for 10 minutes [38].
414 Microbiology of Fruits and Vegetables
18.2.8 Effects of Cl0 2 Gas Treatment on
Quality of Produce
Effects of C10 2 gas treatments on quality of produce have not been extensively
studied. However, some researchers have shown minimal quality effects on
several types of produce, including green peppers [37], apples [40,41,43],
cantaloupes [42], strawberries [38], and potatoes [39]. Han et al. reported
[37,38] that no aerobic microorganisms (aerobic plate count, APC) were
detected after treatment of strawberries with 3.0mg/l C10 2 gas for 10 minutes
followed by a 1-week storage period at 4°C and after treatment of green
peppers with 0.6mg/l C10 2 gas for 10 minutes followed by a 4-week storage
period. Additionally, the color of both strawberry and green pepper surfaces
did not change significantly (/?>0.05) during the storage period after C10 2
gas treatments.
Residues of C10 2 and chlorite on strawberries treated with 3 mg/1 C10 2 gas
for 10 minutes were 0.19±0.33mg C10 2 /kg and 1.17 ± 2.02 mg Cl 2 /kg; while
after 1 week of storage no C10 2 residues were detected, and residual chlorite
levels were reduced to 0.07 ±0.12mg Cl 2 /kg. Residues of C10 2 and chlorite on
peppers were 0.13 ± 0.05 mg C10 2 /kg and 0.39 ± 0.49 mg Cl 2 /kg after treatment
with 0.6 mg/1 C10 2 gas for 10 minutes and 0.02 ± 0.04 mg C10 2 /kg and ± mg
Cl 2 /kg after a 4-week further storage of treated products. No significant color
changes (/?>0.05) were observed after 5.5 mg/1 C10 2 gas treatment for both
strawberries and green peppers. Tsai et al. [39] found that, after treating
potatoes with vaporized C10 2 by purging air through lOOppm C10 2 solution
for 1 hour, the residuals of chlorite and chlorate were less than 0.07 ppm,
measured using ion chromatography. Additionally, no significant color changes
(p>0.05) were observed on cantaloupes after 5.5 mg/1 C10 2 gas treatment [42]
and on apples after 7.8 mg/1 C10 2 gas treatment for 30 minutes [41]. However,
Sapers et al. [43] observed darkening of lenticels on apples when treated with
0.3 mg/1 C10 2 gas for 20 hours. Discoloration (bleaching effect) of lettuce [49]
and green cap on strawberries [38] was observed. It appears that C10 2 gas
treatment may have a negative effect on the color of leafy vegetables. However,
more studies evaluating the effects of C10 2 treatments on leafy vegetables are
needed.
18.3 OZONE GAS
18.3.1 Properties of Ozone
Ozone is a gas at ambient (22°C) and refrigerated (4 to 7°C) temperatures.
This gas is colorless with a pungent odor readily detectable at concentrations
as low as 0.02 to 0.05 ppm (v/v). Important properties of ozone are summarized
in Table 18.1. Ozone is partially soluble in water (12.9 mg/1 ozone can be
dissolved in the water when 3% ozone gas flows through water at 20°C), and
solubility can be affected by partial pressure, flow rate of ozone, tempera-
ture, purity of water, and contact time [52]. Ozone has the unique property of
Gas-/Vapor-Phase Sanitation (Decontamination) Treatments 415
autodecomposition, producing numerous free radical species, the most
prominent being the hydroxyl free radical. Ozone reacts with organic and
inorganic compounds in aqueous solutions either directly with whole molecular
ozone or by its radicals [53]. Ozone is a powerful oxidant with an oxidation
potential of —2.07 V. The half-life of molecular ozone in air is relatively long
(~12 hours), but in aqueous solutions the half-life can be as short as seconds
when organic compounds exist. Decomposition of ozone is so rapid in water
that its antimicrobial properties take place mainly at the microbial surface [54].
The PEL of ozone in the workplace is specified by the Occupational Safety and
Health Administration (OSHA) at 0.1 ppm in air on an 8 hours/day basis, for
a 40-hour work week.
18.3.2 Potential Applications of Ozone in the
Food Industry
Ozone is a strong antimicrobial agent in both gaseous and aqueous phases.
It is well known that ozone is an effective disinfectant in water and waste-
water treatments [55]. Ozone is 1.5 times more effective as an antimicrobial
agent than chlorine. Additionally, ozone is much more effective for a wider
spectrum of microorganisms than chlorine and other disinfectants [56]. It
reacts up to 3000 times faster than chlorine with organic materials and pro-
duces no harmful decomposition products [57]. Numerous studies have focused
on the inactivation of pathogens and spores by aqueous ozone, including
Cryptosporidium parvum [58,59], Giardia spp. [60], bacillus and Clostridium
spores [61, 62], Salmonella Typhimurium, E. coli, Yersinia enter ocolitica,
Pseudomonas aeruginosa, Staphylococcus aureus, and L. monocytogenes [63].
Applications of ozone for enhancing the microbiological safety and quality
of foods have been reviewed by Kim et al. [12] and Khadre et al. [52]. Most
applications for the food industry focus on the use of ozonated water for
sanitation of food-contact surfaces and foods, including fruits and vegetables,
meat and poultry, fish, cheese, and eggs. Although ozonated water has been
shown to be an alternative to chlorinated water for decontamination of
produce, its effectiveness in reducing microorganisms is less than 3 log CFU per
gram or surface [12,52,65].
For more than half a century gaseous ozone as a disinfectant and/or
preservative has also been applied in many areas in the food industry. Such
applications include preservation of perishable foods, including fruits, vegeta-
bles, meat, poultry products, fish, cheese, spices, and eggs [12,52,56,66],
grains [6,7,67], spices [12,68], decontamination of packaging materials [64,69],
and decontamination of environmental air in food plants [12].
18.3.3 Generation of Ozone
Ozone is commonly produced from oxygen or air by utilizing ultraviolet (UV)
light or corona discharge generators. UV light systems use radiation at 185 nm
wavelength emitted from high-transmission UV lamps. These systems are
416
Microbiology of Fruits and Vegetables
relatively low cost and do not require dry air for ozone production. The corona
discharge generators can produce larger concentrations of ozone, up to 4%,
compared to UV light systems. They mainly consist of two electrodes separated
by a dielectric or nonconducting material, providing a narrow discharge gap.
When a high-voltage alternating current is applied across this gap, the air or
oxygen passing through the gap is partially ionized, and the oxygen molecules
are dissociated. The split oxygen atoms combine with other oxygen molecules
to form ozone. Dried air is required for this system to prevent corrosion of
metal surfaces. Ozone can also be produced by cold plasma and electro-
chemical methods [12]. Some commercial ozone generator manufacturers
include IN USA, Inc. (Needham, MA), Lenntech, Inc. (College Station, TX),
Ozone Solution, Inc. (Sioux Center, IA), Ozomax Ltd (Quebec, Canada),
Longevity Resources, Inc. (British Columbia, Canada), and Ozone Services
& Yanco Industries Ltd (Burton, B.C., Canada).
18.3.4 Treatment Systems
A continuous laboratory-scale 3 gas treatment system (Figure 18.8) has been
used for decontamination of fruits and vegetables [66]. This system includes a
corona discharge ozone generator (Clear Water Tech, Inc., San Luis Obispo,
CA), a 10 1 Irvine Plexiglass treatment chamber with a stainless steel shelf, a
humidifier, a diaphragm vacuum pump, a thermo-hydro recorder, an ozone
neutralization unit, and a continuous ozone monitor (model 450H, Advanced
Pollution Instrumentation, Inc., San Diego, CA). Ozone gas from the genera-
tor is first humidified by flowing it through water in a 125 ml gas-washing
bottle. The gas then continuously passes through the treatment chamber.
Thermo-hygro meter
3 gas
monitor
Treatment
cylinder
3 gas
generator
i
t
Neutralization
Fruits or
vegetables
Pump
Humidifier
FIGURE 18.8 Schematic of a continuous laboratory ozone gas treatment system for
decontamination of fruits and vegetables.
Gas-/Vapor-Phase Sanitation (Decontamination) Treatments 417
Meanwhile, the gas in the chamber is circulated by the pump. The ozone gas
is finally neutralized by passing through a solution of reducing agents, such as
sodium sulfite. The concentration of ozone gas is continuously monitored
using a high-concentration ozone monitor. The concentration of ozone in the
air can be recorded as mg/1 or ppm. One mg/1 ozone gas is approximately
equivalent to 467 ppm in volume under standard conditions (0°C, 1 atm) and
509 ppm under normal conditions (25°C, 1 atm).
Ozone concentration can be measured using the indigo colorimetric
method, which has been approved by the Committee on Standard Methods for
the Examination of Water and Wastewater in 1988 [70]. For in-line monitoring
gaseous ozone, a UV spectrophotometric method can be used [12], such as
continuous monitors manufactured by Advanced Pollution Instrumentation,
Inc. (San Diego, CA) and IN USA (Needham, MA).
18.3.5 Mechanisms of Inactivation of Microbes
The oxidizing mechanisms of ozone may involve direct reactions of molecular
ozone and free radical-mediated destruction [12,52]. Inactivation of micro-
organisms by ozone may be due to the oxidation of a number of cellular
components. The oxidation and disruption of cell membranes is considered to
be one of the most important inactivation mechanisms. Ozone can oxidize
polyunsaturated fatty acids, membrane-bound enzymes, glycoproteins, and
glycolipids, and cause a decrease in cell permeability and disruption of normal
cellular activity [52,71,72]. It has also been reported that bacterial inactivation
may be due to inactivation of cellular enzymes, such as dehydrogenating
enzymes in E. coli cells [73], (3-galactosidase in the cytoplasm and alkaline
phosphatases in the periplasm of E. coli [74], and destruction of genetic
materials, such as DNA of E.coli [75,76], circular plasmid DNA [77], phage
DNA and RNA [78,79], and viral DNA and RNA [52,80]. However, there is
very limited information about microbial inactivation mechanisms by gaseous
ozone, and the primary mechanism by both aqueous and gaseous ozone still
needs to be clearly identified and investigated.
1 8.3.6 Factors Influencing Sanitation Treatment
by Ozone Gas
The efficacy of gaseous ozone in reducing microbes on produce can be affected
by many factors, such as ozone concentration, treatment time, temperature,
RH, and surface properties of produce [66,81-83].
The effects of ozone gas concentration (2 to 8 mg/1), RH (60 to 90%), and
treatment time (10 to 40 minutes) on inactivation of E. coli 0157:H7 on green
peppers were studied [66]. Ozone gas concentration, RH, and treatment time
were all significant (P < 0.01) factors for the inactivation of E. coli 0157:H7
(Figure 18.9). Among the three factors, the effect of ozone gas concentration
was the greatest. The interaction between ozone gas concentration and RH
exhibited a significant and synergistic effect (P < 0.05).
418
Microbiology of Fruits and Vegetables
2 4 6 8
3 concentration (mg/l)
60 65 70 75 80 85 90
RH (%)
10 15 20 25 30 35 40
Time (min)
FIGURE 18.9 Effects of ozone gas concentration, % RH, and treatment time on
log reductions of E. coli 0157:H7 on green peppers at 22°C. (From Han, Y. et al.,
J. Food ScL, 67, 1188, 2002. With permission.)
Ishizaki et al. [82] reported that the efficacy of ozone gas for inactivation of
bacillus spores on filter paper increased as ozone concentration (0 to 3.0 g/1),
time (0 to 6 hours), and RH increased (54 to 90%). The spores were more
resistant on a glass fiber filter than on filter paper. At a RH of 50% or below,
there was no appreciable decrease in the number of survivors within 6 hours
exposure to 3.0 mg/l ozone. Other researchers also reported that RH is an
important factor for microbial inactivation by ozone gas, and ozone is less
effective to inactivating dehydrated microorganisms [64].
Moreover, Liew and Prange [84] reported that temperature played an
important role in the storage of carrots treated by ozone gas. The ozone con-
centration at 2°C was higher than that at 16°C, hence providing a greater
reduction in fungal growth rate on carrots. A different linear effect of ozone
concentration was found for each temperature. Significant (P < 0.05) effects
of temperature and linear and quadratic effects of ozone on the growth rate
of Botrytis cinerea on carrots were also observed.
18.3.7 Efficacy in Reducing Foodborne
Microorganisms on Produce Surfaces
The efficacy of ozone gas in reducing foodborne pathogens and spoilage
microorganisms has been studied on produce, including green peppers, carrots,
black peppers, grapes, strawberries, and lettuce (Table 18.5). High concentra-
tions (>1000ppm or 2 mg/l) of gaseous ozone treatments have been shown to
be extremely effective in reducing E. coli 0157:H7 [85] and bacillus spores
[82] on filter paper. Han et al. [66] found that a greater than 5 log reduction
(CFU/site) of E. coli 0157:H7 on green pepper surface could be achieved by a
continuous ozone gas treatment at 5 mg/l for 25 minutes under > 70% RH and
at 22°C. Sarig et al. [87] reported that microbial counts on grapes were signi-
ficantly reduced after an 8 mg/l ozone gas treatment for 20 minutes. Zhao and
Cranston [68] treated ground black peppers with 6.7 mg/l ozonized air (6 1/min).
They found a greater than 3 log reduction of E. coli and Salmonella spp. after
TABLE 18.5
Efficacy of Ozone Gas Treatments in Reducing Microorganisms on Different Surfaces
Surface
Filter paper
Filter paper
Green pepper
Black pepper
Carrot
Blackberry
Lettuce
Microorganism
Bacillus sub t His spores
E. coli OIST.H7
E. coli 015T.H7
E. coli, Salmonella spp.,
Penicillium spp.,
Aspergillus spp.
Botrytis cinerea
Fungi
Total aerobic plate count
Black peppercorn E. coli, Salmonella spp.,
Penicillium spp., Aspergillus spp.
Ozone gas treatment conditions
Continuous gas flow, 3mg/l (1527 ppm)
for 1 h at 95% RH and 22°C
Continuous gas flow at 3 1/min for 5 min,
1000 ppm at4°C
Continuous gas treatment, 5 mg/1
(2545 ppm) for 25 min at >70% RH
and 22° C
Continuous gas sparge at 6 1/min for
10-60 min, 6.7 mg/1 (3410 ppm) at
room temperature
Continuous gas flow at 0.5 1/min for 8h
daily for 28 days, 60 ppm at 2, 8, or 16°C
Gas in storage room, 0.3 ppm for 12 days
at 2°C and 90% RH
Bubbling gaseous ozone (4.9%, v/v; 0.5 1/min)
in a lettuce— water mixture (1:20, w/w)
for 5 min
Bubbling gaseous ozone (6.7 mg/1; 6 1/min)
in a peppercorn— water mixture (2:5, w/w)
for 10 min
Efficacy Ref.
> 5 log CFU/paper reduction 82
> 5 log CFU/paper reduction 85
> 5 log CFU/sample reduction 66
3-6 log CFU/g reduction 68
50% reduction of daily growth rate 84
No visible fungal decay 86
1 .9 log CFU/g reduction 64
3-4 log CFU/g reduction 68
O
O
CO
r-t"
r-t-
o'
=3
o
co
n
o
ST
3
i— h
o
H
—,
CO
(a
i— h
3
CO
=3
i— h
t/5
420
Microbiology of Fruits and Vegetables
O
10 20 30 40
Duration of ozone treatment (min)
U)
O
2 4 6 8 10
Duration of ozone treatment (min)
FIGURE 18.10 Kinetics of microbial reduction in ground black pepper after 6.7mg/l
ozone gas treatment at a flow rate of 61/min. (From Zhao, J. and Cranston, P.M.,
/. Sci. Food Agric, 68, 11, 1995. With permission.)
60 minute exposure and more than a 3 to 4 log reduction of Penicillium
spp. and Aspergillus spp. after 10 minutes. Figure 18.10 shows the kinetics of
microbial reduction in ground black pepper after the ozone gas treatment.
Zhao and Cranston [68] also treated black peppercorns by bubbling gaseous
ozone (6.7mg/l; 61/min) in a peppercorn-water mixture (2:5, w/w) for 10
minutes. This treatment reduced the microbial population by 3 to 4 log
numbers. Kim et al. [64] reported that bubbling gaseous ozone (4.9%, v/v;
0.51/min) in a lettuce-water mixture under sonication and high-speed stirring
was the most effective ozonation method that inactivated up to a 1.9 log of the
natural microbial load in 5 minutes.
The effectiveness of ozone gas treatments is summarized in Table 18.5.
Low concentrations (<100ppm) of ozone gas treatments with long exposure
times (days) have been used for growth inhibition and inactivation of spoilage
microorganisms on fresh fruits and vegetables. Barth et al. [86] found that
fungal development was suppressed when blackberries were stored at 2°C
for 12 days in the air with 0.3 ppm ozone, with 20% of control fruits show-
ing decay. Growth rates of yeast surviving ozone treatment were markedly
decreased under longer exposure times and higher concentrations, RH, and
temperatures [83]. Constant exposure to ozone throughout storage has also
been reported to be effective in inhibiting storage pathogens on lemons and
oranges at 1 ppm (14°C, 85% RH) [88] and peaches at 0.25 ppm (4 to 15°C)
[89]. However, ozone was reported to be ineffective in preventing fungal decay
in strawberries after 4-day treatment with 0.35 ppm ozone at 20°C [90]. Other
Gas-/Vapor-Phase Sanitation (Decontamination) Treatments 421
early reports also indicated that ozonated apples, cantaloupes, and cranberries
demonstrated more decay or damage than those not ozonated [91-94].
18.3.8 Effects of Ozone Gas Treatments on
Quality of Fruits and Vegetables
Ozone gas can be used to prevent fungal decay and rot of fruits and vegetables
during cold storage [55]. Those products include bananas, citrus fruits, apples,
berries, peaches, and potatoes. Ozone can also retard the ripening process of
fruit and vegetables by oxidation of ethylene released during storage. Ewell [95]
indicated that the shelf life of strawberries, raspberries, and grapes could be
doubled when 2 to 3 ppm ozone is applied continuously for a few hours per
day. Barth et al. [86] reported that 0.3 ppm treatment suppressed fungal
development for 12 days at 2°C, and did not cause observable injury or defects
on thornless blackberries. By the 12th day, anthocyanin content and surface
color were maintained; however, peroxidase activity was reduced. Norton et al.
[92] found that 0.6 ppm ozone at 15°C was effective in controlling fungus rot
on Early Black and Howe varieties of cranberries, but caused weight loss and
quality damage by the second and third week. Perez et al. [90] also reported
that ozone was ineffective in preventing fungal decay in strawberries and also
was detrimental to strawberry aroma after 4-day treatment with 0.35 ppm
ozone at 20°C. Another study by Kute et al. [96] evaluating ozone-treated
strawberries suggested that 0.3 or 0.7 ppm ozone did not affect the ascorbic
acid levels, but significantly increased total soluble solid levels after 1 week of
treatment and storage. The shelf life of apples treated by 2 to 3 ppm ozone for a
few hours per day could be increased by several weeks, but damage on apples
was observed with 10 ppm ozone [97]. The shelf life of potatoes exposed to
3 ppm ozone could be extended to as long as 6 month at 6 to 14°C and 93 to
97% RH [98]. Liew and Prang [84] observed some physiological and quality
changes in ozone-treated carrots, such as higher respiration rate, electrolyte
leakage, and lower color, compared to the control samples. Skog and Chu [99]
studied the effect of ozone on quality of fruit and vegetables in cold storage.
They found that 0.04 ppm ozone treatment under 95 to 98% RH appears to
have the potential for extending the storage life of broccoli and seedless
cucumber at 3°C. Response to ozone was minimal for mushrooms stored at
4°C and cucumbers stored at 10°C. The ethylene level in vegetable storage
rooms was reduced from 1.5-2 ppm to a nondetectable level after the
0.04 ppm ozone treatment. The treatment did not affect the quality of apples
and pears. Artes-Hernandez et al. [100] studied the effects of ozone enriched air
treatment for improving quality of seedless table grapes during cold storage at
0°C for 60 days followed by 7 days of shelf life at 15°C in air. Compared to
control grapes, gas-treated samples had superior texture and visual appearance
after the shelf life study.
422 Microbiology of Fruits and Vegetables
18.4 ALLYL ISOTHIOCYANATE GAS
1 8.4.1 Properties
Allyl isothiocyanate (AITC) is the major component in essential oils of cruci-
ferous plants, such as black mustard seeds (Brassica nigra), brown mustard
seeds {Brassica juncea), cabbage (Brassica oleracia), and horseradish
(Armor acia rusticana) [101,102]. AITC is a specific compound from the
isothiocyanate (ITC) group that has been shown to have bactericidal,
bacteristatic, and antifungal activities. AITCs are released upon disruption
or injury of plant tissue due to hydrolysis of glucosinolates by cell wall-bound
myrosinase [103,104]. Other important properties of ITC compounds are their
high volatility, extreme pungency, and low water solubility.
18.4.2 Mechanisms and Factors Influencing
Sanitation Treatment
The proposed mechanisms for inactivation and inhibition of microbes have
focused on the nonspecific inactivation of enzymes through cleavage of
disulfide bonds of proteins; interference with specific enzymes (carriers) in the
electron transport chain, such as cytochrome C oxidase; uncoupling oxidative
phosphorylation; nonspecific reactions with key enzymes or proteins; and
reactions with free amino groups [104-106]. Even though there are several
proposed antimicrobial mechanisms, the true antimicrobial mechanism(s) of
ITCs are not fully understood.
18.4.3 Efficacy in Reducing Foodborne
Pathogens on Produce Surfaces
Gaseous and vaporized AITC has been shown to be an effective antimicrobial
agent for produce and grain surfaces (Table 18.6). Table 18.6 shows different
treatment parameters (antimicrobial concentration, treatment time and tem-
perature, container size) to achieve a log reduction of 3 log CFU/g or higher.
Additionally, data from this table suggest that microorganisms are more
difficult to inactivate on a rough surface than on a smooth surface.
1 8.4.4 Effects of AITC Vapor Treatment on
Quality of Fruits and Vegetables
Detrimental effects on product quality (lettuce texture and crispness) have been
shown when lettuce is treated with high doses (>300ul) of AITC vapor [102].
Additionally, Lin et al. [102] showed that tomato and apple texture did not
change when exposed to low levels of AITC. However, when the apples and
tomatoes were exposed to high levels of AITC vapors (>300 ul), texture started
to soften within 2 days following treatment. Residual pungent odor and off-
flavors on the produce existed for 12 hours when product was treated with
Gas-/Vapor-Phase Sanitation (Decontamination) Treatments
423
TABLE 18.6
Efficacy of Allyl Isothiocyanate in Reducing Foodborne Pathogens on
Produce Surfaces
Surface
Microorganism
Treatment conditions
Efficacy
Ref.
Lettuce
Listeria monocytogenes
400 ul in a 1 gal bag with
1 00 g lettuce; 96 h
-4 log CFU/g
102
Salmonella Montevideo
400 ul in a 1 gal bag
with lOOg lettuce; 48 h
81ogCFU/g
E. coli 0157:H7
400 ul in a 1 gal bag with
1 00 g lettuce; 48 h
7 log CFU/g
Tomato skin
Salmonella Montevideo
400 ul in a 4 1 container with
3 tomatoes; 24 and 48 h
5 log CFU/g
102
Tomato scar
Salmonella Montevideo
500 ul in a 4 1 container with
3 tomatoes; 48 h
5 log CFU/g
102
Apple stem scar
E. coli Ol 51 :H7
600 ul in a 4 1 container with
3 apples; 48 h
3 log CFU/g
102
Wet alfalfa seeds
E. coli 0157:H7
50 ul /950 ml jar; 24 h; 25°C
~4 log CFU/g
107
Alfalfa sprouts
Salmonella mixture
200 and 500mg/l; 48 h at 10°C
7 log CFU/g
108
Alfalfa seeds
Salmonella mixture
1000mg/l; 7hat60°C
~2 log CFU/g
108
AITC vapors < 300 ul; however, when high levels of AITC vapors (500 ul) were
used, off- flavors and odors lasted up to 24 hours after product treatment [102].
An additional side effect that has been seen after treatment of alfalfa seeds with
AITC (up to 500mg/l) is reduced seed germination [108]. Treatment of alfalfa
seeds with 200 and 500 mg AITC/1 of air adversely affected sensory quality
attributes (appearance, color, aroma, and overall acceptance) [108].
1 8.5 OTHER GASES/VAPORS
18.5.1 Hydrogen Peroxide Vapor
The use of hydrogen peroxide (H 2 2 ) has been approved by the U.S. Food and
Drug Administration (FDA) as an antimicrobial for several food processing
applications, such as disinfection of aseptic packaging materials and equip-
ment, processing of cheese and modified whey, and production of thermophile-
free starch [109]. Washes with aqueous H 2 2 , or commercial sanitizers
containing H 2 2 , have been studied in laboratories for disinfection and/or
decontamination of fresh or minimally processed fruits and vegetables [109-
115]. Additionally, vapor-phase H 2 2 has been evaluated for the decontamina-
tion of fruits and vegetables, due to its sporicidal properties against bacillus
spores and food spoilage microbes [116-118]. Forney et al. [119] treated table
grapes inoculated with Botrytis cinerea spores with H 2 2 vapor generated from
30 to 35% H 2 2 solution at 40°C for 10 minutes. After the treatment, and a
24-hour storage period at 10°C, the number of germinable spores was reduced
by 60% or more, and the incidence of decay was also reduced after 12 day
424 Microbiology of Fruits and Vegetables
storage at 10°C. In another study, Simmons et al. [120] applied 3.1 mg/1 H 2 2
vapor to dried prunes for to 60 minutes at ambient room temperature (20 to
26°C). More than 2 log CFU/g reductions in total plate count (TPC), yeast, and
mold counts were observed after the treatment for 10 minutes. However, some
oxidation damage was observed in prunes treated for greater than 20 minutes.
Other studies have shown that H2O2 vapor is effective in reducing spoilage
microbial counts on whole cantaloupes, raisins, and walnut nut meat [109].
Sapers et al. [121] also evaluated the use of H 2 2 vapor to extend the shelf life
of various other fresh and fresh-cut commodities. Produce samples were
exposed to H 2 2 vapor for 2 to 15 minutes at injection rates of 2.5 or 5g
of H 2 2 /min. The treatment appeared to delay or diminish the severity of
bacterial soft rot in fresh cut cucumber, green bell pepper, and zucchini. No
effect on spoilage of fresh-cut broccoli, carrot, cauliflower, or celery, or fresh
raspberries and strawberries was observed. More recently, Sapers et al. [110]
studied decontamination of apples using H 2 2 vapor generated from 35%
H 2 2 solution at 150°C in a pressurized vessel. The treatment led to a
1.7 log CFU/g reduction of is. coll on apples when three treatment cycles were
applied over 5 minutes, which was less than those that would be obtained with
a 5% H 2 2 wash [112].
18.5.2 Acetic Acid Vapor
Acetic acid is known for its preservative properties [122] and has been used
extensively in foods such as pickles, salad dressing, tomato products, and
mustards. Vaporized acetic acid has also shown biocidal effects for decon-
tamination of fruits and vegetables. Researchers have demonstrated that
fumigation with acetic acid vapor or vinegar vapor could control postharvest
decay of fruits and vegetables such as apples, grapes, stonefruit (peaches,
nectarines, and apricots), strawberries, oranges, kiwifruit, tomatoes, and
coleslaw made from cabbage [123-127]. They also demonstrated that
fumigation treatments with 242 ppm (v/v) gaseous acetic acid in air for
24 hours at 22°C or for 12 hours at 45°C could reduce 3 to 5 log CFU/g
Salmonella Typhimurium, E. coli 0157:H7, and Listeria monocytogenes on
mung bean seed without significant reduction of seed germination rates [128].
Recently, Sapers et al. [43] evaluated the use of pressurized acetic acid vapor to
decontaminate apples inoculated with E. coli. In their vacuum-pressure cycle
system, a vacuum (508 to 686mmHg) was applied to a 61 stainless steel
treatment, where inoculated apples were placed, and then acetic acid vapor
generated from glacial acetic acid at 60° C was applied to the vessel to achieve a
pressure of 34.5, 68.9, or 103.4 kPa. The treatment time was 5 to 30 minutes,
and temperatures ranged from 40 to 60°C. After the treatment, the apples were
immediately rinsed with tap water for 30 seconds, observed for treatment-
induced injury, and prepared for microbiological analysis. They found that
the vapor treatment with three vacuum-pressure cycles at 60° C provided
population reductions exceeding 3.5 log CFU/g. However, these conditions
induced discoloration. When a total treatment time of 5 minutes was used, log
Gas-/Vapor-Phase Sanitation (Decontamination) Treatments 425
reductions increased with increasing treatment temperature and the number of
vacuum-pressure cycles applied. Treatment pressure did not appear to affect
the bacterial log reduction. During storage for several hours, the treated
Golden Delicious apples developed dark lesions surrounding the lenticels
which penetrated several millimeters into the flesh beneath the skin, indicating
that acetic acid vapor treatment may not be useful for apples under these
conditions.
18.5.3 Other Natural Plant Volatiles
Some natural plant volatiles, such as methyl jasmonate, /rafts-anethole,
carvacrol, cinnamic aldehyde, eugenol, linalool, and thymol, have been used
as antimicrobials in reducing microbial contamination and extending shelf
life of fruits and vegetables [129]. Methyl jasmonate (MJ) is known for its
properties to enhance resistance to chilling temperature of fruits and vegetables
[130]. MJ vapor, in combination with modified atmosphere packaging, can
reduce loss of firmness, fungal decay, and development of chilling injury and
increase retention of organic acids in papayas [131]. MJ vapor from a 10 -4 or
10" ^ mol source in a 1 1 container also retarded deterioration of celery sticks
for 2 weeks at 10°C and reduced the bacterial load by approximately 3 logs
after 1 week storage [130]. Wang [132] reported that postharvest quality
of raspberries was enhanced with treatments of MJ, AITC, tea tree oil, or
absolute ethyl alcohol during storage at 10°C. Wang and Buta [133] also found
that 2.24 to 22.4 |il/l MJ vapor maintained good quality of fresh-cut kiwifruit
for up to 3 weeks, as did absolute ethanol and isopropyl alcohol.
Weissinger et al. [108] evaluated nine natural volatile compounds for
their ability to destroy salmonella on alfalfa seeds and sprouts. In this study,
vapor-phase acetic acid, AITC, fraws-anethole, carvacrol, cinnamic aldehyde,
eugenol, linalool, MJ, or thymol were applied to inoculated alfalfa seeds at
1000 mg/1 of air concentration for 1, 3, and 7 hours at 60°C. Only acetic acid,
cinnamic aldehyde, and thymol caused significant reductions in salmonella
populations (>3 logCFU/g) compared to the untreated control (1.91ogCFU/g)
after treatment for 7 hours. Treatment of seeds at 50°C for 12 hours with
acetic acid (100 and 300 mg/1) and thymol or cinnamic aldehyde (600 mg/1)
led to a 1.71ogCFU/g reduction of salmonella on seeds without affecting
germination percentage. Treatment of seeds at 50°C with AITC (100 and
300 mg/1) and cinnamic aldehyde or thymol (200 mg/1) did not significantly
reduce populations compared with the untreated control. Seed germination
percentage was largely unaffected by treatment with gaseous acetic acid, AITC,
cinnamic aldehyde, or thymol for up to 12 hours at 50°C. Acetic acid at 200
and 500 mg/1 reduced an initial population of 7.50 log CFU/g of alfalfa sprouts
by 2.33 and 5.72 log CFU/g, respectively, within 4 days at 10°C, whereas AITC
at 200 and 500 mg/1 reduced populations to undetectable levels. However, both
treatments caused deterioration in sensory quality. Treatment of sprouts with
1 or 2 mg/1 AITC also adversely affected sensory quality and did not reduce
salmonella populations after 11 days of exposure at 10°C.
426 Microbiology of Fruits and Vegetables
18.6 PRESENT AND FUTURE APPLICATIONS OF
GASEOUS/VAPOR-PHASE ANTIMICROBIALS FOR
DECONTAMINATION OF FRESH PRODUCE
As an alternative to traditional sanitizers and fumigation agents, gaseous/
vapor-phase antimicrobials certainly have potential for use to improve micro-
bial safety and to extend shelf life of fresh and minimally processed fruits and
vegetables. Some of the possible applications are presented below.
Technologies could be designed and optimized to treat produce in larger
scale operations within a treatment chamber, a storage room, or a continuous
belt tunnel using C10 2 or ozone gas that is continuously supplied by generators
and monitored throughout the process. The treated produce may be most
appropriate in production of fresh-cut products or as ingredients used in juice
production. One such application is an automated continuous CIO2 gas
treatment system that is being studied and evaluated for decontamination
of produce at Purdue University (Figure 18.5). Natural Sterilization &
Fumigation, Inc. (Sparks, NV) has also developed a PureOx ozone gas
treatment system for decontamination of fruits and vegetables. Products are
treated in a vacuum chamber with controlled ozone generation. McCabe [134]
has also patented a continuous process apparatus and method to treat delicate
vegetables, such as potato chips and dehydrated onions, by ozone gas. In this
system, products are continuously passed through a treatment zone containing
ozone. The products, air, and byproducts (oxygen) of the ozone treatment are
separated from the ozone by gravity in a separation zone located above the
treatment zone.
Treatments could also be designed and optimized to treat produce in a
chamber or a storage room using vapor-phase antimicrobials, such as C10 2 .
Tsai et al. [39] evaluated a system for the prevention of potato spoilage during
storage using vaporized C10 2 . In this system, C10 2 is vaporized by purging
air through different concentrations of acidified Oxine (ACD, Bio-Cide
International Inc., Norman, OK) solutions, in which C10 2 is the active
ingredient. C10 2 vapor is delivered to a treatment chamber or storage facility
where the C10 2 treatment is applied to the potatoes based on a calculation
dealing with the amount of C10 2 loss in the ACD solution. Using this system
with 500 ppm ACD Oxine solution, a 3 to 4 log reduction of Erwinia carotvora
and natural flora (aerobic bacteria, yeast, and mold) were found after purging
air through the Oxine solution at a flow rate of 6 1/hour for 1 hour.
A closed chamber or package in which C10 2 gas is introduced could also be
developed. Using this approach, the gas could be slowly released by sachets,
such as sachets that are currently manufactured by ICA TriNova LLC
(Figure 18.3). One advantage of this technology is that there is no need for
costly and space-consuming C10 2 gas generating systems. Sapers et al. [43]
studied decontamination of apples in a 24.61 high-density polyethylene pail
using sachets that generate 0.03 to 0.3 mg/1 C10 2 gas in the pail. They found a
3.24 and 4.42 log CFU/g reduction of E. coli after treatment with 0.3 mg/1 C10 2
Gas-/Vapor-Phase Sanitation (Decontamination) Treatments 427
for 3 hours at 4 and 20°C, respectively, while product quality was minimally
affected. After 20 hours' exposure, the microbial reduction approached nearly
5 logs, but darkening of lenticels was also observed. Lee et al. [135] evaluated
the same C10 2 sachets, which slowly released 11, 18, and 26 mg C10 2 gas within
0.5, 1, and 3 hours, respectively, for the decontamination of lettuce in a 20 1
chamber. They reported a 4 to 6 log reduction of E. coli, L. monocytogenes,
and Salmonella Typhimurium on lettuce leaves.
There may also be an opportunity to treat produce within a plastic film
package where the packaging material is designed to allow for a slow and con-
tinuous release of the antimicrobial gas or vapor in the package. Bernard
Technologies Inc. (BTI, Chicago, IL) has developed two proprietary,
controlled-release technologies called Microsphere® and Microlite® that focus
on preventing and eliminating biological contamination. Both the Microsphere
and Microlite patented sustained release systems [136] enable the creation of
an active Microatmosphere® environment that inhibits the growth of micro-
organisms and provides an extended shelf life for fruits and/or vegetables.
BTFs Microsphere-containing films have been affirmed as a GRAS product
by the FDA and U.S. Department of Agriculture (USD A) for use in
preserving fresh fruits, vegetables, meat, and poultry products.
The effects of combining antimicrobial vapors or gases with a modified
atmosphere packaging strategy to extend the shelf life of fruits and vegetables
could also be explored further. Ozen et al. [69] studied the effects of ozone gas
on mechanical, thermal, and barrier properties of several plastic films used in
food packaging, including linear low-density polyethylene (LLDPE), oriented
polypropylene (OPP), and biaxially oriented nylon (BON) films. Exposure to
2.1 to 4.3 mg/1 ozone gas for 2 to 24 hours caused a decrease in tensile strength
of OPP, decreased melting temperature of OPP and BON, and reduced oxygen
permeability of LLDPE and BON.
18.7 REGULATORY CONSIDERATIONS
Although the FDA has approved the use of aqueous C10 2 (3 ppm residual) to
wash fruits and vegetables, it has not granted permission to use the gaseous
form of C10 2 as a sanitizer for decontaminating produce. It is assumed that
gaseous C10 2 treatment would be very similar to aqueous C10 2 , producing
similar oxychloro byproducts (chlorine, C10 2 , chlorate, and chlorite) as an
aqueous C10 2 treatment. Based on existing scientific literature and data related
to exposure to drinking water containing 1 mg/kg of the oxidant species
(chlorine, C10 2 , chlorate, and chlorite), the use of aqueous chlorine dioxide is
not likely to lead to chemical byproducts that are harmful to human health
[10]. The research group at Purdue University has shown that residuals of
C10 2 and chlorite on green peppers were not detectable after a 4-week storage
period and after a 1-week storage period for strawberries [37,38]. However, in
order to pursue regulatory approval for the use of C10 2 gas for deconta-
mination of fresh and cut produce, more information and research are needed
428 Microbiology of Fruits and Vegetables
on production of byproducts on fruit and vegetable surfaces after C10 2
treatment.
In the food industry, ozone was first permitted by the FDA to disinfect
bottled water [137]. In 1997 ozone was affirmed GRAS status as a disinfectant
and/or sanitizer for broad-based food usage by an expert panel sponsored by
the Electric Power Research Institute (EPRI) in the U.S. [57]. In 2001 the FDA
approved the use of ozone as a direct food additive for the treatment, storage,
and processing of foods in gaseous and aqueous phases. Acetic acid
and hydrogen peroxide have been considered as GRAS food additives by the
FDA; however, currently there are no regulations in the U.S. on the usage of
their vaporous forms or of other natural plant volatiles (AITC, MJ, trans-
anethole, carvacrol, cinnamic aldehyde, eugenol, linalool, and thymol) as
antimicrobial agents for preservation or sanitation of fruits and vegetables.
Regulatory approval of the usage of these antimicrobials for produce will
depend upon many different factors. Certainly, treatment effectiveness and
efficacy data, worker safety and human health exposure data, and industrial
and commercial needs will all play very important roles as this technology
moves forward.
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19
Modified Atmosphere
Packasin
B.G. Werner and J. H. Hotchkiss
CONTENTS
19.1 Introduction 437
19.1.1 Definitions 438
19.2 Antimicrobial Activity of MAP Gases 439
19.2.1 C0 2 439
19.2.2 Superatmospheric O2 440
19.3 Packaging and Films for MAP Produce Systems 441
19.3.1 Film Permeability and C0 2 /0 2 Permselectivity 441
19.3.2 Active Packaging: Antimicrobial Films 442
19.3.2.1 Synthetic Polymer Films 442
19.3.2.2 Edible and Biodegradable Films 443
19.4 An Integrated Approach: Multiple Barriers and MAP 445
19.4.1 Background 445
19.4.2 Biopreservation and Protective Cultures 445
19.4.3 2 /C0 2 Absorbers and Generators 447
19.4.4 Pretreatments and Miscellaneous Strategies 447
19.5 Microbiology of Map Fruits and Vegetables 448
19.5.1 Minimally Processed Fruits and Vegetables 448
19.5.2 Spoilage Organisms and Commodity Shelf Life 450
19.5.3 Pathogenic Organisms and Shelf Life 451
19.5.3.1 Food Safety Risk of MAP Produce 451
19.5.3.2 Psychrotrophic Pathogens 452
19.5.3.3 Other Pathogens of Concern 453
19.5.4 Microbial Ecology of MAP Systems 454
19.6 Mathematical Predictive Modeling 455
19.7 Future Directions 456
References 456
19.1 INTRODUCTION
Modified atmosphere packaging (MAP) has been successfully and widely used
commercially, for both whole and fresh-cut or minimally processed (MP) fruits
and vegetables, as a packaging strategy for maintenance of product safety and
437
438 Microbiology of Fruits and Vegetables
extension of shelf life. MAP technology and systems development has striven
to parallel the increasing demand for longer shelf life, improved food safety,
and expanded variety of convenient MP ready-to-eat (RTE) or ready-to-use
(RTU) fruits and vegetables. MAP systems have been shown to have the
potential to increase the shelf life of specific produce commodities by 50 to
400% [1], particularly when used in concert with hurdle technologies such
as active packaging, postharvest handling strategies and other newly evolving
technologies in an integrated approach.
19.1.1 Definitions
MAP generally utilizes an internal package atmosphere of something other
than air (air can be approximated as <0.1% C0 2 , 21% 2 , 78% N 2 ) in a
hermetically sealed package of suitable permeability in order to extend product
shelf life and maintain food safety. While other gases have been explored for
use in MAP systems, 2 , C0 2 , and N 2 are most commonly employed; 2 levels
are commonly reduced below and C0 2 increased above atmospheric levels
(with a balance of N 2 ) in order to reduce the commodity respiration rate,
retard ripening and senescence, and reduce microbial activity. Microorganisms
are affected indirectly by reductions in ripening and senescence and directly by
restriction of 2 and antimicrobial activity of C0 2 ; superatmospheric 2 has
also been shown to be antimicrobial but is not currently commonly employed.
MAP is a dynamic process where environmental and packaging charac-
teristics and the contained product interact to create an equilibrated internal
atmosphere (EMA). The EMA is achieved when the rate of 2 consumption
and C0 2 generation as a result of respiration by a particular commodity
equals the rate of gas transmission through the packaged material. Generally,
an EMA of 3 to 6% 2 and 2 to 10% C0 2 achieves microbial control and
extension of shelf life for a wide variety of whole and MP produce, although
other atmospheres are also used with commodities that are not physiologically
sensitive to high 2 or C0 2 . Package EMA can be created actively, where
a target internal atmosphere is established initially upon packaging by actively
flushing with the desired atmosphere, or, more commonly, passively, where
the package atmosphere is allowed to reach the desired gas mixture around
the commodity during the course of storage at a particular temperature; a
longer time period is required to achieve a target EMA for passive than for
active MAP.
The EMA is dependent upon both extrinsic and intrinsic factors, includ-
ing product respiration rate, packaging film permeability to gases and water
vapor, package dimensions, and fill weight. The intrinsic respiratory activity of
produce is in turn influenced by the particular commodity, the cultivar, stage of
maturity at harvest, type of tissue, mass, condition, and whether the product is
whole or minimally processed. The mass or produce size indirectly influences
respiratory activity by affecting 2 diffusion rates into tissues, which sub-
sequently directly influence respiration rates; alternatively, the stage of maturity
or age has a direct impact on metabolic activities and rates. The extent of film
Modified Atmosphere Packaging 439
permeability to gases per unit thickness, the effect of relative humidity on this
permeability, the package surface area, seal integrity, free volume inside the
package, and relative humidity around the package will also affect the EMA
achieved. Temperature is the most influential extrinsic factor to consider as
it affects both commodity respiration rate and film gas and water vapor per-
meability. MAP systems will generally be exposed to a dynamic environment
during distribution, storage, display, and consumer purchase. Thus, while a
particular MAP system should be optimized for a particular storage tem-
perature, the effect of significant temperature changes on the system should be
considered [2].
19.2 ANTIMICROBIAL ACTIVITY OF MAP GASES
19.2.1 C0 2
Dissolved C0 2 has been found to inhibit microbial growth [3-5], affecting the
lag phase (A), maximum growth rate (|i m ax) m and/or maximum population
(iV max ) densities reached; levels in excess of 5% in MAP systems have been
found to be bacteriostatic [6]. The mode of action, although not yet fully
understood, is thought to be due to a number of effects, including changes in
intracellular pH, alteration of microbial protein and enzyme structure and
function, and alteration of cell membrane function and fluidity. The partial
pressure and concentration of CO2, package headspace gas volume, tem-
perature, pH, water activity, specific microorganism and growth phase, and
growth medium (produce commodity) all influence the inhibitory effect of C0 2 .
The antimicrobial effect of C0 2 is enhanced as temperatures decrease and C0 2
becomes increasingly soluble.
Low to moderate levels of C0 2 have been shown to inhibit growth of
many common aerobic produce spoilage bacteria. Moderate C0 2 levels of 20
to 60% have been found to reduce the \x max and N max of Pseudomonas spp.
and Moraxella spp., two predominant spoilage bacteria found on produce [7].
Low C0 2 levels below 20% were found to primarily increase A, with slight
reductions in |i max an d no changes in jV max . C0 2 is not antimicrobial towards
all microorganism strains or species, and may in some cases actually promote
growth. Lactobacillus spp. are generally unaffected by C0 2 ; however, some
levels can enhance growth, and 100% C0 2 environments have inhibited growth
of some strains. In absence of 2 , it has been generally shown that the growth
and toxin production of Clostridium hotulinum is only minimally affected by
C0 2 concentrations less than 50%; 100%) C0 2 has been reported to delay toxin
production compared to a 100% N 2 atmosphere [1] and decreases growth at
5 and 10°C [8]. Levels of 10% C0 2 have been found to be inhibitory to growth
of Yersinia enter ocolitica while 40% C0 2 increased A, and 100% C0 2 both
increased X and decreased [x max HI- There is no agreement on the effect of CO?
on Listeria monocytogenes; however, generally it has been found that C0 2 does
not affect or in some cases promotes growth. L. monocytogenes has been found
440 Microbiology of Fruits and Vegetables
to grow well under atmospheres of both 100% N 2 and 3% 2 /97% N 2 ; growth
was enhanced by increasing levels of C0 2 in either atmosphere [9].
19.2.2 SUPERATMOSPHERIC 2
Superatmospheric 2 as an MAP atmosphere is a new concept not yet
commercially in use due to an incomplete understanding of its effects on MAP
systems and mode of action towards microbial populations. Additionally, 2
in excess of 25% is considered explosive, and practical safety issues that need
to be employed in its use may not be feasible. Conventional MAP systems
that commonly target an EMA of 3 to 6% 2 may be exposed to fluctuating
temperatures or temperature abuse conditions during handling, resulting in
complete or near depletion of 2 . Under these conditions, growth of some
pathogens such as C. botulinum may be enhanced due to anaerobiosis, or
unrestricted growth of psychrotrophic facultative anaerobic pathogens such as
L. monocytogenes may occur due to removal of competitive aerobic micro-
organisms. Under certain atmospheric conditions, Staphylococcus aureus,
Vibrio spp., E. coli, Bacillus cereus, and Enter ococcus faecalis have also been
shown to grow with restricted or zero 2 [10]. As an alternative MAP atmos-
phere strategy, high oxygen atmospheres (typically above 70%) that surpass
optimal levels for growth of aerobes (21%) and anaerobes (0 to 2%) could
generally result in growth inhibition of both anaerobic and aerobic
microorganisms, resolving some of the food safety issues possible with lower
2 EMA.
Few reports exist of the effects on specific microorganisms of superatmos-
pheric 2 in MAP systems, and some data are conflicting. Jacxsens and others
[11], in a study on RTE mushrooms, grated celeriac, and shredded chicory
endive, found that growth of Pseudomonas fluorescens, Candida lambica,
Botrytis cinerea, Aspergillus flavus, and Aeromonas caviae was retarded by high
2 MAP atmospheres (70, 80, or 95% 2 , bal. N 2 ), an effect that increased
with increasing levels of 2 ; increasing 2 levels extended X of L. mono-
cytogenes. Using an agar-surface model system, Amanatidou and others [12]
found 90% 2 (bal. N 2 ) extended A of L. monocytogenes and Salmonella
typhimurium, reduced |i max of E. coli and S. enteritidis, and significantly
increased |i max of P. fluorescens, E. agglomerans, Candida guilliermondii, and
C. sake. Combined applications of high 2 and 10 to 20% C0 2 generally
both increased X and reduced N max for all strains tested. On mixed vegetable
salad, Allende and others [13] found that yeast growth was stimulated by MAP
atmospheres of 95kPa 2 while growth of psychrotrophic bacteria and
L. monocytogenes was unaffected. Generally, greater levels of lactic acid
bacteria were found on the mixed salad during storage under conventional
MAP gas mixtures than with superatmospheric 2 MAP. Salads treated with
superatmospheric 2 also exhibited a longer shelf life, retaining acceptable
visual characteristics longer than conventional MAP treatments; the authors
did not report whether any other significant organoleptic changes occurred.
While superatmospheric 2 has the potential to extend shelf life and maintain
Modified Atmosphere Packaging 441
produce marketable qualities, these effects may vary depending upon the
commodity. Wszelaki and Mitcham [14] found that superatmospheric storage
of Camarosa strawberries resulted in acceptable product firmness, titratable
acidity, external color, ethylene production, respiration, and soluble solids,
but unacceptable odors and flavors developed as a result of increased pro-
duction of volatile fermentative metabolites (ethanol, acetaldehyde, and ethyl
acetate).
The mode of action of high 2 is thought by some [12] to be due to oxidative
stress and reduction of cell viability due to the generation of intracellular
reactive oxygen species such as peroxides or superoxides. Some microorgan-
isms may adapt by producing radical scavengers or inducing 2 decomposing
enzymes; repair proteins have been identified for S. typhimuriurn, E.coli, and
L. lactis [11]. It is clear that significantly more work is needed to examine
and clarify the effects on the growth parameters of individual spoilage
microorganisms and food pathogens of superatmospheric 2 , alone or in
combination with C0 2 , applied to different produce commodities and MAP
systems. Additionally, a more complete understanding of the underlying basic
biological mechanisms of superatmospheric 2 is necessary prior to successful
commercialization of this technology [15].
19.3. PACKAGING AND FILMS FOR MAP PRODUCE
SYSTEMS
19.3.1 Film Permeability and C0 2 /0 2
Permselectivity
The specific gases employed, the gas permeability coefficients of the film, and
the EM A desired will in part dictate the packaging films utilized. Concentra-
tions of the two most commonly metabolically active gases employed in
MAP, 2 and C0 2 , will impact produce quality and levels of both should be
optimized within the package. C0 2 /0 2 permselectivity, the ratio of C0 2 to 2
permeation coefficients, will vary for different films and can be selected or
altered to concurrently optimize levels of both C0 2 and 2 in MAP systems
[16]. Most commercial packaging films available have C0 2 /0 2 permselectivities
between 4 and 8, allowing greater diffusion of C0 2 than of 2 [17]; anaero-
biosis and less than optimal C0 2 levels can result, depending upon the
commodity and MAP system employed. Nitrogen is metabolically inert but
can also be important as a filler gas to prevent package collapse. MAP produce
subjected to temperature abuse or temperature changes along the normal
distribution chain may result in increased respiration and depletion of
in-package 2 ; subsequently, the effective target EMA will not be maintained,
and premature spoilage or increased food safety risks may occur [18]. High-
barrier films that further restrict diffusion of C0 2 can result in excessive
buildup of C0 2 as well as anaerobiosis, altered EMA, and lowered product safety
and quality. Thus, there is a demand for films with C0 2 :0 2 permselectivities
closer to 1, or engineered packaging systems that use novel technologies or
442 Microbiology of Fruits and Vegetables
films to increase effectively or finely manipulate the 2 flux. Particularly for
higher respiring produce, permselectivites >2 increase the likelihood that the
atmosphere will rapidly become anaerobic [16]. This is exacerbated as storage
temperature increases. Polymer technology has been developed (Landec
Corporation, Inc., CA) that allows the 2 transmission rate of films to
increase more rapidly than the C0 2 transmission rate in response to
temperature, resulting in an adjustable C0 2 /0 2 permeability ratio [19].
Use of a composite film comprising ethylene vinyl acetate (EVA),
low-density polyethylene (LDPE), and oriented polypropylene (OPP) can
enhance or improve gas permeability characteristics. Shredded cabbage and
grated carrot stored in this composite material achieved an extension of shelf
life of 2 to 3 days over that achieved in OPP alone [20]. Films have been
developed with pores (micro-perforations) or holes (macro-perforations) to
increase the 2 transmission rate, resulting in more equal rates of movement of
2 and C0 2 between the internal package atmosphere and external atmos-
phere, achieving permeability ratios of C0 2 to 2 near 1. Micro-perforated
films may be appropriate for products with a high respiration rate, such as
strawberries, where finer control of package atmosphere is desired, where
internal package 2 depletion is a concern, or where temperature fluctuations
may be anticipated.
The number, perimeter, and total effective area of perforations affects the
rate of gas exchange [21,22]; the application and level of atmosphere control
will determine these factors. Lee and others [21] developed a model to describe
and predict changes in atmosphere and humidity in micro-perforated packages,
verifying the model on refrigerated MAP peeled garlic. The number, cross-
sectional area, and placement of perforations as well as the thickness of the
film affect the EMA attained and alter gas and moisture exchange rates across
package film. Long, narrow channels are more difficult for the gases to move
through than wide, short-path perforations. Perforated films can be applied as
an overwrap to a nonpermeable formed container or attached as a patch or
label to a selectively permeable or nonpermeable bag. Used as a patch or label,
only a small percentage of the package area is utilized to achieve the desired
effects, creating a wide range of gas atmospheres, particularly when used in
combination with a selectively permeable film.
19.3.2 Active Packaging: Antimicrobial Films
19.3.2.1 Synthetic Polymer Films
Antimicrobial packaging is an active packaging strategy that can serve a
variety of distinct barrier functions in MAP systems, such as package self-
sterilization, sterilization of produce, or reduction of growth of spoilage
organisms and/or pathogens on packaged produce. Synthetic polymer films are
most commonly researched for this functionality. Antimicrobial packaging
films can be grouped into two general categories, nonmigratory and migratory.
Nonmigratory packaging incorporates antimicrobials into the polymer or
immobilizes them on the surface of the film in such a way that the compounds
Modified Atmosphere Packaging 443
are not released; food must be in direct and intimate contact with such films in
order for antimicrobial activity to occur. Migratory antimicrobial packaging
incorporates the antimicrobial into or on the surface of the film in such a way
that migration can occur to the food product where activity then occurs;
unlike nonmigratory antimicrobial films, the antimicrobial becomes part of the
foodstuff that eventually is ingested. Migratory antimicrobials can be released
in aqueous solution (e.g., nisin, organic acids), or as a vapor (e.g., allyliso-
thiocyanate, chlorine dioxide). The latter method of release is perhaps most
suited to MAP systems where headspace between package and product is
maintained and intimate contact between package and product does not
typically occur. Release of migratory preservatives must be finely controlled
for useful and effective activity that persists over the course of a defined or
desired package shelf life.
The development of antimicrobial films has been significantly restricted
by the legal status of antimicrobial compounds available for food contact use
or as food preservatives or additives; currently only a limited number of such
approved compounds exist, and approval varies among countries. Silver-
substituted zeolites, a broad-spectrum high-activity antimicrobial with low
human toxicity, has been extensively used commercially in Japan as a thin
laminant on packaging film surfaces; its use on food contact surfaces in the
European Union and the U.S., however, is unclear [23]. Some U.S. Food and
Drug Administration (FDA) generally regarded as safe (GRAS) materials
that have been considered for use as antimicrobials in synthetic polymer
films include organic acids (benzoic, lactic, propionic, malic, succinic, tartaric,
sorbic), enzymes (lactoperoxidase, lactoferrin, lysozyme, chitinase, glucose
oxidase, ethanol oxidase), isothiocyanates (allylisothiocyanate), bacteriocinsc
(nisin, pediocin, sakacin, subtilin, carnocin), and essential oils (thymol,
cinnamic acid, eugenol) [6,24]. Natural plant extracts such as grapefruit seed
extract have been shown to be effective against Staphylococcus aureus and
E. coli on MAP lettuce and bell pepper, and when used in combination with
imazalil could also provide protection against growth of molds, yeasts, and
lactic acid bacteria [15]. Antimicrobials that are volatile, such as chlorine
dioxide and allylisothiocanates, have an advantage in that they can be
distributed within the closed package.
Any antimicrobial must not only be approved for food use, it must also be
compatible with the packaging material and the package/film manufacturing
process as well as maintain activity in the particular food matrix and MAP
system [23]. Thus, different strategies may be employed in creating anti-
microbial films and designing antimicrobial packaging systems from synthetic
polymers versus more natural materials as are used in edible and biodegradable
films.
19.3.2.2 Edible and Biodegradable Films
Biodegradable and edible films mainly comprise one or more proteins, lipids,
or polysaccharides; each of these base materials has unique strengths and
444 Microbiology of Fruits and Vegetables
weaknesses as packaging materials and vehicles for antimicrobial compounds,
and is thus usually employed in combinations. Polysaccharide (cellulose, gums,
starch) or protein (gelatin, corn zein, soy protein, whey, etc.) films are highly
sensitive to moisture and are poor barriers to water vapor; however, they
exhibit suitable mechanical and optical properties. Films composed of lipids
(waxes, lipids) have good water vapor barrier characteristics, but do not exhibit
suitable mechanical and optical properties (are opaque and may be brittle).
Wheat gluten and soy protein isolate films are effective 2 barriers at low
relative humidity, but have limited vapor barrier ability. Addition of lipid
components to protein-based films improves the characteristics of both
materials by optimizing both permeability to moisture and structural strength.
Polarity of these natural films will determine compatibility with a particular
antimicrobial and application or incorporation method.
Use of antimicrobials in edible films and coatings concentrates active
compounds at the produce surface where protection is needed; thus small levels
of additive are needed. This type of treatment is attractive to the increasing
population of consumers who desire minimally preserved fruits and vegetables.
Antimicrobials as edible film or coating components must be approved for
food use. Waxes, with incorporated antimicrobials such as imazalil and
benomyl, have been successfully used to minimize water loss and to inhibit
microbial growth through gas exclusion and wound protection on fruit;
however, benomyl is not an FDA GRAS substance and cannot be directly
applied to food, and imazalil has limited FDA approval.
Some biodegradable film components such as chitosan naturally exhibit
antimicrobial properties. Chitosan, a polysaccharide derived from shellfish and
some fungi, has been found to exhibit broad antimicrobial activity towards a
range of yeasts, molds, and bacteria and thus shows potential for application in
MAP systems. Lee and others [25] found that chitosan, applied as a paper
packaging coating, inhibited growth of E. coli 0157:H7 in orange juice.
Srinivasa and others [26] found that a chitosan film employed as a lid on a
cardboard container for MAP storage of whole mango fruit inhibited fungal
growth and extended shelf life of the product from 9 to 18 days at 27°C as
compared to use of LDPE as an overwrap.
As with synthetic films, 2 permeabilities of edible films and coatings
generally can be very low and C0 2 /0 2 permselectivities can be quite high. In a
selection of edible films [27] including pectin, wheat gluten, chitosan and
bilayer gluten, and beeswax films, at 25°C the C0 2 /0 2 permselectivity ranged
between 6 and 28.4, and 2 permeability ranged between 2 and 258.8 p0 2
mlmm/(m dayatm). Thus the same risk for development of anoxic conditions
and reduced food safety conditions exists for edible natural films as for
synthetic films. Appropriate combinations of coatings and antimicrobial
compounds may compensate for these effects. For example, a wax-based
coating may incorporate nisin to reduce the risk of growth of Clostridium
botulinum and/or L. monocytogenes [15].
Modified Atmosphere Packaging 445
19.4. AN INTEGRATED APPROACH: MULTIPLE
BARRIERS AND MAP
19.4.1 Background
Active packaging strategies and other technologies may be used in MAP
systems, where multiple barriers combining two or more technologies at
inhibitory levels provide integrated and enhanced control of microbial growth.
Barrier technologies may be selected to serve different roles, such as main-
tenance of activity even under temperature abuse conditions or failure of MAP
atmospheres. Barrier technologies may reduce initial microbial populations on
produce prior to packaging and MAP storage, or may be selected specifically
to reduce the incidence of a target pathogen of concern.
The path of produce from field to the point of packaging involves many
stages where handling and environments can be controlled and optimized to
avoid contamination with pathogens or spoilage organisms and to reduce
initial microbial load. Good hygiene practices during harvest and storage,
optimal postharvest storage and transportation/distribution temperatures,
and HACCP (hazard analysis critical control point) implementation during
processing are basic steps that have been historically incorporated into a
multiple barrier approach to microbial control and should include the use of
MAP. Use of one or more active packaging technologies, discussed in the
previous section, can be excellent additions to a multiple barrier approach;
addition of biopreservatives, antagonistic or protective microbial cultures,
inclusion of gas absorbers or generators, ultraviolet C (UVC) treatments, and
combination atmosphere technologies such as CA and MAP or dual MAP
packaging systems can also serve as effective barrier technologies.
1 9.4.2 BlOPRESERVATION AND PROTECTIVE CULTURES
A growing demand for minimally preserved or preservative-free fresh produce
has led to a search for alternatives to more traditionally used food anti-
microbial compounds. Biopreservation, the use of antagonist or protective
cultures, has shown potential for extension of produce storage life; natural
microflora such as lactic acid bacteria (LAB) or bacterial metabolic byproducts
such as organic acids or bacteriocins can serve as natural inhibitors of spoilage
organisms. Application of antagonist organisms along with use of MAP tech-
nology and additional microbial control strategies can be synergistic in effect.
For example, a prepackaging application to sweet cherry of the antagonist
yeast Cryptococcus infirmo-miniatus (CIM) Pfaff and Fell followed by modified
atmosphere storage at 2.8°C for 20 days or — 0.5°C for 42 days resulted in
significant reduction of the causal agent of brown rot, Monilinia fructicola
G. Wint., an effect that was enhanced when a preharvest application of
propiconazole was incorporated [28]. Prepackage application of organic acids
to vegetables or fruits such as melon, papaya, or avocado, which are typically
446 Microbiology of Fruits and Vegetables
low-acid, results in a pH decline that is inhibitory to groups of spoilage
organisms that grow best under neutral or near neutral pH environments.
Protective cultures such as LAB can be found naturally on produce,
and thus may not significantly alter the typical or expected product taste or
cause significant spoilage if applied directly. The most studied LAB
bacteriocinogenic strains include Lactococcus lactis, Pediococcus acidilactici,
and Lactobacillus sakei, which produce the antimicrobials nisin, pediocin, and
sakacin, respectively [29]. Bacteriocins produced by LAB are typically
antimicrobial towards Gram-positive spoilage organisms and pathogens,
including L. monocytogenes and C. botulinum. Bacteriocins do not affect
Gram-negative bacteria, which are a major spoilage group of concern; however
LAB and associated bacteriocins may be used to target specific Gram-positive
pathogens of concern or be used in combination with other technologies
that reduce Gram-negative bacterial growth for a broader overall range of
microbial control.
Some LAB strains may not grow well enough on produce at refrigeration
temperatures in order to produce levels of bacteriocin necessary for
antimicrobial activity; additionally, bacteriocins can be inactivated by bacterial
proteolytic enzymes or by binding to food components, and target bacteria
may become resistant. Bennik and others [30] isolated bacteriocinogenic strains
of Pediococcus parvulus and Enter ococcus mundtii from MAP endive and
evaluated the ability of these strains to produce bacteriocin on mung bean
sprouts at refrigeration temperatures of 4 to 8°C. E. mundtii was able to
produce the bacteriocin mundticin on inoculated mung beans stored under
MAP (1.5% 2 , 20% C0 2 , balance N 2 ) at 8°C, while P. parvulus did not
survive under these conditions. When mundticin was extracted and used as
a dip (200BUml _1 ) or incorporated into an alginate film (200BUml _1 ) on
mung bean, the bacteriocin exhibited antimicrobial activity under refrigerated
MAP storage [30]. Cai and others [31] isolated a strain of Lactococcus lactis
subsp. lactis from mung bean sprouts that contained a gene for nisin-Z, an
antilisterial compound. This isolate could survive on fresh-cut RTE Caesar
salad at levels of 8 log 10 CFU/g at 3 to 4.5°C for up to 20 days and could grow
at 4°C and produce nisin-Z at 5°C. When co-incubated with 2 log 10 CFU/g
cells of Listeria monocytogenes on salad, L. monocytogenes populations were
reduced by 1 to 1.4 logio CFU/g after 10 days' storage at 7 and 10°C. Thus
bacteriocinogenic strains should be assessed for their ability to grow and
produce bacteriocin on a target commodity under specific MAP conditions
and storage temperatures. Additionally, bacteriocins directly applied should
be assessed for their persistence and activity on a specific commodity during
MAP shelf life.
Other microorganisms have been found to exhibit antimicrobial activity
due to competition for nutrients, rapid growth rates, or production of inhibi-
tory metabolites. Enterobacteriacea have been found to limit growth of
L. monocytogenes on endive, most likely due to competition for nutrients.
Mixed populations of nonbacteriocinogenic strains of Lactobacillus brevis and
Leuconostoc citrium have been shown to inhibit competitively the growth of
Modified Atmosphere Packaging 447
L. monocytogenes on MAP MP vegetables [32]; Enterobacter cloacae and
E. agglomerans were also found to be competitively inhibitive. This research
group also found in challenge studies with MAP lettuce that increasing C0 2
atmospheres decreased this inhibitory effect; when CO2 levels increased from
5 to 10 to 20%, a delayed inhibitory effect was increasingly observed [33]. The
use of nonpathogenic strains naturally found on produce that competitively
inhibit spoilage organisms and/or pathogens on produce under MAP storage
conditions warrants further study as a promising biopreservative hurdle
strategy.
19.4.3 2 /C0 2 Absorbers and Generators
Passively achieved MAP systems may be slow to reach a target EMA, creating
a sufficient lag time for significant growth of psychrotrophic aerobic spoilage
organisms such as Pseudomonas spp. 2 scavengers incorporated into packag-
ing materials as sheets, labels, trays, or films can be used as an active strategy
to more rapidly reach EMA. Commercially available 2 scavengers such as
Ageless® (Mitsubishi Gas Chemical Co., Japan) and Freshpax® (Multisorb
Technologies, Inc., USA) [23] are based on iron oxidation.
2 scavenging technology has been used successfully in MAP stored bakery
and dairy products, and applications in MAP stored produce are being explored.
Charles and others [34] created a mathematical model based on the respiration
rate of produce, film permeability, and oxygen absorption kinetics of the
scavenger. Validation using LDPE pouch packaged tomato and a commercial
iron-based 2 scavenger system at 20° C showed that target EMA was actively
established within 50 hours; without the absorber, the EMA was passively
reached within 100 hours. When using 2 absorbers, the possibility exists that
anaerobiosis may occur. In order to optimize MAP produce safety, more
information is needed about how 2 scavengers function or respond in
different MAP environments with different commodities [10].
Alternatively, C0 2 generators can be used to achieve high levels of C0 2
(60 to 80%), which can inhibit microbial growth on produce surfaces. C0 2
generators may pose a safety risk; moderate to high levels of C0 2 will inhibit
growth of aerobic spoilage organisms that usually warn consumers of spoilage,
and growth of pathogens may be enhanced due to lack of competition and the
altered environment [1,10].
19.4.4 Pretreatments and Miscellaneous
Strategies
Treatment of produce with methyl jasmonate prior to MAP has been found
to be successful in suppressing fungal decay in a number of commodities,
including fresh-cut celery and peppers, grapefruit, papaya, strawberries,
zucchini squash, mango, and avocado [35]; the effects and mode of action of
jasmonates in reducing disease development differ among various crops and
pathogens. Synergistic activity between methyl jasmonate treatments and MAP
448 Microbiology of Fruits and Vegetables
has been found for several commodities, including papaya. Gonzalez-Aguilar
and others [35] found that exposure of papaya to methyl jasmonate vapor
(10 -5 or 10 _4 M) for 16 hours at 20°C inhibited growth of Collectotrichum
gloeosporioides and fruit decay in papaya, an effect that enhanced a MAP
treatment of 14 to 32 days at 10°C followed by 4 days at 20°C in a modified
atmosphere of 3 to 5 kPa 2 and 6 to 9 kPa C0 2 .
As a treatment prior to MAP packaging, nonionizing, artificial UVC
radiation has the potential to be effective in reducing the initial microbial load
on produce, providing shelf life extension. UVC has been shown to damage
microbial DNA, an effect that weakens or kills microbial cells. Some bacteria
have been found to utilize repair mechanisms to overcome DNA damage, and
some cells may mutate. Thus typically UVC treatment results in a reduction of
microbial load but not complete sterilization. Allende and Artes [13] found that
treatments of 254 nm UVC doses up to 8.14kJ/m on Red Oak Leaf lettuce,
subsequently stored at 5°C for 9 to 10 days, significantly decreased the growth
of psychrotrophic bacteria, yeast, and coliforms. UVC has been shown to
reduce postharvest diseases and decay in a variety of whole produce including
strawberries, apples, carrots, sweet potatoes, zucchini squash, tomatoes, and
onions [13,36]. These different produce typically are smooth surfaced and
simple in shape; UVC would not be fully effective on produce with naturally
convoluted, rough, or inaccessible surfaces, as radiation would not penetrate
into shadowed regions of these types of surfaces.
Some antimicrobial compounds have been found naturally in fruits and
vegetables and can be used as additional hurdles in MAP systems. Raw carrots
produce compounds antimicrobial towards L. monocytogenes, an antimicrobial
effect that is more pronounced in shredded than in whole carrots, and is absent
in cooked carrots. Mixing shredded raw carrots with other MP vegetables that
did not produce the antilisterial compounds resulted in overall reductions
in populations of L. monocytogenes during storage, and a coleslaw mix of
shredded carrot and cabbage stored under MAP conditions had less spoilage
than either product stored individually [37]. Red chicory has been found to
inhibit growth of Pseudomonas spp. and Aeromonas hydrophylla [38], and
capsaicinoids found in green bell pepper were hypothesized to be antimicrobial
towards Shigella spp. [39]. More knowledge is needed about antimicrobial
compounds naturally found in fresh fruits and vegetables in order to utilize
their benefits.
19.5 MICROBIOLOGY OF MAP FRUITS AND
VEGETABLES
19.5.1 Minimally Processed Fruits and
Vegetables
MP produce includes fresh fruits and vegetables that may be washed, chopped,
trimmed, peeled, sliced, or shredded prior to packaging and storage at
Modified Atmosphere Packaging 449
refrigeration temperatures. There is increasing consumer demand for MP
produce, due to the level of convenience offered by pre-use processing and
availability as a fresh RTE or RTU food. MP produce typically is not washed
or cooked prior to ingestion, increasing the risk of food poisoning. Thus the
level of quality and safety of MP produce must be quite high for the shelf life
achieved. MAP has great potential as a strategy to achieve this goal, and much
research and development effort has been initiated to develop useful MAP
systems for a variety of MP produce.
Commercial MAP systems have been developed for a wide range of whole
fruits and vegetables. However, these same systems cannot be used in parallel
commodities that have been processed; processed produce deteriorates and
metabolizes much differently from whole produce. Processing steps such as
chopping, slicing, and dicing rupture tissues and cells, releasing nutrients and
degradative enzymes such as oxidases. Plant cells are less physically resistant to
microbial invasion, nutrients are made more available for microbial growth,
respiration rates increase, and surface area increases, allowing for greater
incidence of spoilage. Processing thus significantly reduces shelf life, producing
a highly perishable product compared to whole fruits and vegetables; whole
produce that may be stored for several weeks under refrigerated MAP storage
when processed may only have a 1- to 2-day shelf life.
It is a challenge to create a MP produce commodity that exhibits high
quality and safety over a reasonable amount of time for feasible distribution
and sale. MAP technology has the potential to provide adequate shelf life for
MP produce, particularly when used in combination with additional hurdle or
control strategies. MAP strategies must be created for each specific commodity
and preparation method, as commodity characteristics and indirect effects of
preparation steps can influence package EMA, microbial growth, and shelf
life. Allende and others [40] looked at microbial levels on commercial fresh
processed red Lollo Rosso lettuce after reception and processing steps of
shredding, washing, draining, rinsing, centrifugation, and packaging and found
that shredding, rinsing, and centrifugation significantly increased bacterial
counts. Improvements made to reduce microbial levels during each of these
three steps resulted in further extensions to shelf life when the product was
stored under MAP conditions. Others have found that some processing
methods can increase the respiration activity of some produce commodities by
1.2- to 7-fold or more. Hand peeled carrots exhibited a 15% increase in
respiration rate while machine peeled carrots exhibited a 100% increase in
respiration rate; the respiration rate during storage also differed between the
two processing methods [20]. Pretel and others [41] found significantly different
respiration rates and mesophilic bacterial growth on MAP-stored enzymati-
cally peeled versus manually separated oranges; manually separated oranges
generally exhibited a higher level of bacterial growth and production of C0 2
than enzymatically peeled oranges. These differences could be ameliorated to
some extent by manipulating packaging film permeabilities and storage
temperature, achieving similar shelf lives. MP fruits will pose different storage
challenges from vegetables, due to differences in inherent composition,
450 Microbiology of Fruits and Vegetables
physiology, biochemistry, and microbiology as well as differences in processing
procedures and equipment. Thus the MAP strategy must be matched to not
only a particular commodity, and whether whole or processed, but also to the
specific processing method.
19.5.2 Spoilage Organisms and Commodity
Shelf Life
The spoilage microorganisms present on produce in MAP storage systems will
be influenced by the particular commodity and by the atmospheres and
temperatures employed. Initially, Gram-negative bacteria predominate in the
microflora of typically low-acid vegetables while LAB, molds, and yeasts
predominate on high-acid fruits. Indigenous microflora on vegetables that
cause spoilage include a majority of Gram-negative bacteria, predominantly
Pseudomonas spp., Enterobacter spp., and Erwinia spp. as well as Flavobacte-
rium spp. and Xanthomonas spp. and Gram-positive LAB such as Leuconostoc
mesenteroides and Lactobacillus spp. Indigenous yeasts and molds that
cause spoilage include Cryptococcus spp., Candida spp., Rhodotorula spp.,
Fusarium spp., Rhizopus spp., Cryptococcus spp., Botrytis spp., Mucor spp.,
and Penicillium spp., among others.
Zagory [38] reported that for a majority of fresh vegetables, Pseudomonas
spp. comprised 50% or more of the total initial spoilage microflora in MAP
stored product. Jacxsens and others [42] reported that MAP spoilage of
leafy greens and cucumber was primarily due to growth of members of the
Enterobacteriacea family while spoilage of celeriac and green bell peppers was
due to LAB and yeasts. The diversity of spoilage organisms initially found on
MAP produce upon packaging may dynamically change during the course of
shelf life and establishment of EMA. Bennik and others [43] found lowest
counts of pseudomonads under 0% O2 compared to 21% 2 atmospheres,
irrespective of C0 2 levels. Pseudomonads were predominant at 21% 2 , while
enterics were more predominant under 0% 2 . Differences in sensitivities to
modified atmospheres among strains, availability of nutrients, nutrient
requirements, and/or physiological state of the produce can result in shifts in
microbial populations during storage. Bennik and others [44] examined the
microbial composition of MP mung bean sprouts and chicory endive stored
under MAP (atmospheres of 1.5 or 21% 2 with 0, 5, 20 or 50% C0 2 ) at 8°C.
On mung bean sprouts, the predominant species before and after storage were
Enterobacter cloacae, Pantoea agglomerans, Pseudomonas fluorescens, Ps.
viridilivida, and Ps. corrugata. Predominant species on chicory endive before
storage were Rahnella aquaatilis and several Pseudomonas spp.; after storage,
E. vulneris and Ps. fluorescens predominated.
Generally, MAP utilizing mixed atmospheres of 2 /C0 2 /N 2 is most
inhibitory towards aerobic bacteria and molds and may not inhibit or only
minimally inhibit many spoilage yeasts and LAB. Exceptions have been
discovered using specific MAP atmospheres and produce commodities.
Martinez-Ferrer and others [45] found that MAP atmospheres of 4% 2 ,
Modified Atmosphere Packaging 451
10% C0 2 , balance N 2 significantly reduced total yeast populations on
prepared mango and pineapple stored for up to 30 days at 5°C, as compared
with storage under vacuum or air. Piga and others [46] found that in cactus
pear fruit stored for 9 days at 4°C under atmospheres of 17%C0 2 , < 1% 2 ,
balance N 2 , fungal mycelia were not visible on produce surfaces, but these
conditions did not inhibit mold growth; molds increased in number from 20
to 5x 10 2 CFU/g.
19.5.3 Pathogenic Organisms and Shelf Life
19.5.3.1 Food Safety Risk of MAP Produce
When competitive microflora are eliminated by MAP atmospheres, some
pathogens may grow unimpeded. Certain MAP systems can produce anoxic
conditions which, while inhibiting growth of spoilage organisms such as
aerobic bacteria and molds, can allow growth of obligate anaerobic pathogens
such as nonproteolytic C. botulinum even at refrigeration temperatures; tem-
perature abuse conditions that increase product respiration can also result in
anaerobiosis. Where high levels of C0 2 alone restrict growth of susceptible
microorganisms, selection of pathogens that can survive under these conditions
may also occur [47]. At 13 or 22°C, C0 2 was reported to not inhibit growth
of E. coli on shredded lettuce; in fact, atmospheres of 5% 2 and 30% C0 2
(balance N 2 ) actually enhanced growth over storage in air. Atmospheres
containing 40 to 50% C0 2 were only slightly inhibitory towards Yersinia
enter ocolitica at 4°C, although inhibition increased as storage temperature
decreased [9]. Bennik and others [44] observed extended X for this
psychrotrophic pathogen under conditions of 50% C0 2 and 21% 2 (balance
N 2 ), but no effect under decreasing C0 2 concentrations of 5 or 20%. Their
results suggest that typically employed MAP conditions of 1 to 5% 2 and 5
to 10% C0 2 at 8°C may not inhibit growth of the pathogens Aeromonas
hydrophila, L. monocytogenes, or cold-tolerant strains of Bacillus cereus.
The behavior of a particular pathogen in a MAP system is influenced by
the fruit or vegetable type as well as by the nature of the particular microbial
strain. Francis and O'Beirne [48] found that acid-adapted L. monocytogenes
grew on mung bean sprouts at 8°C and atmospheres of 2 to 5% 2 and to
15% C0 2 , while nonacid-adapted strains did not grow. In further work [49]
these authors assessed the effects of vegetable type and strain on survival
and growth of different pathogens under different modified atmospheres at 4
and 8°C. Different growth responses were observed between strains of E. coli
0157:H7 on different RTU vegetables (lettuce, swedes, dry coleslaw, soybean
sprouts), while no difference was observed on these same vegetables among
multiple strains of L. monocytogenes.
Different methods can be utilized to determine the safety of foods stored
under specific MAP systems. Challenge studies, where survival and growth of
inoculated pathogens are followed over time, can be performed in isolation
or in combination with natural produce microflora. Challenge studies using
452 Microbiology of Fruits and Vegetables
C. botulinum can be used to examine toxin production as well as occurrence of
spoilage. Predictive models can be generated to determine microbial growth
or toxin development in produce. To consider the interactions of fluctuating
populations and ratios of spoilage organisms and pathogens, a safety index
ratio may be used to indicate relative spoilage and pathogenicity. The ratio of
a specific pathogenic organism to a food spoilage organism over MAP storage
time can be used as a practical safety index, created for any pathogen of
concern where levels required to produce illness may significantly differ. Such
an index would not represent an absolute measurement of the safety of a food
product: it would quantitatively depict the relationship between spoilage and
pathogenicity [47].
19.5.3.2 Psychrotrophic Pathogens
A potential consumer safety risk may occur due to MAP inhibition of the
aerobic microorganisms that usually warn consumers of spoilage, resulting
in reduction in growth competition, and creation of an altered environment,
allowing enhanced or unrestricted growth of anaerobic or facultative anaero-
bic pathogens capable of growing under MAP conditions at refrigeration
temperatures. These include L. monocytogenes, C. botulinum, Yersinia
enter ocolitica, and Aeromonas hydrophila.
L. monocytogenes, ubiquitous in the environment, is naturally found on
many fruits and vegetables. A facultative anaerobe capable of growing under
temperatures as low as — 1.5°C and under C0 2 -enriched environments, this
pathogen can feasibly grow on MAP refrigerated produce. Beuchat [50]
reported little inhibitory effect of MAP at 4 to 15°C on growth of L. mono-
cytogenes on broccoli, cauliflower, and asparagus. Bennik and others [37]
found that the extent of growth of L. monocytogenes on chicory endive was not
influenced by MAP atmospheres; the initial inoculum level, cultivar of chicory
endive, and population of competitive spoilage microorganisms were primary
growth influences. L. monocytogenes grew better on chicory disinfected with
chlorine than on chicory left untreated prior to MAP storage, most likely due
to reduction of competitive indigenous microflora after treatment. Francis and
O'Beirne [33] found that survival and growth of L. innocua (as a model of L.
monocytogenes) was affected by the indigenous microflora; Enterobacter
cloacae and LAB reduced growth of L. innocua while pseudomonads had
little effect. Thus MAP treatments have the potential to change the dynamics
of microbial populations and alter product safety. Berrang and others [51]
increased shelf life and reduced spoilage microorganisms of cut asparagus,
broccoli, and cauliflower by MAP; however, in later studies they found that the
growth of L. monocytogenes and A. hydrophila was unaffected. Thus shelf life
extension feasibly allowed a longer time period for the pathogens to grow by
removing competitive microorganisms [52,53].
Aeromonas spp. generally grow at temperatures between 1 and 45°C, and
under low 2 atmospheres. Aeromonas spp. can grow at low temperatures
under vacuum but are inhibited by high concentrations of C0 2 . Researchers
Modified Atmosphere Packaging 453
have found A. hydrophila to be present on 100% of 12 different produce items
surveyed, recovering the pathogen from green salad, coleslaw, salad samples,
and mixed salad greens [9]. A. hydrophila was found to survive but not grow on
vegetable salads stored under MAP at 4°C, but rapidly grew at 15°C. Others
[54] have found that A. hydrophila would grow on cucumber slices but not on
mixed lettuce under MAP conditions at 2°C. Bennik and others [44] found that
growth of A. hydrophila was the same under MAP conditions of 1.5 or 21% 2 ;
Mmax decreased with increasing C0 2 concentrations; however, N max was not
affected until C0 2 levels were above 50%.
At 4°C, Y. enterocolitica has been found to grow in air, under vacuum, and
under MAP and atmospheres containing 40 to 50% C0 2 [9]. Yersinia can
grow at temperatures as low as 1°C with a 40-hour doubling time. Farber [1]
reported that 10% C0 2 stimulated growth of Y. enterocolitica, but 40% C0 2
increased X, and 100% C0 2 increased X and decreased |i ma x-
C. botulinum poses a significant food safety risk in MAP produce, as
previously discussed in this chapter. MAP conditions that extend product shelf
life may create an organoleptically acceptable consumer product, but may pose
a food safety hazard not immediately visible to the consumer. Macura and
others [55] found that anaerobic conditions developed under a range of MAP
atmospheres and temperatures employed for storage of ginseng roots; at 10°C,
C. botulinum toxin was detected in roots while overall product quality was still
acceptable. Nonproteolytic strains of C. botulinum have a growth potential
between 3.3 and 45°C and are minimally affected by C0 2 concentrations
<50% [15]. Toxigenesis has been detectable at 8 and 5°C [9] and at 2
concentrations up to 10%; however, it has been reported that toxin production
is dependent as well on the produce commodity [56]. Of all vegetables tested
(butternut, onion, mixed greens, lettuce, rutabaga), at 5°C nonproteolytic
strains could only produce toxin on butternut squash; proteolytic strains could
produce toxin on all vegetables tested at temperatures >15°C. While acidic
environments such as those produced by high C0 2 packaging atmospheres,
acid treatments, and/or low pH produce can inhibit growth of C. botulinum,
microbial diversity and dynamics may increase product pH. Growth of mold
on tomato may increase typical product pH from about 4 to 5-9 [57], creating
microenvironments suitable for growth of C. botulinum. Thus the effects of
atmospheres, temperatures employed, temperature abuse, influences of other
organisms, as well as commodity type should be assessed when designing MAP
systems to reduce risk of food poisoning due to C. botulinum.
19.5.3.3 Other Pathogens of Concern
Salmonella, E. coli, and shigella can survive but are unable to grow at tem-
peratures lower than 4°C; however, growth can resume if temperature abuse
occurs. At higher refrigeration temperatures, growth may be possible, and
atmospheres used in MAP will differently influence growth parameters of these
pathogens. Amanatidou and others [12] found that 8°C and 10 to 20% C0 2 in
N 2 reduced the growth rate of Salmonella enteritidis but had no effect on the
454 Microbiology of Fruits and Vegetables
growth rate of S. typhimurium or E. coli. Combined atmospheres of high 2
and C0 2 increased X and decreased |i max and N max for S. enter it idis and E. coli,
but had no effect on S. typhimurium. High 2 alone reduced u. max or ^V max for
S. typhimurium and S. enteritidis, but had little effect on E. coli. Another study
found that Shigella sonnei and S. flexneri growth was not affected by
atmospheres of 3% 2 and 5 to 10% C0 2 at 7 and 12°C. Growth and survival
was predominantly affected by the type of vegetable tested (grated carrot,
chopped bell pepper, mixed lettuce) [47] as well as by temperature. Francis and
O'Beirne [49] determined that survival and growth of E. coli 0157:H7 under
MAP was dependent upon type of vegetable (swedes, lettuce, soybean sprouts,
dry coleslaw mix), temperature, atmosphere, and strain; the pathogen grew
better under atmospheres of 30% C0 2 and 5% 2 compared to air, and 9 to
12% C0 2 and 2 to 4% 2 were not inhibitory at 8°C. Reduction of storage
temperatures from 8 to 4°C prevented growth and reduced survival of E. coli.
Others [58] have shown that E. coli 0157:H7 can survive on fresh-cut apples
under >15% C0 2 at abusive temperatures (15 and 20°C). These studies
emphasize the important effect of temperature in maintaining MAP produce
safety.
Campylobacter jejuni requires 5% 2 , 10% C0 2 , and 85% N 2 for optimal
growth, atmospheres that may commonly occur in MAP systems. Even under
refrigeration temperatures, MAP atmospheres may create conditions more
hospitable to the survival of this pathogen than under air; populations of C.
jejuni on cilantro, green pepper, and romaine lettuce packaged under MAP for
15 days at 4°C were reported to be reduced by 2 logio CFU/g by day 9 while a
much greater reduction of 3 to 4 log 10 CFU/g occurred under air and vacuum
storage [15]. Campylobacter spp. have a low infective dose (100 to 500 CFU);
while they do not typically grow below 30°C, they can survive. Thus, if the
pathogen was initially present on the commodity upon packaging, only short
intervals of temperature abuse in a typical MAP atmosphere might be needed
to allow enough growth of the pathogen to cause food poisoning. Phillips
[59] reported that 22.2% of mixed salad vegetable MAP products tested were
contaminated with between 80 and 170 CFU/g Campylobacter spp., levels that
could potentially produce illness, particularly if several grams of salad were
ingested.
19.5.4 Microbial Ecology of MAP Systems
Interactions between indigenous microflora and pathogens on produce gen-
erally have not been well studied. Indigenous LAB can be antagonistic due to
organic acid production, generation of H 2 2 , bacteriocin production, or
competition for nutrients. Naturally present in low numbers on vegetables,
they can reach high numbers in MAP where high levels of C0 2 are employed.
MAP gas combinations may be manipulated to encourage growth of these
antagonists, which may indirectly control growth of spoilage organisms or
pathogens. Research has shown that growth of some organisms can result in
enhanced growth of others. Salmonella spp. co-inoculated with a soft-rot
Modified Atmosphere Packaging 455
bacterium or Pseudomonas spp. on potato, carrot, and pepper grew
significantly better than when inoculated alone [57]. Pathogens may grow on
biofilms naturally formed on produce, where the environment may be altered
such that it is more favorable for microbial growth compared to the direct
produce surface. Work has shown that L. monocytogenes can grow on multi-
species biofilms on meat; comparable studies have not yet been performed
on produce. Biofilms have been found to constitute between 10 and 40% of
bacterial populations on endive and parsley, and more work is needed to
determine the extent of biofilm development and microbial interactions at the
biofilm surface on other whole produce [57].
19.6. MATHEMATICAL PREDICTIVE MODELING
The evaluation of a MAP system for any given commodity should be accom-
plished by a systematic and comprehensive approach by first establishing an
initial predictive theoretical model to represent and manipulate underlying
principles, followed by validation through empirical study. Empirical study
unsupported by theoretical models and performed through trial and error is a
lengthy and expensive process that does not take into consideration microbial
ecology, product safety, or interaction of underlying MAP system variables;
shelf life and associated product quality are the primary factors examined.
Mathematical models of interactions among variables that affect MAP
package atmospheres have been proposed and used to design MAP systems.
However, improvements are needed to create more comprehensive models,
and little work has been done to create models for different MP fruits and
vegetables.
Additionally, models are needed for more sophisticated MAP systems such
as multiple pack nested or multiple commodity packaging, multiple barrier
systems, or where the behavior of specific MAP system variables may be
expected to be different, as with perforated films [60], films incorporating gas
scavengers or generators, or systems utilizing superatmospheric 2 or novel gas
mixtures. Models should generally consider the packaging internal and external
environments, the product under storage, and the storage gases and packaging
materials employed. Temperature, product respiration rate (both consumption
of 2 and production of C0 2 ), product weight, package headspace, film per-
meability to gases and water vapor, film surface area and thickness, product
diffusion resistance, and product tolerance to low 2 and high C0 2 are all
important variables to consider. Typically, a film with specific gas and water
vapor characteristics is selected to achieve the particular target EMA for a
stored product having a specific respiration rate, at a specific temperature.
Predictive models should be validated by empirical studies incorporating the
particular produce commodity and testing one or more variables to optimize.
456 Microbiology of Fruits and Vegetables
19.7 FUTURE DIRECTIONS
The future direction of MAP system design must rely on significant advances in
understanding the controlling variables and underlying factors influencing
product safety and shelf life, particularly for MP fruits and vegetables. A
broader knowledge base is needed to more fully understand the microbial
ecology of MAP stored MP produce and the effects of processing and different
atmospheres employed. More information is needed about the microbiological
safety of MAP whole and MP produce, the effects of MAP on growth of
psychrotrophic pathogens, interactive effects of microorganisms in MAP
produce systems, effects of MAP system failures, and effects of varying storage
conditions and temperature abuse. The interactions between MAP and other
preservation methods should be defined to enable development of effective
multiple barrier preservation systems. As we come to a more comprehensive
understanding, more effective and applicable predictive models will be
constructed for designing MAP systems for high-quality, safe produce.
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20
Hot Water Treatments for
Control of Fungal Decay
on Fresh Produce
Elazar Fallik
CONTENTS
20.1 Introduction 461
20.2 Technologies 463
20.3 Heat Treatments 464
20.3.1 In Vitro Studies 464
20.3.2 In Vivo Studies 465
20.3.3 Heat Damage 469
20.4 Mode of Action 470
20.5 Conclusions 472
Acknowledgments 472
References 472
20.1 INTRODUCTION
Fresh fruits and vegetables have been a part of the human diet since the
dawn of history, while farmers and food sellers have been concerned about
losses since agriculture began. While fruits and vegetables have always
provided variety in the diet through differences in color, shape, taste, aroma,
and texture [1], their full nutritional importance has only been recognized in
recent times.
Contamination of fresh produce with pathogenic agents may occur at any
point during production, harvesting, packing, processing, distribution, or
marketing. Therefore, all fresh harvested commodities need to be free of
disease agents, insects, synthetic chemicals, and cleaned of dirt or dust before
being sent to the markets. The problem of how much food is lost after harvest
This chapter is dedicated to the late Mr. Erwin Fisher: an expert on scanning electron microscopy
analysis, who contributed significantly to the understanding of the mode of action of hot water
rinsing and brushing.
461
462 Microbiology of Fruits and Vegetables
by inefficient processing, spoilage, insects and rodents, or other factors takes
on greater importance as world food demand grows. Marketing of produce has
also benefited from an international trend towards fresh natural foods, which
are perceived to be superior to processed foods and to contain fewer chemical
additives.
Although fresh produce is generally not considered a common source
of foodborne illness, the incidence of this problem is increasing [2]. In recent
years the number of cases of illness linked with eating fruits and vegetables
has risen from 2% to about 8% of reported cases. The increased incidence
may be related to changing patterns of food consumption, recognition of new
means for transmission of disease organisms, emergence of pathogens that can
cause infections at very low doses, an expectation that most foods distributed
in any country are safe, and/or a perception that foodborne illness does
not occur at home.
For many years chemical treatments have been the basis for ensuring post-
harvest quality [3]. Although government authorities in each country regulate
fungicide use to ensure that chemicals are not toxic at the concentrations used
[4], there is still growing concern and apprehension by the public about the
use of synthetic pesticides. Pressure is building for the use of alternative
"nonchemical" means of disease control by the horticultural and agricultural
industries.
Several chemical-free technologies to extend the storage and shelf life
of fresh produce are being investigated. Among these technologies are modi-
fied atmosphere packaging [5], irradiation [6,7], use of materials that are
generally regarded as safe (GRAS), such as bicarbonate salts [8] or hydrogen
peroxide [9,10], hypobaric treatment [11], biological control [12], or prestorage
heat treatments [13]. Heat treatment appears to be one of the most promising
means for postharvest control of decay [13,14]. Prestorage heat treatments
to control decay development during storage and marketing period are often
applied for a relatively short time (minutes), because the targeted decay-
causing agents are found on the surface or in the first few cell layers under
the skin of the fruit or vegetable [14]. Heat treatments against pathogens
may be applied to the fresh harvested produce in several ways: by hot water
dips, by vapor heat, by hot dry air [13], and by a short hot water rinse and
brush [15,16]. Hot water is an effective heat transfer medium and, when
properly circulated through the load of fruit, establishes a uniform tempera-
ture profile more quickly than either vapor or dry heat [17]. Hot water
treatments were originally used to control fungal diseases such as brown
rot (Phytophthora spp.) on citrus fruits [18,19], but their use has been exten-
ded to achieve disinfestations from insects and alleviation of physiological
deterioration [13].
Several reviews have been published on the effect of both dry and wet
heat on maintenance of quality in fresh harvested crops [13,14,17,20,21]. This
chapter summarizes recent research on the technologies used in hot water
treatments and their effects on decay development in fruits, vegetables, and
minimally processed products.
Hot Water Treatments for Control of Fungal Decay on Fresh Produce 463
20.2 TECHNOLOGIES
There are three basic designs for administering hot water treatments: the batch
system, the continuous system (hot water treatment, HWT) [22], and the hot
water rinsing and brushing system (HWRB) [15].
Most of the hot water immersion treatment facilities that are in commercial
use currently are of the batch system. In this system, baskets of produce are
loaded onto a platform, which is then lowered into the hot water immersion
tank, where the fruits or vegetables remain at the prescribed temperature for
a certain time before being taken out, usually by means of an overhead hoist.
In the continuous system the produce is submerged (either loosely or in a wire
or plastic mesh basket) on a conveyor belt, which moves slowly from one end
of the hot water tank to the other. The belt speed is set to ensure that the
produce is submerged for the required length of time. This system requires
an instrument to monitor the speed of the conveyor belt.
The main components in these two systems are an insulated treatment
tank of several hundred liters, a heat exchange unit operated by gas, diesel, or
electricity, a pump and water circulation system to provide uniform water
temperatures throughout the treatment process and to avoid the formation
of cool pockets during treatment, and temperature sensors to control and
monitor water temperature during treatment [22,23]. An inexpensive hot water
immersion system can be assembled easily; the machinery can even be made
mobile with little difficulty [23,24].
In contrast to hot water immersion, a new technology based on a brief
HWRB for simultaneous cleaning and disinfestation of fresh produce was first
introduced commercially in 1996 [20]. The fourth generation of the HWRB
machine (Figure 20.1) contains 18 to 22 parallel brushes, all of which are
controlled by a single motor. All components in the machine, including the hot
water tank (300 to 500 1), are made from stainless steel materials. The produce
is prewashed by nonrecycled tap water (ambient temperature) for about 5 to
10 seconds while revolving on cylindrical brushes. A speed-adjustable con-
veyor belt is connected to the simultaneous cleaning and disinfecting stage,
and controls the duration of exposure to hot water, which is heated with a
thermostatically controlled gas or electric heating element. Fruit or vegetables
are rinsed with the pressurized hot water, from nozzles that point down either
vertically or at predetermined angles onto the produce, which rolls on brushes
made from medium-soft synthetic bristles. The produce is exposed to water
at temperatures between 48 and 63°C for 10 to 25 seconds, depending upon
produce type and cultivar [25-34]. The water is filtered and then recycled, while
being supplemented from time to time with new water to compensate for loss
due to evaporation, adsorption, spilling, etc. At the end of the hot washing
treatment, forced-air fans, or hot forced air is used to dry the produce inside a
4 to 6 m long tunnel for 1 to 2 minutes. The estimated cost of the HWRB
system, including the drying tunnel, is $15,000 to $30,000 for units with
washing capacities of 1 to 25 tons/h (further information can be obtained from
the author).
464
Microbiology of Fruits and Vegetables
JO
FIGURE 20.1 Hot water rinsing and brushing machine, fourth generation, made of
stainless steel: (1) conveyor; (2) tap water rinsing and brushing unit; (3) hot water
rinsing and brushing unit with adjustable nozzles, with and without fruits. Water is
recycled. (4) Drying tunnel equipped with forced air fans; (5) heat unit (120,000 kcal);
(6) hot water container equipped with water pump (P) to pressurize and recycle the hot
water and filter (F).
20.3 HEAT TREATMENTS
20.3.1 In Vitro Studies
There is considerable variation in sensitivity to high temperature among
various fungi [14]. Vegetative cells and conidia of most fungi are inactivated
when exposed to a temperature of 60° C for 5 to 10 minutes in vitro [35].
Spore germination and germ tube elongation in vitro was inversely related to
the duration of exposure or the range of temperature used [36]. Hot water
treatments were found to be ineffective in killing dormant spores [37,38].
Ranganna et al. [39] reported that bacterial infection (Erwinia caratovora)
was more sensitive to hot water treatments than was the fungal pathogen
Fusarium solani. Colletotrichum gloeosporiodes was more heat sensitive than
Dothiorella dominicana in mango fruits [40]. Botrytis cinerea was found to be
Hot Water Treatments for Control of Fungal Decay on Fresh Produce 465
more susceptible to hot water treatment than Alternaria alternata in sweet
bell pepper [36]. The effective time to kill 50% of the spores (ET 50 germination)
for B. cinerea was 3.2, 1.5, and 0.8 minutes at 45, 50, and 55°C, and for
A. alternata was 8.8, 4.2, and 1.4 minutes, respectively, at those temperatures.
The ET 50 for germ tube elongation for B. cinerea was 2.6, 0.9, and 0.5 minutes
at 45, 50, and 55°C, and for A. alternata was 7.2, 2.5, and 1.6 minutes
at the same temperatures [36]. Percentage spore germination of A. alternata
and F. solani was inversely proportional to the length of exposure to
55 and 60°C. Exposing fungal spores of A. alternata and F. solani to 60°C
for about 15 seconds, in vitro, resulted in 48 and 42% reduction in spore
germination, respectively [27]. The ET 50 for A. alternata was 25 and 16 seconds
at 55 and 65°C, respectively, whereas for F. solani the ET 50 was 18 seconds
at 60°C. None of the temperature/time regimes tested completely inhibited
spore germination, although A. alternata was slightly more susceptible to
heat treatment than F. solani [27]. A minimum exposure period of 20 seconds
at 56° C was required to inhibit Fenicillium digitatum spore germination
in vitro [30]. No surviving spores of B. cinerea were observed after 15 minutes
at 45°C [7]. Monilinia fructigena was more sensitive and a thermal treatment
of 3 minutes at 45° C resulted in complete spore inactivation [7]. In vitro studies
showed Monilinia fructicola to be more sensitive than Fenicillium expansum
to high temperature (60° C for 20 seconds) [41].
The viability of five pathogens was decreased by treatment with hot water
when tested in vitro. Polyscytalum pustulans was most sensitive and Rhizoctonia
solani least sensitive. The temperatures that killed 50% (LT50) and 95% (LT95)
of spores of Fenicillium digitatum in 15 seconds were about 5.2°C higher
than those for arthrospores of Geotrichum citri-aurantii, and those for spores
of P. digitatum in 30 seconds were about 3.4°C higher [34].
20.3.2 In Vivo Studies
Fruit responses to heat treatments depend on the condition of the fruit prior
to treatment, the commodity concerned, the temperature and duration of
treatment, as well as the mode of heat application. The physiological respon-
ses of different fruits, vegetables, or flower species to hot water treatments
can vary by season and growing location, and can be due to differences
in climate, soil type, season, production practices, fruit maturity at harvest,
and fruit size [24,42-45]. Hot water treatments prevent rot development
in numerous temperate, subtropical, and tropical fruits and vegetables
[13,14,16,17,46,47].
A dip time of 1 to 2 minutes in 55°C water for Galia melons (Cucumis
melo L.) was an optimal antifungal treatment, while a higher temperature
or longer exposure time resulted in heat injury to the fruits [48]. However,
the optimal HWRB treatment to reduce decay while maintaining fruit quality
after prolonged storage and marketing was 59 ± 1°C for 15 seconds [15].
Hot water treatment at 44 or 46°C for 15 minutes delayed Botrytis cinerea
proliferation on artificially inoculated or naturally infected strawberries
466 Microbiology of Fruits and Vegetables
(Fragaria x ananassa Duch. Tudla) [49,50]. Dipping strawberries inoculated
with botrytis at 63°C for 12 seconds, followed by controlled atmosphere (CA)
storage (15kPa CO2) was found to reduce rot development during a short or
long storage regime [51]. Peaches and nectarine infected with Monilinia
fructicola were immersed in hot water at 46 or 50° C for 2.5 minutes to control
decay. These treatments reduced the incidence of decayed fruit from 82.8 to
59.3 and 38.8%, respectively [52]. Mature green plums (Prunus salicina Lindl.
cv. Friar) were treated in water at 40, 45, 50, and 55°C for 40, 35, 30, and 25
minutes, respectively, and stored at 0°C for 35 days plus 9 days of ripening at
20 to 25°C. Decay symptoms were retarded in fruits treated at 45 and 50°C,
while decay symptoms were severe in fruits of the control and those treated
at 55 and 40°C [53].
HWRB at 55°C for 15 seconds significantly reduced decay development
in Penicillium expansum-'moculated apple fruit after 4 weeks at 20° C, or
in naturally infected (P. expansum) apple fruit after prolonged storage
of 4 months at 1°C plus 10 days at 20° C [27]. Recently, Lunardi et al. [54]
reported that prestorage hot water immersion at 47°C for 3 minutes signi-
ficantly reduced rot development caused by white rot (Botryosphaeria dot hided)
on Fuji apples after prolonged CA storage. In vivo studies of inoculation
of peach and nectarine fruit with Monilinia fructicola followed by HWRB at
55 or 60° C for 20 seconds gave 70 and 80% decay inhibition, respectively,
compared with the control [41]. The inhibition percentages of M. fructicola
with HWRB were similar, if HWRB was applied shortly after inoculation or
24 hours later. In contrast, the sensitivity of P. expansum spores inoculated
into wounds increased when the fruit were treated with HWRB 24 hours
after the inoculation, compared with treatment just after inoculation [41].
Treating fruit with HWRB at 60° C for 20 seconds and then dipping them into
a cell suspension (10 cells/ ml) of Candida spp. 24 hours after inoculation
with P. expansum reduced decay development by 60% compared with the
controls, but did not reduce rot development caused by M. fructicola [41].
Many types of fresh produce from the Solanaceae benefit from hot water
treatments. Potato tubers were dipped at 55°C for 5 minutes in a commercial
continuous hot water treatment plant [55]. The frequency of eyes colonized by
Pseudomonas pustulans, Helminthosporium solani, and Rhizoctonia solani was
reduced to virtually zero and the effect persisted on tubers subsequently stored
at 4 and at 15°C for up to 16 weeks. Results with Colletotrichum coccodes were
inconclusive. Treatment suppressed Penicillium spp. which, however, rapidly
colonized the eyes during storage, leading to higher contamination levels in
the treated than in the untreated tubers. With tubers inoculated with Phoma
foveata, good control was achieved when the incubation period before treat-
ment was 10 days but not when the fungus was more established 42 days after
inoculation [55]. Potato tubers inoculated with Erwinia carotovora and
Fusarium solani were safely stored for 12 weeks at either 8 or 18°C without
spoilage if dipped in a 57.5°C hot water bath for 20 to 30 minutes [39].
The effectiveness of hot water dipping on the control of grey mould, caused
by Botrytis cinerea, and black mould, caused by Alternaria alternata, and on
Hot Water Treatments for Control of Fungal Decay on Fresh Produce 467
sweet red pepper quality was investigated. Dipping naturally infected
or artificially inoculated fruit at 50°C for 3 minutes completely inhibited or
significantly reduced decay development caused by B. cinerea and A. alternately
respectively [36]. Heat damage was observed on fruit dipped for 5 minutes
at 50°C, or at 55°C for 1 minute or longer [36]. Significant differences in
the incidence of decay were found between temperatures and in the time-
temperature interaction, but not between times of dipping [36]. Treatment of
peppers with hot water at 53°C for 4 minutes was found to be effective in
reducing decay after 14 and 28 days of storage at 8°C. Treatment at 45°C for
15 minutes was less effective in maintaining pepper quality during storage
[56,57]. HWRB of red and yellow sweet bell pepper cultivars at 55°C for
about 12 seconds significantly reduced decay incidence while maintaining
quality compared both to untreated control and to most other commercial
treatments [25].
A hot water dip at 50° C for 2 minutes was more effective than 1 kGy
of gamma radiation in reducing Botrytis cinerea and Rhizopus stolonifer
decay in inoculated light-red tomatoes (Lycopersicon esculentum Mill, cv
F-121) [58]. Mature green tomatoes (L. esculentum Mill, cv Sunbeam) were
treated in water for 1 hour at 27, 39, 42, 45, or 48°C, and then ripened for
14 days at 20 or 2°C. Treatment at 42°C reduced decay by 60%, whereas
other water temperatures were less effective [59]. HWRB of freshly harvested
tomatoes at 52°C for 15 seconds, or dipping the fruit at 52°C for 1 minute,
significantly reduced decay development and chilling injury after 3 weeks'
storage at 2 or 12°C and additional 5 days at 20°C [33]. These two prestorage
heat treatments did not affect other quality parameters such as fruit firmness,
total soluble solids, or acidity [33].
Fresh broccoli floret (Brassica oleracea L. Italica group) is a highly
perishable fresh vegetable when held at ambient temperatures; it becomes
unmarketable within 1 to 3 days. Immersion in hot water at 50 or 52°C for
2 minutes was most effective in controlling decay development and reducing
yellowing [75].
Several recent studies have shown that hot water immersion for 2 to
3 minutes at 53° C significantly reduced decay development in a wide variety
of citrus cultivars [43,61-64]. Hot water dipping at 53°C for 3 minutes
gave beneficial effects on decay control of Tarocco blood oranges (Citrus
sinensis L. Obsek) fruit harvested in February and March but was detrimental
for fruit harvested in April [43]. No decay symptoms were detected after
90 days' storage at 4°C in March grapefruit treated with 45° C water for 3 hours
[65]. A hot water dip for 2 minutes at 52 to 53°C inhibited the development
of decay in lemons inoculated with P. digitatum [63]. When the fruit were
heat treated 1 day after inoculation, no decay occurred within 6 days after
the treatment, whereas fruit dipped in water at 25° C reached 100% decay
after 4 days. When the hot water dip was applied 2 days after inoculation, 90%
of the treated fruit remained healthy [63]. The optimal HWRB treatment to
reduce decay development while maintaining fruit quality of kumquat was
55°C for 20 seconds [28]. When organic citrus fruits were treated with HWRB
468 Microbiology of Fruits and Vegetables
at 56, 59, and 62°C for 20 seocnds after artificial inoculation with P. digitatum,
decay development in infected wounds was reduced to 20, 5, and less than
1%, respectively, of that in untreated control fruits or fruits treated with tap
water [30]. A 20-second HWRB treatment at 59 or 62°C reduced decay in Star
Ruby grapefruit that was artificially inoculated with P. digitatum, by 52 and
70%, respectively, compared with control unwashed fruit. Tap water wash
(~20°C) or HWRB at 53 or 56°C were ineffective [31]. Green mold incidence
caused by P. digitatum was reduced from 97.9 and 98% on untreated lemons
and oranges, respectively, to 14.5 and 9.4% by a brief 30-second HWRB
treatment at 62.8°C [34].
Hot water immersion also inhibits decay development on tropical
and subtropical fruits. Dipping Kensington Pride mango fruits in hot water
at 52°C for 5 minutes together with the fungicide benomy gave good control
of stem end rot caused by Dothiorella dominicana and Lasiodiplodia
theobromae [40]. Treatments consisting of hot water only or hot water fol-
lowed by the fungicide prochloraz gave only partial control of stem-end rot.
All treatments gave good control of anthracnose caused by Colletotrichum
gloeosporioides [40]. The effectiveness of different postharvest treatments to
control different levels of quiescent infections of Alternaria alternata caus-
ing alternaria rot in mango fruits during storage was compared. A combined
HWRB treatment at 48 to 62°C (depending on the cultivar) for 15 to 20
seconds with 225mg/ml prochloraz was the most effective treatment for
control of alternaria rot in fruit with a high relative quiescent infected
surface [66]. However, the effectiveness of postharvest HWRB and prochloraz
applications are dependent on the quiescent infected area of the fruit by
A. alternata at harvest [66]. Hot water treatments reduced body rot caused
by Colletotrichum spp. in ripe avocado fruit with 40 and 41°C for 30 minutes
[67]. However, treatment at 42°C for 30 minutes increased body rot com-
pared to the other HWTs in one season, but there was no benefit of HWT
times longer than 30 minutes [67]. Stem rot caused mainly by Dothiorella
spp. was also reduced by HWT at 40 and 4PC [67]. HWRB at 55°C for
20 seconds significantly reduced chemical use (prochloraz) to control decay
development caused mainly by Penicillium spp. in litchi fruit [29].
Food safety has become a very important issue for fresh and minimally
processed products [68]. Minimal processing of vegetables provides conve-
nience to the food industry and retail consumers, but may result in limited
shelf life and marketing because of undesirable physiological and patho-
logical changes [69,70]. Very little research has been done to evaluate the
efficacy of hot water treatment on minimally processed products. A 4-minute
water wash of green onion at 52.5°C reduced the aerobic plate count by
1 to 2 logs compared with water wash at 20° C [70]. A similar reduction in
microbial population of soybean sprouts and watercress after a 30-second
water dip at 60°C was reported by Park et al. [71].
Immersing spot-inoculated apple fruits at 80 and 95°C for 15 seconds
produced a reduction of more than 5 log in Escherichia coli 0157:H7 [72].
Several pasteurization procedures for alfalfa (Medicago sativa) seeds were
Hot Water Treatments for Control of Fungal Decay on Fresh Produce 469
investigated to disinfect completely inoculated Escherichia coli (Migula)
Castellani and Chalmers ATCC 25922 [73]. Hot water treatments (85°C
for 9 seconds) were equally or more effective than 20,000 ppm calcium hypo-
chlorite treatments, yielding a reduction of 2 log CFU/g [73]. Li et al. [74]
reported that the population of Listeria monocytogenes on cut iceberg lettuce
treated at 50°C for 90 seconds steadily increased throughout storage at 5°C
for up to 18 days.
20.3.3 Heat Damage
Hot water immersion or rinsing while brushing may result in commodity
damage, which typically is manifested as browning on fruit surfaces, uneven
ripening, breakdown of the fruit flesh, and even enhanced rot development
if the technique is not properly applied [13]. Incidence of HWT-associated
damage varied between regions, harvest dates, and orchards [76]. Immersion
of guavas (Psidium guajava L.) for 35 minutes in water at 46.1 ± 0.2°C delayed
ripening by 2 days, but increased susceptibility to decay [77]. Immersion
of Marsh grapefruit at 48° C for 2 or 3 hours significantly increased decay
(>30%). However, if fruit were treated at 45°C for 3 hours, no decay
symptoms were detected after 90 days' storage at 4°C [56].
Heat treatment has been associated with increased susceptibility to decay
in a number of crops, such as nectarine [78] and papaya [79]. The increase
in the incidence of rot is likely to be due to pathogens invading areas on
the fruit injured by the heat treatment. Hot water dips at 45°C for 2.5 minutes
did not control mold development caused by P. digitatum in Clementines [78].
The significant water loss and softness of fruit dipped at 55°C for 5 minutes
was due to heat damage causing cracks and pitting on the surface of the
treated fruit and the expansion and collapse of the hypoderm cells [36]. HWRB
at 60°C for 25 seconds caused heat damage as irregular reddish pits of 0.5
to 1.5 mm in diameter [16]. Heat damage on HWRB-treated apples (60° C
for 15 seconds) appeared as round brown sunken pits [32].
Hot water dips can change some biochemical properties in minimally
processed (peeled and trimmed) onions. Dipping prepeeled onions in 80° C
for 1 minute resulted in irreversible membrane damage [80]. Dipping heads
of fresh broccoli (Brassica oleracea L. Italica group cv. Paragon) at 52° C for
3 minutes enhanced off-odor development and caused visual damage to newer
buds [81].
Hot water treatment of mandarins at 56 and 58° C for 3 minutes induced
heat damage in the form of rind browning [62]. Hot water treatments for
10 minutes at 48°C and below were noninjurious to both yellow and green
lemon fruit, with injury in the form of lesions on the rind beginning to occur
at 50°C (yellow) and 52°C (green), and increasing in severity up to 58°C, the
highest temperature tested. Emanation of d-limonene increased correspond-
ingly with increasing injury. Green lemons were injured more severely than
yellow and tended to release more d-limonene, especially at higher tem-
peratures [82]. Heat damage was evident in avocado fruit as hardening of the
470 Microbiology of Fruits and Vegetables
skin when fruit ripened [83]. However, Obenland and Aung [84] reported that
sodium chloride at a concentration of 200 mM reduced hot water damage in
nectarine cultivars by effectively reducing the amount of water entering the
fruit during hot water treatment.
20.4 MODE OF ACTION
Heat treatments can interact directly or indirectly with pathogens and/or fresh
harvested produce via several responses. The efficacy of heat on pathogens
is usually measured by reduced viability of the heated propagules [14,40].
Heat effects may be lethal or sublethal, and pathogen kill is not always
proportional to the temperature-time product of the treatment [85,86].
Heat treatments may cause changes in nuclei and cell walls, denature proteins,
destroy mitochondria and outer membranes, disrupt vacuolar membranes,
and form gaps in the spore cytoplasm, which lead to reducing inoculum
level [14].
Applying HWRB to melon and citrus fruits resulted in a 3 to 4 log
reduction of the total microbial colony forming units (CFU) of the epiphytic
microorganism population, compared to untreated control fruit [27,30,34].
Scanning electron microscopy (SEM) showed that HWRB removed fungal
spores from the fruit surface, and partially or entirely sealed natural openings
in the epidermis (Figure 20.2) [25,27,30,33]. As a result of heat treatments that
reduce fungal viability, the effective inoculum concentration that causes decay
development is reduced, thus reducing rot development [86]. In addition,
sealing epidermal cracks with heat treatment could reduce sites of fungal
penetration into the fruit, thus reducing decay incidence [13]. Schirra and
D'Hallewin [62] and Ben-Yehoshua [64] reported that hot water dips of
grapefruit and mandarins redistributed the epicuticular wax layers, which
sealed or partially sealed cracks, thus improving physical barriers to pathogen
invasion.
Hot water immersion or rinse was found to inhibit ripening processes as
measured by relatively low respiration rate and ethylene evolution, and slow
color development, compared with nonheated control fruit. In addition, heat
treatment prevented postharvest geotropic curvature of vegetables [13,87,88].
Fruits that ripen slowly have less susceptibility to fungal attack during
storage [47]. Ben-Yehoshua [64] reported that heat treatment induced
resistance of grapefruits to decay caused by P. digitatum by delaying the
breakdown and disappearance of preformed antifungal compounds. The
heated citrus fruit had higher concentrations of the phytoalexin scoparone,
which in turn was correlated with antifungal activity in the fruit extract [64].
However, a 10-minute hot water dip caused a faster decrease of antifungal
compounds and the earlier appearance of rot symptoms in treated avocado,
compared to nonheated fruit [89].
Hot water treatments caused a delay in spore germination and fungal
growth in citrus fruit [37]. This was explained by a building up or improvement
Hot Water Treatments for Control of Fungal Decay on Fresh Produce
471
•
rM r\r\
CI dp
•
...
ri *
1
■ j
10KV x200 100|_im 000006 |
FIGURE 20.2 Scanning electron micrographs of untreated tomato (Al), pepper calyx
(Bl), and melon (CI) compared to HWRB-treated tomato (A2), pepper calyx (B2), and
melon (C2) (s, fungal spores; h, hyphae; dp, dirt particles).
in the defense systems against pathogens that slowed fungal development as a
result of heat-induced changes in the fruit tissue. Heat treatments induced the
biosynthesis of lignin-like polymers that were bound to walls of cells adjacent
to wound sites in citrus peel [64]. HWRB treatment of 52°C for 15 seconds
induced tomato resistance when fruit was artificially inoculated with B. cinerea
24 hours after treatment [90]. HWRB treatment at 62°C for 20 seconds was
most effective in inducing disease resistance against green mold (P. digitatum)
when grapefruit was inoculated after 1 and 3 days after treatment [91]. The
HWRB treatment induced the accumulation of proteins that cross-reacted
with heat shock proteins and of proteins that cross-reacted with chitinase
and 1,3-glucanase antibodies [61]. The increases in the accumulation of
chitinase and glucanase proteins, which were detected 1 and 3 days after
472 Microbiology of Fruits and Vegetables
HWRB treatment, may be part of the complex fruit resistance mechanisms
induced by this technology [31,91]. The mode of action of the hot water dip in
reducing the decay of lemon fruit is partly related to the temporary thermal
inhibition of pathogen growth that allowed the infected fruit to build up
production of lignin-like material at the inoculation site, followed later by
accumulation of the phytoalexins scoparone and scopolitin [63].
20.5 CONCLUSIONS
Interest in alternative methods for postharvest decay control of horticultural
crops in order to minimize pre- or postharvest treatments with agrochemicals
has been growing continuously. Prestorage heat treatment is one of the most
promising and simple technologies to reduce rot development on fresh har-
vested fruits, vegetables, and minimally processed products. However, further
research is needed to determine the most heat-sensitive life-stage of disease-
causing agents of economic importance and to obtain the intrinsic kinetics
information of this life stage for developing hot water treatment protocols.
There is also a special need to obtain information regarding time-temperature
effects on the quality of fresh harvest produce. A better understanding of both
direct and indirect modes of action of heat treatments on pathogens and
on fresh produce tissue will enable development of optimal, successful, and
relatively cheap hot water dip or rinsing treatments and equipment that will
control decay-causing agents without affecting the overall quality of the fruit
or vegetable.
ACKNOWLEDGMENTS
This paper was prepared under grant no. 406-0813-03 from the Chief Scientist
of the Ministry of Agriculture, Israel, with a contribution from the Agricultural
Research Organization, The Volcani Center, Bet Dagan, Israel, no. 402/04.
The author thanks Dr. Joshua D. Klein for his critical review and Michael
Fallik for the graphic work.
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476 Microbiology of Fruits and Vegetables
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478 Microbiology of Fruits and Vegetables
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21
Surface Pasteurization
with Hot Water and Steam
Bassam A. Annous and Michael F. Kozempel
CONTENTS
21.1 Introduction 479
21 .2 Surface Pasteurization with Hot Water 480
21.3 Surface Pasteurization with Steam 483
21.3.1 Thermosafe Process 485
21.3.1.1 Process Operation 485
21.3.1.2 Process Effectiveness 485
21.3.1.3 Product Quality 486
21 .3.2 University of Bristol Process 486
21.3.2.1 Process Operation 486
21.3.2.2 Process Effectiveness 486
21.3.2.3 Product Quality 487
21.3.3 Ventilex Continuous Steam Sterilizing System 487
21.3.3.1 Process Operation 487
21.3.3.2 Process Effectiveness 487
21.3.3.3 Product Quality 488
21.3.4 Vacuum-Steam-Vacuum (VSV) Process 488
21.3.4.1 Process Operation 491
21.3.4.2 Process Effectiveness 491
21.3.4.3 Product Quality 493
21.4 Conclusions 493
References 494
21.1 INTRODUCTION
The demand by consumers for fresh and fresh-cut fruits and vegetables has
steadily increased due to nutritious qualities associated with fresh produce
and the convenience of ready-to-eat fresh foods. This increased demand has
resulted in increased per capita consumption of fresh produce. Although
Mention of trade names or commercial products in this chapter is solely for the purpose of
providing specific information and does not imply recommendation or endorsement by the U.S.
Department of Agriculture.
479
480 Microbiology of Fruits and Vegetables
fresh produce is generally considered safe, it has been implicated in numerous
foodborne outbreaks in recent years. The Centers for Disease Control and
Prevention reported that foodborne outbreaks associated with fresh produce
doubled between the period 1973 to 1987 and 1988 to 1992 [1]. Contami-
nation of fresh produce, often grown on the ground and/or in areas adjacent
to animal production, with human pathogens may occur during growth,
harvesting, handling, and processing. Conventional washing and sanitizing
treatments have limited efficacy in inactivating and/or removing pathogens
on the surface of produce. Survival of human pathogens and other bacteria
during washing and sanitizing treatments is attributed to their attachment to
inaccessible sites on produce surfaces such as within the netting of a cantaloupe
[2], infiltration within the stem scar of tomatoes and the calyx region of apples
[3,4], and incorporation into biofilms, as seen with apples [3], cantaloupes [2],
and leaf surfaces [5,6]. Inadequate decontamination of fresh produce can
result in the survival of human pathogens on the surface with the possibility
of subsequent transfer of the pathogen from the surface, such as the rind of
a cantaloupe or the peel of an orange, to the flesh during fresh-cut processing
or juice extraction, respectively. Thus, the safety of fresh and fresh-cut produce
in supermarkets and salad bars, as well as the safety of freshly squeezed
unpasteurized juices, especially those served in fresh juice bars, is of concern.
Although experimental approaches to washing produce, such as vacuum
infiltration of sanitizers and application of abrasives during washing, have
resulted in greater microbial reductions compared to conventional treatments
[7], these new treatments are not capable of adequately inactivating the
pathogenic bacteria in their protective attachment states on produce surfaces.
Furthermore, inactivation of sanitizing agents by organic material such as soil
and debris in the washing solution, prior to contact with microorganisms, may
limit their sanitizing effectiveness [8].
An alternative approach to chemical sanitizers is surface pasteurization
with steam or hot water. Of all the agents used to sanitize the surface of foods,
water is probably the most readily acceptable to the public.
21.2 SURFACE PASTEURIZATION WITH HOT WATER
Unlike chemical sanitizers that only affect the surface of produce, hot water
(heated potable city water) washing can inactivate bacteria below the produce
surface [8], and thus is potentially more effective than chemical washes [2,8,9].
Hot water immersion provides excellent heat transfer between the produce
and the heating medium [10] and can quickly establish a uniform temperature
profile on the surface of produce [2,10]. Hot water surface pasteurization
has been used to control insects and is the most effective method for destroying
microorganisms, including postharvest plant pathogens that cause spoilage
(Chapter 20). While surface pasteurization, using hot water or steam, has been
shown to be effective in reducing levels of human pathogens on the surface
of meat and poultry [11,12] and intact eggs [13], it has only limited use in the
fresh and fresh-cut produce industries. Fresh fruits and vegetables investigated
Surface Pasteurization with Hot Water and Steam
481
TABLE 21.1
Effect of Washing Treatment (2 minutes) on Log Reduction 3 in Escherichia coli
0157:H7 Cell Concentration Applied to the Skin Region of Apples
Washing temperature
Washing solution
(logio
CFU/g)
25C (log 10 CFU/g)
60°C (log 10 CFU/g)
Tap water
6.37
3.71 ±0.25 AB
4.23±1.24AB
5% hydrogen peroxide
5.24
3.97 ± 1.20 AB
3.74±0.68AB
1200ppm Sanova c
5.49
4.38 ± 0.45 AB
4.83 ±0.75 A
400 ppm chlorine (pH d =
= 6.5)
5.39
3.00 ±1.23 ABC
4.84±0.15A
Acidified electrolyzed water
4.65
1.64±0.19C
4.07 ± 0.37 AB
a
Log reduction = mean cell population of untreated inoculated control (duplicate samples) minus
mean cell population following washing treatment (duplicate samples). Means with no letter in
common are significantly different at p < 0.05.
b Mean populations of untreated inoculated control samples.
c Sanova (acidified sodium chlorite) solution was prepared according to the manufacturer's
specifications.
d The pH of the chlorine solution was adjusted to 6.5 using concentrated hydrochloric acid.
for surface pasteurization include apples, melons, mangoes, lemons, oranges,
cucumbers, pears, tomatoes, and alfalfa seeds.
Immersion of apples in hot water or sanitizing solutions (60°C for
2 minutes) resulted in >4 log CFU/g reductions in Escherichia coli 0157:H7
populations inoculated on the skin surface (Table 21.1). However, these
treatments were not effective in inactivating cells inoculated in inaccessible
sites (stem and calyx) of apples (Table 21.2 and Table 21.3) [3,4]. Fleischman
et al. [14] reported similar results for surface pasteurization of apples using
water at 95°C for up to 60 seconds. Hot water immersion of apples can result
in heat damage resulting in browning of the skin at temperatures above 60°C
and softening of the subsurface flesh above 70 to 80°C [7,15].
Reductions in Salmonella Poona populations on cantaloupe surfaces were
>51ogCFU/cm following commercial-scale hot water immersion at 76°C for
3 minutes (Table 21.4) [2]. Also, this hot water commercial-scale treatment
maintained the fresh quality and increased the shelf life of this commodity.
The use of laboratory-scale hot water or heated hydrogen peroxide treatments
(70 or 97° C for 1 minute) to inactivate salmonella cells on cantaloupe rind
surface resulted in fresh-cut product with enhanced microbiological qualities
[16] and extended shelf life [17]. Hot water immersion (70°C for 2 minutes or
80°C for 1 minute) was shown to be effective in reducing populations of E. coli
0157:H7 on orange surfaces [18]. Also, hot water (>57°C for 5 minutes) immer-
sion was effective in reducing populations of S. Stanley on alfalfa seeds [19].
Hot water treatment of a variety of fruits and vegetables greatly improves
their microbiological quality and shelf life, while maintaining their sensory
qualities. Over-processing of produce, however, can significantly reduce seed
germination and cause thermal injury to apples and to juice extracted from
482
Microbiology of Fruits and Vegetables
TABLE 21.2
Effect of Washing Treatments (2 minutes) on Log Reduction 3 in Escherichia
coli 0157:H7 Cell Concentration Applied to the Calyx Region of Apples
Washing temperature
Inoculated control 6
(log 10 CFU/g) 25°C (log 10 CFU/g) 60C (log 10 CFU/g)
Washing solution
Tap water
5% hydrogen peroxide
1200ppm Sanova c
400 ppm chlorine (pH d = 6.5)
Acidic electrolyzed water
6.71
5.64
5.80
6.11
5.18
0.19±0.18AB
0.39 ± 0.08 AB
0.48 ± 0.09 AB
0.66 ± 0.37 AB
-0.04 e ±0.20B
0.43 ±0.1 5 AB
0.80 ± 0.44 AB
1.06±0.14A
0.95 ±0.28 A
-0.09 e ±0.28B
a Log reduction = mean cell population of untreated inoculated control (duplicate samples) minus
mean cell population following washing treatment (duplicate samples). Means with no letter in
common are significantly different at;? < 0.05.
b Mean populations of untreated inoculated control samples.
c Sanova (acidified sodium chlorite) solution was prepared according to the manufacturer's
specifications.
d The pH of the chlorine solution was adjusted to 6.5 using concentrated hydrochloric acid.
e Negative numbers indicate no reduction in cell populations was detected following washing
treatment.
TABLE 21.3
Effect of Washing Treatment (2 minutes) on Log Reduction 3 in Escherichia coli
0157:H7 Cell Concentration Applied to the Stem Region of Apples
Washing temperature
Washing solution
(log™
CFU/g)
25C (Iog 10 CFU/g)
60C (log 10 CFU/g)
Tap water
6.37
-0.10 C ±0.12D
0.11±0.0.12D
5% hydrogen peroxide
5.50
1.83 ± 0.17 AB
0.96 ± 0.72 BC
1200 ppm Sanova d
5.66
2.24 ±0.68 A
2.04 ± 0.62 AB
400 ppm chlorine (pH e = 6
■5)
6.53
0.49 ±0.51 CD
1.56 ±0.26 ABC
Acidic electrolyzed water
5.19
-0.20 C ±0.27D
-0.30 C ±0.30D
a Log reduction = mean cell population of untreated inoculated control (duplicate samples) minus
mean cell population following washing treatment (duplicate samples). Means with no letter in
common are significantly different at/7 < 0.05.
b Mean populations of untreated inoculated control samples.
c Negative numbers indicate no reduction in cell populations was detected following washing
treatment.
d Sanova (acidified sodium chlorite) solution was prepared according to the manufacturer's
specifications.
e The pH of the chlorine solution was adjusted to 6.5 using concentrated hydrochloric acid.
Surface Pasteurization with Hot Water and Steam 483
TABLE 21.4
Efficacy of Surface Pasteurization Process Using Hot Water Immersion on
Salmonella Poona Populations 3 on Inoculated Cantaloupes b
Storage temperature
Treatment 4°C 20°C
2 h control 3.66 ± 0.43 3.66 ± 0.43
24h control 3.31 ±0.16 5.54±0.09
76°Cfor3min 0.10±0.00 d 0.16±0.08 d
Room temperature wash for 3 min 4.23 ±0.32 5.08 ±0.20
a S. Poona populations were selectively isolated on XLT4 agar medium, and reported as
log CFU/cm" rind.
b Data are reported as the mean ± standard deviation for three separate cantaloupes.
c Cantaloupes were dip inoculated with S. Poona for 5 min, allowed to air dry under biosafety
cabinet for 2h, and were stored at either room temperature or 4°C for 24 h prior to washing
treatments.
d Although two of three cantaloupes tested showed no survivors, 0.1 log CFU/cm 2 (minimum
detection level) was used in place of no survivors for determining the mean and standard deviation.
treated oranges. These adverse effects can be controlled by limiting treatment
temperatures and times. Since individual commodities have different thermal
tolerances, the hot water immersion treatment should be tailored to each
commodity. While the rind of a cantaloupe [2] and the peel of an orange [18]
effectively insulate the flesh from thermal damage at temperatures above
70°C, the peel of an apple does not protect the flesh from thermal damage
at temperatures above 60° C [7]. Accordingly, the tolerance to hot water
immersion over a range of temperatures must be determined for individual
commodities at different maturity stages [15].
Following hot water immersion, produce should be rapidly cooled to
reduce the risk of heat damage to the commodity [2]. This cooling process
must be carefully controlled, for it is known to induce infiltration of the cool-
ing solution, including any possible contaminating microorganisms, into the
commodity [20-23]. Therefore, the cooling water to be used for this purpose
should be free of human pathogens and spoilage microorganisms. A forced
cold air tunnel could be used for rapid cooling of the commodity. The use of
sanitizing agents during washing treatments, which includes a hot water wash,
is recommended to reduce the microbial load in the washing solution. This
prevents possible cross contamination in the washing tank, which could result
in internalization during the subsequent cooling treatment.
21.3 SURFACE PASTEURIZATION WITH STEAM
Steam is a gas — gaseous water. Because water vapor molecules are many
orders of magnitude smaller (about 2 x 10~ urn) than bacterial cells such as
salmonella (4 urn long and 0.7 urn thick), and the mean free path length of water
484 Microbiology of Fruits and Vegetables
vapor molecules (0.4 urn) is smaller than bacterial cells, steam should be able
to enter any crevices or pores that bacteria can enter [24]. Steam is a unique
fluid for pasteurizing food surfaces. It is sufficiently hot to kill virtually all
bacterial vegetative cells on contact. However, much like hot water, treatment
with steam may damage heat-sensitive foods like fruits and vegetables.
Much of the research on the use of steam for surface pasteurization has
been on meat rather than fruits and vegetables. Of course, the meat-related
research can be relevant to fruits and vegetables, but meats, except poultry,
are generally more thermally resistant and forgiving than fruits and vegetables.
Unfortunately, most of the information found on steam treatment of fruits
and vegetables is not in the peer-reviewed literature but on web sites and
company brochures.
In 1970 Klose and Bayne [25] experimented with steam to kill bacteria on
the surface of chicken. Chicken samples were hung inside a three-necked
flask, and steam was introduced under vacuum at 70 to 75°C. They obtained a
3 log reduction of naturally present bacteria with a 2-minute exposure, and a
5 log reduction after 16 minutes. Unfortunately, treatment above 60°C resulted
in partial cooking of the outer layers of the samples.
In a follow-up study, Klose et al. [26] developed a cylindrical metal vacuum
chamber to treat whole chicken carcasses with steam. Reductions of 3 logs of
inoculated S. Typhimurium were achieved by application of subatmospheric
pressure steam at 75°C for 4 minutes. However, "the cooked breast meat was
almost twice as tough for steam treated as for controls (5.4 versus 3.0 kg shear)
and was similarly judged by a trained taste panel," presumably because the
surface was cooked.
Davidson et al. [27] used a double-walled steel plate steam chamber to
treat whole chicken carcasses and chicken parts with 180 to 200°C steam for
20 seconds. They realized a 1 to 2 log reduction of the aerobic plate count
(APC) on whole carcasses and breasts. The kill on legs and wings was 2 logs.
They reported "evidence of fat separation in the skin and a lightly cooked
appearance of skin and exposed muscles."
Steam has been used commercially as a surface treatment for meats [28].
Nutsch et al. [29] reported that the bacterial reduction in a commercial beef
processing plant using atmospheric pressure steam for 6 or 8 seconds was
1.35 logs.
When steam is brought into contact with food surfaces, it displaces the air
while compressing a very thin film of air against the food surface. This film
of air insulates the food surface against direct contact by the steam. The steam
is hot enough to kill bacteria instantly, but to do so it must transfer its thermal
energy to the bacterial cell. With a film of air present, the steam cannot contact
the bacteria directly and must transfer the energy across the compressed
air film to the bacteria. This is a relatively slow process compared to conden-
sation of steam directly onto the bacteria cell walls. The process is so slow,
in fact, that the steam will cook the surface before killing the bacteria, which
is detrimental to the quality of thin-skinned and heat-sensitive commodities.
However, for some thick-skinned fruits and vegetables which are destined
Surface Pasteurization with Hot Water and Steam 485
for subsequent processing, such as for production of juice or fresh-cuts, this
might not be a problem since the thermal injury would not extend into the
edible portion of the commodity.
In the following sections, new steam surface pasteurization technologies
applicable to fresh produce are described.
21.3.1 Thermosafe Process
Thermosafe is a patented [30,31] process of Biosteam Technologies, Inc. that
uses condensing steam to kill bacteria on the surface of fruits and vegetables.
Steam raises the surface temperature of fruits and vegetables to a preset value
for a preset hold time. Chilled water follows the steam treatment to quench
cooking. Bacterial reductions of 5 logs or greater can be realized with this
process. The resultant product is acceptable for produce destined for further
processing. It is not acceptable for the fresh food market because the steam
cosmetically degrades the surface.
21.3.1.1 Process Operation
The equipment is relatively inexpensive and mechanically simple. The pro-
cess consists of a chamber or steam tunnel which is designed to be integrated
into a fruit or vegetable process line. The fruit or vegetable enters the chamber,
usually on a conveyer, or the produce can be inserted batch mode. Pressur-
ized saturated steam is injected through vents into the chamber to bring the
surface temperature up to 74°C. Surface temperature can be monitored by
contacting the surface with a thermocouple or by inserting a thermocouple
into the produce 6 mm below the surface. Since this might not be reliable or
practical in a continuous operation, surface temperature can also be moni-
tored with a remote infrared sensor. After reaching 74°C, steam injection
continues for a 60-second hold time. The actual time and temperature can
be adjusted, but a surface temperature of 84°C degrades the organoleptic
properties of fruits and vegetables. Following the hold period, chilled water
at 2 to 5°C quenches the surface for another 60 seconds. The unit comes with
its own steam supply and self-contained water system, including chilled water,
making it easy to install and operate.
21.3.1.2 Process Effectiveness
Food Safety Net Services Ltd conducted a large-scale validation study of the
process [32]. The study concluded that the "process can be effective in reducing
the microbial load of Listeria monocytogenes, Salmonella spp., E. coll 0157:H7,
and more thermoduric Lactobacillus spp. on the surface of fruits and
vegetables by at least 5 logs and in some cases up to 9 logs.'' The data on canta-
loupes show a 5 log reduction for salmonella and E. coli, a 7 log reduction
for listeria, and a 4 log reduction for Lactobacillus. For oranges the data
show almost total kill, 9 logs, for salmonella, listeria, and E. coli and an 8 log
reduction for Lactobacillus. For apples, there was a 5 log reduction for
486 Microbiology of Fruits and Vegetables
salmonella, listeria, and E. coli and a 7 log reduction for Lactobacillus. Using
a combination of steam and hot air, bacterial reductions in excess of 7 logs
were realized on peppers.
21.3.1.3 Product Quality
The quality of the interior portions of treated products was successfully
maintained. Sensory evaluations were made on citrus juice that was extrac-
ted from fruit heated in the range of 65 to 88°C. The results of triangle tests
indicated no significant differences (p < 0.05) in flavor between treated and
untreated product. Therefore, the Thermosafe process effectively pasteurizes
the surface of the tested fruits and vegetables with no significant sensory
damage to the interior. These fruits and vegetables are suitable for further
processing into processed products such as juice but typically would not be
suitable for the fresh food market [33].
21.3.2 University of Bristol Process
The University of Bristol investigated the use of pressurized steam, atmos-
pheric pressure steam, and vacuum steam for reducing the bacterial con-
tamination of meat and fruits and vegetables [34]. The process consists of three
stages: (1) noncondensable gases (air) are removed with vacuum, (2) steam is
applied to the surface of the produce to reach a pasteurization temperature,
and (3) the surface is evaporatively cooled under vacuum to quench cooking.
Steam, with its high latent heat of condensation, gives a rapid rise in the surface
temperature which minimizes thermal exposure time.
21.3.2.1 Process Operation
The pressure and subatmospheric pressure process systems consist of a steam
boiler, a processing chamber, and a vacuum pump. The system operates in
batch mode. The pressure process chamber is 1 m by 0.6 m in diameter. The
flushing action tends to remove noncondensable gases. The subatmospheric
steam chamber is 0.45 m high by 0.3 m in diameter. Steam is injected in the
top, and air and condensate are removed from the bottom.
With a chamber pressure of 2.3 bar, product exposure time was a nomi-
nal 90 seconds. Initial vacuum time was of the order of 10 minutes, and the
evaporative cooling was about 5 minutes. Exposure times in the atmospheric
pressure process were 2 to 6 seconds. Times for the subatmospheric steam
process were not reported.
21.3.2.2 Process Effectiveness
Although the pressure pilot plant process was applied to peppers and soft
fruits, no detailed results were reported. The manufacturer reported
1.2 to 3.4 log reductions in APC of beef treated at temperatures of 100, 120,
or 135°C, depending on whether it was lean or fat. In a comparison of various
Surface Pasteurization with Hot Water and Steam 487
decontamination methods, pressurized steam gave a 1.5 to 5 log reduction in
APC on peppers. Reductions in APC for chilled raspberries and blackberries
were 3 to 5 logs. The subatmospheric steam unit was used for peppers, apples,
and lettuce. Bacterial reductions up to 2 logs were achieved after exposure
for 10 seconds at 65°C, and up to 4 logs were achieved following exposure to
80 to 85°C for 40 seconds.
21.3.2.3 Product Quality
There is no published assessment of the quality for the processed produce.
Because of the temperature and time conditions of treatment, the authors
suspect that the products should be suitable for further processing.
21.3.3 Ventilex Continuous Steam
Sterilizing System
The Ventilex process uses saturated steam to decontaminate or sterilize
herbs, spices, and seeds [35]. The product enters a horizontal steam chamber
through a patented rotary valve, designed to prevent buildup of product within
the valve. Once in the chamber, the herbs, spices, or seeds are contacted by
saturated steam for a given time appropriate to reduce or eliminate pathogenic
bacteria. The treated product exits through a second rotary valve. Following
steam treatment, the herbs, spices, or seeds are dried and cooled.
21.3.3.1 Process Operation
A description of the process is available at the Ventilex web site [35]. The
process is a high-temperature/short-time treatment for herbs, spices, and seeds
using saturated steam. Small particle products such as these tend to clump in
valves and clog the process. This is prevented by using a continuous scraping
action within the valve to dislodge any adhering product.
The treatment chamber is horizontal with a vibrating belt. The belt moves
the material through the chamber in plug flow at a set speed to achieve the
desired residence time. The frequency of the vibrating belt is variable and
governs the flow rate that determines the dwell time for each product. Products
are treated with saturated steam over the range 107 to 123°C. Typical treat-
ment times are 25 to 50 seconds depending on the commodity, contamination
level, and final use of the product. The treated product drops off the vibrat-
ing belt into a second rotary valve using the same scraping action. The
herbs, spices, or seeds then go to a fluidized bed dryer/cooler. The condensed
steam flashes off, and the herbs, spices, or seeds are dried with indirectly heated
sterile air.
21.3.3.2 Process Effectiveness
According to the manufacturer, products treated with this process often have
APC counts below 1 log. Salmonella is eliminated. Mold and yeast populations
488
Microbiology of Fruits and Vegetables
TABLE 21.5
Effectiveness of the Ventilex Process on Natural Microbial Flora (log CFU/g)
of Paprika and Rosemary
Aerobic Aerobic
plate spore Bacillus
count formers Enterobacteriaceae ceteus Yeast Molds
Commodity
count
form
Paprika
Before
treatment
6
6
After
3
3
treatment
Rosemary
Before
treatment
5
5
After
<2
treatment
<2
Note: — , not determined.
From van Gelder, A., Personal communication, 2003.
are below 2 logs. Spores of Bacillus cereus, Clostridium perfringens, and
Staphylococcus aureus have counts below 2 log. Specific results for paprika
and rosemary are shown in Table 21.5 [36]. There is a 3 log or better reduction
in total APC and aerobic spore formers. Yeasts and molds are essentially
eliminated.
21.3.3.3 Product Quality
There is little or no product degradation. For example, the color value for
paprika differed from the control by only 2 ASTA units after treatment
(reduced from 95 to 93). Volatile oils for rosemary did not change (0.7 ml/ 100 g).
In addition, the process inactivates enzymes.
21.3.4 Vacuum-Steam- Vacuum (VSV) Process
Thermal damage is a major problem for steam surface-pasteurized fruits and
vegetables destined for the fresh market. Conventional wisdom seems to dictate
that if the steam exposure time is sufficient to kill the bacteria, the produce is
thermally damaged. The treated produce may be suitable for the processed
fruit or vegetable market but not for the fresh market. If fresh quality is to be
maintained by using a shorter exposure time, the bacterial population will not
be sufficiently reduced. One solution to this problem is the U.S. Department
of Agriculture's (USDA) novel VSV process [37].
To circumvent the problem of thermal damage, the film of air and moisture
on the commodity surface is removed so that steam can rapidly contact
the bacteria directly. It is a simple concept but difficult to achieve in practice.
One approach was the concept proposed by Morgan et al. [24,38,39]. In this
Surface Pasteurization with Hot Water and Steam 489
method, the food is exposed to vacuum to remove air and moisture. Next,
saturated steam is applied to the surface. When the saturated steam contacts
the product, it condenses to form a water film on the fruit or vegetable surface
which impedes further bacteria reduction. Therefore, the food is exposed to a
vacuum again to remove the condensate and to evaporatively cool the surface.
Kozempel et al. [40] showed that cycling between vacuum and steam to remove
the condensate enhanced the population reduction of Listeria innocua on
hot dogs. This concept of alternating vacuum and steam is the basis of the VSV
process.
Initial research used a stainless steel device consisting of a rotor and
stator. The 150 mm long and 150 mm in diameter [24,38] rotor was turned
rapidly around its horizontal axis, stopping at precisely determined angular
positions, exposing the sample alternately to vacuum or steam. A
25 mm x 75 mm x 75 mm deep treatment chamber was milled into the surface
of the rotor.
The treatment consisted of four steps: (1) air was removed by exposure
to vacuum; (2) the sample was flushed with low-temperature saturated steam
(this flush was later abandoned); (3) the sample was exposed to pressurized
saturated steam; and (4) the sample was evaporatively cooled with vacuum.
Bacterial reductions on chicken meat inoculated with nonpathogenic L. innocua
were about 2 to 2.5 logs. Steam exposure time was 0.1 to 0.2 seconds [24,38].
This prototype proved the concept, but was not practical with actual fruits
and vegetables such as cantaloupes. For mechanical reasons it was preferable
to move the machinery and not the food sample. Therefore, a new prototype
pilot plant unit was designed and fabricated. The surface intervention proces-
sor was designed to process chicken carcasses, specifically broilers. However,
the design is also suitable for many fruits and vegetables, especially canta-
loupes. The performance requirements of a surface intervention processor
are to accept the individual food sample and enclose it in a chamber within
a rotor; to evacuate that chamber; to pressurize the chamber with steam; to
vacuum cool it; and, finally, to eject the sample into a clean environment. The
simplest execution of this prototype, one chamber in one rotor, was designed
and constructed [41]. Figure 21.1 shows the processor, and Figure 21.2
shows details of the product treatment section. The chamber is cylindrical,
about 200 mm in diameter and 240 mm deep, and is provided with an 8-inch
ball valve.
To admit vacuum or steam into the closed chamber, two opposing 200 mm
holes were bored through the stator at right angles to both the axis of rotation
of the ball and to the centerline of the open chamber. Two platter valves,
consisting of a flat disk rotating against an inlet header that holds poly-
etheretherketone (PEEK) seals, were close-coupled to the 200 mm ports. Each
disk contained two holes, which when stopped at one of the ports in the inlet
header permitted steam flow into or vacuum evacuation from the treatment
chamber. Multiple holes reduced the rotor angular movement necessary for
valve action and increased the cross-sectional area for gas flow. Each disk was
programmed independently and moved by its own servomotor. To expose all
490
Microbiology of Fruits and Vegetables
Servo drive
Vacuum
pump
Vacuum
' ader
Platter
Ives
Product
valve and
mandrel
FIGURE 21.1 Vacuum-steam-vacuum processor.
Product valve
Product goes in and
comes out here
Shaft seals (4) .Vacuum inlet (2)
Steam inlet (2)
Servo drive
Platter (disk)
Inlet header
Drive coupling
FIGURE 21.2 Schematic of the product treatment section of the Vacuum-steam-
vacuum processor.
Surface Pasteurization with Hot Water and Steam 491
exterior surfaces of the test sample to treatment, a screen was installed at the
midpoint of the treatment chamber to hold the sample.
21.3.4.1 Process Operation
Each sample of fruit or vegetable is manually inserted into the treatment
chamber of the VSV processor. A computer-controlled servomotor is used to
rotate the ball valve 90° to seal the chamber from the atmosphere. The platter
valves rotate to expose alternately the sample to vacuum, then steam, and
then vacuum again. With multiple cycles, the sequence of vacuum, then steam,
is repeated multiple times. After treatment, the ball valve rotates back 90° to
expose the sample to the atmosphere. After treatment, the fruit or vegetable
sample is removed manually with sterile gloves.
21.3.4.2 Process Effectiveness
Three different kinds of produce (uninoculated) were processed to assess
thermal damage and bacterial reduction. The commodities were chosen to
represent aerial fruits (grapefruits), fruits growing on the ground (cantaloupes),
and vegetables that grow in the ground (carrots). Table 21.6 summarizes
the results. There was no visual thermal damage, and the bacterial popula-
tion reductions were 3.4 to > 5 logs, but these process conditions were not
optimized. The optimum conditions give maximum bacteria reduction with
minimal or no thermal damage to the product. Therefore, a series of optimiza-
tion experiments were conducted to determine the best processing conditions
[41]. Beets were substituted for the in-ground crop because the shape of the
treatment chamber was more amenable to spherical foods and tended to chop
off the ends of carrots. (A VSV processor for carrots or other cylindrical crops
would require a differently shaped treatment chamber.) Papayas were added
to the list of products tested. Table 21.7 lists the optimum process condition
and bacterial reduction for cantaloupes, grapefruits, papayas, and beets.
Because of the high natural bacteria count on beets, they were not inoculated;
cantaloupes, grapefruits, and papayas were inoculated with Listeria innocua.
TABLE 21.6
Initial Results for the VSV Intervention Process for Uninoculated Produce
Commodity Steam temperature (°C) Control Population reduction
Carrots 130 5.6 >5.0
Grapefruits 130 3.6 3.6
Cantaloupes 138 5.6 3.4
Note: Vacuum time = 0.25 sec, and steam time = 0.25 sec.
492
Microbiology of Fruits and Vegetables
TABLE 21.7
Optimization Results for the VSV Surface Intervention Process on Inoculated
and Uninoculated Produce
Steam
Steam
Vacuum
Number of
Bacterial reduction
Commodity
temp. (°C)
time (sec)
time (sec)
cycles
(log CFU/ml)
Cantaloupes
143
0.1
0.1
2
3.4 L. innocua
Grapefruits
138
0.1
0.1
2
3.6 L. innocua
Papayas
138
0.2
0.1
2
3.6 L. innocua
Beets
143
0.2
0.1
3
2.5 aerobic plate count
TABLE 21.8
Application of the VSV Surface Intervention Process to Other Fruits and
Vegetables
Commodity Bacteria
Steam Control Treated Bacterial
Steam time per No. of (log (log reduction
temp (°C) cycle (sec) cycles CFU/ml) CFU/ml) (log CFU/ml)
Mangoes
L. innocua
138
0.1
2
5.4
1.4
4.0
Avocados
L. innocua
138
0.1
2
4.1
1.0
3.1
Kiwis
L. innocua
138
0.1
3
6.4
1.6
4.8
Bananas
a
104
0.1
1
Mutilated
Carrots
Aerobic
plate count
138
0.1
3
5.7
1.6
4.1
Cucumbers
Aerobic
plate count
138
0.1
3
5.4
1.6
3.8
Peaches
L. innocua
138
0.1
2
5.0
1.4
3.6
Cauliflower
a
127
0.1
1
Color change
Broccoli
a
116
0.1
1
Color change
Peppers
a
116
0.1
1
Mutilated
Note: Vacuum times =0.1 sec.
a No microbiology analyses were performed
on prod
ucts that
were thermally damaged.
Several tropical fruits were tested at the general optimum conditions of
138°C steam for 0.1 seconds using one, two, or three cycles and a vacuum time
of 0.1 seconds. The results for kiwis, mangoes, and avocados are listed in
Table 21.8. All samples were inoculated with L. innocua for 10 minutes and
dried under ambient conditions for 1 hour. The log reduction for kiwis was 4.8;
for mangoes, 4.0; and for avocados, 3.1.
Another tropical fruit, banana, was tested. However, the process caused
the peel to split and the fruits to darken immediately. Milder conditions (104°C
for 0.1 seconds and one cycle) were tried with green bananas, but the samples
still were destroyed. No microbiological analyses were performed on products
that were thermally damaged.
Surface Pasteurization with Hot Water and Steam 493
Other products processed without success were peppers, broccoli, and
cauliflower. When subjected to vacuum, the peppers exploded. Upon exposure
to steam the delicate florets on broccoli turned a bright green indicative
of blanching or heat treatment. Although the flower part of cauliflower was
essentially unscathed, the stalk and the remnants of the leaves turned bright
green as in blanching.
Other fruits and vegetables were tested at the conditions stated above.
The results are listed in Table 21.8. The bacterial reduction (APC) on uninocu-
lated carrots was 4.1 logCFU/ml. Treatment of cucumbers, inoculated for
10 minutes with L. innocua and dried at ambient conditions for 1 hour, resulted
in 3.81ogCFU/ml reduction with three cycles. Peaches were inoculated for
10 minutes with L. innocua and were allowed to dry for 1 hour under ambient
conditions. Using two cycles, the reduction for L. innocua was 3.61ogCFU/ml
with no thermal damage.
In addition to bacteria, some insects such as red scale infest the surface
of fruits. Red scale is a major problem on citrus fruits. Currently, methyl
bromide is used to eliminate insects such as red scale, but the impending loss
of methyl bromide in 2005 requires alternative methods of quarantine
treatment for disinfestations of produce imported or exported each year.
The VSV process was used to process lemons infested with red scale [42].
No scale insects survived the process. The process resulted in 100% kill
of insects at all stages of development. As a bonus, up to 96% of first molt
scales were physically removed, but the process was much less effective
in removing other stages from the fruit, especially those that had advanced
beyond the second instar. However, the process was completely effective in
killing the scales.
21 .3.4.3 Product Quality
To date, evaluation of thermal damage has been only qualitative. Except
for bananas, broccoli, and peppers that were not amenable to this process,
there was no thermal damage observed. Most of the treated produce samples
(uninoculated) were consumed and found to be indistinguishable from the
untreated controls.
21.4 CONCLUSIONS
Steam and hot water surface pasteurization are both promising technologies
that are capable of achieving more than 5 log reductions in target pathogens as
well as greatly reducing populations of spoilage microorganisms on the surface
of fruits and vegetables. However, hot water immersion treatment of fresh
produce appears to be a gentler process and has better control over the surface
temperature of produce during treatment as compared to steam treatment.
Steam processes are acceptable treatments for produce intended for further
processing due to thermal damage of the produce surface. The VSV process
produces good results with a number of commodities with bacterial reductions
494 Microbiology of Fruits and Vegetables
up to 4.8 logCFU/ml (Table 21.8), depending on the fruit or the vegetable.
The VSV is a rapid process requiring less than 2 seconds for treatment and with
little or no thermal damage.
Even though highly promising, surface pasteurization technology is in
need of further research to determine thermal penetration profiles and heat
sensitivity at different temperatures for individual commodities. There is
also a need to obtain thermal inactivation data for human pathogens of
concern, attached to surfaces of commodities that have subsurface sites
(e.g., pores) and other sites providing protection as well as exposed sites.
Furthermore, research is needed to determine the temperature-time effect of
surface pasteurization on sensory qualities, storability, and processability
of fresh produce at different maturity stages. Results from such research would
enable the development of cheap, safe, and environmentally sound disinfec-
tion treatments for controlling pathogens and/or spoilage microorganisms
on fresh produce.
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22
Novel Nontherma
Treatments
Dongsheng Guan and Dallas G. Hoover
CONTENTS
22.1 Introduction 498
22. 1 . 1 Nonthermal Processing Methods 498
22.1.2 Advantages and Disadvantages of Application 498
22.2 High Hydrostatic Pressure Processing (HPP) 499
22.2.1 Introduction 499
22.2.1.1 Definition and Historical Perspective 499
22.2. 1 .2 Equipment 500
22.2.1.3 Critical Processing Factors 500
22.2.2 Inactivation of Problematic Microorganisms 501
22.2.2.1 Spores and Vegetative Bacteria 501
22.2.2.2 Viruses 504
22.2.2.3 Parasites 505
22.2.3 Summary 505
22.3 Irradiation 505
22.3. 1 Introduction 505
22.3.2 Application to Fruits, Vegetables, and Juices 506
22.3.2.1 Spores and Vegetative Bacteria 506
22.3.2.2 Parasites 508
22.3.2.3 Viruses 508
22.3.3 Summary 509
22.4 Pulsed Electric Fields in Juice Processing 509
22.4.1 Introduction 509
22.4.2 Application to Juices 511
22.4.3 Summary 512
22.5 Ultrasonic Waves for Preservation of Fruit and
Vegetable Products 512
22.5.1 Introduction and Description of Process 512
22.5.2 Microbial Inactivation 513
22.5.3 Summary 514
22.6 Electrolyzed Water 514
22.6.1 Introduction 514
497
498 Microbiology of Fruits and Vegetables
22.6.2 Application as a Novel Disinfectant for Fruits
and Vegetables 515
22.6.3 Summary 516
22.7 Final Remarks and Future Perspectives 516
Acknowledgment 517
References 517
22.1 INTRODUCTION
22.1.1 Nonthermal Processing Methods
In recent years there has been a growing and sustained interest in the group of
food processing methods commonly referred to as the nonthermal processing
technologies. These processes usually employ a somewhat novel application
of energy to reduce or eliminate problematic microorganisms without the
generation of high levels of heat normally found in conventional thermal
processing. Ideally with limited exposure to high temperatures, the food
possesses sensory qualities and nutrient content more closely resembling
the raw, fresh, or minimally processed counterpart while retaining the desired
shelf life and safety.
22.1.2 Advantages and Disadvantages of
Application
In the thermal processing of foods, heat inactivates both microorganisms and
enzymes to extend the shelf life of the treated foods. With application and
incorporation of nonthermal processing methods the same microorganisms
and enzymes in the food are usually targeted with the same expectation
of inactivation or control; however, the actual mechanisms of microbial
inactivation and protein denaturation are usually different when comparing
thermal processing to nonthermal processing methods. Different mechanisms
of inactivation mean the rates of inactivation are different and the degree of
effectiveness is also usually different. Consequently, the usual primary goal
in adapting the nonthermal methods for commercial use is to maintain
the same high degree of safety enjoyed with thermal processing while minimiz-
ing changes to the desirable sensory qualities and nutrition in the product.
That can be an immense challenge depending on the food product. Each
nonthermal process is somewhat distinct with its own set of limiting factors
that can include an inability to denature browning enzymes, limited or non-
existent inactivation of viruses or bacterial endospores, regulatory hurdles,
high costs for equipment and maintenance, and a lack of background infor-
mation usually provided by past industrial experience. Examples of areas
of insufficient information for these new technologies include standardization
of industrial process procedures, and methods and surrogate identification for
Novel Nonthermal Treatments 499
process validations. These factors make the commercialization of new food
products utilizing nonthermal processes complex and daunting.
With the exploding demand for fresh fruits and vegetables by consumers
in North America comes the necessary importation of produce from areas of
the world where crops can be grown and harvested all year round. For
example, the Produce Marketing Association stated that imports of fresh
produce increased from 13.8 billion pounds in 1993 to 20.2 billion pounds in
2000. The quality of fruit in winter is now nearly equivalent to fruit sold in the
warmer months due to significant improvements in storage, transportation,
and distribution. Unfortunately, the incidence of foodborne illness also
coincides with this increased bounty of fresh fruits and vegetables (both
domestic and imported). For example, in the marketplace the most dangerous
food related to acute illness is not derived from meat, milk, eggs, or seafood,
but is a plant product: sprouts [1]. In fact, while meat is the most regulated and
monitored food commodity, in comparison fruits and vegetables receive only a
fraction of government attention. Federal health surveillance of foodborne
diseases from 1993 to 1997 documented 2,751 outbreaks that involved 12,537
individual cases of foodborne illness related to contaminated fruits and
vegetables, compared with 6,709 cases involving meat products. Outbreaks
linked to fruits and vegetables are often the result of fecal contamination
caused by inadequate hygiene during production and harvest. Imported fruits
and vegetables now appear to harbor exotic and emerging parasites once
unknown in North America, and traditional pathogens, such as salmonellae,
hemorrhagic Escherichia coli, and Listeria monocytogenes, can be anticipated in
both domestic and imported produce as a consequence of poor agricultural
practices involving the composting and distribution of manure. Landmark
outbreaks involving cyclospora in Guatemalan raspberries, salmonella-
contaminated sprouts, E. coli in fresh cider, and Mexican scallions tainted
with hepatitis A have become well-known reference points to American
consumers who worry about the safety of the foods they eat.
The aim of this chapter is to furnish an overview of the use of nonthermal
processing technologies applied for the preservation of fruits, vegetables, and
their byproducts, most notably juices. The nonthermal processing methods
that are covered in this chapter include high hydrostatic pressure processing,
and applications of ionizing irradiation, high-intensity pulsed electric fields,
ultrasonic waves, and electrolyzed oxidizing water.
22.2 HIGH HYDROSTATIC PRESSURE
PROCESSING (HPP)
22.2.1 Introduction
22.2.1.1 Definition and Historical Perspective
Historically, applications of HPP for the preservation of foods were first con-
ducted by Bert Hite who adapted high-pressure processing to a variety of foods
500 Microbiology of Fruits and Vegetables
and beverages in the late 1890s and early 20th century [2,3]. From Hite's work
until the 1980s, only scattered attempts were made to investigate the potential
commercial application of HPP to foods. With the sustained demand for high-
quality foods that are minimally processed and additive-free in developed
countries, HPP has attracted interest from research institutions, food com-
panies, and regulatory agencies over the last two decades in the pursuit
of producing better quality foods more economically.
HPP subjects foods to pressures between 100 and 800 MPa with exposure
times ranging from a millisecond pulse to over 20 minutes, although most
commercial treatments times are 7 minutes or preferably less [4,5]. The
temperatures of products and media during pressure processing can be below
0°C or above 100°C, depending on the product requirements; however, current
commercial HPP uses ambient temperatures.
The first commercialized food product employing pressure preservation
was fruit preserves marketed in Japan in 1991. These pressure-treated jams
and jellies continue to be sold in Japan in addition to salad dressings and a
wide range of fruit juices. Pressure-treated guacamole successfully entered the
U.S. marketplace in 2001, followed by HPP salsa. These products have been
available nationwide, but pressurized guacamole and salsa remain most
popular in the southwestern region of the U.S. In 2004 it is anticipated that
pressure-processed chopped onions will be sold as an ingredient in premium
salad dressings. This onion product will be available in fresh chopped form
months after the normal season ends. They will be available in 8 oz stand-up,
resealable bags and as 2.51b pouches for club and superstores. The intended
refrigerated shelf life is 45 days, but 90 days of storage has been demonstrated
without detrimental quality changes. Pressure-processed chopped onions
are said to have a "sweeter taste that isn't bitter, with a fresher, crunchier
texture." Also anticipated in 2004 from a Canadian venture is applesauce and
applesauce/fruit blends packaged as eat-on-the-go single-serve flexible tubes:
"Our Way of Preserving Nature," and from Mexico, fruit "smoothie" products
for North American distribution. This company produces juices, nectars, and
bottled drinks.
22.2.1.2 Equipment
Typical HPP equipment usually consists of a pressurized vessel, two end
closures at each end of the vessel, a low-pressure pump, an intensifier to
generate higher pressures, and system controls. HPP systems can be designed
to treat either unpackaged liquid foods semicontinuously or packaged foods
in a batch manner. A schematic of a batch HPP system is presented in
Figure 22.1.
22.2.1.3 Critical Processing Factors
Critical process factors in HPP include, but are not limited to, treatment
pressure, holding time at pressure, come-up time to achieve treatment
Novel Nonthermal Treatments
501
Safety Pressure Safety Control
light beam vessel light beam cubicle
Customer supplied
work platform
Service area access hatch
9 ft 10 in x 6 ft 7 in (3 m x 2 m)
Switchgear
cubicle
Vessel temperature Water module 25X pump Service area
control module
TM
FIGURE 22.1 Fresher Under Pressure high-pressure processing systems (215 L)
from Avure Technologies Inc., a wholly owned subsidiary of Flow International
Corporation.
pressure, decompression time, initial temperature of food products, treatment
temperature, the temperature distribution in the vessel at pressure as a result
of adiabatic heating, product properties (e.g., pH, composition and water
activity, food compressibility), packaging material, and the microbiota of
the food [6]. Package size and shape are not critical factors in process
determination because pressure acts instantaneously and uniformly throughout
the chamber and the food mass. For pulsed pressure processing, additional
process factors are pulse shape (i.e., the waveform), frequency, and pulse
pressure magnitudes.
22.2.2 Inactivation of Problematic Microorganisms
22.2.2.1 Spores and Vegetative Bacteria
Most fungal conidiospores and ascospores can usually be inactivated at
pressures between 300 and 450 MPa at ambient temperature, but exceptions
exist. For example, in a study on dormant Talaromyces macrosporus asco-
spores, mild treatment (200 to 500 MPa, 20°C) activated dormant ascospores
but caused little or no inactivation. Higher pressures (500 to 700 MPa, 20°C)
were required to inactivate the ascospores; however, a treatment of 700 MPa
after 60 minutes only reduced the spore population by less than 21og 10 units,
indicating the resistance of the ascospores to high pressure [7].
502 Microbiology of Fruits and Vegetables
Hayashi [8] found that pressures of 200 MPa effectively killed yeasts
and molds in freshly squeezed orange juice at ambient temperature. Ogawa
et al. [9] stated that after HPP (400 MPa and 23°C) both freshly squeezed
orange juice and orange juice that had been inoculated with yeasts and molds
showed no increase in total counts after 17 months of storage at 4°C. Aleman
et al. [10] reported treatment at 340 MPa for 15 minutes extended the shelf
life of fresh-cut pineapple. Populations of Gram-negative bacteria, yeasts, and
molds could be reduced by at least 1 logio at pressures of 300 and 350 MPa
for inoculated lettuce and tomatoes, even though the tomato skins loosened
and peeled away and lettuce browned in this range of pressures [11]. Raso et al.
[12] used pressure to inactivate ascospores and vegetative cells of Zygosac-
charomyces bailii suspended in apple, orange, pineapple, cranberry, and grape
juices. HPP at 300 MPa for 5 minutes reduced the population of vegetative cells
and ascospores by almost 51og 10 units and 0.5 to 1 log 10 units, respectively.
Parish [13] applied pressures between 500 and 350 MPa to nonpasteurized
Hamlin orange juice (pH 3.7) inoculated with Saccharomyces cerevisiae and
found that the D values for ascospores and vegetative cells inoculated into the
pasteurized orange juice were from 4 to 76 sec and from 1 to 38 sec, respec-
tively. The native microbiota in the orange juice had D values from 3 to 74 sec
in the pressure range of 500 and 350 MPa. The corresponding z values were
123, 106 and 103 MPa for ascospores, vegetative cells, and native microbiota,
respectively.
Zook et al. [14] used fruit juices and a model juice buffer (pH 3.5 to 5.0) as
suspension media to determine pressure inactivation kinetics of S. cerevisiae
ascospores. Approximately 0.5 x 10 6 to 1.0 x 10 6 ascospores/ml were pressur-
ized at 300 to 500 MPa in juice or buffer. D values were 8 sec to 10.8 min at 500
and 300 MPa, respectively; the corresponding z values were 115 and 121 MPa.
No differences (P > 0.05) in D values (at constant pressure) or z values among
buffers or juices at any pH were determined, suggesting little influence of pH in
this range.
Bacterial endospores are the most difficult life forms to eliminate with
hydrostatic pressure; spores of bacillus have been exposed to > 1,724 MPa
(250,000 psi) and remained viable [15]. Applications of pressure alone will
not inactivate bacterial endospores. Hurdle technology that utilizes pressure
in combination with other process technologies (including pressure pulsing)
are proposed to improve spore inactivation rates. Mild elevated heat (e.g.,
40 to 55°C) with pressure treatment is required for substantial reduction
of spore loads [16,17]. Sterilization requires higher temperatures resulting in
a definite cooked appearance of the food.
Bacterial spores have been shown to demonstrate variable pressure
resistances with respect to sporulation conditions. The anhydrous structure and
dimensions of the spore are believed to contribute to the pressure resistance
of bacterial spores, causing a major challenge to produce shelf-stable low-acid
food products [18]. Spores can germinate at different combinations of tem-
perature and pressure [19]. The initiation of spore germination results in loss
of resistance. Two-exposure treatments (i.e., twin pressure pulse) have been
Novel Nonthermal Treatments 503
proposed to enhance the inactivation of spores by HPP [20]. The concept is that
the first exposure at low pressure results in spore germination, and the second
exposure at a higher pressure inactivates the germinated spores and vegetative
cells. Unfortunately, it appears that not all spores are germinated by pressure
and not all germinated spores appear to be inactivated by pressure [21].
Oh and Moon [22] investigated the effect of pH on the initiation of spore
germination and inactivation of Bacillus cereus KCTC 1012 spores using pres-
sures to 600 MPa. The pH of the sporulation medium affected inactivation of
B. cereus spores under pressure more than the suspension medium pH. B. cereus
spores obtained through sporulation at pH 6.0 showed greater resistance to
pressure than those sporulated at pH 7.0 and 8.0 at 20, 40, and 60°C.
To date, successful commercial preservation of foods utilizing HPP depends
upon the use of post-treatment refrigeration or a product pH below 4.5 to
block the germination of spores of C. botulinum and other sporeforming
bacteria. Production of commercially sterile low-acid foods such as meat, milk,
and vegetables must overcome the extreme pressure resistance of spores.
Similar as found in fungi, vegetative forms of bacteria are normally
more easily inactivated by pressure than spores. Linton et al. [23] investigated
the inactivation of a pressure-resistant strain of Escherichia coli 0157:H7
(NCTC 12079) in orange juice over the pH range 3.4 to 5.0. The sterile
orange juices were adjusted to various pH levels (3.4, 3.6, 3.9, 4.5, and 5.0)
and inoculated with E. coli 0157:H7 at 10 CFU/ml. A 61og 10 inactivation was
obtained after 5 minutes at 550 MPa and 20°C at every pH evaluated except
pH 5.0 (~5.51ogio); this pressure combined with mild heat (30°C) resulted in a
61ogio inactivation at pH 5.0.
There were considerable variations in bacterial pressure resistance in dif-
ferent types of fruit juices. Teo et al. [24] reported HPP treatment at low
temperature (15°C) had the potential to inactivate E. coli 0157:H7 strains.
A three-strain cocktail of E. coli 0157:H7 (SEA13B88, ATCC 43895, and
932) was found to be most sensitive to pressure in grapefruit juice (8.3 log i
reduction) and least sensitive in apple juice (0.41og 10 reduction) when pres-
surized at 615 MPa for 2 minutes at 15°C. The resistance difference might come
from the various pH values and the presence of natural antimicrobials in
different fruit juices.
Wuytack et al. [25] applied pressures of 250, 300, 350, and 400 MPa to
reduce the microbial loads of garden cress, sesame, radish, and mustard seeds
that were immersed in water and treated at 20°C for 15 minutes. The percen-
tages of seeds germinating on water agar were recorded to 1 1 days after
pressure treatment. Radish and garden cress seeds were the most pressure-
sensitive and pressure-resistant types, respectively. For example, after a
250 MPa treatment, radish seeds displayed 100% germination nine days later
than untreated controls, while garden cress seeds attained 100% germination
one day after the controls. Garden cress seeds were inoculated with suspensions
of seven different kinds of bacteria (starting inocula 10 7 CFU/g). Treatment at
300 MPa for 15 minutes and 20°C resulted in 61ogi reductions of Salmonella
Typhimurium, E. coli MG1655, and Listeria innocua, >41og 10 reductions of
504 Microbiology of Fruits and Vegetables
Shigella flexneri and the pressure-resistant stain E. coli LMM1010, and a
21ogio reduction of Staphylococcus aureus; however, Enter ococcus faecalis was
not inactivated.
Ramaswamy et al. [26] applied 150 to 400 MPa to apple juices inocu-
lated with E. coli 29055 at 25°C for to 80 minutes. The surviving cells with
and without injury were differentiated through the use of brain-heart infusion
agar (BHIA) and violet-red bile agar (VRBA). It was found that D values of
E. coli decreased with an increase in pressure, and pressure D values from
BHIA (survivors including injured cells) were higher than from VRBA
(survivors excluding injured cells), indicating that a greater number of cells
were initially injured than killed with HPP treatment. The associated z values
(pressure range to result in a decimal change in D values) were 126 and
140 MPa on BHIA and VRBA, respectively.
22.2.2.2 Viruses
The first examination of the pressure sensitivity of viruses was by Giddings et
al. [27] who found that a 920 MPa exposure was required to inactivate tobacco
mosaic virus (TMV). Since that early work, it now appears that most human
viruses are substantially more pressure-sensitive than TMV. Human immuno-
deficiency viruses (HIV) can be reduced by 10 4 to 10 5 viable particles after
exposure to 400 to 600 MPa for 10 minutes [28], but some viruses can be inacti-
vated at even lower levels of pressures. For example, Brauch et al. [29] reported
that pressures of 300 to 400 MPa significantly killed bacteriophage (</>x, X and
T4), and Shigehisa et al. [30] found that an 81og 10 plaque-forming unit (PFU)
population of herpes simplex virus type 1 could be eliminated by a 10-minute
exposure to 400 MPa, and a 51og 10 PFU population of human cytomegalo-
virus was inactivated by a 10-minute exposure to 300 MPa. Shigehisa et al. [31]
later reported that a 5.51ogio tissue culture infectious dose of HIV type 1 was
eliminated after a 10-minute exposure to 400 MPa at 25°C.
According to Kingsley et al. [32], a 71og 10 PFU/ml hepatitis A virus
(HAV) stock in tissue culture medium was reduced to nondetectable levels
after exposure to > 450 MPa for 5 minutes. Titers of HAV were reduced in
a time- and pressure-dependent manner between 300 and 450 MPa, but
poliovirus titer was unaffected by a 5-minute treatment at 600 MPa. Salts
had a protective effect on viruses because dilution with seawater increased the
pressure resistance of HAV. Experiments involving RNase protection indi-
cated that viral capsids might remain intact during pressure treatment,
suggesting that inactivation was due to subtle alterations of viral capsid
proteins. A 71og 10 tissue culture infectious dose of feline calicivirus, a Norwalk
virus surrogate, was completely inactivated by exposure to 275 MPa or above
after 5 minutes, indicating that HAV and feline calicivirus could be inactivated
by pressure.
Currently, there are few publications available addressing pressure
inactivation of viruses in fruit and vegetables products. It can be anticipated
that investigations will more closely evaluate inactivation of viruses by HPP
Novel Nonthermal Treatments 505
given the recent food safety issues concerning fecally contaminated fresh
produce.
22.2.2.3 Parasites
Human feces are not just a source of human viruses, but also a source of
human parasites. Raw fruits and vegetables can become fecally contami-
nated with parasites that include the protozoans Giardia intestinalis,
Cryptosporidium parvum, Cyclospora cayetanensis, and the helminth parasites
Fasciola hepatica, Ascaris lumbricoides, and Ascaris suum [33]; however,
few articles are available regarding pressure inactivation of parasites in or on
fresh fruits and vegetables. Slifko et al. [34] applied 550 MPa to apple and
orange juices in which Cryptosporidium parvum oocysts were suspended. After
a 30-second exposure, C. parvum oocysts were inactivated by at least 3.4 log 10 ,
and an exposure to 550 MPa for more than 60 seconds efficiently rendered the
oocysts nonviable and noninfectious.
Recently, HPP was used to inactivate parasites from muscle tissues and
fish. A pressure of 200 MPa for 10 minutes inactivated all anisakis larvae
isolated from fish tissues either in distilled water or in a physiological isotonic
solution between and 15°C; when exposed to 140 MPa for 1 hour, all larvae
were killed [35]. Dong et al. [36] pressure-inactivated Anisakis simplex larvae
inoculated in king salmon and arrowtooth flounder. Complete kill of the
larvae (ranging from 13 to 118) contained in fish fillets was obtained by
treatments of 414 MPa for 0.5 to 1 minute, 276 MPa for 1.5 to 3 minutes, and
207 MPa for 3 minutes; however, it was stated that the application of HPP
to raw fish was limited because of the significant whitening of the flesh of
HPP-treated fish fillets (P < 0.05).
22.2.3 Summary
Because of its capacity to inactivate pathogenic microorganisms with minimal
application of heat and quality loss, HPP is continuing to gain attention as
one of the viable alternative nonthermal methods to thermal processing.
HPP-treated products maintain the nutritional and sensory quality with
extended shelf life. Standardization and commercialization of HPP seem very
promising for a range of food and beverage products.
22.3 IRRADIATION
22.3.1 Introduction
Studies on the effect of ionizing radiation upon living organisms started after
the discoveries of X-rays in 1895 and radioactivity in 1896. The first patent
for the use of irradiation as a food processing technology was filed in 1905,
but sustained effort to use radiation to preserve foods did not begin until
the end of World War II. The first commercial use of food irradiation occurred
506
Microbiology of Fruits and Vegetables
Electron accelerator
Electron beams
ooooooooo
Product
FIGURE 22.2 Schematic of an electron accelerator.
in 1957 on a spice in Germany [37]. Irradiation is now widely used to
inhibit tuber sprouting, delay fruit and vegetable ripening, control insects in
fruits and grains, and reduce parasites in products of animal origin [38].
Through the interaction of chemically active species (i.e., free radicals)
induced from irradiation and direct damage to microbial DNA caused by
high-energy particles, irradiation is also used to reduce or eliminate foodborne
microorganisms [39].
Typical ionizing radiation facilities use either gamma rays from the radio-
active isotopes 60 Co or 137 Cs or electron beams as well as X-rays generated
in electron accelerators [40]. Strict safety measures are required for gamma
ray facilities due to continuous emission from 60 Co and 137 Cs, such as use of
thick concrete walls to construct the irradiation chamber. In contrast, electron
accelerators, as shown in Figure 22.2, have few leakage problems because they
produce no high-energy electrons when not in use. As recommended by a joint
FAO/IAEA/WHO expert committee on food irradiation (JECFI) in 1980, the
absorbed dose or amount of energy absorbed by a food product has a limit of
lOkGy (1 Gy is a dose equal to 1 J/kg of absorbing material). After reviewing
toxicological, nutritional, and microbiological data on foods irradiated at
doses over lOkGy, the committee concluded that foods are both safe and
nutritious to consumers when irradiated to any dose adequate to obtain
the intended technological objective; however, most foods exposed to dosages
above lOkGy will lose sensory quality to some extent [41].
22.3.2 Application to Fruits, Vegetables,
and Juices
22.3.2.1 Spores and Vegetative Bacteria
Al-Bachir [42] investigated the effect of irradiation (0 to 2.5 kGy) on the quality
of two cultivars of Syrian grapes {Vitis vinifera) stored at 1 to 2°C for two
Novel Nonthermal Treatments 507
weeks. The irradiation treatment decreased spoilage caused by Botrytis cinerea
and improved the quality of both varieties. The optimum doses were 0.5 to
1.0 kGy for Helwani grapes and 1.5 to 2.0 kGy for Baladi grapes. The storage
periods were extended by 50% after irradiation at optimal doses for both
varieties.
Aziz and Moussa [43] studied the effect of irradiation on the viable
population of fungi and production of mycotoxins in randomly collected fruits
that included strawberries, apricots, plums, peaches, grapes, dates, figs, apples,
pears, and mulberries. Analysis of these fruits detected the mycotoxins
penicillic acid, patulin, cyclopiazonic acid, citrinin, ochratoxin A, and aflatoxin
B]. Irradiation of fruits at doses of 1.5 and 3.5 kGy significantly decreased the
total fungal counts compared with unirradiated controls. The corresponding
occurrence of mycotoxins in fruits decreased with increasing irradiation dose
and was not detected after treatments at 5.0 kGy.
Niemira et al. [44] irradiated frozen broccoli, corn, lima beans, and peas at
subfreezing temperatures ranging from —20 to — 5°C and determined the
influence of irradiation temperature on quality factors of frozen vegetables
as well as irradiation sensitivity of inoculated L. monocytogenes. The irradia-
tion resistance of L. monocytogenes changed significantly with the type of
vegetable and the treatment temperature. The levels of irradiation necessary
to reduce the bacterial population by 90% (D values) for L. monocytogenes
increased with decreasing temperature for all the vegetables that were
evaluated. D values ranged from 0.505 kGy for broccoli to 0.613 kGy for
corn at — 5°C and from 0.767 kGy for lima beans to 0.916 kGy for peas
at -20°C.
Lettuce inoculated with 1x10 CFU/g of acid-adapted E. coll 0157:H7 was
chlorinated at 200|ig/ml and irradiated at 0.15, 0.38, or 0.55 kGy by Foley
et al. [45]. The viability of E. coli 0157:H7, aerobic mesophiles, yeast, and
molds was measured over 10 days. Chlorination alone reduced counts of E. coli
0157:H7 by 1 to 21og 10 CFU/g. Chlorination combined with irradiation at
0.55 kGy produced 5.41og 10 reductions in E. coli 0157:H7 levels. When stored
at 1 and 4°C after irradiation at 0.55 kGy, standard plate counts and yeast
and mold counts were reduced by 2.51og 10 CFU/g for samples storage on day
17 without obvious softening of the lettuce or any other adverse effect on
sensory quality.
Niemira et al. [46] irradiated leaf pieces and leaf homogenate of endive
{dehor ium endiva) inoculated with L. monocytogenes (pathogen) or Listeria
innocua (nonpathogenic surrogate). Similar radiation sensitivity was obtained
for the two strains, but L. innocua was more sensitive to irradiation in
leaf homogenate than on the leaf surface. A dose of 0.42 kGy reduced
L. monocytogenes on inoculated endive by 99%; however, the pathogen grew
after 5 days of refrigerated storage until it exceeded the bacterial levels of
the control after 19 days of storage, but a dose of 0.84 kGy, equivalent to a
99.99% reduction, suppressed L. monocytogenes throughout refrigerated
storage. When increasing the doses up to 1.0 kGy, no significant change of
color was observed for endive leaves taken either from the leaf edge or the leaf
508 Microbiology of Fruits and Vegetables
midrib. Dose tolerances for acceptable texture of leaf edge and midrib material
were a maximum of 1.0 and 0.8 kGy, respectively.
Niemira et al. [47] also irradiated orange juices with varying levels
of turbidity and inoculations with Salmonella Anatum, Salmonella Infantis,
Salmonella Newport, or Salmonella Stanley at 2°C. Neither the resistance of
each isolate (D value) nor the pattern of relative resistance among isolates
was altered in orange juice. S. Anatum (D = 0.71 kGy) was significantly
more resistant than the other species in orange juice, followed by S. Newport
(D = 0.48 kGy), S. Stanley (D = 0.38 kGy), and S. Infantis (£> = 0.35 kGy).
Van Gerwen [48] analyzed the irradiation resistance of both spores
and vegetative bacteria based on the data available from the literature. As
expected, spores were found to have significantly higher D values with an
average of 2.48 kGy compared to the average D value for most vegetative
bacteria of 0.76 kGy. The notoriously radiation-resistant nonpathogenic vege-
tative bacterium Deinococcus radiodurans, had the highest D value of 10.4 kGy.
The average irradiation resistances for spores and vegetative bacteria were
further estimated to be 2.11 and 0.42 kGy after excluding specific conditions
showing extreme D values.
22.3.2.2 Parasites
According to Dubey et al. [49], outbreaks of cyclospora-associated gastro-
enteritis in humans have been epidemiologically linked to the ingestion of
fecally contaminated fruits (raspberries), vegetables (lettuce), or herbs
(basil). Also, Cryptosporidium oocysts have been demonstrated on vegetables.
Unsporulated and sporulated T. gondii oocysts were used as a model system to
determine the effect of irradiation on fruits contaminated with other coccidia
such as cyclospora or Cryptosporidium. Unsporulated oocysts of T. gondii
irradiated at 0.4 to 0.8 kGy sporulated, but were not infective to mice; however,
sporulated oocysts irradiated at doses greater than 0.4 kGy were able to encyst.
Sporozoites were infective but not capable of inducing a viable infection in
mice. T. gondii was detected in histological sections of mice up to 5 days,
but not 7 days after feeding oocysts irradiated at 0.5 kGy. Raspberries inocu-
lated with sporulated T. gondii oocysts were rendered nonviable after irradia-
tion at 0.4 kGy. An irradiation of 0.5 kGy was recommended in this study
to inactivate coccidian parasites on fruits and vegetables.
22.3.2.3 Viruses
Against viruses the effectiveness of irradiation is dependent on the size
of the virus, the suspension medium and/or type of food product, and the
temperature of exposure [50,51]. Because of their smaller size and genetic
makeup (often single-stranded RNA), most viruses are more resistant to
irradiation than bacteria, parasites, or fungi [51]. According to Bidawid et al.
[52], only a few studies have examined the efficiency of irradiation on viruses
in or on food products, including work on poliovirus in fish fillets [53],
Novel Nonthermal Treatments 509
coxsackievirus B in ground beef [54], and rotavirus and HAV in clams
and oysters [55]. Lettuce and strawberries were inoculated with HAV and
irradiated with doses ranging between 1 and lOkGy at ambient temperature
[52]. Plaque assays of HAV after irradiation showed a linear pattern of
inactivation, i.e., a linear decrease in virus titer occurred when the irradiation
dose was increased. Data analysis by a linear model indicated that D values
were 2.72 ±0.05 and 2.97 ±0.1 8 kGy for HAV in lettuce and strawberries,
respectively. These data were similar to those reported of 2.0 kGy for HAV
in both clams and oysters [53]. No noticeable deterioration was observed in the
texture and appearance of lettuces and strawberries, even at the highest dose
of 10 kGy.
22.3.3 Summary
Ionizing (gamma) radiation can be used to control microbiological spoilage
agents and pathogens and parasites and viruses in fruits, vegetables, and
juices, while increasing the shelf life without major damage to the physical or
chemical properties (texture, appearance, and sensory palatability); however,
the widespread use of irradiation is still limited mostly by concerns from
consumers, costs, and effect on the product quality. Irradiation, possibly in
combination with other processes such as mild heat or modified atmosphere
packaging, can provide a suitable means to improve produce safety.
22.4 PULSED ELECTRIC FIELDS IN JUICE PROCESSING
22.4.1 Introduction
Pulsed electric field (PEF) processing applies high voltage pulses to foods
located between a series of electrode pairs. The electrical fields (generally at 20
to 80 kV/cm) are achieved through capacitors that store electrical energy from
DC power supplies. During PEF treatment, the applied electric field increases
membrane permeability of microbial cells by either forming transmembrane
pores (electrical breakdown) or temporarily destabilizing the lipid bilayer and
proteins of cell membranes (electroporation), causing inactivation of micro-
organisms [56-58].
PEF units usually consist of three major parts: the PEF generation unit,
treatment chamber, and process control system (Figure 22.3). A PEF can be
generated in the form of exponentially decaying, square-wave, bipolar, instant-
charge-reversal, or oscillatory pulses, depending on the circuit design of the
generating device. The treatment chamber in a PEF unit holds two electrodes
in position with insulating materials that form a chamber. Electrochemical
reactions can occur at the electrode surfaces, causing partial electrolysis of
medium solution, electrode corrosion, and introduction of small particles
of electrode material into the liquid medium [59]. Use of very short pulses
or bipolar pulses is recommended to avoid the cumulative buildup of charges
and thus minimize electrode corrosion.
510 Microbiology of Fruits and Vegetables
High voltage pulse generator
Control system Treatment chamber
FIGURE 22.3 Schematic of a PEF unit.
Another major issue for the design of PEF treatment chambers is to
provide a relatively uniform electric field. For example, uniform electric fields
can be achieved with parallel plate electrodes if the distance between the
electrodes is sufficiently smaller than the electrode surface dimension. For
operation safety, pressure relief devices are necessary to avoid the destruction,
or the explosion, of PEF chambers due to the possibility of the buildup of
pressure, which can arise either from the expansion of dissolved air or partial
vaporization promoted from local heating within the PEF chamber after a
spark [60].
Critical processing factors for PEF include electric field intensity, pulse
width, treatment time, temperature, and pulse wave shape. The induced
potential difference across the cell membrane of a microorganism is pro-
portional to the applied electric field (electroporation theory). A lethal effect
to living cells is observed when the induced potential or transmembrane electric
potential exceeds by a large margin a critical value of approximately 1 V. Qin
et al. [61] stated that microbial inactivation increases with an increase in
the electric field intensity above the critical transmembrane potential. Pulse
width influences the critical electric field and the efficiency of microbial
inactivation [62]. Treatment time, defined as the product of pulse numbers and
pulse duration, affects microbial inactivation when either of the two variables
changes [63]. The efficiency for microbial inactivation varies with different
pulse wave shapes. Oscillatory pulses are the least efficient for microbial
inactivation; square wave pulses are more efficient than exponential decaying
pulses; and bipolar pulses are more lethal than monopolar pulses [64,65].
Treatment temperatures can change cell membrane fluidity and permeability,
thus affecting the susceptibility of cells to mechanical disruption [66]. Targeted
microorganisms (type, growth stage, and initial concentration) and properties
of the PEF treatment medium (pH, conductivity, and medium ionic strength)
also influence the microbial inactivation efficiency. Critical processing
factors of PEF treatment need to be monitored and recorded to ensure the
microbiological safety of the processed food products while maintaining
food quality with acceptable energy efficiency.
Novel Nonthermal Treatments 511
22.4.2 Application to Juices
PEF research had been mostly focused on the inactivation of microorganisms
suspended in foods, including semisolid and liquid foods such as pea soup,
milk, liquid eggs, and juices, particularly orange and apple juices. Sitzmann [67]
obtained a 31og 10 reduction of native microbiota for freshly squeezed orange
juice using a continuous PEF process with an electric field of 15kV/cm. There
was no significant change in quality. Zhang et al. [68] found that total aerobic
counts of reconstituted orange juice were reduced 3- to 4-log 10 cycles when
treated with an integrated PEF pilot plant system operating at less than 32 kV/
cm. Raso et al. [69] investigated PEF inactivation of ascospores and vegetative
cells of Zygosaccharomyces bailii suspended in apple, orange, pineapple,
cranberry, and grape juices. Two pulses of 32 to 36.5kV/cm decreased the
population of vegetative cells or ascospores 3.5 to 51og 10 cycles for each fruit
juice studied. Evrendilek et al. [70] treated fresh apple juice inoculated with
E. coli 0157:H7 and E. coli 8739 using bipolar PEF. A 51og 10 reduction was
obtained for each culture when the treatment temperature was below 35°C.
The lethality for fresh apple cider inoculated with E. coli 0157:H7 was also
reported for PEF treatment with instant charge reversal pulses [71]. In orange
juice, McDonald et al. [72] inactivated Leuconostoc mesenteroides, E. coli, and
L. innocua by as much as 51og 10 cycles at 30kV/cm and 50°C. A maximum of
2.51og 10 cycle reduction was achieved for Saccharomyces cerevisiae ascospores
at 50kV/cm and 50°C.
The synergy of PEF, pH, water activity, ionic strength, temperature,
antimicrobial agents (e.g., nisin, lysozyme), and other combinations of hurdle
technology (ozone treatment or high hydrostatic pressure) can increase micro-
organism inactivation [73-75]. Using the hurdle approach, Hodgins et al. [76]
studied the effect of temperature, acidity, and number of pulses on microbial
inactivation in orange juice. A 61ogi reduction in the natural microbiota
was obtained under optimal conditions consisting of 20 pulses of an electric
field of 80kV/cm, at pH 3.5, and a temperature of 44°C with a dose of 100 U
nisin/ml. The process was most influenced by a change in temperature
(p < 0.0001). There was a 97.5% retention of vitamin C along with a 92.7%
reduction in pectinmethylesterase activity after PEF treatment. The shelf life of
the orange juice was at least 28 days when stored at 4°C without aseptic
packaging. Gas chromatography revealed no significant differences in aroma
compounds before and after pulsing.
Liang et al. [77] applied PEF to pasteurized and freshly squeezed orange
juices (with and without pulp) and determined the reduction of Salmonella
Typhimurium at moderately high temperatures (<60°C). The effect of anti-
microbial compounds (nisin and lysozyme) was examined. PEF treatment
(90 kV/cm and 20 pulses) did not have a notable effect on cell viability or injury
until the temperature reached 46°C or above. Presence of nisin, lysozyme, or
a mixture of nisin and lysozyme increased cell viability loss by an additional
0.04 to 2.751ogio cycles for PEF treatment. The combination of nisin and
512 Microbiology of Fruits and Vegetables
lysozyme had a more pronounced bactericidal effect than did either nisin or
lysozyme alone.
The inactivation of enzymes also accompanies pasteurization of juices
using PEF technology. In general, higher electric field strengths and longer
total treatment times are more effective for the purpose of enzyme inactiva-
tion [78]. Decrease of polyphenoloxidase (PPO) activity in peach juices was
reported to follow an exponential decay kinetic model [79]. For those
orange juices that had a similar shelf life (196 days at 4°C) after thermally
processing (90°C and 90 seconds) and PEF processing (40kV/cm and 97
milliseconds), ascorbic acid, flavor, and color of PEF-treated juice were
found to be superior to that of thermally processed juice (P < 0.05) [78]. This
is also true for tomato juices processed either by PEF at 40 kV/cm
(57 milliseconds) or thermally processed (92°C and 90 seconds) and stored
at 4°C for 112 days [80]. In both cases, sensory evaluations indicated that the
flavor of PEF-processed juices was preferred to that of thermally processed
juices (P < 0.01) [78,80].
22.4.3 Summary
PEF technology is effective in the inactivation of microorganisms in liquid
foods, particularly in orange and apple juices, without significant changes to
sensory quality. Advantageous preservative effects of PEF with other process
treatments, including ozone treatment, pressure, pH, water activity, ionic
strength, temperature, antimicrobial agents (e.g., nisin, lysozyme), has been
demonstrated.
22.5 ULTRASONIC WAVES FOR PRESERVATION OF
FRUIT AND VEGETABLE PRODUCTS
22.5.1 Introduction and Description of Process
The oscillation of a vibrating body can cause a periodic disturbance that
travels through an elastic medium (air, ground, or water) and radiates outward
in straight lines in the form of a pressure wave perceived as sound. Based
on whether or not it can be heard by the human ear, sound can be divided
into communication waves (audible) and ultrasonic waves (or ultrasound,
inaudible). Ultrasound, having little or no effect on the ear even at high
intensities, vibrates at frequencies greater than 20 kHz and is produced by
a transducer, which contains a piezoelectric substance such as a quartz
crystal oscillator and converts high-frequency electric current (an input of
energy) into vibrating ultrasonic waves (an output of energy) with a fixed
relationship.
Ultrasound was first developed in World War II to locate submerged
objects. Additional uses have been developed for industrial applications in the
field of nondestructive testing, cleaning, welding, and sonochemistry [81].
Novel Nonthermal Treatments 513
22.5.2 Microbial Inactivation
The fluctuating pressures induced by an ultrasonication process produce
and break microscopic bubbles, creating micromechanical shocks to disrupt
cellular structural and functional components up to the point of cell lysis
[82]. Intracellular cavitations make ultrasound capable of inactivating micro-
organisms [83]. The inactivation effect depends on the control of critical factors
including the amplitude of ultrasonic waves, the exposure or contact time
with the microorganisms, the type of microorganism, the volume of food to
be processed, the composition of the food, and the temperature of treatment
[82]. The mechanism of inactivation of vegetative bacteria appears to be
intracellular cavitations that lead to cellular lysis, but ultrasound alone has
no effect on spores. Cavitations may play an auxiliary role and allow ultra-
sound to assist other methods in spore inactivation. This limits the singular
use of ultrasound as a preservation method, requiring the use of a combin-
ation of ultrasound with other preservation processes (e.g., heat and mild
pressure) for industrial applications.
Palacios et al. [84] examined the effect of ultrasound on the heat resis-
tance of spores. After ultrasound treatment (20 kHz, 120 W, 12°C, 30 minutes),
several substances were detected to be released from B. stearothermophilus
spores to the surrounding aqueous medium, including calcium, dipicolinic
acid, a glycopeptide of 7kDa, fatty acids, acyl glycerols, and glycolipids
(but no phospholipids). The release of low-molecular-weight substances from
the spore protoplast and the consequent modification of its hydration state
led to the heat resistance reduction.
The presence of spoilage bacteria, yeasts, molds, and the occasional
pathogen on fresh produce is not uncommon. Seymour et al. [85] examined
the potential of ultrasound in cleaning minimally processed fruits and vege-
tables. Cut iceberg lettuce (100 g) inoculated with S. Typhimurium (10 CFU/g)
was washed for 10 minutes with tap water (control), a 25ppm free chlorine
dip only, ultrasound (32 to 40 kHz, 10 to 15W/1) only, and ultrasound
combined with the 25ppm free chlorine dip. The control reduction was
0.71og 10 , while reductions of 1.61ogio and 1.71og 10 were obtained from wash-
ing treatment by ultrasound and chlorine individually. Reductions obtained
from the combined washing treatment were 2.6 to 2.71og 10 , corresponding to
a 99.8% reduction in total bacteria. The cleaning action of cavitations
appeared to remove cells attached to the surface of fresh produce, rendering
the pathogens more susceptible to the sanitizer. The frequency of ultrasound
(25, 32 to 40, 62 to 70 kHz) showed no significant effect on decontamination
efficiency (P > 0.69).
Yeasts such as S. cerevisiae and Zygosaccharomyces spp., including Z. bailii
and Z. rouxii, and pathogenic bacteria like L. monocytogenes can cause
significant spoilage and affect the safety of nonpasteurized fruit juice products.
Traditional thermal pasteurization methods can detrimentally affect the
organoleptic properties when they are used to extend the shelf life for fruit
juices. Mincz et al. [86] explored the potential use of ultrasound combined with
514 Microbiology of Fruits and Vegetables
refrigeration to extend the shelf life of fresh juice products. Freshly squeezed
lemon and pineapple juices inoculated with S. cerevisiae, Z. bailii, Z. rouxii,
and L. monocytogenes were immediately sonicated at 20 kHz at 45°C and an
amplitude of 95 um. The treated samples were stored in sterilized glass
containers (10 ml) at 7°C for 15 days. The microbial population and color
of the inoculated samples was monitored at preset intervals during storage.
The combined ultrasound and refrigeration treatment significantly suppressed
microbial growth in fresh lemon and pineapple juices with improved color
retention. No significant change in pH and a w was observed.
22.5.3 Summary
Ultrasonic waves have the potential to inactivate microorganisms in fruits,
vegetables, and juices. In most cases, a combination of ultrasound with
other preservation processes is probably more realistic to achieve an effective
degree of microbial inactivation. Further efforts are required for its application
as a component in a commercially feasible preservation process.
22.6 ELECTROLYZED WATER
22.6.1 Introduction
Electrolyzed oxidizing (EO) water, also called strongly oxidizing water,
strongly acidic electrolyzed water, or acidic oxidative potential water, has
recently attracted interest in medicine, agriculture, and food processing for
purposes of sanitation.
An EO water generator usually contains a power supply, and a pair
of electrodes (i.e., anode and the cathode) installed in two individual cells
that hold sodium chloride solutions separated by a specialized membrane
(Figure 22.4). When electrolyzing saline solutions, hydrogen is generated in the
cathode side and chlorine is generated in the anode side. The chlorine further
reacts with water to form HOC1 and HC1. EO water is then produced in the
cell installed with anodes and electrolyzed reducing (ER) water is produced
in the cell installed with cathodes.
EO water contains free chlorine and has a high oxidation-reduction
potential (ORP, above lOOOmV) and low pH (around 2.3). ER water exhibits a
high pH (above 11.0), low redox potential (RP, below 800 mV), low levels
of dissolved oxygen, and high levels of dissolved molecular hydrogen. The
chlorine gas, HOC1, and OCP ions contained in EO water contribute to
the availability of uncombined chlorine radicals or free available chlorine, the
primary component responsible for the disinfection ability of EO water
[87-89]. A generator without a separating membrane produces water at pH
6.8 because HC1 formed on the anode side neutralizes NaOH on the cathode
side [90]. EO water can be preserved for one year under shaded and sealed
conditions [91], but EO water becomes inert after three days when exposed
to light.
Novel Nonthermal Treatments
515
+
HoO
hi
Na
+
CI
H
+
Cathode
OCI
H 2
HOCI
Cl 2
2
Anode
FIGURE 22.4 Schematic of an EO water generator.
22.6.2 Application as a Novel Disinfectant for
Fruits and Vegetables
EO water has been extensively applied to fresh or fresh-cut vegetables and
fruits because of its strong bactericidal effects [88,92]. Izumi [93] utilized EO
water to reduce the total microbial counts of fresh-cut carrots, bell peppers,
spinach, and potatoes and found microbial counts on all cuts reduced by
0.6 to 2.61og 10 CFU/g. The bactericidal effect of EO water increased with
available chlorine in the range of 15 to 50ppm for fresh-cut carrots, spinach,
or cucumbers. Tissue pH, surface color, and general appearance of fresh-cut
vegetables were not affected after treatment.
Park et al. [94] examined the efficacy of EO water and acidified chlorinated
water (45 ppm residual chlorine) against E. coli Ol 51:117 and L. monocytogenes
on lettuce. Each leaf was surface-inoculated and immersed in 1.51 of EO or
acidified chlorinated water for up to 3 minutes at 22°C. Compared to water
washes, a 3-minute EO water rinsing significantly decreased mean populations
of E. coli 0157:H7 and L. monocytogenes by 2.4 and 2.71og 10 CFU per lettuce
leaf, respectively (p < 0.05). There was no significant difference between the
bactericidal activity of EO water and acidified chlorinated water (p > 0.05),
and no obvious quality change was observed during two weeks of storage
after washing.
Fresh tomatoes were surface-inoculated with E. coli 0157:H7,
S. Enteritidis, L. monocytogenes, and nonpathogenic E. coli and rinsed in
neutral EO water for up to 60 seconds [95]. EO water rinsing reduced the
surface population from 51og 10 to < 1 log 10 CFU/cm independent of the type
of microorganism and treatment time. No cells were detected in the washing
solution. There was no significant difference in organoleptic qualities
compared to untreated tomatoes.
516 Microbiology of Fruits and Vegetables
Ratna and Demirci [96] applied EO water to alfalfa seeds and sprouts
inoculated with a five-strain cocktail of nalidixic acid-resistant E. coli
0157:H7. Reductions were in the range 0.2 to 1.6 and 1.1 to 2.71ogio CFU/g
for treated seeds and sprouts, respectively, corresponding to a percentage
reduction of 38.2 to 97.1 and 91.1 to 99.8%, respectively. Germination of
the treated seeds was reduced from 92 to 49% when soaking time and the
electric current used to generate EO water were increased. No visible damage
occurred to the sprouts.
Besides the inactivation of bacteria, EO water can also serve as an effective
fungicide on fruits and the foliage and flowers of bedding plants. Al-Haq et al.
[97] immersed peach inoculated with Monilina fructicola in EO water up to
5 minutes to examine its effectiveness against postharvest brown rot. EO water
did not control brown rot in wound-inoculated peaches, but did reduce the
incidence and severity in nonwounded inoculated fruit. No chlorine-induced
phytotoxicity was observed on treated products. In this case, EO water was
an effective surface sanitizer that delayed disease development. Al-Haq et al.
[98] also evaluated the effects of EO water on suppressing fruit rot of pear
caused by Botryosphaeria berengeriana. Pears with wounds necessary to cause
ut bot rot" were inoculated with spore suspensions of B. berengeriana and
immersed in EO for 10 minutes. No chlorine-induced phytotoxicity was
observed on the treated fruits, and EO water suppressed the incidence and
severity of disease, suggesting that EO water can be used as surface sanitizer
to possibly reduce postharvest fungal rot development.
22.6.3 Summary
EO water has demonstrated disinfecting ability against bacteria and fungi. EO
water is easy to use and environmental friendly; however, further study
regarding quality changes after EO water rinsing is necessary for commercial
use on fresh-cut fruits and vegetables.
22.7 FINAL REMARKS AND FUTURE PERSPECTIVES
The success of any fruit and vegetable preservation technology depends on a
complete and correct understanding of the reasonable causes of food spoilage
and associated foodborne illness. To ensure safety and prolong shelf life,
conventional thermal processing is a mainstay of the food industry; however,
driving forces from the market, and development and introduction of
nonthermal processing treatments by the food industry and research institu-
tions have provided a well-accepted platform to deliver safe products processed
at lower temperatures with minimum quality losses. The potential use of each
of the described nonthermal treatments in combination with other procedures,
including established manipulations employing temperature, water activity
adjustment, oxidation-reduction potential and pH controls, and modified
atmosphere packaging, is worth considering in food product development.
A good indication of this potential is the commercial success now being
Novel Nonthermal Treatments 517
realized with pressurization methods to preserve fruits and vegetables. With
pressure-treated jams, jellies, juices, salsa, guacamole, and chopped onions
available on the market, one can assume that more products will follow.
ACKNOWLEDGMENT
The authors are with the Department of Animal & Food Sciences, University
of Delaware, Newark, DE. The authors wish to acknowledge the support
provided by the USDA (grant no. 2001-35201-09947).
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23
Biological Control of
Microbial Spoilage
of Fresh Produce
Julien Mercier and Pamela G. Marrone
CONTENTS
23. 1 Introduction 523
23.2 Approaches to Biocontrol in Postharvest Situations 524
23.2.1 Use of Naturally Occurring Antagonists for
Colonization of Infection Sites 524
23.2. 1 . 1 Postharvest Applications 524
23.2. 1 .2 Preharvest Applications 525
23.2.1.3 Possible Mechanisms for Biocontrol 526
23.2.2 Use of Mutant Pathogen Strains 527
23.2.3 Biological Fumigation 528
23.3 Advantages and Limitations of Postharvest Biocontrol 528
23.3. 1 Advantages of Postharvest Biocontrol 528
23.3.2 Disadvantages of Biocontrol Agents 529
23.4 Enhancing Biocontrol Activity 530
23.4. 1 In Combination with Other Treatments 530
23.4.2 Improvement in Formulation 531
23.4.3 Screening and Selection of the Microorganism 532
23.4.4 Collaborative Research Among Industry, University
Researchers, Government, and Growers/Packers 532
23.5 Regulatory Process for Biocontrol Agents 533
23.5.1 U.S. Environmental Protection Agency (EPA) 533
23.5.2 California and International Regulations 534
23.6 Concluding Remarks 534
References 535
23.1 INTRODUCTION
With the registration and commercialization of biocontrol products such as
Aspire (Candida oleophila) and BioSave (Pseudomonas syringae) in the U.S.
523
524 Microbiology of Fruits and Vegetables
and YieldPlus {Cryptococcus alhidus) in South Africa, biological control has
become a new tool for managing storage diseases, which until recently were
only controlled by chemical and cultural means. With the large volume of
recent publications on this subject from several on-going research programs,
it is possible that more products will be submitted to regulatory agencies
and commercialized in the future. The needs for alternatives to chemical
control have in a large part been responsible for this effort. Not only is there
demand for more organic or "chemical-free" produce, but also many countries
have lowered the residue tolerance for many chemical pesticides, putting
pressure on exporters to have low residues on the fruit they ship abroad.
Furthermore, risk assessments for fungicides make postharvest applications
less attractive for pesticide companies, as this might cause them to limit
the amounts that can be sold in larger, more profitable markets, such as
field crops. Finally, with the prevalence of fungicide resistance in certain
pathogen populations [1], biofungicides can be used to manage such resistance
and help extend the commercial life of some chemical products. This chapter
provides an overview of the different uses of biocontrol agents for the
management of postharvest decay, discussing their possibilities and limitations,
as well as possible modes of action.
23.2 APPROACHES TO BIOCONTROL IN
POSTHARVEST SITUATIONS
23.2.1 Use of Naturally Occurring Antagonists
for Colonization of Infection Sites
23.2.1.1 Postharvest Applications
So far, the biological control of postharvest diseases with naturally occurring
microorganisms has relied essentially on the inundative approach, that is,
the mass introduction of a microbe to establish an antagonistic population on
wounds and other possible infections sites on fruits or tubers. Little work
has been done on other approaches such as the enhancement of the existing
surface microflora. While many antagonistic microbes were initially selected
from the fruit microflora [2], their natural populations are likely to be too
low to have a significant impact on plant pathogens [3]. Mass introduction
of yeasts or bacteria permits achieving an instant antagonistic population
that would never be attained under normal conditions. This approach has
several advantages since a drench or spray treatment of several commodities
can easily be applied as the harvested commodities are brought in from the field
before packing or storage. If the process is performed in a timely fashion, most
wounds sustained during harvesting and handling should become protected
before significant pathogen development can occur. Treatment after harvest
can also be more economical than treating a whole orchard or field and
allows use of more concentrated cell suspensions.
Biological Control of Microbial Spoilage of Fresh Produce 525
The purpose of antagonists is to colonize rapidly possible infection sites
and protect them from infections. Usually, populations of effective antagonists
increase rapidly initially and stabilize thereafter. Such a colonization pattern
can be seen in fruit wounds treated with yeasts such as Candida oleophila
[3,4], Cryptococcus albidus [5], C. laurentii [6], and Pichia membranefaciens [7]
and bacteria such as Pseudomonas syringae [2]. However, wounds can have
a narrow window for optimal colonization as they dry out [8]. This requires
that application takes place as soon as possible after harvest and handling, as
antagonists usually have little curative activity [2]. Also, wounds on oil glands
of citrus fruit were found to be more difficult to colonize by C. oleophila,
resulting in poor decay control [4].
Research has shown that there may be limitations to the postharvest use
of antagonists [2], although results comparable to those obtained with syn-
thetic fungicides can sometimes be achieved [9]. Often, a lack of curative
activity is the main problem, as efficacy becomes much reduced or nil when
antagonists arrive on wounds after pathogens [2,5,10]. Also, antagonists
may have different efficacy depending on the fruit species [11] or the type of
decay [12]. These limitations likely result from the mode of action of a given
antagonist or differing ability to colonize and establish on various commodi-
ties. For these reasons, antagonistic yeasts or bacteria cannot be considered
as ''silver bullets,'' and as biological systems are more sensitive to environ-
mental conditions than chemical agents. Depending on the disease system,
improvements in formulation or combination with other treatments
may help increase decay control and meet the requirements of the horticultural
industry. Most probably, further improvements in efficacy are likely to
come from a better understanding of the mode of action and ecology of these
antagonists.
23.2.1.2 Preharvest Applications
While most of the research on naturally occurring antagonists has focused
on postharvest treatment, there have been positive reports recently on the use
of preharvest applications of antagonists to control postharvest diseases
[13-15]. Such application can be done periodically during the growth of the
fruit, up to the day of harvest. Field application can help achieve an early
colonization of possible infection sites and reduce incipient infections from
the field. Also, it can make biological control possible in crops that are too
fragile or incompatible with postharvest drenching or spraying, such as
grapes and soft fruits. However, field application of antagonists will expose
them to possibly adverse environmental conditions such as desiccation
and solar radiation, which they will have to withstand in order to be effective.
In this situation, the selection process must be different than for postharvest
application and take into account the ability of antagonists to survive on
the intact fruit surface. Benbow and Sugar showed that certain yeasts are
naturally adapted to field conditions and could maintain their populations
on pear fruit for three weeks [13]. Using another strategy, Teixido et al. used
526 Microbiology of Fruits and Vegetables
culture media with low water activity to help Candida sake adapt to water
stress [15]. Such physiologically modified yeasts were better adapted to
colonize apples in the orchard. Our own field research on preharvest appli-
®
cations with Bacillus subtilis (Serenade AS) has shown promise on stone fruit
for control of monilinia. On apricots artificially inoculated after harvest with
Monilinia, there was only 8% infection when sprayed preharvest with Serenade
(applied as 4Qt in 100 gal of water per acre) compared to 52% infection
in untreated fruit. A rate of 4oz per 100 gal of Elite 45WP (tebuconazole) had
0% infection. In a second test, there was 14% infection with Elite, 28% with
Serenade, and 71% in the untreated fruit.
Inoculated trials are the most severe test, and in an actual postharvest
situation the results are likely to be better. A trial was conducted with the
University of California at Davis on Bing cherries inoculated postharvest with
brown rot (monilinia) and gray mold {Botrytis cinerea) after preharvest
treatment (air blast sprayer, 100 gal water per acre) with Serenade (6 lb) and an
adjuvant (Sylgard) and chemical pesticides. The incidence of brown rot decay
with the Serenade treatment was 5.6%, compared to 4.5% for triflumizole
(Procure 4SC) (rate of 12fl oz), 0.4% for iprodione (Rovral) 4F (1 Qt), and
32% in the untreated fruit. Against gray mold, there was a 1% incidence of
decay (percent of fruit with decay) with Serenade treatment compared to 0%
incidence for both Procure and Rovral, and 2.9% incidence for untreated fruit.
23.2.1.3 Possible Mechanisms for Biocontrol
There has been no systematic study of the mode of action of any given
postharvest biocontrol agent, and most possible inhibition mechanisms remain
unproven at this time. Antagonists could act in passive ways, simply using space
or nutrients needed by pathogens, or directly interact with the pathogen to
cause inhibition through parasitism or the synthesis of inhibitory molecules,
such as antibiotics or hydrolytic enzymes. Finally, the triggering of defense
responses in the host, resulting in enhanced resistance, could also be part of
the biocontrol mechanism. It is quite possible that in many cases, a number of
active and passive mechanisms are involved and act together which make
it even more difficult to decipher the basis of the biocontrol phenomenon.
Many antagonistic yeasts that are good wound colonizers are not asso-
ciated with any obvious inhibitory mechanism. By simply colonizing and
forming a cell layer on the surface of wounds, these antagonists may act by
competitive or pre-emptive exclusion, blocking access to the infection site.
This mechanism is difficult to prove but its occurrence is plausible, especially
when a colonization period is required for the antagonist to be effective.
Along with competitive exclusion, competition for nutrients is often claimed in
the absence of other more obvious or active mechanisms and is supported by
the fact that yeasts or yeast-like organisms were able to remove amino acids or
sugars in nutrient wells or in wounds [16,17]. Also, the addition of nutrients
was shown to cancel antagonistic activity [18]. However, in many cases, the
question remains as to whether nutrients in wounds are really limiting for
Biological Control of Microbial Spoilage of Fresh Produce 527
pathogens. Furthermore, yeasts that effectively remove nutrients in wounds
are not necessarily good antagonists [16]. As in leaf surface bacteria, where
nutrient-regulated reporter genes were used to study nutrient consumption
on leaves [19,20], such molecular tools in antagonists or pathogens could be
useful for elucidating the question of nutrient competition in wounds.
The involvement of active mechanisms such as antibiotic production
by Pseudomonas syringae [21], production of cell wall degrading enzymes
by Aureobasidium pullulans [18] or Pichia anomala [22], or attachment to
pathogens by various bacteria and yeasts [23,24] have been associated with
biocontrol activity on fruits. Again, the definite role of these mechanisms in
biocontrol is difficult to demonstrate, and the use of molecular tools might
be the best approach to elucidate the role of those antifungal factors.
Such a molecular approach was used by Grevesse et al. to elucidate the role
of (3-1,3-glucanase produced by the antagonistic yeast P. anomala [25].
The biocontrol activity of the yeast remained unaffected by the shut down of
P-l,3-glucanase production from the disruption of a gene involved in the
production of the enzyme, thus dismissing its role in antagonism.
So far, the induction of disease resistance in stored fruits and vegetables
by antagonists has been little studied. In most cases, it is not known whether
biocontrol agents can induce such defense responses. Defense enzymes such
as P~l,3-glucanase, chitinase, and peroxidase were induced in apple wounds
by A. pullulans [26]. In oranges, Arras reported the accumulation of the phyto-
alexins scoparone and scopoletin in response to Candida famata [27]. While
these defenses could contribute at least in part to the biocontrol activity, the
importance of induced resistance in postharvest biocontrol remains unknown
and its possible role is yet to be demonstrated. It is possible that many more
antagonists can trigger defense responses and enhance host resistance. It is
likely that biocontrol action relying on induced defenses would be rather host-
specific, as harvested fruits and vegetables vary in their ability to respond to
elicitors and produce defense responses. More advances in the development
of biofungicides are likely to come when we better understand mechanisms of
biological control on stored commodities.
23.2.2 Use of Mutant Pathogen Strains
Little work has been done on the use of pathogens for biological control.
Mutant or attenuated plant pathogens could be used to induce disease
resistance or to compete against wild pathogen populations on the host. One
promising avenue for the control of aflatoxin production in grain, nuts, and
dry fruit involves the use of Aspergillus flavus strains that cannot produce
toxins [28,29]. These strains are applied in the field, where they compete
and help suppress the wild populations of Aspergillus spp. While we know
that localized inoculation with Sclerotinia sclerotiorum and Botrytis cinerea
can induce systemic disease resistance in cold-stored carrot [30,31], to our
knowledge, there has been no study on the use of attenuated pathogens for
decay control in fresh fruits and vegetables.
528 Microbiology of Fruits and Vegetables
23.2.3 Biological Fumigation
The production of volatile antibiotics is rare among microorganisms and has
been reported only in a few soilborne organisms such as Trichoderma spp. and
Bacillus spp. [32,33]. A recently discovered fungus, Muscodor albus, produces
about 28 volatile compounds, mainly alcohol, ester, ketone, and acid
derivatives, which together can inhibit or kill fungi, bacteria, and oomycetes
[34]. The fungus, which was isolated from a cinnamon tree in Honduras, was
described as a new genus and is related to endophytes of the family Xylariaceae
(Ascomycetes) [35]. Recently, the possibility of controlling postharvest decay
by biological fumigation with M. albus was demonstrated by Mercier and
Jimenez [36]. Biofumigation was performed passively by placing a grain culture
of the fungus in the presence of inoculated fruits. Diseases controlled by such
biofumigation treatment were gray mold of apples and grapes, caused by
Botrytis cinerea, blue mold of apples, caused by Penicillium expansum, brown
rot of peaches, caused by Monilinia fructicola, and green mold and sour rot of
lemons, caused by P. digitatum and Geotrichum citri-aurantii, [36,37]. Several
storage pathogens belonging to species of botrytis, colletotrichum, geotrichum,
monilinia, penicillium, and rhizopus were also killed in vitro by exposure to
volatile compounds produced by a potato dextrose agar colony of M. albus
[36]. This suggests that pathogens are not merely inhibited but are killed in fruit
wounds. In some cases, there was effective decay control when biofumigation
was performed 24 hours after inoculation. Fumigation at low storage
temperature was also effective in grapes [37] and apples (J. Mercier,
unpublished data). Besides controlling postharvest decay, biofumigation with
M. albus also reduced populations of pathogenic bacteria such as Escherichia
coli 0157:H7, salmonella serotypes, Shigella spp., and Listeria monocytogenes
on the surface of cucurbit and tomato fruits [38]. Biofumigation treatment with
M. albus would be applicable to different stages of storage and shipping in
most commodities. It could also be used with commodities that are too fragile
to be handled or to receive a liquid fungicide treatment, such as strawberries or
grapes. Exposure to the volatiles does not cause any off-flavor of the treated
produce in informal taste tests. There were no detectable volatile compounds in
the skin of any fruit tested (apples, peaches).
23.3 ADVANTAGES AND LIMITATIONS OF
POSTHARVEST BIOCONTROL
23.3.1 Advantages of Postharvest Biocontrol
Biocontrol agents have the following advantages:
1. Complex mode of action: low chance for resistance development.
Biocontrol agents work in complex ways, rather than having a single
site of action as for some chemical pesticides. For example, a
Biological Control of Microbial Spoilage of Fresh Produce 529
biocontrol agent may act via antagonism and antibiosis (production
of multiple compounds by the organism). If used in a program
with chemical pesticides as tank mixes, biocontrol agents could delay
development of resistance to chemical products, which has been
documented and can be a problem in commercial production [39,40].
2. Reduction or elimination of chemical residues. The production of
fruit and vegetables is now a global enterprise, and products are
shipped around the world for export markets. As such, chemical
residues are a consumer concern as well as nontariff trade barriers.
Products can be rejected due to the presence of residues of specific
chemical products not allowed in importing countries or because
residue levels exceed limits allowed by importing countries and
companies [41]. In addition, the Food Quality Protection Act of
1996 requires that the registration and use of a product take into
account the amount of pesticide residue that occurs in foods eaten
frequently by children. If a product is also used preharvest, the "risk
cup" (total amount of allowable residues on all crops) from
postharvest use may result in restrictions of quantity and frequency
of applications for preharvest use. This may not be economically
feasible to companies who make more money on preharvest markets.
Biocontrol agents are exempt from residue tolerances and thus not
subject to international rules regulating chemical residues.
3. Safety to workers and the environment. Worker safety is a significant
worldwide concern. Postharvest handling of fruits and vegetables
may result in worker exposure to chemical residues of products that
are carcinogens or acutely toxic. For example, certain governments
in Central America have strongly encouraged banana producers
to reduce the use of chemicals due to worker exposure [42].
Use of biocontrol agents will reduce the worker exposure to these
toxic chemicals. Waste chemicals from fruit dipping operations are
an environmental issue. Biocontrol agents are biodegradable, leave
no chemical residues, and do not pollute the environment and
ground water.
23.3.2 Disadvantages of Biocontrol Agents
Biocontrol agents have not met with significant commercial success for post-
harvest applications and have remained relegated to small niches despite
the market need for methods to reduce the development of resistance to
chemical products. Biocontrol agents have the following disadvantages:
1. Special handling and timing and use restrictions. Some biocontrol
agents cannot be tank mixed with other fungicide products because
the fungicides will kill (and thus deactivate) the biocontrol agent.
This limits their use and negates the most positive reason to use
530 Microbiology of Fruits and Vegetables
them: resistance management. Also, when a biocontrol agent is
an antagonist to the pathogen, timing the application is crucial.
The microorganism must be applied shortly before or at the same
time as exposure to the pathogen for efficacy. This reduces the
agent's practicality in packinghouses.
2. Efficacy. Efficacy reports are mixed largely because users expect
to use a biocontrol product like a chemical without special regard
to timing and handling. Efficacy can equal or approach chemi-
cals if the biocontrol agent is used exactly as required and
directed, with an understanding and appreciation of the mechanism
of action.
3. Shelf stability. Some biocontrol agents do not produce spores
and consist only of bacterial cells or fungal mycelia, and as a result
the shelf life of a biocontrol product may not match that of a
chemical product. Maintaining a high degree of viability and efficacy
in formulated biocontrol agents can be a challenging task. As with
most pesticides, biofungicides are commonly dehydrated into
powder or concentrated into liquid formulations. Such processes
can have a negative impact on disease control efficacy. As most
information on the formulation of biopesticides is proprietary, there
is limited literature on the subject. For example, freeze-drying of
Candida sake caused cell mortality and greatly affected control of
blue mold of apple, compared to that obtained with fresh cells [43].
Protective agents and additives, such as skimmed milk, peptone,
or lactose, at the time of freeze-drying and rehydration can somewhat
help reduce cell mortality of C. sake [43]. As mentioned above,
growing yeast cells at low osmotic potential can be used for better
desiccation adaptation [15].
23.4 ENHANCING BIOCONTROL ACTIVITY
23.4.1 In Combination with Other Treatments
Under the experimental conditions used by some researchers, there have
been occurrences of disappointing results when the currently registered
biocontrol products were used as stand-alone treatments [44-46]. Research
on biocontrol organisms, whether for pre- or postharvest uses, is typically
reductionist — testing one product versus another in stand-alone trials. When
the biocontrol agent does not perform equally well as the chemical in these
stand-alone trials, the biocontrol agent may be pronounced inferior, and that
is usually the end of the story. Research shows that mixtures of biocontrol
agents with other biorational products can provide excellent efficacy not seen
in stand-alone trials. For example, Conway et al. showed that they could
eliminate postharvest decay caused by Collet otrichum acutatum on apples with
a combination of either of two antagonists and heat [47]. Either antagonist
alone eliminated decay caused by Penicillium expansum, but the two were
Biological Control of Microbial Spoilage of Fresh Produce 531
more effective together. Combinations with chemical agents should be tested
as well to prevent resistance, increase efficacy, and reduce the amount of
chemicals that could be used. Continued funding for research of this type will
increase the probability that biocontrol agents will be more widely adopted
for postharvest uses. For this reason, combination with other treatments might
be envisaged to achieve more consistent and robust disease control. Biocontrol
agents are not necessarily incompatible with chemical fungicides, which can
often be used at reduced rate in such situations. The use of several antagonistic
bacteria and yeasts, including the agents in BioSave and Aspire, has been
shown to be compatible with postharvest chemical fungicides at full or reduced
rates, and such combinations have resulted in higher efficacy than biocontrol
agents alone [5,7,12,44-46,48,49]. Lowering the use rates of chemical fungi-
cides, while helping reduce chemical residues on fruit, can also compensate
for possible shortcomings of biofungicides against early or incipient infections
and sensitivity to environmental conditions.
The combination of biocontrol agents with other storage technologies
is not only desirable, but necessary. Biocontrol agents should be compatible
with low temperature and controlled or modified atmosphere storage, as these
methods are commonly used to extend the shelf life of fruits and vegetables.
So far, reports of combining antagonistic yeasts with controlled or modified
atmospheres have been positive, improving the performance of yeasts in high
C0 2 and low 2 compared to storage in air [11,12]. Several antagonistic
yeasts are capable of growth and survival on fruit held in cold storage
[3,5,49,50], which make them compatible for use on commodities that require
rapid chilling and storage at low temperatures. Combination of biocontrol
agents with other cultural disease control methods like fruit curing [51] or heat
treatment [52,53] could also be advantageous depending on the commodity.
Finally, the combination of two or more antagonists with different charac-
teristics, which could be considered as mixtures of microorganisms, can be
more effective in controlling decay than single agents [3,10]. While it has
been suggested that the postharvest environment can be manipulated some-
what to accommodate biological control, it is likely that the most successful
biofungicides will be effective under the normal handling and storage
conditions used for each commodity.
23.4.2 Improvement in Formulation
The addition of nontoxic substances in biofungicide formulations or to
spray preparations could also be used to improve disease control. Although
their mode of action is often poorly understood, these "inert" ingredients
can act in several ways, such as reducing disease susceptibility, inhibiting
pathogens, or improving wound colonization or the inhibitory action of
antagonists. Combining yeasts with calcium salts [5,7,54], sodium bicarbonate
[55,56], or ammonium molybdate [56,57] resulted in better control of fruit
diseases. Since salts are inexpensive additives, more research should be
done on their mode of action and their compatibility with biofungicides.
532 Microbiology of Fruits and Vegetables
Other chemicals such as sugar analogs like 2-deoxy-D-glucose [58] and
chitosan [59] also improved decay control by yeasts. Some amino acids can
enhance biocontrol activity and improve colonization of wounds by
antagonists [50,60]. Thus, further enhancement of the activity of biocontrol
agents can be expected with benign chemicals likely to be suitable for use on
food crops.
23.4.3 Screening and Selection of the
Microorganism
Relatively few microorganisms have been screened for postharvest use.
AgraQuest (Davis, CA) screened over 20,000 microorganisms of diverse
taxonomies for preharvest uses and found many microorganisms that equal
or approach the efficacy of chemical pesticides. The same approach could be
taken with screening for postharvest use.
There are many microorganisms that have a long shelf life due to hardy
spores (bacillus, some actinomycetes, and some fungi). Efforts to date
have focused largely on microorganisms wherein the living cells act as antago-
nists by competing with the plant pathogen, as opposed to microorganisms
that work through other mechanisms such as antibiosis. Microorganisms
are known to produce compounds that can disrupt the membranes of plant
pathogens [61]. By releasing these compounds, such microbes can be effective
in tank mixes and perform more like chemical pesticides.
23.4.4 Collaborative Research Among Industry,
University Researchers, Government, and
Growers/Packers
Canada's Biocontrol Network (www.biocontrol.ca) is a model of how to
increase adoption of biocontrol agents through a coordinated research and
testing effort by all stakeholders.
We work on replacing pesticides with effective and economically viable biocontrol
treatments based on the natural enemies of insect pests and disease pathogens,
used in coordinated and synergistic ways. We establish effective and mutually
beneficial research partnerships with the private sector (pest management
products and services companies and grower associations) as well as other
stakeholders in the field of plant protection.
The Biocontrol Network efforts are organized around a seven-stage
product-oriented process:
1 . Identification of users' needs
2. Ecological studies
3. Screening
Biological Control of Microbial Spoilage of Fresh Produce 533
4. Product development: production
5. Product development: efficacy
6. Product development: environmental impact and toxicology
7. Registration (if needed), marketing, training, and education
If a similar approach were taken in the U.S. or other countries, adoption
of new biological control agents would increase, to the benefit of the
environment, workers, and consumers.
23.5 REGULATORY PROCESS FOR BIOCONTROL
AGENTS
23.5.1 U.S. Environmental Protection
Agency (EPA)
Under FIFRA (Federal Insecticide, Fungicide, and Rodenticide Act), biologi-
cal pesticides (also known as biopesticides) are regulated by the EPA's
Biopesticide Pollution and Prevention Division. Biopesticides fall into the
categories of "microbials" and "biochemicals."
Microbial pesticides contain a microorganism (e.g., a bacterium, fungus,
virus, or protozoan) as the active ingredient. The most widely used micro-
bial pesticides are various types of the bacterium Bacillus thuringiensis, or Bt.
Biochemical pesticides are naturally occurring substances that control pests
by nontoxic mechanisms. Biochemicals include products, such as pheromones,
that interfere with pests' growth or mating patterns, and certain plant growth
regulators that increase the productivity of many crops.
Products can be registered as biochemicals provided the active ingre-
dients are natural or derived from a natural source, show no direct toxic
effects, and have a specific, nontoxic mode of action. Biopesticides (microbials
and biochemicals) can take considerably less time and money to bring to
market than synthetic chemicals (3 years and $3 million to $6 million versus
>10 years and $185 million for synthetic chemicals).
Each new biopesticide must go through Tier I toxicology, ecotoxicology,
and end product (final formulation) tests. If there are no direct toxic effects in
these tests, no further testing is required.
The following data are required for registering a biopesticide active
ingredient:
• Product chemistry, batch analysis
• Microbiology/human pathogens
• Acute toxicity/pathogenicity
• Ecological effects (nontarget birds, fish, invertebrates, insects, plants)
• Primary dermal and eye irritation
534 Microbiology of Fruits and Vegetables
The following data are required for registering a biopesticide "end use"
(formulated):
• Product chemistry/storage stability
• "Acute 6-pack":
Acute oral LD50
Acute dermal LD50
Primary eye irritation
Primary dermal irritation
Hypersensitivity
Acute inhalation
The FFDCA (Federal Food, Drug, and Cosmetic Act) requires a tolerance
for all chemical pesticides (a limit on the amount of chemical allowed in a fresh
or processed food). All biopesticides registered so far have been exempt from
tolerance because of their safety.
23.5.2 California and International
Regulations
California EPA's Department of Pesticide Regulation (DPR) regulates
biopesticides. Currently, biopesticides are given favorable "fast track" treat-
ment. A biopesticide application submitted concurrently to the U.S. EPA and
DPR may be approved by both agencies at the same time, or DPR approval
may come before the EPA approval. Sales cannot occur until there is federal
approval, however. California currently requires efficacy data for approval.
The EPA may ask for efficacy data, but typically does not.
Canada has coordinated its biopesticide regulations with the U.S. so
that biopesticides can be registered concurrently in the U.S. and Canada if
a registrant chooses to submit the applications at the same time. Otherwise, a
Canadian registration will take one year or more for approval after submitting
a separate application. In Mexico registration can take six months to years
after U.S. approval, although there is currently a NAFTA (North American
Free Trade Association) harmonization initiative. In Japan there is an EPA-
like tiered system, which takes approximately 12 months after registration
submission for approval. In Europe, the European Union is harmonizing
biopesticide regulations, and the regulations for biopesticides are becoming
more favorable, but registration still takes several years. Most countries
outside the U.S. require two years of official field trials conducted by
government entities, making the timeline longer than the U.S. process.
23.6 CONCLUDING REMARKS
Progress on postharvest biocontrol has been accomplished in the last 20 years,
but biocontrol agents have much more unrealized potential. A better
Biological Control of Microbial Spoilage of Fresh Produce 535
knowledge of antagonist modes of action, screening of additional microorgan-
isms, improvements in biofungicide formulation, and the ecology of
antagonists on fruits and vegetables will help in the design of better application
strategies and ensure more reliable disease control. Land grant and U.S.
Department of Agriculture research should increase their focus on practical
implementation of biocontrol agents in integrated programs to reduce the
chemical load on postharvest commodities. Setting up a postharvest bio-
control network like the Canadian network can provide a dedicated focus on
discovery, development, and adoption.
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Section V
Microbiological Evaluation
of Fruits and Vegetables
24
Sampling, Detection,
and Enumeration of
Pathogenic and Spoilage
Microorganisms
Larry R. Beuchat
CONTENTS
24.1 Introduction 543
24.2 Pathogen or Spoilage Microorganism Under Study 545
24.2.1 Media for Routine Microbiological Analyses 546
24.2.2 Selection of Test Strains for Sanitizer Efficacy
and Challenge Studies 548
24.3 Types of Produce and Methods for Preparing Samples 551
24.4 Procedures for Inoculation 556
24.5 Efficiency of Retrieval 557
24.6 Efficacy of Decontamination Treatment 558
24.7 Procedures for Detection and Enumeration 558
24.8 Number of Samples Analyzed and Reporting the Results 559
References 561
24.1 INTRODUCTION
Fruits and vegetables can become contaminated with spoilage and pathogenic
microorganisms at several points from the field through to the time they
are consumed. Given sufficient time at an appropriate temperature, some
pathogens can grow on produce to populations exceeding 10 7 CFU/g, resulting
in increased risks of human infections. Outbreaks of human illnesses associated
with the consumption of raw fruits and vegetables and unpasteurized fruit
juices have been documented with increased frequency in recent years. [1-3].
Conditions affecting survival and growth of pathogens and spoilage micro-
organisms on raw produce have been studied extensively. A wide range of
chemical and physical treatments have been evaluated for their effectiveness in
543
544 Microbiology of Fruits and Vegetables
killing microorganisms on raw fruits and vegetables [4]. Substantial variations
in conditions used to prepare inoculum, methods for inoculation, storage of
samples after inoculation, and application of treatments have been used.
Procedures used to sample, detect, and enumerate pathogens and spoilage in
microorganisms on raw produce have also varied across laboratories, making
it difficult to compare the results.
There is a need to develop and validate standard methods to determine
accurately the presence and numbers of pathogenic and spoilage bacteria,
yeasts, molds, parasites, and viruses on raw fruits and vegetables. These
TABLE 24.1
Considerations When Developing Standard Method(s) for Determining
the Efficacy of Sanitizers in Killing Pathogenic Microorganisms, and
Survival and Growth of Pathogens on Raw Fruits and Vegetables
Type of produce
Whole or cut
Washed, brushed, waxed, or oiled
Botanical part (fruit, leaf, stem, flower, root, tuber)
Pathogen of interest
Gram-negative or Gram-positive bacteria, parasite, or virus; mixture of strains or a single
strain
Marker or no marker
Conditions for preparing inoculum
Number of cells in inoculum
Procedure for inoculation
Composition of carrier
Temperature of produce and inoculum
Dip, spray, or spot inoculum
Temperature and relative humidity between time of inoculation, testing, and analysis
Procedure for evaluating test condition
Define treatment, condition, or sanitizer
Method for measurement of concentration and activity
Temperature of produce and treatment condition or sanitizer
Dipping, spraying, fogging, or atmospheric
Agitated, rubbed, or static condition during exposure
Time of exposure of inoculated produce to sanitizer or condition
Ratio of sanitizer to produce sample
Blending, homogenizing, macerating, or washing
Time of treatment
Composition of neutralizer (for sanitizer studies)
Detection and enumeration media
Conditions for incubating plates and broth
Confirmation procedures
Reporting results
Number of replicates and samples/replicate
CFU/g, CFU/cm", CFU/piece, fraction negative
Appropriate statistical analysis and interpretation
Sampling, Detection, and Enumeration of Pathogenic and Spoilage Microorganisms 545
methods can then be used in studies focused on determining survival and growth
characteristics in challenge studies and efficacy of antimicrobial treatments in
killing specific pathogenic and spoilage microorganisms that may be present
on raw produce. The objective would be to develop, validate, and recommend,
through an appropriate authoritative body, a basic experimental protocol or
protocols that could be modified according to specific applications to various
groups of fruits and vegetables.
Some of the factors that should be considered when developing a
standard method(s) for determining the effectiveness of sanitizers in killing
microorganisms or, in the case of challenge studies, to determine the survival
and growth characteristics of microorganisms on raw fruits and vegetables are
listed in Table 24.1. These include the type of produce to be examined,
anticipated population of pathogenic or spoilage microorganism or group of
microorganisms to be used in the inoculum or naturally present on produce,
composition of the carrier for the inoculum, and conditions for storing
produce between the time of inoculation and treatment or sampling. The
time produce is exposed to chemical or physical treatment, the temperature
of the produce and treatment solution, procedures for washing produce
after treatment, and procedures for removing and enumerating viable cells
of pathogenic and spoilage microorganisms after treatment should be
standardized.
Modifications of a basic analytical method for groups of fruits and
vegetables may be necessary to enable the most accurate detection
or enumeration of microorganisms of interest and to determine accurately
the efficacy of sanitization treatments. These modifications will be neces-
sary for a yet to be determined number of groups of fruits and vegetables
to be defined according to similarities and differences in surface morpho-
logy and hydrophobicity, internal tissue composition, and conditions
of processing, e.g., washing, brushing, or waxing, to which they had been
previously subjected. Observations on current methods and those under
development, with options and suggestions concerning directions that might
be taken to establish standard methods to detect accurately or enumerate
pathogenic and spoilage microorganisms on raw fruits and vegetables, are
presented here.
24.2 PATHOGEN OR SPOILAGE MICROORGANISM
UNDER STUDY
An evaluation of the efficacy of treatments to sanitize fruits and vegetables
or the appropriateness of experimental protocols for challenge studies to
determine the survival or growth characteristics of pathogenic or spoilage
microorganisms must be preceded by the development and validation of
standard methods for detection and enumeration. Broad considerations that
need to be assessed in evaluating or developing standard protocols for patho-
genic and spoilage microorganisms must be addressed.
546 Microbiology of Fruits and Vegetables
24.2.1 Media for Routine Microbiological
Analyses
Methods have been developed and validated to analyze raw and processed
foods of animal origin and processed foods of plant origin for the presence and
populations of pathogenic and spoilage microorganisms. Methods for analysis
of raw fruits and vegetables, in contrast, are not well defined. Procedures
for enrichment and direct plating of raw produce samples in the U.S. are
generally modifications of those outlined for processed fruits and vegetables
in the Bacteriological Analytical Manual (BAM) of the U.S. Food and Drug
Administration [5] or the Compendium of Methods for the Microbiological
Examination of Foods published by the American Public Health Association
(APHA) [6]. A 25 g analytical unit diluted at a 1:9 ratio (weight: volume)
of sample:diluent or sample:broth is prescribed for most foods. The food
and diluent or broth are then mixed, swirled, soaked, or blended before
withdrawing samples for direct plating or incubated for a specified time at
a given temperature for preenrichment or enrichment. In part because of the
lack of a standard method(s) to analyze raw fruits and vegetables for
pathogens and spoilage microorganisms, researchers have modified BAM
and APHA methods to suit their needs to analyze specific types of produce,
often without validation of the efficiency of detection or recovery of the test
microorganism or group of microorganisms under investigation. Substantial
variations in methods to select and process samples for preenrichment, enrich-
ment, and direct plating have been used to analyze raw produce for the
presence and populations of pathogens. To illustrate these variations,
Table 24.2 gives some examples of procedures used by researchers to analyze
produce for salmonella.
The inability of injured or stressed cells of pathogenic bacteria to
resuscitate and grow on selective media is too often not recognized. Selective
media recommended for foods other than raw fruits and vegetables have been
formulated and evaluated for that purpose. The same media may not perform
well for detection and enumeration of pathogens or other microorganisms on
produce. The number of stressed salmonella recovered from tomatoes
[24,31,32], lettuce [33], alfalfa sprouts and seeds [32,35], and parsley [33], for
example, is significantly less on selective versus nonselective media. Recovery
of Escherichia coli 0157:H7 and Listeria monocytogenes from tomatoes [31],
lettuce, and parsley [33] treated with sanitizers has also been shown to be less
on selective media.
While progress has been made in developing media for enumerating yeasts
and molds in a wide range of foods and beverages [36], less attention has been
given to evaluating the performance of diluents in removing and dispersing
fungal propagules on fruits and vegetables. Standard methods for determining
yeast and mold counts in foods recommend 0.1% peptone as a diluent
[5,37,38]. A study done by Beuchat et al. [39] was aimed at determining
the retention of viability of mycoflora recovered in seven diluents used to
wash seven types of raw fruits as affected by composition of diluents.
Sampling, Detection, and Enumeration of Pathogenic and Spoilage Microorganisms 547
TABLE 24.2
Examples of Variations in Weight, Diluent Composition, and Volume, and
Processing Methods Used to Prepare Raw Fruits and Vegetables to Analyze
for Populations of Salmonella
Diluent
or
wash so
lution
Process
Vol.
Produce type (no.)
Weight (g)
Type 3
(ml)
type
Time
Ref
Alfalfa seeds
10
NB
90
Stomach
1 min
7
5±1
BPB
45
Stomach
90 sec
8
Alfalfa sprouts
50
PW
450
Stomach
2 min
9
50
PW
100
Hand
massage
1 min
10
25
PBS
25
Stomach
5 min
11
Apples
Whole (1)
180
PW
20
Hand rub
40 sec
12
Skins
5 apples
PBS
250
Homogenize
Not stated
13
Broccoli
25
BPW
225
Stomach
2 min
14
Cantaloupe
1 (whole)
BPBT
200
Shake
10.5 min
15
25
PW
75
Blend
1 min
16
Carrot
20-30
PWT
99
Shake
1 min
17
Lettuce
50
PW
50
Shake
20 sec
12
20
BPB
180
Blend
30 sec low
speed plus
30 sec high
speed
18
25
BPW
225
Stomach
Not stated
19
10
PWS
90
Stomach
2 min
20
15-25
PW
200
Stomach
2 min
21
Melons
25
BPB
18
Stomach
1 min
22
Radish
50-75
PWT
90
Shake
1 min
17
Spinach
50
PW
50
Stomach
1 min
23
Strawberry
25-30
Several
30
Stomach
or shake
2 min;
25 min
24
Tomatoes
Whole (1)
75
PW
20
Hand
massage
2 min
25
180-200
PW
20
Hand rub
40 sec
26
110-140
PW
20
Hand rub
1 min
27
Chopped
50
PW
50
Stomach
1 min
27
Small pieces
20
Saline
180
Stomach
30 sec
28
Various
Vegetables (9)
25
BPW
225
Stomach
2 min
29
Fruits/vegetables
10-600
Water
500
Agitate
30 min
30
(401)
a BPB, Butterfield's phosphate buffer (pH 7.2); BPBT, Butterfield's phosphate broth + 1% Tween
80; BPW, buffered peptone water; NB, neutralizing broth; PBS, phosphate-buffered saline (pH 7.2);
PW, peptone water (0.1%); PWS, peptone water (0.1%) + 0.85% sodium chloride; PWT, peptone
water (0.1%)+ 1% Tween 80.
548 Microbiology of Fruits and Vegetables
The performance of recovery media for supporting colony development was
also evaluated. The composition of diluents had little effect on the number of
yeasts and molds recovered from fruits. Dichloran rose bengal chloramphe-
nicol agar and plate count agar supplemented with chloramphenicol were
equivalent in supporting colony formation. A study to determine the effect
of diluent composition on recovery of yeasts from grape juice and passion
fruit pulp showed that dilution in 0.1% peptone gave higher counts compared
to dilution in distilled water, saline, or phosphate buffers [40].
Media selective for yeasts and molds found on raw fruits and vegetables,
as well as other types of foods, have been reviewed [36,41]. Formulations
have been developed to select for acid-resistant yeasts and detection of proteo-
lytic and lipolytic enzyme activity. Media for detecting mycotoxigenic molds
and selecting for xerophilic and xerotolerant fungi have been developed.
Release of tissue juices from produce, particularly those with high sugar
content, followed by drying on the produce surface can result in reduced water
activity that is selective for these yeasts and molds. Some considerations when
analyzing produce and other foods for the presence of xerophilic fungi are
recommended by Beuchat and Hocking [42].
24.2.2 Selection of Test Strains for Sanitizer
Efficacy and Challenge Studies
The strain or strains of a particular microorganism selected for studies
designed to determine the efficacy of a decontamination treatment or survival
and growth in challenge studies are extremely important. The use of well-
characterized reference strains enhances the comparative assessment of a given
method among laboratories. Five or more strains, preferably recently isolated
from produce or other plant materials, and from patients suffering from illness
associated with consumption of a raw fruit or vegetable, are preferred.
Approximately equal populations of each strain in a mixed inoculum should be
used. If there are differences in the ability of one or more of these strains to
survive or grow on produce subjected to various environmental conditions
during storage, or if there are differences in susceptibility to decontamination
treatments, the most robust strain(s) will prevail. If only one strain is used in
the inoculum, it should be first evaluated against several other strains for
its ability to survive or grow under the proposed test conditions. The use of
a single strain that may be less tolerant to test conditions could result in an
inaccurate assessment of the behavior of the test microorganism. Strains used
to prepare mixed-strain suspensions should be examined for potential reactions
against each other that may be caused by bactoriocins, killer proteins, and
other inhibitors they may produce.
Test microorganisms should be cultured in a standard broth or on a
defined agar medium at a specific temperature for a specific time. The
temperature at which microorganisms are grown for preparing inocula should
be representative of the temperature at which they had grown before
contaminating produce or the temperature at which inoculated produce will
Sampling, Detection, and Enumeration of Pathogenic and Spoilage Microorganisms 549
be stored after inoculation, in the case of a challenge study. Several transfers of
cultures should be made preceding the day of inoculum preparation. The time
elapsed between the last transfer and collecting cells to prepare the inoculum
will depend on the test microorganism. Although this practice may result in
strains with reduced environmental stress tolerance as a result of adaptation
to a nutrient-rich medium, it is desirable to prepare inoculum of uniform
cell type. The type of study being conducted should be considered in terms
of potential impact of genetic selection of test strains on the predictive value of
the results. This is particularly important for pathogenic bacteria that may
have originated from diverse sources such as clinical specimens, foods, or the
environment. Stationary phase bacterial and yeast cells are generally more
tolerant than are logarithmic growth phase cells to environmental stresses
[43]. For this reason, cells in stationary growth phase should be used in studies
to develop optimum procedures to assess their behavior on or in inoculated
produce.
The use of markers such as antibiotic resistance may be desirable
to facilitate the recovery of cells in enrichment broth or counting colonies on
selective or nonselective direct plating media. Otherwise, these media may
support the growth of large numbers of background microflora which interfere
with growth of the test microorganism. Adaptation of Gram-negative patho-
gens to nalidixic acid (50 Jig/ml) has been used to achieve this objective. In
a study to determine survival of five strains of nalidixic acid-resistant and
refampicin-resistant Salmonella Poona on cantaloupes it was observed that
average reductions in the number of control and antibiotic-resistant cells were
not significantly different (P > 0.05) [44]. Resistance of test cells to rifampicin
(80 ug/ml) can also be successfully used as a marker, particularly for isolat-
ing pathogens from inoculated fruits and vegetables that have significant
adhering soil. Plasmid-borne or chromosomally stabilized markers such as
fluorescent proteins with various chromophoric properties have also been used.
It is important to assess the impact of markers on the growth rate, stress
tolerance, and recovery efficiency of cells on enumeration media before subjec-
ting them to sanitizer efficacy or challenge studies. Characterization of the
stability of the marker over at least ten generations, without selection, is needed
for challenge studies in which growth may occur and for recovery methods
that include preenrichment or enrichment procedures.
Different strains of the same bacterial species may release byproducts
that inhibit or kill other strains. Colicins produced by Escherichia coli and killer
toxins produced by some species of yeasts are examples. When an inoculum
containing several strains of the same microorganism is used, each strain
should be tested for its potential to inhibit all other strains in the inoculum.
This can be done by cross streaking cultures of individual strains on an
appropriate agar medium and examining incubated plates for inhibition of
growth at intersections of the streaked cultures.
Determination of the survival characteristics of viruses and parasites
on produce poses unique problems. Survival of enteric viruses has been
studied but obstacles still remain in standardizing methodology to determine
550 Microbiology of Fruits and Vegetables
the efficacy of sanitizers in killing or removing viruses that may occasionally
contaminate produce. Freshly isolated viruses such as hepatitis A and noro-
viruses are not culturable, and thus cannot be propagated in sufficient
quantities to prepare inocula, nor can they be quantitated by plaque assay.
A few viruses, e.g., poliovirus, have been adapted to grow in tissue culture
in the laboratory and can be quantitated by plaque assays. Although some
of these viruses belong to the same family, they can vary greatly in their level of
resistance to chemical and physical stresses. Adapted strains may be repre-
sentative of their parental wild type but not other members in the same family.
Propagation of foodborne viruses has been limited to only a few adapted
strains. The behavior of these strains may or may not be similar in behavior
to other isolates of the same virus. Rapid molecular methods to detect
viruses in foods are sensitive but cannot be used to quantitate viruses or to
differentiate between infectious and noninfectious strains [45]. These attributes
pose unique challenges in developing and standardizing methodology for
detecting and quantitating viruses on produce.
Survival of parasites on raw produce as affected by treatment with sani-
tizers or exposure to various environmental stress factors during storage has
not been well defined. A major constraint to investigating survival charac-
teristics is the limited supply of oocysts [46]. Infected humans are the only
source of Cyclospora cayetanensis oocysts in quantities needed in studies
to determine susceptibility to stress or lethal conditions that may be imposed
by decontamination treatment or storage conditions. The lack of sensitive
laboratory methods for quantitating and assessing the viability of oocysts
hampers progress in developing methods to determine the efficacy of saniti-
zation treatments and the influence of processing, packaging, and storage
conditions on their survival.
Vehicles of pathogens and spoilage microorganisms for contaminating
fruits and vegetables include dust, rain water, irrigation water, sewage, soil,
feces, decayed plant material, contact surfaces, workers at any point from
harvesting through preparation in foodservice, and home settings [47]
Vegetative cells, spores, cysts, and other propagules of microorganisms are
likely to be entrapped in organic material. To simulate practical conditions
of surface contamination of produce, the carrier for the inoculum should
contain organic material. Horse serum (5%) and aqueous peptone solution
(0.1%) have been used as carriers with fairly defined composition in studies
to determine the efficacy of sanitizers. Buffer solutions and other carriers
containing salts or other chemicals that could be detrimental to cells after the
inoculum has dried on the surface of test produce are not recommended for use
as carriers. Cells in broth cultures of bacteria or yeasts should be washed
in peptone water and resuspended in the organic carrier shortly before using
as an inoculum. For challenge studies designed to determine the survival
or growth of pathogenic and spoilage microorganisms on or in produce, the
carrier may provide a source of nutrients, thus complicating interpretation
of results. The use of two carriers, one with and one without organic material
(deionized or distilled water) for test cells may be useful in generating
Sampling, Detection, and Enumeration of Pathogenic and Spoilage Microorganisms 551
information to enable the effects of carrier nutrients on survival and growth
of test microorganisms to be discerned.
The desired population of test cells in the inoculum depends on the
objective of the study. Two or three levels of inocula, ranging from 10° to
10 CFU/g or CFU/cm , may be applied to facilitate the determination of
efficiency of retrieval, efficacy of sanitizers, or survival and growth during
subsequent storage. High numbers of cells in the inoculum are needed in
decontamination studies to enable measurement of several log 10 reductions
in population. Challenge studies require inocula containing low numbers
of cells to enable measurement of growth during storage under conditions
simulating practices to which produce is subjected in commercial distribution,
retail, foodservice, and home settings.
24.3 TYPES OF PRODUCE AND METHODS FOR
PREPARING SAMPLES
A single method to remove efficiently microbial cells or spores from all types
of fruits and vegetables for the purpose of detection or enumeration would be
ideal, but this may not be an achievable goal. Differences in size, shape,
and surface morphology of fruits and vegetables complicate the protocol. The
ratio of surface area to weight of individual produce items varies substantially,
raising the need to establish a basis (CFU/g or CFU/cm ) to be used to record
and report data.
The procedure for preparing the sample for analysis may affect the effi-
ciency of retrieval of microbial cells from produce, as well as dispersal before
preenrichment, enrichment, or direct plating. Homogenization of a standard
weight of a fruit or vegetable using a standard volume of diluent would be
a simple procedure for selecting sample size and method of preparation of
samples. Problems, however, may be associated with homogenized, blended,
or macerated plant tissues. These include the potential lethal effect of naturally
occurring antimicrobial compounds against pathogens or other microflora
targeted for detection or enumeration. When microbial cells on the surface of
produce tissues come in contact with organic acids or other antimicrobials
naturally present in tissue fluid, or produced in the form of phytoalexins
as a result of rupture of cells or invasion with insects or molds, death may
occur [48].
Acids and phenolic compounds are naturally present in plant stems, leaves,
flowers, and fruits. These compounds may interfere with detection and enumer-
ation of pathogenic and spoilage microorganisms. The low pH of produce
tissues, particularly those in many fruits, is attributable to a wide range
of organic acids they may contain. Garlic, onion, and leek are probably the
most widely consumed vegetables that have antimicrobial activity. Allicin, a
diallyl thiosulfate, is not present in intact tissues but is produced when the
tissues are disrupted. Plant tissues used largely as seasoning agents may also
be inhibitory to pathogenic and spoilage microorganisms (Table 24.3). Spices
552
Microbiology of Fruits and Vegetables
TABLE 24.3
Plants Used Largely as Seasoning Agents That Also Contain Antimicrobials
Achiote
Cinnamon
Licorice
Rosemary
Allspice (pimenta)
Citronella
Mace
Sage
Angelica
Clove
Marjoram
Sassafras
Anise
Coriander
Musky bugle
Savory
Basil (sweet)
Dill
Mustard
Spearmint
Bay (laurel)
Elecampane
Nutmeg
Star anise
Bergamot
Fennel
Onion
Tarragon (estragon)
Calmus
Fenugreek
Oregano
Thyme
Cananga
Garlic
Paprika
Turmeric
Caraway
Ginger
Parsley
Vanillin
Cardamom
Horseradish
Pennyroyal
Verbena
Celery
Leek
Peppermint
Wintergreen
Chenopodium
Lemongrass
Pimento
and herbs prepared from plant parts owe some of their desired sensory attrib-
utes to these antimicrobials [49]. Like organic acids, these compounds are
released from cut tissues and may be lethal to microorganisms naturally
occurring or intentionally inoculated onto fruits and vegetables.
Compounds involved in plant defense mechanisms have been classified as
prohibitins, inhibitins, postinhibitins, or phytoalexins, depending on preinfec-
tion or postinfection factors [50]. These compounds may also kill or inhibit
test microorganisms or microflora naturally present on produce. Table 24.4
lists some antimicrobials other than major flavor and aroma compounds
that are known to be naturally present in raw fruits and vegetables or produced
as a result of breakage of tissues or infections with bacteria and molds. These
antimicrobials may interfere with detection and enumeration of bacteria
capable of causing human illnesses.
Several studies have investigated inhibitory or lethal activities of naturally
occurring antimicrobials against foodborne pathogenic and spoilage micro-
organisms. A study to compare washing in 0.1% peptone, stomaching, and
homogenizing for their influence on recovery of salmonella inoculated onto
26 types of fruits, vegetables, and herbs, revealed that, overall, no significant
differences in recovery of the pathogen could be attributed to a particular
sample processing method [52]. In an attempt to determine if exposure of
salmonella to low pH of tissue fluids as a result of stomaching or homogenizing
samples was lethal to the pathogen, the 26 types of produce were arbitrarily
separated into groups based on pH. Significantly higher percent recoveries
were obtained in produce in the pH 5.53 to 5.99 range compared to produce
in lower pH ranges. Reduced percent recoveries from herbs (pH 5.94 to 6.34)
were attributed in part to antimicrobials released from plant cells during
sample preparation. The mean pH of herbs was 6.08. Lethality caused by anti-
microbials other than acids in herbs may have been masked by the minimal
inhibitory affect of this slightly acidic pH, causing a reduction in the number
Sampling, Detection, and Enumeration of Pathogenic and Spoilage Microorganisms 553
TABLE 24.4
Antimicrobials Other Than Major Flavor and Aroma Compounds That Are
Naturally Present in Edible Plant Tissues or Produced as a Result of Infection
or Rupture of Tissues
Common name
Botanical name
Antimicrobial produced
Alfalfa
Medicago sativa
Medicarpin
Apple
Malus spp.
Phloretin, hydroxybenzoic
acid, anthocyanidins
Avocado
Per sea spp.
Borbonol
Beet (red)
Beta vulgaris
Beta vulgarin
Broad bean
Vica faba
Wyerone acid
Cabbage
Brassica oleracea
Rapine, sinigrin
Carrot
Daucus carota
Falcarindiol, 6-methoxymellein
Chick pea
Cicer are tie turn
Medicarpin
Eggplant
Solarium melongena
Aubergenone
French bean
Phaseollus vulgaris
Phaseollin
Garlic
Allium sativum
Allyl sulfoxides
Grape
Vitis spp.
Yiniferin
Mulberry
Morus alba
Mulberrofuran, albafuran, moracin
Olive
Olea europaea
Oleuropein
Onion
Allium cepa
Protocatechoic acid
Parsley
Petroselinum spp.
Begapten, graveolone, isopimpinellin,
psoralen, xanthotoxin
Passion fruit
Passifiora mollissima
Passicol
Pea (shoot)
Pisum sativa
Pisatin
Peanut
Arachis hypogaea
Resveratrol
Pear
Pyrus spp.
Arbutin
Pepper (sweet)
Capsicum annuum
Capsidiol
Pigeon pea
Cajanus cajan
Stilbene-2-carboxylic acid, glyceollin
Potato
Solarium tuberosum
Rishitin, hydroxylubimin,
caffeic acid, scopoletin, a-tomatine
Radish
Raphanus sativus
Raphanin
Sweet potato
Ipomoea batatas
Impomeamarone
Soybean
Glycine max
Pterocarpan, glyceollin,daidzein,
coumestrol
Tomato
Ly coper sicon esculentum
Alkaloids
Yam
Discorea rotundata
Hicicol, isobatasin
Adapted from Walker, J.R.L., Antimicrobial compounds in food plants, in Natural Antimicrobial
Systems and Food Preservation, Dillon, V.M. and Board, R.G., Eds., CAB International,
Wallingford, U.K., 1994, p. 181; Whitehead, I.M. and Threlfall, D.R., /. Biotechnoi, 26, 63, 1992.
of salmonella recovered. While blending, homogenizing, or macerating may
be acceptable in preparing samples of some types of fruits and vegetables,
a simple surface washing without rupturing of plant cells may be required for
other types.
The presence of inhibitory or protective residues from crop management
practices and variations in surface morphology unique to specific fruits
or vegetables should also be a consideration when selecting a procedure for
554 Microbiology of Fruits and Vegetables
preparing samples for analysis. Microorganisms may be most effectively
retrieved by washing the surface of fruits and vegetables such as tomatoes,
mangoes, avocados, watermelons, oranges, and other produce with a relatively
smooth, rigid surface. Even produce with hard, apparently blemish-free
surfaces, however, can harbor microorganisms in areas that are not easily
accessible by washing or homogenization [53]. Infiltration of microbial cells
into stomata, lenticels, broken trichomes, and cracks in the skin surface can
occur. The porous stem scar tissue of tomatoes, for example, offers a relatively
easy port of entry for microorganisms compared to intact skin [27,54].
Microorganisms are also known to partition into the cut tissues of produce.
Infiltration of E. coli 0157:H7 into cut tissue of lettuce is affected by
temperature [55]. Microorganisms harbored in subsurface and other protected
areas should be considered when selecting a method to retrieve them from
produce tissues for the purpose of detection and enumeration.
Sonication of samples may be an alternative method for removal of micro-
organisms from the surface of produce with minimal tissue disruption,
although this approach has not been thoroughly researched. Seymour et al. [20]
evaluated the use of ultrasound to promote decontamination of raw vegetables.
Cavitation caused by treatment appeared to enhance the release of Salmonella
Typhimurium. Release of salmonellae and E. coli 0157:H7 from inoculated
alfalfa seeds is enhanced by ultrasound treatment [56]. These observations
suggest that ultrasound treatment, perhaps in combination with other methods
for removing microorganisms from produce tissues, may result in a more
accurate assessment of populations. Tissues of leafy and floret vegetables,
strawberries, raspberries, blackberries, and other produce with complex surface
tissues are easily ruptured by rubbing, thus exposing surface microflora to
stress conditions imposed by reduced pH or other factors associated with tissue
juice. Agitation using a mechanical shaker or by manually shaking in a wash
fluid with standard composition and volume for a set period of time may be the
most suitable method for removing microbial cells from these produce items.
To avoid too many modifications of a standard sample preparation proto-
col, the sample weight or number of pieces and the volume of wash solution
or diluent should be standardized for each type or group of produce. Results
of analysis can be reported as CFU/g of sample or be converted to CFU/cm
using a conversion table listing estimated values for specific fruits and vege-
tables categorized as spheres, cylinders, two-sided planes, or perhaps other
geometric shapes. An alternative would be to calculate microbial populations
on the basis of CFU/piece of fruit or vegetable, although this method has little
meaning if the weight of each produce piece is not reported.
Removal and disposal of microbial cells from surface tissues of fruits and
vegetables that have been mechanically cleaned by brushing or that have been
waxed or oiled may be more difficult, compared with retrieval from untreated
produce. Microorganisms entrapped in bruised tissue, waxes, and oils may be
more difficult to remove and disperse in homogenates or wash fluids, resulting
in an underestimation of populations. Dip inoculation of bruised, unwaxed
apples in a suspension of E. coli 0157:H7 is known to result in lodging and
Sampling, Detection, and Enumeration of Pathogenic and Spoilage Microorganisms 555
infiltration of cells in broken tissues, the waxy cutin layer, and lenticels
(Figure 24.1). Cells can be harbored in lenticels at depths up to 24|im, making
their retrieval difficult [57]. It may be necessary to modify the sample
preparation protocol to maximize release of cells from tissues, as well as from
FIGURE 24.1 (Color insert follows page 594) Confocal laser scanning microscopy
(CLSM) images showing attachment of E. coli 0157:H7 to various sites on the surface
of apples. (A) Bruised tissue of unwashed, unrubbed, bruised apple at a junction
(4.8 urn depth) between wax platelets: edge of wax platelets (open arrow); most cells
attached to the edge of the wax platelets (filled arrow). (B) Bruised tissue of
unwashed, unrubbed, bruised apple at a junction (6.6 urn depth) between wax
platelets: heavy colonization of junctions between wax platelets (arrow). (C) Bruised
tissue of unwashed, rubbed, bruised apple showing a cuticular crack on surface of
apple (16.6 um depth): cells are trapped within the cuticular crack (arrow).
(D) Bruised tissue of unwashed, unrubbed, bruised apple showing a lenticel (9.2 um
depth): cells are within lenticel (arrow) at a depth of 20.6 um below the surface of the
apple. (From Kenney, S.J., Burnett, S.L., and Beuchat, L.R., /. Food Prot., 64, 132,
2001. With permission. Copyright International Association for Food Protection,
Des Moines, IA.)
556
Microbiology of Fruits and Vegetables
the naturally occurring waxy cutin layer and waxes or oils that may be applied
to enhance appearance or extend the shelf life of some fruits and vegetables.
24.4 PROCEDURES FOR INOCULATION
Surface inoculation of fruits and vegetables with pathogens or spoilage micro-
organisms can be done by dipping or spraying with a suspension of cells or
by applying a known volume of suspension containing a known population
(spot inoculation). Some of the advantages and disadvantages of these methods
are listed in Table 24.5. The inoculation method should ideally simulate vari-
ous contamination events and postcontamination conditions prior to washing
and sanitization. If contamination of produce is suspected to occur by an
immersion process, dipping the produce in the test cell suspension may be an
appropriate method for inoculation. A problem associated with inoculation
by dipping or spraying is that the number of cells actually applied or adhering
to the produce is not known. The population remaining on the surface and
in subsurface tissues after dip or spray inoculation is not consistent among
TABLE 24.5
Some Advantages and Disadvantages of Dip, Spot, and Spray Inoculation
Methods for Determining the Efficacy of Sanitizer Washes on Produce
Inoculation
method
Dip
Spot
Spray
Advantages
Mimics contamination from
highly contaminated irrigation,
run-off, or flume water
Delivered volume of inoculum
known. Population in inoculum
can be accurately calculated.
Efficacy of sanitizer can be
compared on different tissues
within the same produce item.
Most consistent inoculum
applied of three inoculation
methods
Mimics contamination from
aerosols
Disadvantages
Volume of inoculum and number of
cells delivered to each produce item
are unknown. Some inoculum may
be internalized, complicating
interpretation of data. Large volumes
of high inoculum containing
populations of pathogens are
difficult to manage safely, even in
highly experienced laboratories
May not reflect contamination that
would occur from contaminated
irrigation, run-off, or flume water
Accurate delivery of inoculum is
difficult, especially with smaller
produce items. Aerosols generated are
difficult to manage safely, even in
highly experienced laboratories
Sampling, Detection, and Enumeration of Pathogenic and Spoilage Microorganisms 557
various types of produce and between different types of tissues on the same
produce. Infiltration of the inoculum into cavities, wounded tissues, or porous
areas on the produce surface, e.g., cut tissue, stem scar tissue, stomata, or
lenticils [58,59], can result in conditions that may inhibit or enhance growth.
Cells lodged in these areas may be protected against contact with sanitizers,
particularly those with little or no surfactant activity. Analysis of produce
inoculated by dip or spray methods requires a large number of units for each
treatment, as random error values can be unpredictably large. Thus, efficiency
of recovery or logi changes in viable populations during subsequent storage
or as a result of treatment with a sanitizer cannot be accurately calculated.
Alternatively, fruits and vegetables can be inoculated by applying a known
volume of cell suspension, e.g., up to 100 ul, of known population to the
surface. Spot inoculation represents contamination from a point source such as
contact with soil, workers' hands, or surfaces of equipment and has been
recommended for testing the efficacy of sanitizers in killing foodborne
pathogens on tomatoes [31], lettuce, and parsley [33].
Temperature differentials between produce and inocula can affect the
number of cells that infiltrate tissues. A negative differential, i.e., when the
temperature of the produce is higher than the temperature of the inoculum,
can result in enhanced infiltration of microbial cells [27,54,60]. A standard
temperature at which both the produce and the inoculum are adjusted before
inoculation should be selected for sanitizer efficacy studies. Otherwise, expo-
sure of test cells to sanitizers and the efficiency of retrieval of cells may be
affected.
In studies to determine the efficiency of retrieval of cells or efficacy of
sanitizers, the inoculum applied to produce should be dried for a set period
of time at a controlled temperature and relative humidity before treatment is
applied and samples are analyzed. Fluctuations in temperature and relative
humidity should be minimized between the time of drying and treatment
or analysis. Three or more replicate experiments, each including four or more
samples for each set of test parameters in each replicate, should be done.
More samples may be needed, depending on specific objectives. Negative
controls should always be included.
24.5 EFFICIENCY OF RETRIEVAL
Development of a standard protocol for detecting or enumerating a specific
microorganism or group of microorganisms on or in produce should include
experiments to validate the efficiency of recovery based on a known number
of cells applied. This can be done using a known volume of inoculum con-
taining a known number of test cells. Although some cells may die during
the drying period following application of inoculum, efficiency of retrieval
can be more accurately measured using spot inoculation than dip or spray
inoculation, which do not enable measurement of the number of cells adhering
to the produce.
558 Microbiology of Fruits and Vegetables
The efficiency of retrieval of microbial cells naturally occurring on produce
is not easily determined, simply because the actual number of retrievable cells is
not known [61]. The presence of a surfactant in peptone water used to remove
pathogens from produce, e.g., cantaloupes, may enhance the number detected
[44]. A comparison of various combinations of sample weights, wash fluids,
diluents, homogenization or washing treatments, and neutralizers (in the case
of chemical sanitizer tests) should be made before choosing test parameters
that give the highest percentage of viable microorganisms recovered. Some
protocols have been demonstrated to be more efficient than others, and a single
basic protocol should be selected for analysis of specific fruits, vegetables, or
groups of produce in all laboratories.
24.6 EFFICACY OF DECONTAMINATION TREATMENT
A protocol for efficient recovery of pathogens or groups of microorganisms
from fruits and vegetables must be established before proceeding with
experiments designed to determine the efficacy of treatment with sanitizers
or changes in populations as affected by storage conditions. Procedures for
chemical decontamination should use standard weight-to-volume ratios
(produce:treatment solution or atmosphere), whether applied as a dip, spray,
or fog. A standard concentration of treatment solution applied for a standard
time at a standard temperature, followed by neutralization of the active
component using a standard volume and concentration of neutralizer should
be defined. Whether the produce should be static, agitated, or hand rubbed
during chemical treatment should be stated. Agitation, e.g., by placing the
produce and treatment solution on a mechanical shaker or manually shaking,
should be standardized. Conditions for separating the produce from the
chemical treatment solution, washing with a specific neutralizer, and
subsequent homogenization or washing in a specific volume of a given diluent
should also be standardized. Controls that will reveal the effect of rinsing after
treatment should also be included.
For physical decontamination treatments, standardization of conditions,
e.g., temperature, irradiation, or pressure, would facilitate comparison of
observations across laboratories. A neutralization step is not necessary in a
standard protocol to measure the efficacy of physical treatments but, like
protocols for determining the efficacy of chemical sanitizer treatments,
standardization of diluent composition, ratio of produce weight:diluent
volume, homogenization or washing procedure, preenrichment, enrichment,
and direct plating media, and incubation conditions is necessary.
24.7 PROCEDURES FOR DETECTION AND
ENUMERATION
The selection of preenrichment, enrichment, and/or direct plating media, as
well as conditions for incubation and procedures for confirmation of isolates
Sampling, Detection, and Enumeration of Pathogenic and Spoilage Microorganisms 559
will differ, depending on the microorganism or group of microorganisms
targeted for detection or enumeration. Media, incubation conditions, and
confirmation techniques selected for each microorganism or group of micro-
organisms that may be inoculated onto or naturally present in produce should
be the same across laboratories. Optimum protocols for retrieving pathogens
and nonpathogens from fruits and vegetables may differ, depending upon
whether analysis of the surface, tissue, or a composite of both is desirable.
Washing, rubbing, blending, homogenizing, stomaching, macerating, and
grinding, or a combination of one or more of these procedures, are among
the choices to process samples for preenrichment, enrichment, or direct plating.
One piece of fruit or vegetable, several pieces, or only a portion of the
whole or cut produce may be selected for analysis, but the procedure needs
to be standardized in terms of sample weight and/or excision technique.
The composition and pH of the diluent and ratio of diluent to sample need to
be consistent, at least within each type of fruit and vegetable. The time
and temperature for processing samples for preenrichment, enrichment, or
direct plating should be standardized. The likelihood of stressed or injured
microbial cells being present on or in fruits and vegetables should be
recognized, and appropriate resuscitation conditions should be considered
and applied. Repair of cells on the surface of produce that, for example,
may be debilitated by desiccation or as a result of exposure to a harsh
chemical environment, is important if these cells are to be detected or
enumerated. Adjustment of the pH of homogenates of highly acidic fruits and
vegetables may be necessary to protect microorganisms against exposure
to potentially lethal conditions during preparation of samples for inoculation
of recovery media.
24.8 NUMBER OF SAMPLES ANALYZED AND
REPORTING THE RESULTS
Conditions intrinsic to fruits and vegetables, as well as variation in types
and numbers of microorganisms and amount of soil and organic matter
present on produce surfaces, are variable, necessitating a standard procedure
for selecting samples for sanitizer efficacy or challenge studies. A sufficient
number of replicates with a sufficient number of whole fruits or vegetables,
or cut produce samples will be necessary to enable appropriate types of
statistical analysis to be applied to the data generated. The experimental design
should enable statistical analysis to be done at a level rigorous enough to deal
with the complexities associated with microbiological testing. Traditional
methods of bacteriological or mycological analysis of foods and beverages
report results on the basis of CFU/g, CFU/ml, or CFU/cm . Treatment
with sanitizers or application of processing technologies may be designed
to achieve a certain log 10 reduction in the number of a specific pathogen,
several pathogens, or a spoilage microorganism, based on weight or volume of
the product. Substantial variation in the weight-to-surface area ratio (g:cm )
560
Microbiology of Fruits and Vegetables
Tomato
O)
■5 200
Iceberg lettuce
100 200 300 400 500 600 700
.2>
Surface area (cm )
FIGURE 24.2 Relationship between weight and surface area of tomato fruit and lettuce
leaf. (From Beuchat, L.R., Farber, J.M., Garrett, E.H., Harris, L.J., Parish, M.E.,
Suslow, T.V., and Busta, F.F., /. Food Prot., 64, 1079, 2001. With permission.
Copyright International Association for Food Protection, Des Moines, IA.)
can exist among various types of produce, making this approach unreasonable
in terms of assessing log 10 reductions against risk of illness that may result from
consumption of a given weight of produce. Relationships between the weight
(g) and surface area (cm ) of iceberg lettuce and tomato (Figure 24.2) illustrate
this point. Recognizing that the weight: surface area will vary, depending on the
thickness of the lettuce leaf and variations in shape of both vegetables, this
figure simply shows that large differences in ratios in weight: surface area can
exist among fruits and vegetables. Ratios for other fruits and vegetables with
geometric configurations other than a two-sided plane (lettuce) or a sphere
(tomato) would fall somewhere between these extremes. A decontamination
process designed to achieve, for example, a 31og 10 reduction in CFU/g of
lettuce or tomato would theoretically result, respectively, in approximately
0.11 and 181ogio reductions in CFU/cm ; a 31ogi reduction in CFU/cm of
lettuce or tomato would result, respectively, in approximately 79 and 0.51og 10
reductions in CFU/g [62].
A standard procedure for calculating and reporting populations of micro-
organisms on fruits and vegetables needs to be established and, if guidelines
or limits for maximum populations of pathogens are to be considered, calcu-
lation should be done on a standard basis (CFU/g or CFU/cm ). The number
of log 10 reductions in CFU resulting from a processing or decontamination
treatment should likewise be based on a standard procedure for calculation.
Regardless of the procedure used, if guidelines or limits and log 10 reductions
for specific pathogens are established, differences in weight and geometric
configuration of fruits and vegetables should be considered. Data need to
be subjected to appropriate statistical analysis to determine significant differ-
ences in populations of pathogens or spoilage microorganisms recovered from
produce that has been subjected to various treatment or storage conditions.
Sampling, Detection, and Enumeration of Pathogenic and Spoilage Microorganisms 561
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25
Rapid Detection of
Microbial Contaminants
Daniel Y.C. Fung
CONTENTS
25.1 Introduction 565
25.2 Sample Preparation and Treatments 566
25.3 Total Viable Cell Count Methodologies 567
25.4 Advances in Miniaturization and Diagnostic Kits 572
25.5 Immunological Testing 575
25.6 Instrumentation and Biomass Measurements 579
25.7 Genetic Testing 583
25.8 Biosensors 588
25.9 U.S., World Market, and Testing Trends (1999-2008) 590
25.10 Predictions of the Future 592
Acknowledgment 593
References 593
25.1 INTRODUCTION
Rapid methods and automation in microbiology is a dynamic area in
applied microbiology dealing with the study of improved methods for the
isolation, early detection, characterization, and enumeration of microorgan-
isms and their products in clinical, food, industrial, and environmental samples.
In the past 20 years this field has emerged into an important subdivision of
the general field of applied microbiology and is gaining momentum nationally
and internationally as an area of research and application to monitor the
numbers, kinds, and metabolites of microorganisms related to food spoilage,
food preservation, food fermentation, food safety, and foodborne pathogens.
Medical microbiologists began involvement with rapid methods around the
mid-1960s. In the 1970s developments started to accelerate and continued to
do so into the 1980s, 1990s, and up to the present day. Food microbiologists
were lagging about 10 years behind the medical microbiologists but in
the past decade they have greatly increased their activities in this field [1].
565
566 Microbiology of Fruits and Vegetables
This chapter presents rapid microbiological methods for food in general with
emphasis in fruit and vegetable microbiology.
25.2 SAMPLE PREPARATION AND TREATMENTS
One of the most important steps for successful microbiological analysis of any
material is sample preparation. With the advancement of microbiological
techniques and miniaturization of kits and test systems to ever smaller sizes,
proper sample preparation becomes critical. Chapter 24 discusses in detail
various sample preparation, detection, and enumeration methods for fruits
and vegetables. Some novel methods are discussed in this chapter.
Fruits and vegetables are considered solid food. The most efficient method
to prepare the samples for enumeration and detection is to use the Stomacher
instrument where a known weight of solid sample is placed in the stomacher
bag, and a volume of sterile diluent is added to make a 1:10 dilution of the
sample. Then the sample is "stomached" for one to two minutes before an
aliquot is taken out for viable cell count, differential count, or pathogen count
and detection. Dr. Anthony Sharpe invented the Stomacher about 25 years
ago, and now more than 40,000 units are in use worldwide. Recently he
introduced a new instrument called the Pulsifier (Microgen BioProducts Ltd,
Surrey, U.K.) for dislodging microorganisms from foods without excessively
breaking the food structure. The Pulsifier has an oval ring that can house
a plastic bag with sample and diluent. When the instrument is activated
the ring will vibrate vigorously for a predetermined time (30 to 60 seconds).
During this time microorganisms on the food surface or in the food will be
dislodged into the diluent with minimum destruction of the food. Fung et al. [2]
evaluated the Pulsifier against the Stomacher with 96 food items (including
beef, pork, veal, fish, shrimp, cheese, peas, a variety of vegetables, cereal, and
fruits) and found that both systems gave essentially the same viable cell count
in the food, but the 'pulsified" samples were much clearer than the
"stomached" samples. Kang et al. [3] found that the Pulsifier and Stomacher
had a correlation coefficient of 0.971 and 0.959 for total aerobic count and
coliform count, respectively, with 50 samples of lean meat tissues. More
recently, Wu et al. [4] made a comprehensive study of the Pulsifier versus the
Stomacher on 30 vegetables and reported no difference in total count and
coliform between the two methods (Table 25.1 for total count). However, there
were distinct differences in the liquids between the methods with pulsified
samples having less turbidity, less total solids, and higher pH than the
stomached samples (Table 25.2). The superior quality of microbial suspensions
with minimum food particles and inhibitors from the Pulsifier has positive
implications for general microbial analysis such as ease of pipetting samples
and ease of filtration through bacteriological membrane filters, as well as for
techniques such as adenosine triphosphate (ATP) bioluminescence tests,
DNA/RNA hybridization, polymerase chain reaction (PCR) amplifications,
enzymatic assays, etc.
Rapid Detection of Microbial Contaminants
567
TABLE 25.1
Comparison of Total Viable Cell Counts Obtained from Stomached and
Pulsified Samples of All Vegetables
Log CFU/g
Pulsifier (P)
Stomacher (S)
(P-S)
(P/S) ratio
1. Head lettuce
4.78
5.09
-0.31
0.94
2. Onions, green
6.20
6.18
0.02
1.00
3. Fresh carrot
5.83
5.85
-0.02
1.00
4. Radish-radicchio
6.07
6.04
0.03
1.00
5. Parsley
5.96
6.16
-0.20
0.97
6. Green leaf lettuce
5.85
6.13
-0.28
0.95
7. Romaine
5.82
4.03
1.79
1.44
8. Red leaf lettuce
6.12
6.12
0.00
1.00
9. Boston lettuce
5.99
5.79
0.20
1.03
10. Spinach
5.76
6.11
-0.35
0.94
11. Endive
6.11
4.82
1.29
1.27
12. Orange pepper
4.14
4.36
-0.22
0.95
13. Cucumber
6.03
4.25
1.78
1.42
14. Celery hearts
5.04
4.39
0.65
1.15
15. Broccoli
5.61
4.90
0.71
1.14
16. Cauliflower
3.29
5.31
-2.02
0.62
17. Snow peas
4.91
5.70
-0.79
0.86
18. Turnips
5.41
5.58
-0.17
0.97
19. Cilantro
6.07
6.10
-0.03
1.00
20. Zucchini squash
6.20
6.01
0.19
1.03
21. Rhubarb
5.65
4.38
1.27
1.29
22. Parsnips
6.12
6.12
0.00
1.00
23. Asparagus
6.10
5.72
0.38
1.07
24. Cabbage
3.54
2.31
1.23
1.53
25. Chinese cabbage
5.74
5.84
-0.10
0.98
26. Green beans
5.34
4.21
1.13
1.27
27. Tomato (hot house)
2.01
2.31
-0.30
0.87
28. Potato (baking)
5.75
5.48
0.27
1.05
29. Walla Walla jumbo
0.00
yellow onion
30. Eggplant
5.64
5.91
-0.27
0.95
Average
0.20
1.02
From Wu, V.C.H., Jitareerat,
P., and Fung,
D.Y.C., /. Rapid Methods Automat.
Microbiol, 11,
145, 2003. With permission fr<
Dm Food Nutrition Press, Trumbull, CT.
25.3 TOTAL VIABLE CELL COUNT METHODOLOGIES
One of the most important factors concerning food quality, food spoilage,
food safety, and potential implication of foodborne pathogens is the total
viable cell count of food, water, food contact surfaces, air, and environments
in food plants. The conventional standard plate count method has been in
use for the past 100 years in applied microbiology. The method involves
sample preparation, dilution, and plating with a nonselective or selective agar,
568 Microbiology of Fruits and Vegetables
TABLE 25.2
Comparison of Chemical Attributes of the Liquid Obtained from Stomached
(S) and Pulsified (P) Samples of All Vegetables
Turbidity 3
pH
TSS b
P
S
P
S
P
S
1. Head lettuce
0.01
0.073
6.66
6.48
0.2
0.2
2. Onions, green
0.031
0.148
6.46
6.29
0.4
0.6
3. Fresh carrot
0.016
0.365
6.76
6.61
0.25
0.9
4. Radish-radicchio
0.021
0.037
6.42
6.61
0.1
0.4
5. Parsley
0.033
0.322
6.94
6.46
0.15
0.4
6. Green leaf lettuce
0.063
0.192
6.88
6.6
0.05
0.4
7. Romaine
0.063
0.222
6.75
6.39
0.15
0.4
8. Red leaf lettuce
0.138
0.32
6.55
6.55
0.4
0.3
9. Boston lettuce
0.047
0.177
6.73
6.53
0.4
0.3
10. Spinach
0.282
0.56
6.66
6.45
0.4
0.4
11. Endive
0.102
0.283
6.63
6.45
0.4
0.5
12. Orange pepper
0.001
0.125
6.57
5.64
0.22
0.7
13. Cucumber
0.003
0.095
6.67
6.21
0.4
0.5
14. Celery hearts
0.003
0.123
6.78
6.45
0.2
0.5
15. Broccoli
0.024
0.271
6.96
6.86
0.3
0.5
16. Cauliflower
0.004
0.078
6.94
6.96
0.3
0.5
17. Snow peas
0.012
0.227
6.78
6.44
0.5
1.0
18. Turnips
0.001
0.019
6.81
6.64
0.3
0.6
19. Cilantro
0.14
0.765
6.67
6.4
0.1
0.7
20. Zucchini squash
0.003
0.094
6.85
6.73
0.3
0.5
21. Rhubarb
0.079
0.224
4.66
3.75
0.3
0.5
22. Parsnips
0.284
1.158
5.51
6.49
0.5
0.8
23. Asparagus
0.041
0.154
6.49
6.47
0.4
0.7
24. Cabbage
0.001
0.034
6.74
6.6
0.2
0.4
25. Chinese cabbage
0.011
0.01
6.68
6.57
0.22
0.2
26. Green beans
0.232
6.83
6.94
0.22
0.7
27. Tomato (hot house)
0.023
0.05
5.07
4.89
0.5
0.4
28. Potato (baking)
0.018
0.247
6.84
6.59
0.3
0.6
29. Walla Walla jumbo
0.013
0.042
6.74
6.15
0.4
0.8
yellow onion
30. Eggplant
0.082
0.47
6.72
6.3
0.22
0.8
Average
0.052
0.237
6.56
6.35
0.30
0.55
a Optical density measurement.
Total soluble solids.
From Wu, V.C.H., Jitareerat,
P., and Fung, D.Y.C., /.
Rapid Methods Automat. Microbiol., 1 1.
145, 2003. With permission from Food Nutrition Press,
Trumbull,
CT.
incubating the plates at 35°C, and counting the colonies after 48 hours. There
is a great variety of factors to be considered. These include plating media,
incubation time and temperature and incubation environment, and volumes
to be plated. The operation of the conventional standard plate count
method, although simple, is time-consuming both in terms of execution and
data collection. Also, this method utilizes a large number of test tubes, pipettes,
dilution bottles, dilution buffer, sterile plates, incubator space, and related
Rapid Detection of Microbial Contaminants 569
disposable materials and requires resterilizing and clean up of reusable
materials for further use.
Several methods have been developed, tested, and used effectively in
the past 20 years as alternatives to the standard plate count method. Most
of these methods were first designed to perform viable cell counts and relate
the counts to standard plate counts. Later, coliform count, fecal coliform
count, and yeast and mold counts were introduced. Further developments
in these systems include differential counts, pathogen counts, and even
pathogen detection after further manipulations. Many of these methods have
been extensively tested in many laboratories throughout the world and went
through AOAC (Association of Official Analytical Chemists) International
collaborative study approvals. The aim of these methods is to provide reliable
viable cell counts of food and water in more convenient, rapid, simple, and
cost effective alternative formats, compared to the cumbersome standard
plate count method.
The spiral plating method is an automated system to obtain viable
cell count (Spiral Biotech, Bethesda, MD). By use of a stylus, this instrument
can spread a liquid sample on the surface of a prepoured agar plate (selective
or nonselective) in a spiral shape (the Archimedes spiral) with a concentration
gradient starting from the center and decreasing as the spiral progresses
outward on the rotating plate. The volume of the liquid deposited at any
segment of the agar plate is known. After the liquid containing microorgan-
isms is spread, the agar plate is incubated overnight at an appropriate
temperature for the colonies to develop; the colonies appearing along the
spiral pathway can be counted either manually or electronically. The time
for plating a sample is only several seconds compared to minutes used in the
conventional method. Also, using a laser counter an analyst can obtain an
accurate count in a few second as compared with a few minutes in the tiring
procedure of counting colonies by the naked eye. The system has been used
extensively in the past 20 years with satisfactory microbiological results from
many products including meat, poultry, seafood, vegetables, fruits, dairy
products, and spices. Manninen et al. [5] evaluated the spiral plating system
against the conventional pour plate method using both manual count and
laser count and found that the counts were essentially the same for bacteria
and yeast. Newer versions of the spiral plater were introduced as Autoplater
(Spiral Biotech, Bethesda, MD) and Whitley Automatic Spiral Plater
(Microbiology International, Rockville, MD). With these automatic instru-
ments an analyst needs only to present the liquid sample, and the instrument
completely and automatically processes the sample, including resterilizing the
unit for the next sample.
The Isogrid system (Neogen, Lansing, MI) consists of a square filter with
hydrophobic grids printed on the filter to form 1600 squares for each filter. A
food sample is first weighed, homogenized, diluted, and enzymatically treated,
then passed through the filter assisted by vacuum. Microbes are trapped in the
squares on the filter. The filter is then placed on prepoured nonselective or
selective agar and then incubated for a specific time and temperature. Since
570 Microbiology of Fruits and Vegetables
a growing microbial colony cannot migrate over the hydrophobic material,
all colonies are confined to a square shape. The analyst can then count the
squares as individual colonies. Since there is a chance that more than one
bacterium is trapped in one square, the system has a most probable number
(MPN) conversion table to provide statistically accurate viable cell counts.
Automatic instruments are also available to count these square colonies in
seconds. This method also has been used to test a great variety of foods in the
past 20 years.
Petrifilm (3M Co., St. Paul, MN) is an ingenious system involves appro-
priate rehydratable nutrients embedded in a series of films in the unit. The unit
is little larger than the size of a credit card. To obtain viable cell count, the
protective top layer is lifted, and 1 ml of liquid sample is introduced to the
center of the unit, and then the cover is replaced. A plastic template is placed
on the cover to make a round mold. The rehydrated medium will support the
growth of microorganisms after suitable incubation time and temperature.
The colonies are counted directly in the unit. This system has a shelf life of
over one year in cold storage. The attractiveness of this system is that it
is simple to use, small in size, has a long shelf-life, does not require agar
preparation, and provides easy-to-read results. Recently the company also
introduced a Petrifilm counter so that an analyst only needs to place the
Petrifilm with colonies into the unit, and the unit will automatically count and
record the viable cell count in the computer. The manual form of the Petrifilm
has been used for many food systems and is gaining international acceptance
as an alternative to the standard plate count method.
Redigel system (3M Co., St. Paul, MN) consists of tubes of sterile nutrient
with a pectin gel in the tube but no conventional agar. This liquid system
is ready for use, and no heat is needed to "melt" the medium since there is
no agar in the liquid. After an analyst mixes 1 ml of liquid sample with the
liquid in the tube, the resultant contents are poured into a special Petri dish
coated with calcium. The pectin and calcium will react and form a gel which
will solidify in about 20 minutes. The plate is then incubated at the proper
time and temperature, and the colonies can be counted the same way as the
conventional standard plate count method.
The four methods described above have been in use for approximately
20 years. Chain and Fung [6] made a comprehensive evaluation of all four
methods against the conventional standard plate count method on 7 different
foods, 20 samples each, and found that the alternative systems and the con-
ventional method were highly comparable at an agreement of r = 0.95. In the
same study these researchers also found that the alternative systems cost less
than the conventional standard plate count method.
A newer alternative method, the SimPlate system (BioControl, Bellevue,
WA), has 84 wells imprinted in a round plastic plate. After the lid is removed,
a diluted food sample (1 ml) is dispensed onto the center landing pad, and
10 ml of rehydrated nutrient liquid, provided by the manufacturer, is poured
onto the landing pad. The mixture (food and nutrient liquid) is distributed
evenly into the wells by swirling the SimPlate in a gentle, circular motion.
Rapid Detection of Microbial Contaminants 571
Excessive liquid is absorbed by a pad housed in the unit. After 24 hours
of incubation at 35°C, the plate is placed under ultraviolet (UV) light. Positive
fluorescent wells are counted and the number is converted in the MPN table
to determine the number of bacteria present in the SimPlate. The method
is simple to use with minimum amount of preparation. A 198-well unit is
also available for samples with high counts. Using different media, the unit
can also make counts of total coliforms and E. coli counts, as well as yeast and
mold counts.
The above methods are designed to count aerobic microorganisms.
To count anaerobic microorganisms, one has to introduce the sample into
the melted agar, and after solidification the plates need to be incubated in
an enclosed anaerobic jar. In the anaerobic jar, oxygen is removed by the
hydrogen generated by the gas pack in the jar to create an anaerobic envi-
ronment. After incubation, the colonies can be counted and reported as
anaerobic count of the food. The method is simple but requires expensive
anaerobic jars and disposable gas packs. It is of concern that the interior
of the jar needs almost an hour to become anaerobic. Some strict anaero-
bic microorganisms may die during this one-hour period of reduction of
oxygen. Fung and Lee [7] developed a simple anaerobic double-tube system
which is easy to use and provides instant anaerobic condition for the
cultivation of anaerobes from foods. In this system, the desired agar (~23 ml)
is first autoclaved in a large test tube (OD 25 x 150 mm). When needed, the
agar is melted and tempered at 48° C. A liquid food sample (1 ml) is added
into the melted agar. A smaller sterile test tube (OD 16 x 150 mm) is inserted
into the large tube with the food sample and the melted agar. By so doing,
a thin film is formed between the two test tubes. The unit is tightly closed by a
screw cap. The entire unit is placed into an incubator for the colonies to
develop. No anaerobic jar is needed for this simple anaerobic system. After
incubation, the colonies developing in the agar film can be counted and provide
an anaerobic count of the food being tested. The Fung double-tube system
has been used extensively for applied anaerobic microbiology in the author's
laboratory for more than 20 years [8,9]. Recently, the author tested the
double-tube method for Clostridium perfringens in recreational waters, and he
was able to obtain anaerobic C. perfringens counts in about 6 to 8 hours from
the time of sampling to the time of reading the results. By combining the
Isogrid system with the double-tube method, the author can test volumes
of waters ranging from 1 to 100 ml or more.
The above-mentioned methods are designed to grow colonies to visible
sizes for enumeration and report the data as CFU per gram, milliliter, or
square centimeter of the food being tested.
A few "real time' 1 viable cell count methods have been developed
and tested in recent years. These methods rely on using "vital" stains to stain
"live' 1 cells or ATP detection of live cells. All these methods need careful
sample preparation, filtration, selection of dyes and reagents and instrumen-
tation. Usually the entire systems are quite costly. However, they can provide
results in one shift (8 hours) and can handle a large number of samples.
572 Microbiology of Fruits and Vegetables
The direct epifluorescent filter techniques (DFET) method has been tested
for many years and is in use in the U.K. for raw milk quality assurance
programs. In this method, the microorganisms are first trapped on a filter and
then the filter is stained with acridine orange dye. The slide is then observed
by UV microscopy. "Live" cells usually fluoresce orange-red, orange-yellow,
or orange-brown whereas "dead" cells fluoresce green. The slide can be read
manually or by a semiautomated counting system marketed by Bio-Foss,
which can provide a viable cell count in less than an hour.
The Chemunex Scan RDI system (Monmouth Junction, NJ) involves
filtering cells on a membrane and staining cells with vital dyes (Fluorassure).
After approximately 90 minutes of incubation (for bacteria), the membrane
with stained cells is read in a scanning chamber that can scan and count
fluorescing viable cells. This system has been used to test disinfecting solu-
tions against such organisms as Pseudomonas aeruginosa, Serratia marcescens,
Escherichia coli, and Staphylococcus aureus with satisfactory results.
The MicroStar system (Millipore Corporation, Billerica, MA) utilizes
ATP bioluminescence technology by trapping bacteria in a specialized mem-
brane (Milliflex). Individual live cells are trapped in the matrix of the filter
and grow into microcolonies. The filter is then sprayed with permeabilizing
reagent in a reaction chamber to release ATP. The bioluminescence reagent is
sprayed onto the filter. Live cells will give off light due to the presence of ATP,
the light is measured using a CCD camera, and thus the fluorescent particles
(live cells) are counted.
These are new developments in staining technology, ATP technology,
and instrumentation for viable cell counts. The application of these methods
for the food industry is still in the evaluation stage. The future looks promising.
25.4 ADVANCES IN MINIATURIZATION AND
DIAGNOSTIC KITS
Identification of microorganisms constituting normal flora, spoilage organ-
isms, foodborne pathogens, starter cultures, etc., in food microbiology is
an important part of microbiological manipulations. Conventional methods,
dating back more than 100 years, utilize large volumes of medium (10 ml
or more) to test for a particular characteristic of a bacterium (e.g., lactose
broth for lactose fermentation by Escherichia coli). Inoculating a test culture
into these individual tubes one at a time is also very cumbersome. According
to Hartman [10], over the years many microbiologists have devised vessels
and smaller tubes to reduce the volumes used for these tests. This author has
systematically developed many miniaturized methods to reduce the volume
of reagents and media (from 5 to 10 ml down to about 0.2 ml) for microbi-
ological testing in a convenient microtiter plate which has 96 wells arranged
in an 8x12 format. The basic components of the miniaturized system are
the commercially sterilized microtiter plates for housing the test cultures, a
multiple inoculation device, and containers to house solid media (large Petri
Rapid Detection of Microbial Contaminants 573
dishes) and liquid media (in another series of microtiter plates with 0.2 ml
of liquid per well). The procedure involves placing liquid cultures (pure
cultures) to be studied into sterile wells of a microtiter plate (~0.2 ml for each
well) to form a master plate. Each microtiter plate can hold up to 96 different
cultures, 48 duplicate cultures, or various combinations as desired. The
cultures are then transferred using a sterile multipoint inoculator (96 pins
protruding from a template) to solid or liquid media. Sterilization of
the inoculator is accomplished by alcohol flaming. Each transfer represents
96 separate inoculations in the conventional method. After incubation at
an appropriate temperature, the growth of cultures on solid media or liquid
media can be observed and recorded, and the data can be analyzed. These
methods are ideal for studying large numbers of isolates or for research
involving challenging large numbers of microbes against a host of test com-
pounds. Using this miniaturized system the author has characterized thousands
of bacterial cultures isolated from meat and other foods, studied the effects
of organic dyes against bacteria and yeasts, and performed challenge studies of
various compounds against microbes with excellent results.
Other scientists also have miniaturized many systems and developed
them into diagnostic kits in the late 1960s and early 1970s. Diagnostics systems
such as API, Enterotube, Minitek, Crystal ID, MicroID, RapID, Biolog,
and VITEK systems are currently available. Most of these systems were first
developed for identification of enterics (salmonella, shigella, proteus,
enterobacter, etc.). Later, many of these companies expanded the capacity of
their diagnostic systems to identify nonfermentors, anaerobes, Gram-positive
organisms, and even yeast and molds. Originally, an analyst needed to read the
color reaction of each well in the diagnostic kit and then use a manual
identification code to "key" out the organisms. Recently, diagnostic companies
have developed automatic readers interfaced with a computer to provide rapid
and accurate identification of the unknown cultures.
The most successful and sophisticated miniaturized automated identifi-
cation system is the VITEK system (bioMerieux, Hazelwood, MO) which
utilizes a plastic card containing 30 tiny wells in each of which there is a
different reagent. The unknown pure culture in a liquid form is "pressurized"
into the wells in a vacuum chamber, and then the cards are placed in an
incubator for a period of time ranging from 4 to 12 hours. The instrument
periodically scans each card and compared the color changes or gas produc-
tion of each tiny well with the database of known cultures. VITEK can identify
a typical Escherichia coli culture in 2 to 4 hours. Each VITEK unit can auto-
matically scan 120 cards or more simultaneously. There are a few thousand
VITEK units currently in use in the world, and the database is especially good
for clinical isolates.
Biolog system (Hayward, CA) is also a miniaturized system using the
microtiter format for growth and reaction information. Pure cultures are
first isolated on agar and then suspended in a liquid to the appropriate
density (~61og cell/ml). The culture is then dispensed into a microtiter plate
containing different carbon sources in 95 wells and one nutrient control well.
574 Microbiology of Fruits and Vegetables
The plate with the pure cultures is then incubated overnight, after which
the microtiter plate is removed, and the color pattern of the wells with carbon
utilization is observed and compared with profiles of typical patterns of
microbes using computer software to obtain identification. This system is very
ambitious and tries to identify more than 1400 genera and species of environ-
mental, food, and medical isolates from major groups of Gram-positive,
Gram-negative, and other organisms. There is no question that miniaturiza-
tion of microbiological methods has saved much material and operational time
and has provided needed efficiency and convenience in diagnostic micro-
biology. The systems developed by the author and others can be used in
many research and developmental laboratories for studying large numbers of
cultures. These miniaturized systems and diagnostic kits can be used efficiently
in identifying isolates from fruits and vegetables.
The conventional viable cell count method and the MPN (3- or 5-tube
MPN) procedure have been used extensively for water and food testing for
almost 100 years. The conventional methods are too cumbersome, time
consuming, and utilize too many tubes, plates, and media. More than 30 years
ago, Fung and Kraft [11] miniaturized the viable cell count procedure by
diluting the samples in the microtiter plate using 0.025 ml size calibrated
loops in 1:10 dilution series. One can simultaneously dilute 12 samples to
8 series of 1:10 dilutions in a matter of minutes. After dilution, the samples
can be transported by a calibrated pipette and spot plating 0.025 ml on agar;
one conventional agar plate can accommodate 4 to 8 spots. After incubation,
colonies in the spots can be counted, and the number of viable cells in
the original sample can be calculated since all the dilution factors are known.
The accepted range of colonies to be counted in one spot is 10 to 100. The
conventional agar plate standard is from 25 to 250 colonies per plate. This
procedure actually went through an AOAC International collaborative study
with satisfactory results [12]. However, the method has not received much
attention and is waiting to be "rediscovered" in the future.
In a similar vein, Fung and Kraft [13] also miniaturized the MPN
method in the microtiter plate by diluting a sample in a 3-tube miniaturized
series. In one microtiter plate one can dilute 4 samples, each in triplicate
(3-tube MPN), to 8 series of 1:10 dilution. After incubation, the turbidity of the
wells is recorded, and a modified 3-tube MPN table can be used to calculate
the MPN of the original sample. This procedure has recently received renewed
interests in the scientific community.
Walser [14] in Switzerland reported the use of an automated system for
microtiter plate assay to perform classic MPN of drinking water. He used
a pipetting robot equipped with sterile pipetting tips for automatic dilution of
the samples. After incubation, the robot placed the plate in a microtiter plate
reader and obtained MPN results with the use of a computer. The system can
cope with low or high bacterial load from to 20,000 colonies per milliliter.
This system takes out the tediousness and personnel influences on routine
microbiological work and can be applied to determine MPN of fecal organisms
in water as well as other microorganisms of interest in food microbiology.
Rapid Detection of Microbial Contaminants 575
Irwin et al. [15] in the U.S. also worked on a similar system using a
modified Gauss-Newton algorithm and a 96-well micro-technique for calcu-
lating MPN using Microsoft EXCEL spreadsheets. These improvements
are possible today compared with the original work of the author in 1969
because: (1) automated instruments are now available in many laboratories
to dispense liquid into the microtiter plate and automated dilution instru-
ments are also available to facilitate rapid and aseptic dilutions of samples;
(2) automated readers of microtiter wells are now commonplace to read effi-
ciently turbidity, color, and fluorescence of the liquid in the wells for calcu-
lation of MPN; and (3) elegant mathematic models, computer interpretations
and analysis, and printout of data are now available which the author could
not have envisioned back in 1969.
25.5 IMMUNOLOGICAL TESTING
The antigen and antibody reaction has been used for decades for detecting
and characterizing microorganisms and their components in medical, food, and
diagnostic microbiology. This reaction is the basis for serotyping bacteria
such as salmonella, Escherichia coli 0157:H7, and Listeria monocytogenes.
These antibodies can be polyclonal (a mixture of several antibodies in the
antisera which can react with different sites of the antigens) or monoclonal
(only one pure antibody in the antiserum which will react with only one
epitope of the antigens). Both polyclonal and monoclonal antibodies have been
used extensively in applied food microbiology. There are many ways to
perform antigen-antibody reactions, but the most popular format in recent
years has been the "sandwich" enzyme-linked immunosorbant assay, popularly
known as the ELISA test.
Briefly, antibodies (e.g., anti-salmonella antibody) are fixed on a solid
support (e.g., wells of a microtiter plate). A solution containing a suspect
target antigen (e.g., salmonella) is introduced to the microtiter well. If
the solution has salmonella antigens, it will be captured by the immobilized
antibodies.
After washing away food debris and excess materials, another anti-
salmonella antibody complex is added into the solution. The second anti-
salmonella antibody will react with another part of the trapped salmonella.
This second antibody is linked with an enzyme such as horseradish peroxi-
dase. After another washing to remove debris, a chromagen complex such as
tetramethylbenzidine and hydrogen peroxide is added. The enzyme will
react with the chromagen and will produce a colored compound that will
indicate that the first antibody has captured salmonella. If all the reaction
procedures are done properly and the liquid in a microtiter well exhibits a
color reaction, then the sample is considered positive for salmonella.
This procedure is simple to operate and has been used for decades with
excellent results. It should be emphasized that these ELISA tests need about
a million cells to be reactive, and therefore, before performing the ELISA tests,
576 Microbiology of Fruits and Vegetables
the food sample has to go through an overnight incubation so that the target
organism reaches a detectable level. The total time to detect pathogens by
these systems includes the enrichment time of the target pathogens (ca. 24 hrs).
Many diagnostic companies (such as BioControl, Organon Teknika,
and Tecra) have marketed ELISA test kits for foodborne pathogens and toxins
(e.g., salmonella, Escherichia coli) and toxins (e.g., staphylococcal enterotox-
ins). However, the time involved in sample addition, incubating, washing
and discarding of liquids, adding of another antibody complex, washing, and,
finally, adding of reagents for color reaction all contribute to the inconveni-
ence of the manual operation of the ELISA test. Recently several companies
have completely automated the entire ELISA procedure.
VIDAS (bioMerieux, Hazelwood, MO) is an automated system which
can perform the entire ELISA procedure automatically and can complete an
assay in 45 minutes to 2 hours, depending on the test kit. Since VIDAS utilizes
a more sensitive fluorescent immunoassay for reporting the results, the system
is named enzyme-linked fluorescent assay (ELFA). All the analyst needs to
do is to present to the reagent strip a liquid sample of an overnight enriched
sample. The reagent strip contains all the necessary reagents in a ready-to-use
format. The instrument will automatically transfer the sample into a plastic
tube called the solid phase receptacle (SPR) which contains antibodies
to capture the target pathogen or toxin. The SPR will be automatically
transferred to a series of wells in succession to perform the ELFA test. After
the final reaction, the result can be read, and interpretation of a positive
or negative test will be automatically determined by the instrument. Presently,
VIDAS can detect listeria, Listeria monocytogenes, salmonella, E. coli 0157,
staphylococcal enterotoxin, and Campylobacter. Its manufacturers also
market an immuno-concentration kit for salmonella and E. coli 0157.
Currently more than 13,000 VIDAS units are in use internationally.
BioControl (Bellevue, WA) markets an enzyme immunoassay (EIA) system
called Assurance EIA which can be adapted to automation for high-volume
testing. Assurance EIA is available for salmonella, listeria, E. coli 0157:H7,
and Campylobacter. Diffchamb (Hisings Backa, Sweden) has a high-precision
liquid delivery system that can be used to perform a variety of ELISA tests
depending on the pathogens to be tested. Tecra OPUS (International
BioProducts, Redmond, WA) and Bio-Tek (Highland Park, VT) instruments
can also perform ELISA tests automatically as long as the proper reagents
are applied to the system. Many ELISA test kits are now highly standardized
and the test can be performed automatically to increase efficiency and reduce
human errors.
Another exciting development in immunology is the use of lateral flow
technology to perform antigen-antibody tests. In this system, the unit has
three reaction regions. The first well contains antibodies to react with target
antigens. These antibodies have color particles attached to them. A liquid
sample (after overnight enrichment) is added to this well, and if the target
organism (e.g. E. coli 0157:H7) is present, it will react with the antibodies.
The complex will migrate laterally by capillary action to the second region
Rapid Detection of Microbial Contaminants 577
which contains a second antibody designed to capture the target organism.
If the target organism is present, the complex will be captured, and a blue
line will form due to the color particles attached to the first antibody. Excess
antibodies will continue to migrate to the third region which contains another
antibody that reacts with the first antibody (which has now become an antigen)
and will form a blue color band. This is a "control" band indicating that
the system is functioning properly. The entire procedure takes only about 10
minutes. This is truly a rapid test!
Neogen (Lansing, MI; Reveal system) and BioControl (Bellevue, WA;
VIP system) are the two main companies marketing this type of system for
E. coli 0157, salmonella, and listeria. Merck KGaA (Darmstadt, Germany)
developed similar systems using gold particles in the reagent to increase the
sensitivity of the test.
A number of interesting methods utilizing growth of the target pathogen
are also available to detect antigen-antibody reactions. The BioControl 1-2
test (BioControl, Bellevue, WA) is designed to detect motile salmonella from
foods. In this system, the food sample is first preenriched for 24 hours in a
broth, and then 0.1 ml is inoculated into one of the chambers in an L-shaped
system. The chamber contains selective enrichment liquid medium for salmon-
ella. There is a small hole connecting the liquid chamber with a soft agar
chamber through which salmonella can migrate. An opening on the top of
the soft agar chamber allows the analyst to deposit a drop of polyvalent
anti-H antibodies against flagella of salmonella. The antibodies move
downward in the soft agar due to gravity and diffusion. If salmonella is
present, it will migrate throughout the soft agar. As the salmonella and the
anti-H antibodies meet, they will react and form a visible V-shaped "immuno-
band.'" The presence of the immunoband indicates the presumptive positive
for salmonella in the food sample. This reaction occurs after overnight
incubation of the unit. This system is easy to use and interpret, and it has
gained popularity because of its simplicity.
Tecra (Roseville, Australia) developed a detection system (Unique
Salmonella) that combines immuno-capturing, growth of the target patho-
gen, and an ELISA test in a simple-to-use self-contained unit. The food is
first preenriched in a liquid medium overnight and an aliquot is added into
the first tube of the unit. Into this tube a dipstick coated with salmonella
antibodies is introduced and left in place for 20 minutes; at this time the
antibodies will capture salmonella, if present. The dipstick, with salmonella
attached, is then washed and placed into a tube containing growth medium.
The dipstick is left in this tube for 4 hours. During this time, if salmonella is
present, it replicates, and the newly produced salmonella are automatically
trapped by the coated antibodies. Thus, after 4 hours of replication, the
dipstick becomes saturated with trapped salmonella. The dipstick is then
transferred to another tube containing a second antibody conjugated to
enzyme, and the tube contents are allowed to react for 20 minutes. After this
second antigen-antibody reaction, the dipstick is washed in the fifth tube and
placed into the last tube for color development similar to other ELISA tests.
578 Microbiology of Fruits and Vegetables
Development of a purple color on the dipstick indicates the presence
of salmonella in the food. The entire process, from incubation of food sample
to reading of the test results, requires about 22 hours, making it an attractive
system for detection of salmonella. A similar system can now also detect
listeria. An automated system is now being marketed.
The BioControl 1-2 test and the Unique Salmonella test are designed
for laboratories with a low volume of tests. Thus, both the automatic systems
and the hands-on unit systems have their place in different food testing
laboratory situations.
A truly innovative development in applied microbiology is the immuno-
magnetic separation system. Vicam (Somerville, MA) pioneered this concept
by coating antibodies against listeria on metallic particles. Large numbers
of these particles (in the millions) are added into a liquid suspected to contain
listeria cells. The antibodies on the particles will capture the listeria cells while
the mixture is rotated for about an hour. After the reaction has gone to
completion, the tube is placed next to a powerful magnet which will immo-
bilize all the metallic particles at the side of the glass test tube regardless
of whether the particles have or have not captured the listeria cells. The rest of
the liquid will be decanted. By removing the magnet from the tube, the metallic
particles can again be suspended in a liquid. At this point, the only cells in the
solution will be the captured listeria. By introducing a smaller volume of liquid
(e.g., 10% of the original volume), the cells are now concentrated by a factor
of 10. Cells from this liquid can be detected by direct plating on selective
agar, ELISA tests, PCR reaction, or other microbiological procedures in
almost pure culture state. Immunomagnetic capture can save at least one day
in the total protocol of preenrichment and enrichment steps of pathogen
detection in food.
Dynal (Oslo, Norway) developed this concept further by use of very
homogeneous paramagnetic beads that can carry a variety of molecules such
as antibodies, antigens, and DNA. Dynal has developed beads to capture
E. coli 0157, listeria, Cryptosporidium, giardia, and others. Furthermore,
the beads can be supplied without any coating materials, and scientists can
tailor them to their own needs by coating with the necessary antibodies or
other capturing molecules for detection of target organisms. Currently, many
diagnostic systems (ELISA, PCR, etc.) are incorporating an immunomagnetic
capture step to reduce incubation and increase sensitivity of the entire protocol.
Fluorescent antibody techniques have been used for decades for the detec-
tion of salmonella and other pathogens. Similar to the DEFT test designed
for viable cell count, fluorescent antibodies can be used to detect a great variety
of target microorganisms such as E. coli 0157:H7 in milk and juice.
One of the newest and fastest immunological methods to detect food-
borne pathogens is the Pathatrix system (Matrix MicroScience, Golden, CO).
Wu et al. [16] tested a same-day protocol for the detection of Escherichia coli
0157:H7 by the Pathatrix system (which employs a novel immuno-capture
method) and Colortrix (a rapid ELISA test). The Pathatrix system can circulate
a 4.5 hour preenriched 250 ml sample (25 g of food in 225 ml of preenrichment
Rapid Detection of Microbial Contaminants 579
broth) over a sheet of paramagnetic beads coated with antibodies against
E. coli 0157:H7 many times in 30 minutes to capture almost all target patho-
gens. This circulation system increased the concentration of E. coli 0157:H7
from the population after 4.5 hours of enrichment to 1.2 to 2.6 log CFU/25g
higher concentration in 30 minutes. After Pathatrix concentration the beads
with target pathogens are applied to the Colortrix system, a rapid ELISA
system that was able to detect E. coli 0157:H7 in 15 minutes. The results
indicated an excellent correlation (100%) between positive Pathatrix/Colortrix
(5.25 hours) compared with a 30-hour conventional plating method. The
sensitivity of the system is from 0.7 to 2.1 log CFU/25g as the initial concen-
tration of E. coli 0157:H7 in the sample before the 4.5 hours of enrichment.
This system is also able to detect Listeria monocytogenes, Campylobacter,
and other pathogens.
Antigen-antibody reaction provides a powerful system for rapid detec-
tion of all kinds of pathogens and molecules. This section has described
some of the useful methods developed for applied food microbiology. Some
systems are highly automated, and others are exceedingly simple to operate.
It should be emphasized that many of the immunological tests described in
this section provide presumptive positive or presumptive negative screening test
results. For negative screening results, the food in question is allowed to be
shipped for commerce. For presumptive positive test results, the food will
not be allowed for shipping until confirmation of the positive is done by the
conventional microbiological methods.
25.6 INSTRUMENTATION AND BIOMASS
MEASUREMENTS
As the field of rapid methods and automation has developed, the boundaries
between instrumentation and diagnostic tests have begun to merge. Instru-
mentation is now playing an important function in improving the efficiency
of diagnostic kit systems, and the trend will continue. The following discus-
sions are mainly on instrumentation measuring signals related to microbial
growth.
Instruments can be used to monitor changes in a population such as
ATP levels, levels of specific enzymes, pH, electrical impedance, conductance
and capacitance, generation of heat, radioactivity, carbon dioxide, and others.
It is important to note that for the information to be useful, these param-
eters must be related to viable cell counts of the same sample series. In general,
the larger the number of viable cells in the sample, the shorter the detec-
tion time of these systems. A scattergram is then plotted and used for further
comparison of unknown samples. The assumption is that as the number of
microorganisms increases in the sample, these physical, biophysical, and
biochemical events will also increase accordingly. When a sample has 5 or 6 log
organisms/ml, detection time can be achieved in about 4 hours from the time
the sample is placed in the instrument.
580 Microbiology of Fruits and Vegetables
All living things utilize ATP. In the presence of a firefly enzyme system
(luciferase and luciferin system), oxygen, and magnesium ions, ATP will facili-
tate the reaction to generate light. The amount of light generated by this
reaction is proportional to the amount of ATP in the sample. Thus, the
light units can be used to estimate the biomass of cells in a sample. The light
emitted by this process can be monitored by a sensitive and automated
fluorimeter. Some instruments can detect as little as 100 to 1000 femtograms
of ATP (1 femtogram, 1 fg, is —15 log g). The amount of ATP in one
colony-forming unit has been reported as 0.47 fg with a range of 0.22 to 1.03 fg.
Using this principle, many researchers have used ATP to estimate the number
of microbial cells in solid and liquid foods.
Initially, scientists attempted to use ATP to estimate the total viable cell
count in foods. The results are inconsistent due to the fact that (1) different
microorganisms have different amounts of ATP per cell (e.g., a yeast cell can
have 100 times more ATP than a bacterial cell); (2) even for the same organism,
the amount of ATP per cell is different at different growth stages; and (3)
background ATP from other biomass such as blood and biological fluids in the
foods interferes with the target bacterial ATP. Only after much research and
development will scientists be able to separate nonmicrobial ATP from micro-
bial ATP and obtain reasonable accuracy in relating ATP to viable cell counts
in foods. Since obtaining an ATP reading takes only a few minutes, the
potential of exploring these methods further exists. To date, ATP has not been
applied much to estimation of viable cell counts in food microbiology
laboratories.
From another viewpoint, the presence of ATP in certain foods such as
wine is undesirable regardless of the source. Thus monitoring ATP can be a
useful tool for quality assurance in the winery.
There has been a paradigm shift in the field of ATP detection in
recent years. Instead of detecting ATP of microorganisms, systems are now
designed to detect ATP from any source for hygiene monitoring. The idea is
that a dirty food processing environment will have a high ATP level, and
a properly cleansed environment will have a low ATP level regardless of
what contributed to the ATP in these environments. Once this concept is
accepted by the food industry, there will be an explosion of ATP systems being
used in the food industry for hygiene monitoring. In all of these systems, the
key is to be able to obtain an ATP reading in the form of relative light units
(RLUs) and to relate these units to the cleanliness of food processing surfaces.
The scale of RLU readings obtained from different surfaces in food factories
encompasses acceptable, marginal, and unacceptable levels. Since there is no
standard as to what constitutes an absolutely acceptable ATP level in any given
environment, these RLUs are quite arbitrary. In general, a dirty environment
will have high RLUs, and after proper cleaning the RLUs will decrease.
Besides the sensitivity of the instruments, an analyst should consider the
following attributes in selecting a particular system: simplicity of operation,
compactness of the unit, computer adaptability, cost of the unit, support from
the company, and documentation of usefulness of the system.
Rapid Detection of Microbial Contaminants 581
Besides the above mentioned issues, Dreibelbis [17] in a study of five ATP
instruments for hygiene monitoring of a food plant considered the following
attributes to be important as selection criteria of the systems: the ability of the
technicians in the microbiological laboratory to use the ATP bioluminescence
hygiene monitoring system without supervision, the reputation of the ATP
system in the industry, and the quality of services received from the manu-
facturer during the evaluation of the product.
Currently the following ATP instruments are available: Lumac (Landgraaf,
the Netherlands), BioTrace (Plainsboro, NJ), Lightning (BioControl, Bellevue,
WA), Hy-Lite (EM Science, Darmstadt, Germany), Charm 4000 (Charm
Sciences, Maiden, MA), Celsis system SURE (Cambridge, U.K.), Zylux
(Maryville, TN), Profile 1 (New Horizon, Columbia, MD), and others.
As microorganisms grow and metabolize nutrients, large molecules are
metabolized to smaller molecules in a liquid system and cause a change in
electrical conductivity and resistance in the liquid as well as at the interface
of electrodes. These changes can be expressed as impedance, conductance,
and capacitance changes. When a population of cells reaches about 5 log
CFU/ml, it will cause a change in these parameters. Thus, when a food has
a large initial population, the time to make this change will be shorter than
with a food that has a smaller initial population. The detection time of the
test sample, the time when the curve accelerates upward from the baseline,
is inversely proportional to the initial concentration of microorganisms in the
food. In order to use these methods, a series of standard curves must be
constructed by making viable cell counts in food with different initial
concentrations of cells and then measuring the resultant detection time.
A scattergram can then be plotted. Thereafter, in the same food system, the
number of the initial population of the food can be estimated by the detection
time on the scattergram.
The Bactometer (bioMerieux, Hazelwood, MO) has been in use for many
years to measure impedance changes by microorganisms in foods, water,
cosmetics, and similar products. Samples are placed in the wells of a 16-well
module which is then plugged into the incubator to start the monitoring
sequence. As the cells reach the critical number (5 to 6 log/ml), the change
in impedance increases sharply, and the monitor screen shows a slope similar
to the log phase of a growth curve. The detection time can then be obtained to
determine the initial population of the sample. If one sets a cut-off point of
6 log CFU/g of food for acceptance or rejection of the product, and the
detection time is 4 hours ± 15 minutes, then one can use the detection time as
a criterion for quality assurance of the product. Food that exhibits no change
of impedance curve after more than 4 hours and 1 5 minutes in the instrument
is acceptable while food that exhibits a change of impedance curve before
3 hours and 45 minutes will not be acceptable. For convenience the instrument
is designed such that the sample bar displayed on the screen for a food will
flash red for an unacceptable sample, green if acceptable, and yellow for
marginally acceptable. The rapid automated bacterial impedance technique
(RABIT) is a similar system, marketed by Bioscience International (Bethesda,
582 Microbiology of Fruits and Vegetables
MD) for monitoring microbial activities in food and beverages. Instead of the
16-well module used in the Bactometer, individual tubes containing electrodes
are used to house the food samples.
The Malthus system (Crawley, U.K.) uses conductance changes of the
fluid to indicate microbial growth; it generates conductance curves similar to
impedance curves used in the Bactometer. The Malthus system uses indi-
vidual tubes for food samples. Water heated to the desired temperature (e.g.,
35°C) is used as the temperature control instead of heated air as with the
previous two systems. All these systems have been evaluated by various
scientists in the past 10 to 15 years with satisfactory results. All have their
advantages and disadvantages depending on the type of food being analyzed.
These systems can also be used to monitor targeted groups of organisms such
as coliform or yeast and mold using specially designed culture media. In fact,
the Malthus system has a salmonella detection protocol that was approved
by AOAC International.
BacT/Alert Microbial Detection System (Organon Teknika, Durham,
NC) utilizes colorimetric detection of carbon dioxide production by micro-
organisms in a liquid system using sophisticated computer algorithms and
instrumentation. Food samples are diluted and placed in special bottles with
appropriate nutrients for growth of microorganisms and production of carbon
dioxide. At the bottom of the bottle there is a sensor that is responsive to the
amount of carbon dioxide in the liquid. When a critical amount of the gas is
produced, the sensor changes from dark green to yellow, and this change is
detected by reflectance colorimetry automatically. The units can accommodate
120 or 240 culture bottles. Detection time of a typical culture of E. coli is about
6 to 8 hours.
BioSys (BioSys, Inc., Ann Arbor, MI) utilizes color changes of media
(designed for specific target organisms) during the growth of cultures to detect
and estimate organisms in foods and liquid systems. The uniqueness of
the system is that the color compounds developed during microbial growth
are diffused into an agar column situated at the bottom of the unit, and
the changes are measured automatically without the interference of food
particles in the chamber. Depending on the initial microbial load in the food,
microbial information can be obtained during the same production shift that
the sample was taken in a food processing operation. The system is easy to
use and can accommodate 32 samples for one incubation temperature or
128 samples for 4 independent incubation temperatures in different models.
The system is designed for bioburden testing and HACCP (hazard analysis
critical control points) control and can test for indirect total viable cell,
coliform, E. coli, yeast, mold, and lactic acid bacteria counts in swab samples
and environmental samples.
Basically, any type of instrument that can continuously and automatically
monitor turbidity and color changes of a liquid in the presence of microbial
growth can be used for rapid detection of the presence of microorganisms.
There will definitely be more systems of this nature on the market in years
to come.
Rapid Detection of Microbial Contaminants 583
25.7 GENETIC TESTING
Rapid tests discussed earlier for detection and characterization of microorgan-
isms were based on phenotypic expressions of genotypic characteristics of
microorganisms. The phenotypic expressions are subject to growth conditions
such as temperature, pH, nutrient availability, oxidation-reduction potentials,
environmental and chemical stresses, toxins, and water activities. Phenotypic
expression, even including immunological tests, depends on cells' ability to
produce the target antigens to be detected by the available antibodies or vice
versa. The conventional "'gold standards" of diagnostic microbiology rely
on phenotypic expression or traits that are inherently subject to variation.
Genotypic characteristics of a cell are far more stable than its phenotype.
The natural mutation rate of a bacterial culture is about 1 in 100 million cells.
Thus, there has been a push in recent years to make genetic test results
the confirmative and definitive identification step in diagnostic microbiology.
The debate is still continuing, and the final decision has not been reached by
governmental and regulatory bodies for microbiological testing. Genetic-based
diagnostic and identification systems are discussed in this section.
Hybridization of the deoxyribonucleic acid (DNA) sequence of an
unknown bacterium by a known DNA probe is the first stage of genetic
testing. The Genetrak system (Framingham, MA) provides a sensitive and
convenient method to detect pathogens such as salmonella, listeria, Campylo-
bacter, and E. coli 0157 in foods. Initially, the system utilized radioactive
isotopes bound to DNA probes to detect complementary DNA of unknown
cultures. The drawbacks of the first generation of this type of probes are
(1) most food laboratories are not eager to work with radioactive materials in
routine analysis and (2) there are limited copies of DNA in a cell. The second
generation of probes uses enzymatic reactions to detect the presence of the
pathogens and uses RNA as the target molecule. In a cell, there is only one
complete copy of DNA; however, there may be 1,000 to 10,000 copies of
ribosomal RNA. Thus, the new generation of probes is designed to detect
target RNA using color reactions. After enrichment of cells (e.g., salmonella) in
a food sample for about 18 hours, the cells (target cells as well as other
microbes) are lysed by a detergent to release cellular materials (DNA, RNA,
and other molecules) into the enrichment solution. Two RNA probes (designed
to react with one piece of target salmonella RNA) are added into the solution.
The capture probe with a long tail of a nucleotide (e.g., polyadenine tail or
AAAAA) is designed to capture the RNA onto a dipstick with a long tail
of thymine (TTTTT). The reporter probe, with an enzyme attached, will react
with the other end of the RNA fragment. If salmonella RNA molecules
are present, the capture probes will attach to one end of the RNA, and the
reporter probes will attach to the other end. A dipstick coated with many
copies of a chain of complementary nucleotide (e.g., thymine, TTTTT) will
be placed into the solution. Since adenine (A) will hybridize with thymine (T),
the chain (TTTTT) on the dipstick will react with the AAAAA and thus
capture the target RNA complex onto the stick. After washing away debris and
584 Microbiology of Fruits and Vegetables
other molecules in the liquid, a chromagen is added. If the target RNA is
captured, then the enzyme present in the second probe will react with the
chromagen and will produce a color reaction indicating the presence of
the pathogen in the food. In this case, the food is positive for salmonella. The
system developed by Genetrak has been evaluated and tested for many years
and has AOAC International approval of the procedure for many food types.
More recently, Genetrak has adapted a microtiter format for more efficient
and automated operation of the system.
PCR is now an accepted method to detect pathogens by amplification
of the target DNA and detecting the target PCR products. Basically, a DNA
molecule (double helix) of a target pathogen (e.g., salmonella) is first denatured
at about 95° C to form single strands, then the temperature is lowered to
about 55°C for two primers (small oligonucleotides specific for salmonella)
to anneal to specific regions of the single stranded DNA. The temperature is
increased to about 70°C for a special heat-stable polymerase, the TAQ enzyme
from Thermus aquaticus, to add complementary bases (A, T, G, or C) to the
single-stranded DNA and complete the extension to form a new double
strand of DNA. This is called a thermal cycle. After this cycle, the tube will
be heated to 95°C again for the next cycle. After one thermal cycle, one copy of
DNA will become two copies. After about 21 cycles and 31 cycles, one million
and one billion copies of the DNA will be formed, respectively. This entire
process can be accomplished in less than an hour in an automatic thermal
cycler. Theoretically, if a food contains one copy of salmonella DNA, the
PCR method can detect the presence of this pathogen in a very short time.
After PCR reactions, one still needs to detect the presence of the PCR products
to indicate the presence of the pathogen. Four commercial kits for PCR
reactions and detection of PCR products are briefly discussed in the following.
The BAX system (Qualicon, Inc., Wilmington, DE) for screening food-
borne pathogens combines DNA amplification and automated homogeneous
detection to determine the presence or absence of a specific target. All primers,
polymerase, and deoxynucleotides necessary for PCR as well as a positive
control and an intercalating dye are incorporated into a single tablet. The
system works directly from an overnight enrichment of the target organisms.
No DNA extraction is required. Assays are available for salmonella, E. coli
0157:H7, listeria genus, and Listeria monocytogenes. The system uses an array
of 96 blue LEDs as the excitation source and a photomultiplier tube to detect
the emitted fluorescent signal. This integrated system improves the ease-of-
use of the assay. In addition to simplifying the detection process, the new
method converts the system to a homogeneous PCR test. The homogenous
detection process monitors the decrease in fluorescence of a double-stranded
DNA (dsDNA) intercalating dye in solution with dsDNA as a function of
temperature. Following amplification, melting curves are generated by slowly
ramping the temperature of the sample to a denaturing level (95° C). As the
dsDNA denatures, the dye becomes unbound from the DNA duplex, and the
fluorescent signal decreases. This change in fluorescence can be plotted against
temperature to yield a melting curve waveform. This assay thus eliminates the
Rapid Detection of Microbial Contaminants 585
need for gel-based detection and yields data amenable to storage and retrieval
in an electronic database. In addition, this method reduces the hands-on time
of the assay and reduces the subjectivity of the reported results. Further,
melting curve analysis makes possible the ability to detect multiple PCR
products in a single tube. The inclusivity and exclusivity of the BAX system
assays reach almost 100% meaning that false positive and false negative rates
are almost zero. The automated BAX system can now be used with assays for
the detection of Cryptosporidium parvum and Campylobacter jejunilcoli and
for the quantitative and qualitative detection of genetically modified organisms
in soy and corn. The new BAX system is far more convenient than the old
system in which a gel electrophoresis step was required to detect PCR products
after thermal cycling.
The following two methods also have been developed to bypass the elec-
trophoresis step to detect PCR products. These methods are called "real-time
PCR" because they involve a solution in which a fluorescent signal increases if
the target sequence is present in the solution. They rely on the use of fluo-
rescent molecules and can directly measure the amplification products while
amplification is in progress. The more target DNA in the solution, the sooner
the number of PCR products will reach the detection threshold and can be
detected since fewer thermal cycles are needed, compared to a solution with a
smaller number of target DNA molecules. With the use of different fluore-
scent dyes in the same solution, several target DNA molecules can be studied
simultaneously. This is called a multiplex PCR system.
The TaqMan system of Applied Biosystems (Foster City, CA) also ampli-
fies DNA by a PCR protocol. However, during the amplification step a special
molecule is annealed to the single-stranded DNA to report the linear ampli-
fication. The molecule has the appropriate sequence for the target DNA. It
also has two attached particles. One is a fluorescent particle, and another one
is a quencher particle. When the two particles are close to each other no
fluorescence occurs. However, when the TAQ polymerase is adding bases to
the linear single strand of DNA, it will break this molecule away from the
strand (like the PacMan in computer games). As this occurs, the two particles
will separate from each other, and fluorescence will occur. By measuring
fluorescence in the tube, a successful PCR reaction can be determined. Note
that the reaction and reporting of a successful PCR protocol occur in the same
tube. The author's research team developed a TaqMan procedure to detect
rapidly Yersinia enter ocolitica in foods [18].
A new system called Molecular Beacon Technology (Stratagene, La Jolla,
CA) was developed and can be used for food microbiology in the future [19].
In this technology, all reactions are again in the same tube. A Molecular
Beacon is a tailor-made hairpin-shaped hybridization probe. The probe is used
to attach to target PCR products. On one end of the probe there is attached
a fluorophore, and on the other end a quencher. In the absence of the target
PCR products the beacon is in a hairpin shape, and there is no fluorescence.
However, during PCR reactions and the generation of target PCR products,
the beacons will attach to the PCR products and cause the hairpin molecule to
586 Microbiology of Fruits and Vegetables
unfold. As the quencher moves away from the fluorophore, fluorescence
will occur, and this can be measured. The measurement can be done as the
PCR reaction is progressing, thus allowing "real-time" detection of target PCR
products, and thus the presence of the target pathogen in the sample. This
system has the same efficiency as the TaqMan system, but the difference is
that the beacons detect the PCR products themselves, while in the TaqMan
system they only report the occurrence of a linear PCR reaction and not the
presence of the PCR product directly. By using molecular beacons containing
different fluorophores, one can detect different PCR products in the same
reaction tubes, and thus it is possible to perform "multiplex" tests of several
target pathogens or molecules. The use of this technology is very new and not
well known in food microbiology areas.
One of the major problems of PCR systems is contamination of PCR
products from one test to another. Thus, if any PCR products from a positive
sample (e.g., salmonella PCR products in a previous run) enter the reaction
system of the next analysis, they may cause a false positive result. The Probelia
system, developed by Institut Pasteur (Paris, France), attempts to eliminate
PCR product contamination by substituting the base uracil for the base thymine
in the entire PCR protocol. Thus, in the reaction tube there are adenine, uracil,
guanine, and cytosine, and no thymine. During the PCR reaction, the resultant
Probelia PCR products will be AUGC pairing and not the natural ATGC
pairings. The PCR products are read by hybridization of known sequences in
a microtiter plate. The report of the hybridization is by color reaction similar
to an ELISA test in the microtiter system.
After one experiment is completed, a new sample is added into another
tube for the next experiment. In the tube there is an enzyme, uracil-
D-glycosylase (UDG), which will hydrolyze any DNA molecules that contain
a uracil. Therefore, if there are contaminants from a previous run, they will be
destroyed before the beginning of the new run. Before a new PCR reaction,
the tube with all reagents is heated to 56°C for 15 minutes for UDG to
hydrolyze any contaminants. During the DNA denaturization step, the UDG
will be inactivated and will not act on the new PCR products containing uracil.
Currently, Probelia can detect salmonella and Listeria monocytogenes from
foods. Other kits under development include E. coli 0157:H7, Campylobacter,
and Clostridium botulinum.
Theoretically, PCR systems can detect one copy of target pathogen
DNA from a food sample (e.g., salmonella DNA). In practice, about 200 cells
are needed to be detected by current PCR methods. Thus, even in a PCR
protocol, bacteria in the food must be enriched for a period of time, e.g., over-
night or at least 8 hours' incubation of food in a suitable enrichment liquid,
so that there are enough cells for the PCR process to be reliable.
Besides the technical manipulations of the systems which can be compli-
cated for many food product microbiology laboratories, two major problems
need to be addressed: inhibitors of PCR reactions and the question of live
and dead cells. In food, there are many enzymes, proteins, and other com-
pounds that can interfere with the PCR reaction and result in false negatives.
Rapid Detection of Microbial Contaminants 587
These inhibitors must be removed or diluted. Since the PCR reaction amplifies
target DNA molecules, even DNA from dead cells can be amplified, and thus
food with dead salmonella can be declared as salmonella positive by PCR
results. In this situation, food properly cooked but containing DNA of dead
cells may be unnecessarily destroyed because of a positive PCR test. PCR can
be a powerful tool for food microbiology once all the problems are solved,
and analysts are convinced of its applicability in routine analysis of foods.
The aforementioned genetic methods are for detection of target pathogens
in foods and other samples. They do not provide identification of the cultures
to the species and subspecies level which is critical in epidemiological investi-
gations of outbreaks or routine monitoring of occurrence of microorganisms
in the environment. The following discussions will center around developments
in the genetic characterization of bacterial cultures.
The RiboPrinter microbial characterization system (DuPont Qualicon,
Wilmington, DE) characterizes and identifies organisms to genus, species,
and subspecies levels automatically. To obtain a RiboPrint of an organism, the
following steps are followed:
1. A pure colony of bacteria suspected to be the target organism (e.g.,
salmonella) is picked from an agar plate by a sterile plastic stick.
2. Cells from the stick are suspended in a buffer solution by mechanical
agitation.
3. An aliquot of the cell suspension is loaded into the sample carrier to
be placed into the instrument. Each sample carrier has space for eight
individual colony picks.
4. The instrument will automatically prepare the DNA for analysis by
restriction enzyme and lysis buffer to break the cell envelope, release
and cut DNA molecules. The DNA fragments will go through an
electrophoresis gel to separate DNA fragments into discrete bands.
Lastly, the DNA probes, conjugate, and substrate will react with the
separated DNA fragments, and light emission from the hybridized
fragments is then photographed. The data are stored and compared
with known patterns of the particular organism. The entire process
takes eight hours for eight samples. However, at two-hour intervals,
another eight samples can be loaded for analysis.
Different bacteria will exhibit different patterns (e.g., salmonella versus
E. coli), and even the same species can exhibit different patterns (e.g., Listeria
monocytogenes has 49 distinct patterns). Examples of numbers of RiboPrint
patterns for some important food pathogens are: salmonella, 145; listeria, 89;
Escherichia coli, 134; staphylococcus, 406; and vibrio, 63. Additionally, the
database includes 300 lactobacillus, 43 lactococcus, 11 leuconostoc, and 34
pediococcus patterns. The current identification database provides 3267
RiboPrint patterns representing 98 genera and 695 species.
One of the values of this information is that in the case of a foodborne
outbreak, scientists not only can identify the etiological agent (e.g., Listeria
588 Microbiology of Fruits and Vegetables
monocytogenes) but can pinpoint the source of the responsible subspecies. For
example, in the investigation of an outbreak of Listeria monocytogenes,
cultures were isolated from a sheer of the product and also from the drains
of the plant. The question was: which source was responsible for the outbreak?
By matching RiboPrint patterns of the two sources of L. monocytogenes
against the foodborne outbreak culture, it was found that the isolate from
the sheer matched the outbreak culture, thus determining the true source of the
problem. The RiboPrinter system is a very powerful tool for electronic data-
sharing worldwide.
These links can monitor the occurrence of foodborne pathogens and other
important organisms as long as different laboratories utilize the same system
for obtaining the RiboPrint patterns.
Another important system concerns the pulsed-fleld gel electrophoresis
patterns of pathogens. In this system, pure cultures of pathogens are isolated
and digested with restriction enzymes, and the DNA fragments are subjected
to a system known as pulsed-fleld gel electrophoresis which effectively sepa-
rates DNA fragments on the gel (DNA fingerprinting). For example, in
a foodborne outbreak of E. coli 0157:H7, biochemically identical E. coli
0157:H7 cultures can exhibit different patterns. By comparing the gel patterns
from different sources, one can trace the origin of the infection or search
for the spread of the disease and thereby control the problem.
In order to compare data from various laboratories, the Pulse Net System
was established under the National Molecular Subtyping Network for Food-
borne Disease Surveillance at the Centers for Disease Control and Prevention
(CDC). An extensive training program has been established so that all
the collaborating laboratories use the same protocol and are electronically
linked to share DNA fingerprinting patterns of major pathogens. As soon
as a suspect culture is noted as a possible source of an outbreak, all the
collaborating laboratories are alerted to search for the occurrence of the
same pattern to determine the scope of the problem and share information
in real time.
There are many other genetic-based methods, but they are not directly
related to food microbiology and are beyond the scope of this review. It is
safe to say that many genetic-based methods are slowly but surely finding their
way into food microbiology laboratories, and they will provide valuable
information for quality assurance, quality control, and food safety programs in
the future.
25.8 BIOSENSORS
The use of biosensors is an exciting field in applied microbiology. The basic
idea is simple, but the actual operation is quite complex and involves much
instrumentation. Basically, a biosensor is a molecule or a group of molecules
of biological origin attached to a signal recognition material. When an analyte
Rapid Detection of Microbial Contaminants 589
comes in contact with the biosensor, the interaction will initiate a recognition
signal which can be reported in an instrument.
Many types of biosensors have been developed, such as enzymes (a great
variety of enzymes have been used), antibodies (polyclonal and monoclonal),
nucleic acids, cellular materials, and others. Whole cells may also be used
as biosensors. Analytes detected include toxins (staphylococcal enterotoxins,
tetrodotoxins, saxitoxin, botulinum toxin, and others), specific pathogens
(salmonella, staphylococcus, Escherichia coli 0157:H7, etc.), carbohydrates
(fructose, lactose, galactose, etc.), insecticides and herbicides, ATP, antibiotics
(e.g., penicillins), and others. The recognition signals used include electro-
chemical (potentiometry, voltage changes, conductance and impedance,
light addressable, etc.), optical (such as UV, bioluminescence and chemilumin-
escence, fluorescence, laser scattering, reflection and refraction of light, surface
plasmon resonance, and polarized light), and miscellaneous transducers (such
as piezoelectric crystals, thermistors, acoustic waves, and quartz crystals).
An example of a simple enzyme biosensor is the sensor for glucose. The
reaction involves the oxidation of glucose (the analyte) by glucose oxidase
(the biosensor) yielding the end products, gluconic acid and hydrogen
peroxide. The reaction is reported by a Clark oxygen electrode which monitors
the decrease in oxygen concentration amperometrically. The range of meas-
urement is from 1 to 30 mM with a response time of 1 to 1.5 minutes and a
recovery time of 30 seconds. The lifetime of the unit is several months. Some of
the advantages of enzyme biosensors are their strong binding to the analyte,
high selectivity and sensitivity, and rapid reaction time. Some of the
disadvantages are expense, loss of activity when enzymes are immobilized
on a transducer, and loss of activity due to deactivation. Other enzymes
used include galactosidase, glucoamlyase, acetylcholinesterase, invertase, and
lactate oxidase. Excellent review articles and books on biosensors are presented
by Eggins [20], Cunningham [21], Goldschmidt [22], and others.
Recently much attention has been directed to the field of "biochips" and
"microchips" development to detect a great variety of molecules including
foodborne pathogens. Due to advancements in miniaturization technology, as
many as 50,000 individual spots (e.g., DNA microarrays), with each spot
containing millions of copies of a specific DNA probe, can be immobilized on
a specialized microscope slide. Fluorescent labeled targets can be hydridized
to these spots and be detected. An excellent article by Deyholos et al. [23]
described the application of microarrays to discover genes associated with a
particular biological process such as the response of a plant (arabidopsis)
to NaCl stress and detailed analysis of a specific biological pathway such as
one-carbon metabolism in maize.
Biochips can also be designed to detect all kinds of foodborne pathogens
by imprinting a variety of antibodies or DNA molecules against specific
pathogens on the chip for the simultaneous detection of pathogens such as
salmonella, listeria, Escherichia coli, and Staphylococcus aureus on the same
chip. According to Heron writing in 2000 [24], biochips are an exceedingly
important technology in life sciences, and at that time the market value was
590 Microbiology of Fruits and Vegetables
estimated to be as high as $5 billion by the middle of the present decade. This
technology is especially important in the rapidly developing field of proteomics
which requires massive amount of data to generate valuable information.
Certainly, the development of these biochips and microarray chips is
impressive for obtaining a large amount of information for biological sciences.
As for foodborne pathogen detection, there are several important issues
to consider. These biochips are designed to detect minute quantities of target
molecule. The target molecules must be free from contaminants before being
applied to the biochips. In food microbiology, the minimum requirement
for pathogen detection is 1 viable target cell in 25 g of a food such as ground
beef. A biochip will not be able to seek out such a cell from the food
matrix without extensive cell amplification (either by growth or PCR) or
sample preparation by filtration, separation, absorption, centrifugation, etc.,
as described in this chapter. Any food particle in the sample will easily clog the
channels used in biochips. These preparations will not allow the biochips to
provide "real-time" detection of pathogens in foods.
Another concern is viability of the pathogens to be detected by biochips.
Monitoring the presences of some target molecule will only demonstrate
the presence or absence of the target pathogen and will not show the viability
of the pathogen in question. Some form of culture enrichment to ensure growth
is still needed in order to obtain meaningful results. It is conceivable that
the biomass of microbes can be monitored by biochips but instantaneous
detection of specific pathogens such as salmonella, listeria, and Campylobacter
in a food matrix during food processing operations is still not possible.
The potential of biochip and microarrays for food pathogen detection is great,
but at present much more research is needed to make this technology a reality
in applied food microbiology.
25.9 U.S., WORLD MARKET, AND TESTING TRENDS
(1999-2008)
There is no question that many microbiological tests are being conducted
nationally and internationally on food, pharmaceutical products, environ-
mental samples, and water. The most popular tests are total viable cell count,
coliform/is. coll count, and yeast and mold counts. A large number of tests are
also performed on pathogens such as salmonella, listeria and Listeria mono-
cytogenes, E. coli 0157:H7, Staphylococcus aureus, Campylobacter, and other
organisms.
Applied microbiologists working in medical, food, environmental, and
industrial settings in government, academia, and the private sector are inter-
ested in the numbers and kinds of microbiological tests being done annually on
local, regional, national, and international scales.
Strategic Consulting, Inc. (phone: 802-457-9933; e-mail: weschler@
strategic-consult.com; Woodstock, VT) produced three major reports on the
market for microbiological testing [25-27]. This group researched diagnostic
Rapid Detection of Microbial Contaminants 591
testing companies through public records and interviews of hundreds of
practitioners of applied microbiology by phone or other means to obtain
estimated data to compile the reports. Readers are advised to contact Strategic
Consulting, Inc. for details of these reports. Below is information that the
author received permission to use for this article.
In 1998 the number of worldwide industrial microbiological tests was esti-
mated to be 755 million with a total market value of US$1.1 billion, assuming
the average price per test to be US$1.47. They also estimated that 56% of the
tests were for food; 30% for pharmaceuticals; 10% for beverages; and 4% for
environmental water tests [25]. Of these tests, 420 million were done in food
laboratories with 360 million for "routine tests" (total viable cell counts,
coliform counts, and yeast and mold counts) and 60 million for "specific
pathogen tests" (salmonella, listeria, Staphylococcus aureus, E. coli 0157:H7
tests). Approximately one third of all the tests were done in the U.S., another
third in Europe, and the rest were performed in the rest of the world.
It was projected that from 1998 to 2003 there would be a 24.6% increase in
the number of tests; 17% increase in the price per test, and 45.8% increase
in the total revenue of the testing market by 2003. Of the 50 or so diagnostic
companies reviewed, there seems to be no absolute dominance of the field
by any one company, although there are clear leaders in the area [25]. The
situation is quite fluid since some companies are constantly acquiring products
from other companies. Many new companies are also emerging in this area as
new technologies are developed.
The 2000 U.S. food industry market study [26] indicated that the total
number of microbiological tests per year was 144.3 million, total number of
tests for pathogens was 23.5 million, with a market value of US$53.4 million,
and the average selling price per test was US$2.27. These data were obtained
from a survey of 5,979 food processing plants with an average of 464 tests per
plant per week, and 24,128 tests per plant per year. The percentage of
microbiological testing performed on selected food categories was as follows:
processed foods, 36.2%; dairy, 31.8%; meat, 22.3%; fruits/vegetables, 9.7%.
The number of test to be done in the future for fruits and vegetables will
certainly increase due to recent foodborne outbreaks related to these food
commodities.
Another valuable set of data is the proportion of routine to pathogen tests
which is 83.7% versus 16.3%. Further breakdown of these data revealed that
the total viable count represented 37.2% of all tests; coliform/^. coli, 30.8%;
yeast and mold, 15.7%; and pathogens, 16.3%. The percentage for pathogen
testing is an increase from 15% reported in the 1998 review [25]. It is projected
that this number will increase further in the years to come.
Estimation of the use of "rapid methods" versus "conventional methods"
is hard to obtain. From the author's experiences, about 70% of microbial tests
are currently done using manual or conventional methods and 30% using
rapid methods. By 2008, for total testing, about 50% will be using conven-
tional methods, and 50% will be using rapid tests. However, for pathogen
testing 60 to 70% will be some form of rapid test, and 30 to 40% will the
592 Microbiology of Fruits and Vegetables
conventional tests. These projected changes are attributed to the current and
future improvement of rapid methods.
The newest global test volume predictions for industrial microbiological
testing (per year) for 2008 are: food, 715.6 million tests (47.5%, total); bever-
ages, 137.0 million tests (9.1%); pharmaceuticals, 311.1 million tests (20.7%);
personal care products, 249.1 million tests (16.5%); environmental, 55.9
million tests (3.7%); and material processing, 36.5 million tests (2.4%) [27].
It is safe to say that the field of rapid methods and automation in
microbiology will continue to grow in number and kinds of tests to be done
in the future due to the increased concern about food safety.
25.10 PREDICTIONS OF THE FUTURE
It is always difficult to predict the future development in any field of endeavor.
In 1995 the author was honored to present a lecture at the annual meeting
of the American Society of Microbiology as the Food Microbiology Divi-
sional Lecturer concerning the current status and the future outlook of the field
of rapid methods and automation in microbiology. The following is a synopsis
of the ten predictions, with a look into the future made in 1995. A more
detailed description of the predictions can be found in the paper by Fung
published in 1999 [28].
1. Viable cell counts will still be used.
2. Real-time monitoring of hygiene will be in place.
3. PCR, ribotyping, and genetic tests will become a reality in food
laboratories.
4. ELISA and immunological tests will be completely automated and
widely used.
5. Dipstick technology will provide rapid answers (10 minutes).
6. Biosensors will be in place for HACCP programs in the future.
7. Instant detection of target pathogens will be possible by a computer-
generated matrix in response to particular characteristics of
pathogens (microarrays, biochips, microchips).
8. Effective separation and concentration of target cells will greatly
assist in rapid identification.
9. Microbiological alert systems will be in food packages.
10. Consumers will have rapid alert kits for detection of pathogens at
home.
Along with the prediction of the future of rapid testing methods, it is useful
to describe the ten attributes and criteria for an ideal automated microbiology
assay system as follows:
1. Accuracy for the intended purposes. Sensitivity, minimal detectable
limits, specificity of test system, versatility, potential applications,
comparison to reference methods.
Rapid Detection of Microbial Contaminants 593
2. Speed in productivity. Time in obtaining results, number of samples
processed per run, per hour, per day.
3. Cost. Initial, per test, reagents, labor.
4. Acceptability by scientific community and regulatory agencies.
5. Simplicity of operation. Sample preparation, operation of test
equipment, computer versatility.
6. Training. On-site, length of time, qualification of operator.
7. Reagents. Preparation, stability, availability and consistency.
8. Company reputation.
9. Technical services. Speed, availability, cost and scope.
10. Utility and space requirements.
The future looks very bright for the field of rapid methods and automation
in microbiology. The potential is great and many exciting developments will
certainly unfold in the near and far future.
ACKNOWLEDGMENT
This material is based upon work supported by the Cooperative State Research
Education and Extension Service, United States Department of Agriculture,
under Agreement No. 93-34211-8362. Contribution No. 05-85-B Kansas
Agricultural Experimental Station, Manhattan, Kansas.
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1. Fung, D.Y.C., Rapid methods and automation in microbiology, Compr. Rev.
Food Sci. Food Saf. (IFT), 1, 3, 2002.
2. Fung, D.Y.C. et al., The Pulsifier: a new instrument for preparing food
suspensions for microbiological analysis, J. Rapid Methods Automat. Microbiol.,
6, 43, 1998.
3. Kang, D.H., Dougherty, R.H., and Fung, D.Y.C, Comparison of Pulsifier and
Stomacher to detach microorganisms from lean meat tissues, J. Rapid Methods
Automat. Microbiol., 9, 27, 2001.
4. Wu, V.C.H., Jitareerat, P., and Fung, D.Y.C, Comparison of the Pulsifier and
the Stomacher for recovering microorganisms in vegetable, J. Rapid Methods
Automat. Microbiol., 11, 145, 2003.
5. Manninen, M.T., Fung, D.Y.C, and Hart, R.A., Spiral system and laser
counter for enumeration of microorganisms, J. Food Prot., 11, 177, 1991.
6. Chain, V.S. and Fung, D.Y.C, Comparison of Redigel, Petrifilm, Spiral
Plate System, Isogrid and aerobic plate count for determining the numbers
of aerobic bacteria in selected food, /. Food Prot., 54, 208, 1991.
7. Fung, D.Y.C. and Lee, CM., Double-tube anaerobic bacteria cultivation
system, Food Sci., 7, 209, 1981.
8. Ali, M.S. and Fung, D.Y.C, Occurrence of Clostridium perfringens in
ground beef and turkey evaluated by three methods, /. Food Prot., 11, 197,
1991.
594 Microbiology of Fruits and Vegetables
9. Schmidt, K.A. et al., Application of a double tube system for the enumeration of
Clostridium tyrobutyricum, J. Rapid Methods Automat. Microbiol., 8, 21, 2000.
10. Hartman, P. A., Miniaturized Microbiological Methods, Academic Press, New
York, 1968.
11. Fung, D.Y.C. and Kraft, A.A., Microtiter method for the evacuation of viable
cells in bacterial cultures, Appl. Microbiol., 16, 1036, 1968.
12. Fung, D.Y.C. et al., A collaborative study of the microtiter count method and
standard plate count method on viable cell count of raw milk, /. Milk Food
Technol, 39, 24, 1976.
13. Fung, D.Y.C. and Kraft, A.A., Rapid evaluation of viable cell counts using the
microtiter system and MPN technique, J. Milk Food Technol., 32, 408, 1969.
14. Walser, P.E., Using conventional microtiter plate technology for the auto-
mation of microbiology testing of drinking water, /. Rapid Methods Automat.
Microbiol., 8, 193, 2000.
15. Irwin, P., Tu, S., Damert, W., and Phillips, J., A modified Gauss-Newton
algorithm and ninety-six well micro-technique for calculating MPN using
EXCEL spread sheets, </. Rapid Methods Automat. Microbiol., 8, 171, 2000.
16. Wu, V.C.H. et al., Rapid protocol (5.25 H) for the detection of Escherichia
coli 0157:H7 in raw ground beef by an immuno-capture system (Pathatrix)
in combination with Colortrix and CT-SMAC, /. Rapid Methods Automat.
Microbiol., 12, 57, 2004.
17. Dreibelbis, S.B., Evaluation of Five ATP Bioluminescence Hygiene Monitoring
Systems in a Commercial Food Processing Facility, Master's thesis,
Kansas State University Library, Manhattan, KS, 1999.
18. Vishubhatla, A. et al., A Rapid 5' nuclease (TaqMan) assay for the detection
of pathogenic strains of Yersinia enterocolitica, Appl. Environ. Microbiol.,
66,4731, 2000.
19. Robinson, J.K., Mueller R., and Pilippone, L., New molecular beacon
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20. Eggins, B., Biosensors: An Introduction, John Wiley, New York, 1997.
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22. Goldschmidt, M.C., Biosensors: scope in microbiological analysis, in Encyclo-
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Academic Press, New York, 1999, p. 268.
23. Deyholos, M., Wang, H., and Galbraith, D., Microarrays for gene discovery
and metabolic pathway analysis in plants, Life Sci., 2, 2, 2001.
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Strategic Consulting Inc., Woodstock, VT, 2004.
28. Fung, D.Y.C, Prediction in the future of rapid methods and microbiology,
Food Test. Anal., 5, 18, 1999.
26
Methods in Microscopy
for the Visualization of
Bacteria and Their
Behavior on Plants
Maria T. Brandl and J.-M. Monier
CONTENTS
26. 1 Introduction 596
26.2 Visualization of Bacteria on Plants: Available Tools 596
26.2.1 Labeling of Bacteria with Fluorescent Proteins 596
26.2.2 Labeling of Bacteria with Dyes and
Fluorescent Conjugates 598
26.3 Applications 600
26.3.1 Spatial Distribution 600
26.3.2 Cell-Cell Interactions 602
26.3.3 Measurement of Biological Parameters 604
26.3.3. 1 Kogure Assay for Cell Viability 605
26.3.3.2 Indicators of Membrane Integrity 606
26.3.3.3 GFP Fluorescence and Cell Viability 607
26.3.3.4 Other Fluorescent Indicators of Bacterial
Physiology 607
26.3.4 Bacterial Gene Expression In Situ on Plants 608
26.3.4.1 GFP as a Reporter of Gene Expression 608
26.3.4.2 Practical Note on the Use of GFP for Gene
Expression Measurements 610
26.3.4.3 FISH for the Detection of Bacterial mRNA .... 61 1
26.3.4.4 Immunolabeling of Gene Products 611
26.4 Other Types of Microscopy 612
26.4.1 Multiphoton Excitation Fluorescence Microscopy 612
26.4.2 Fluorescence Stereomicroscopy 613
26.4.3 Immunoelectron Microscopy 613
26.5 Concluding Remarks 614
References 615
595
596 Microbiology of Fruits and Vegetables
26.1 INTRODUCTION
Since the discovery of microbes by Robert Hooke and Antonie van
Leeuwenhoek in the 17th century, microscopy has made great strides to
enhance our understanding of the microbial world, including the microflora
of plants. However, despite our increasing ability to probe the minuscule
at high resolution, with instruments such as the electron microscope, bacteria
have remained relatively anonymous because of their lack of morphological
diversity at the cellular scale. In addition, most types of electron microscopy
involve extensive sample preparation that may dislodge or alter bacterial cells,
leaving the microscopist in doubt about the interpretation of the observations
made. It was the discovery of confocal microscopy, and the green fluorescent
protein (GFP) as an intrinsic bacterial label, that spurred a new revolution,
starting in the 1990s, in the use of fluorescence microscopy to study bacteria in
their natural habitat. Because most bacterial species or strains cannot be
distinguished from each other microscopically, intrinsic labeling of bacteria
with GFP or other fluorescent proteins has been used widely to track specific
bacteria in complex environments, including plants.
This chapter focuses on novel experimental approaches in fluorescence
microscopy to detect bacteria and investigate their behavior on plants. Recent
advances in microscope technologies that may be applied to plant micro-
biology research are also discussed.
26.2 VISUALIZATION OF BACTERIA ON PLANTS:
AVAILABLE TOOLS
26.2.1 Labeling of Bacteria with Fluorescent
Proteins
The recent renaissance in the application of microscopy to the study of
bacterial behavior on plants is largely attributable to the discovery of GFP.
The popularity of GFP as a fluorophore lies in its bright and relatively stable
fluorescence, the expression of gfp in most bacterial species, and the ability to
use it as an intrinsic label without the need for a substrate. The latter property
means that samples can be mounted without prior processing for visualization
under the microscope, and thus disruption of bacterial cells in the environment
under study is minimal. This is in sharp contrast with the potential artifacts
generated during sample preparation for immunofluorescence or electron
microscopy. Additionally, mutants of GFP that have enhanced fluorescence
intensity and emit at various wavelengths of the visible spectrum have provided
investigators with versatile tools to circumvent problems with detecting the
GFP signal against autofluorescent backgrounds. This problem is especially
prevalent in plant tissues, which emit with various intensities in different
regions of the visible spectrum [1]. It is exacerbated also by contamination of
the image with stray fluorescence, but may be minimized by the use of confocal
microscopy. Because the confocal laser scanning microscope (CLSM) collects
Methods in Microscopy 597
the fluorescent signal solely from the focal plane by rejecting scattered light
with its pinhole, the CLSM can improve greatly the detection of fluorescently
tagged bacterial cells on plants.
Despite the availability of GFP mutants with enhanced fluorescence
intensity, such as the widely used S65T mutant [2], the detection of GFP
fluorescence at low levels of expression in bacterial cells remains difficult.
Therefore, intrinsic labeling with GFP is commonly achieved by transforming
the bacterial strain of interest with a plasmid that is maintained stably at
a moderate copy number per cell and that harbors gfp expressed from
strong promoters. Adverse effects of GFP on the fitness of bacteria that
were transformed with high copy number plasmids encoding GFP have been
reported [3], presumably because the resultant high concentrations of GFP
overload bacterial metabolism or disrupt cellular functions, and thus use of
such plasmids should be avoided.
GFP production from single insertion of gfp into the bacterial chromosome
may circumvent problems associated with excessive GFP concentrations
or plasmid instability, but may still decrease the competitiveness of the
tagged strain compared to its parental strain under stressful conditions such
as carbon-substrate limitation [4]. In addition, gfp expression from a single
location on the bacterial chromosome may yield a signal-to-noise ratio that
approaches the lower limit of detection for GFP in a strong fluorescent
background. In such cases, GFP fluorescence may be imaged only by high
signal gain, which results in poor definition of the bacterial cell profile and
in grainy images [5]. This is particularly true for imaging of dim GFP-tagged
bacteria on plant tissue that emits in the green range, e.g., the roots of certain
plant species.
Expression of gfp from plasmids requires that plasmid stability in
the transformed bacterial strain be assessed in the plant environment
under study. By comparison of population sizes of E, coli 0157:H7 cells
that showed green fluorescence to those that were detected by immunolabeling,
Takeuchi and Frank demonstrated that E. coli 0157:H7 pEGFP retained
pEGFP, or GFP per se, at higher frequency on lettuce leaves and cauliflower
florets than on tomato [6]. Since few broad-host-range plasmids are completely
stable in any bacterial species, it is expected that the frequency at which a GFP-
encoding plasmid is lost in a cell population will increase as the bacterial
growth rate increases, or as physiological stress impacts that population.
Therefore, the retention of a GFP plasmid among a bacterial population may
vary greatly depending on the experimental conditions and on the plant host
tissue or species studied.
In addition to GFP and its color variants, intrinsic fluorophores emitting
in the near red (DsRed) and far red (HcRed) have been cloned from Discoma
and Heteractis crispa, respectively [7,8]. The availability of fluorophores
that span the visible range allow for multispectral imaging of bacteria within
a same sample. The excitation and emission spectra of the red fluorescent
proteins and of the most widely used variants of GFP are shown in Figure 26.1.
The peaks of their excitation and emission spectra are listed in Table 26.1.
598
Microbiology of Fruits and Vegetables
300
350
400
450
500
550
600
650
300 350 400 450 500 550
Wavelength (nm)
600
650
FIGURE 26.1 Excitation (A) and emission (B) spectra of blue (BFP), cyan (CFP), green
(GFP), and red (DsRed and HcRed) fluorescent proteins suitable for intrinsic labeling
of bacteria. The F-axis represents the normalized fluorescence intensity. (Fluorescence
data courtesy of BD Biosciences Clontech, Palo Alto, CA.)
26.2.2 Labeling of Bacteria With Dyes and
Fluorescent Conjugates
In addition to fluorescent proteins, a wide range of fluorescent dyes
and bioconjugates are available for the visualization of bacteria on plants.
Exhaustive lists of fluorescent probes and their applications in biological
microscopy are beyond the scope of this chapter, but can be found in an excel-
lent review by Kasten [9]. The DNA-intercalating stain acridine orange (AO)
and the newer SYTO® dyes (Molecular Probes, Eugene, OR), which are avail-
able in a wide range of the visible spectrum, are particularly useful to visualize
bacteria against the autofluorescent plant background. 4',6-Diamidino-2-
phenylindole (DAPI), also a nucleic acid stain, requires ultraviolet (UV)
illumination, and thus may cause extensive damage to plant cells. Other dyes,
such as Sypro® Orange, a general protein stain, and Nile Red (Molecular
Probes), which stains lipid-hydrophobic sites , have been applied to the study
of bacterial biofilms [10].
TABLE 26.1
List of Non-Conjugated Fluorochromes Commonly Used in Bacteriology (Color Insert Follows Page 594)
Fluorescent Proteins
Ex. (nm) Em. (ran) Ref. Biological Parameters
Ex. (nm) Em. (nm)
Ref
CD
i— h
=r
Q_
BFP (Blue GFP); eBFP
384
448
71
Kits
LIVE-DEAD flacLight
n
CFP (Cyan GFP); eCFP
434
476
71
SYTO® 9 (total cells)
485
498 n 73
WtGFP
393/473
504
71
Propidium Iodide (dead cells)
536
617 H 73
n
o
GFPuv
399
511
71
LIVE itacLight Gram Stains
"O
GFP (S65T), eGFP
488
508
71
SYTO® 9 (total cells)
485
498 n 73
YFP (Yellow GFP); eYFP
514
527
71
Hexidium iodide (Gram+ cells)
518
600 [J 73
DsRed
558
583
71
ViaGram Red +
HcRed
592
645
71
DAPI (viable cells)
SYTOX® Green (dead cells)
359
504
461 [] 73
523 H 73
Fluorescent Dyes
Ex. (nm)
Em. (nm)
Ref.
Texas Red®-X-WGA (Gram+ cells)
595
615 I 73
Proteins
Nile Red
540
600
73
Viability
CTC
450 a
630 u ■
Sypro® Ruby
280, 450
610
73
Ethidium Bromide
545
610
9
Sypro® Orange
300, 470
570
73
Propidium Iodide
530
615
9
Sypro® Rose
350
610
73
SYTOX® Blue
431
480
73
Sypro® Red
300, 550
630
73
SYTOX® Green
504
523
73
Sypro® Tangerine
300, 490
640
73
SYTOX® Orange
547
570
73
Nucleic acids
Acridine Orange (+DNA)
500
526
73
TO-PRO™-l
515
531
73
Acridine Orange (+RNA)
460
650
73
TO-PRO™-3
642
661
73
DAPI
358
461
73
TO-PRO™-5
747
777
73
Blue SYTO® dyes (40-45)
420-455
441-484
73
pH
BCECF
500
530/620 9
Green SYTO® dyes (11-25)
488-521
509-556
73
SNAFL®-1
508/540
540/623 73
Orange SYTO® dyes (80-85)
530-567
544-583
73
SNARF® (high pH)
574
630 ■ 9
Red SYTO® dyes (17, 59-64)
559-657
619-678
73
SNARF® (low pH)
548
587 \J 9
SYTO® RNASelect
490
530
73
Po ly saccharides
DTAF
492
516
73
a Data provided by Polysciences, Inc, Warring!
on, PA.
10
vfi
600 Microbiology of Fruits and Vegetables
In contrast to general DNA dyes, immunostaining with fluorescent-labeled
antibodies [11] or fluorescence in situ hybridization (FISH) with 16S rRNA
probes [12-16] may provide high specificity to detect a given bacterial species
or strain among the plant microflora. Also, fluorescently labeled lectins have
proven useful to probe bacteria associated with the exopolysaccharide matrix
of aggregates on plants and in biofilms in general [17,18]. While fluorescein and
rhodamine bioconjugates have been popular fluorochromes for
the visualization of bacteria on plant tissue, newer products for labeling of
bioconjugates, such as Alexa Fluor® (Molecular Probes) and Cy™ dyes
(Amersham Biosciences Corp., Piscataway, NJ), exhibit brighter fluorescence
and enhanced photostability.
General stains and bioconjugates can be used alone or in combination with
fluorescent proteins to probe complex microenvironments, processes, or
biological parameters governing bacterial behavior on plants. In the sections
below, we discuss various applications of fluorescence microscopy to the study
of the ecology of plant-associated bacteria and human enteric pathogens in
the plant environment. Table 26.1 lists the excitation and emission peaks, as
well as specific applications, of a variety of fluorescent proteins and stains,
some of which are mentioned in this chapter.
26.3 APPLICATIONS
26.3.1 Spatial Distribution
The microscopic investigation of the localization of human or plant pathogenic
bacteria on plant surfaces has provided new insights into their ecology in that
environment. The observations by confocal microscopy that GFP-labeled
Salmonella enter ica formed microcolonies on the leaves of cilantro plants
provided evidence that this enteric pathogen has the ability to colonize plants
in a preharvest environment and may cause outbreaks due to contamination
that occurred in the field [19]. Furthermore, S. enter ica developed high-density
populations and large heterogeneous aggregates in the vein area of the leaf,
thus following spatial colonization patterns similar to those of the natural
plant microflora on uninoculated plants [20]. In this same study, Brandl and
Mandrell used confocal scans in the xz plane to obtain optical cross-sections
of healthy and diseased cilantro leaves, and demonstrate that S. enterica cells
had gained access to internal tissue while growing in the plant lesion whereas
they remained on the cuticle layer while colonizing a healthy leaf [19]
(Figure 26.2). Optical cross-sectioning by CLSM is an effective way to demon-
strate unequivocally the internalization of bacteria in plant openings or tissue
without the potential artifacts created by mechanical sectioning, which may
contaminate internal tissue with external bacteria.
Confocal microscopy of GFP-labeled cells was used in several studies
to demonstrate that enteric pathogens attach to and multiply at high densities
in the damaged tissue of a variety of fruits and vegetables [6,19,21]. Burnett
et al. combined CLSM and digital image analysis to count GFP-tagged E. coli
Methods in Microscopy
601
FIGURE 26.2 (Color insert follows page 594) CLSM micrograph of GFP-labeled S.
enter ica serovar Thompson cells after their inoculation and incubation on the leaves of
cilantro plants. S. Thompson cells were observed on healthy (A) and diseased (B) leaf
tissue. The lower panels show cross-sections of the tissue in the top panel that were
acquired by optical sectioning in the xz plane. They reveal that the bacterial cells are
located on top of the cuticle on the healthy leaf (A), but have invaded the damaged
tissue of the diseased leaf (B). Bars, 10 urn. (From Brandl, M.T. and Mandrell, R.E.,
Appl. Environ. Microbiol., 68, 3614, 2002.)
0157:H7 cells that attached at various depths in healthy or punctured apple
tissue during inoculation [21]. From this approach, they concluded quantita-
tively that the pathogen infiltrated intact tissue and natural openings to a
greater extent under negative than positive temperature differential.
The ability of plant pathogens and human enteric pathogens to become
internalized in plant tissue, where they are shielded from the adverse effects of
environmental conditions or chemical control agents, has been the focus of
much interest in plant microbiology. Confocal microscopy has been a valuable
means of probing the internal tissue of plants inoculated with GFP-tagged
bacteria. Hallmann et al. were among the first to use the CLSM to observe
the endophytic localization of a bacterial species, Rhizobium etli, within
Arabidopsis thaliana roots [22]. Using a similar approach, endophytic coloni-
zation of A. thaliana and alfalfa roots by E. coli 0157:H7 and S. enterica,
respectively, has been demonstrated in plant systems in vitro [23,24]. A. thaliana
and various other plant species have transparent roots, which lend themselves
602 Microbiology of Fruits and Vegetables
well to optical sectioning. In contrast, opaque plant tissues, e.g., potato tubers,
make visualization of internalized GFP-labeled bacteria more challenging.
Although opaque plant tissue can be cleared with various techniques, these
generally inhibit or destroy GFP fluorescence [22]. In an experimental soil
system that attempted to simulate field conditions, albeit with high inoculum
levels, Solomon et al. found evidence for the transmission of GFP-tagged Exoli
0157:H7 from contaminated manure to the internal tissue of lettuce leaves [25].
However, the low resolution of their published confocal micrograph reveals the
difficulty of imaging fluorescent bacterial cells located under several layers of
opaque leaf tissue. Also challenging is the localization of the internalized
bacterial cells within the plant tissue in the epifluorescence mode during
browsing before image acquisition in the confocal mode; browsing large
numbers of fields of view or samples for the visualization of rare events is
impractical by confocal microscopy. In our experience, this problem occurs
even with brightly fluorescent bacterial cells.
Fluorescence microscopy has been instrumental to the discovery that
bacteria can form biofilms on plants. Morris et al. used epifluorescence micro-
scopy and AO staining of microbial cells to demonstrate the presence of
natural biofilms on leaves of a variety of vegetables [26]. Since this first report,
biofilm formation on plants has been shown in several studies using various
microscopy techniques. Monier and Lindow developed a method to study
the frequency, size, and localization of bacterial aggregates in situ on leaf
surfaces [27]. Quantitative data were obtained by digital image analysis of
epifluorescence micrographs of AO-stained bacteria present on leaf samples.
The analysis was performed based on the outlining of the profiles of single
bacteria or bacterial aggregates, by thresholding on their bright fluorescence
intensity against the less fluorescent plant background. This method was
applied to the quantification of the size of aggregates formed by S. enter ica in
the cilantro phyllosphere using GFP as an intrinsic fluorescent bacterial label
instead of AO [28], and is illustrated in Figure 26.3.
26.3.2 Cell-Cell Interactions
The characterization of individual microbial aggregates isolated from plants
revealed that they harbor a wide range of microorganisms including numerous
species of Gram-negative and Gram-positive bacteria, as well as yeasts and
filamentous fungi [26,29]. The heterogeneous composition of aggregates
suggests that a complex pattern of microbial interactions is possible even at
such small scales in the plant environment. While the spatial organization
of epiphytic bacterial populations had remained obscure until recently, the use
of marker genes conferring the production of fluorescent proteins combined
with fluorescent stains has proven to be a valuable tool and has provided
new insight into our understanding of bacterial interactions on plant surfaces.
Despite the heterogeneity of the plant surface habitat, which makes it a
difficult task to study bacterial interactions in situ, a few studies attempting
to decipher the factors shaping the structure of epiphytic communities have
Methods in Microscopy 603
FIGURE 26.3 Schematic diagram illustrating the basis for digital analysis of
fluorescence images. (A) Epifluorescence micrograph of cellular aggregates of GFP-
labeled S. enterica on a cilantro leaf. Each GFP-labeled S. enterica single cell or
aggregate in the image can be identified by thresholding on the bright pixels originating
from the GFP fluorescence, which is of higher intensity than the background pixels
originating from the leaf surface (inset). This thresholding yields objects (B) for which a
variety of parameters, such as total number of pixels or mean pixel intensity, can be
automatically measured with the image analysis software. Because of the highly
heterogeneous spatial distribution of bacteria on plants, as well as variations between
plants, this type of analysis requires the acquisition of a large number of images from
random fields of view of multiple plant samples in order to yield unbiased data.
been reported. Normander et al. have reported the significance of bacterial
distribution on genetic exchange in the phyllosphere using GFP as an indi-
cator of plasmid transfer [30]. Conjugation was observed under CLSM to
occur primarily in the interstitial spaces of epidermal cells and vein cells, and
in stomata; bacterial aggregation had a great stimulatory effect on plasmid
transfer. Such data are pertinent to assessing the risk associated with the
dissemination of antibiotic resistance genes among bacterial cells on plants.
Monier and Lindow tested the spatial partitioning of cells within aggre-
gates on leaf surfaces by establishing different pairwise mixtures of three
different epiphytic bacterial species that were tagged either with GFP or CFP
[31]. The spatial structure of the resulting aggregates was studied in situ on
leaves by epifluorescence microscopy. Digital image analysis was employed
to quantify the degree of segregation of the GFP- and the CFP-marked strains
and revealed that the fraction of cells in direct contact ranged from 0.2 to
8.0%. The highest segregation occurred between two bacterial species
exhibiting negative interactions (Figure 26.4).
Fluorescence microscopy has proven useful also for the assessment of
various bacterial genes hypothesized to have a role in cell-cell interactions
on plants. For example, by comparing the behavior of GFP-labeled parental
and mutant strains in situ on plants under the microscope, the function of
the adhesin encoding hecA gene in the attachment and aggregation of Erwinia
chrysanthemi on plants was confirmed [32]. In contrast, comparison of
GFP-labeled S. enterica parental and LuxS~ mutant strains by digital image
604 Microbiology of Fruits and Vegetables
FIGURE 26.4 (Color insert follows page 594) Epifluorescence micrograph of
P. agglomerans and P. fluorescens cells labeled with CFP (blue arrow) and GFP (green
arrow), respectively, and inoculated onto the leaves of bean plants incubated under
humid conditions. The dual labeling with fluorescent proteins provided a good means
to quantify the degree of spatial segregation between aggregates of these two bacterial
species by image analysis. Bar, 20 urn.
analysis of their aggregate sizes on leaves revealed that production of the
autoinducer-2 molecule for cell-cell signaling had no detectable role in
aggregate formation by this human pathogen in the cilantro phyllosphere [28].
Besides the fluorescent proteins, or in combination with them, fluorescent
dyes can provide a means to view the microbial composition and possible
interactions on plant surfaces. The LIVE Bachight M bacterial Gram stains
(Molecular Probes) impart green and red fluorescence to Gram-negative
and Gram-positive bacterial cells, respectively. The DNA dye SYTO® 9, which
is included in the assay, also stains fungi, and therefore the assay allows
for easy visualization of the fungal and bacterial composition of the plant
microflora (Figure 26.5) [33]. In a study of the interaction of S. enterica with
the common bacterial epiphyte P. agglomerans in the cilantro phyllosphere, the
SYTO® 62 dye, which emits in the red region of the spectrum, enabled the
observation under CLSM that these two species were part of larger bacterial
aggregates (Chapter 2, Figure 2.3B), and that they attached to fungal hyphae
(Figure 26.6).
26.3.3 Measurement of Biological Parameters
The assessment of bacterial cell viability, although complex in its inter-
pretation, is central to our understanding of how bacteria survive in a
given habitat. Also, there is an increasing need to understand the physiology
of bacteria in the plant environment in order to design efficient strategies
to control plant colonization by human or plant pathogens, or to sanitize fruits
and vegetables. Fluorescent reporters may provide information about the
Methods in Microscopy 605
FIGURE 26.5 (Color insert follows page 594) CLSM micrograph of the microbial
community on a leaf section of field-grown bean plants. The microbes were stained
with LIVE BacLight Gram stain. Single cells and large mixed aggregates composed
of fungi (bright green filaments, white arrow), and putative Gram-negative
(green cells) and Gram-positive (red/orange cells) bacterial cells are present on
the red autofluorescent leaf and in the vicinity of a glandular trichome (yellow arrow).
Bar, 20 urn.
physiological status of bacterial cells in complex ecosystems. They can be used
to determine cell viability via the assessment of basic cell functions such as
reproductive ability, membrane integrity, and respiration, or to measure
cellular parameters, such as pH and levels of various ions.
26.3.3.1 Kogure Assay for Cell Viability
The ability of bacterial cells to grow and multiply has been the gold standard to
demonstrate cell viability. In an approach based on the Kogure assay [34],
Wilson and Lindow used a direct viable count method to examine the viability
of epiphytic populations of Pseudomonas syringae on bean plants under
desiccation stress [35]. The method consisted of incubating cells recovered from
bean leaves in low-percentage yeast extract and in nalidixic acid to provide
substrates for growth and to prevent cell division, respectively. The cells were
then stained with DAPI, and cells that were fluorescent and elongated (growing
cells in which division is inhibited by nalidixic acid) were counted as viable cells
under the epifluorescence microscope. The increasing frequency of viable but
nonculturable cells of the pathogen R. solanacearum during infection of tomato
606 Microbiology of Fruits and Vegetables
FIGURE 26.6 (Color insert follows page 594) CLSM micrograph of GFP-labeled
S. enterica cells (yellow arrow) and DsRed-labeled P. agglomerans cells (white arrow)
attached to a SYTO® 62-stained fungal hypha (blue arrow) in the phyllosphere
of cilantro plants. The bright blue round objects are the chloroplasts of the leaf
vein epidermal cells. The SYTO® 62 stain which emits at 680 max nm was assigned
the pseudocolor blue. The image was acquired by excitation with argon, krypton,
and He/Ne lasers (Leica Microsystems, Wetzlar, Germany). Bar, 20 urn.
plants was reported in a similar study [36]. This method has the advantage of
examining viability directly via the ability of the cells to grow, but provides
little information about the spatial distribution of the viable and nonviable
cells at the micro scale in situ on plants.
26.3.3.2 Indicators of Membrane Integrity
Biological stains that report on bacterial membrane activity can be useful
to probe bacterial cell viability directly on plants. These stains penetrate
only cells that have a compromised cytoplasmic membrane, and therefore
are presumably nonviable. They include the DNA dyes ethidium bromide,
TO-PROTM-3 and SYTOX® Green (Molecular Probes), and propidium
iodide (PI), which is probably the most commonly used. The LIVE/DEAD
BacLight bacterial viability assay (Molecular Probes) is a popular and simple
method for determination of bacterial cell viability in which live cells fluoresce
green due to staining with SYTO® 9, and dead cells fluoresce red due to
staining with PI. The spectra of both stains are sufficiently close to detect both
the green and red cells with a fluorescein filter. Using this assay, Warriner et al.
reported the differential survival of E. coli and S. enterica cells inside and on
the surface of bean sprouts after their treatment with sodium hypochlorite [37].
Also, PI staining was combined with immunostaining to detect specifically
Methods in Microscopy 607
viable and nonviable cells of E. coli 0157:H7 introduced onto cut lettuce and
exposed to sodium hypochlorite [11].
Monier and Lindow developed a protocol based on PI staining, epifluo-
rescence microscopy, and digital image analysis to determine the viability of
individual bacterial cells directly on plants [38]. In their studies, PI was used
to map the distribution of viable and nonviable GFP- or CFP-labeled cells
on leaves (1) before and after they were exposed to desiccation stress, (2) in
homogenous versus heterogeneous aggregates, and (3) after they landed in an
aggregate of the same species versus in that of another species. These studies
demonstrated quantitatively the importance of aggregation in the survival of
epiphytic bacteria [38], the existence of antagonistic interactions at the bacterial
scale within mixed aggregates [31], and the differential fate of immigrant
bacteria to leaf surfaces depending on resident bacteria and on leaf anatomical
features at the landing site [39].
Because the correlation between membrane integrity and physiological
status of the cell is still controversial, cell viability data obtained with stains
such as PI should be interpreted carefully. It is important to emphasize that
the choice of a fluorescent viability probe is critical, and whether a certain
fluorescent probe is suitable for viability assessment under the conditions
tested has to be assessed.
26.3.3.3 GFP Fluorescence and Cell Viability
With the general excitement about GFP as an intrinsic label for bacteria, a
thorny issue that has received little attention is whether GFP or its variants can
serve as an indicator of cell viability. That is, can all fluorescent GFP-tagged
cells observed under the microscope be considered as viable cells? Lowder et al.
reported a strong correlation between Pseudomonas fluorescens cell death and
leakage of GFP from cells; most dead cells (as assessed by viability staining)
were not GFP fluorescent, but a small percentage were dead and retained
green fluorescence [40]. We have made similar observations with cultured
GFP-labeled S. enterica cells that were exposed to the lethal stress of high
temperature, desiccation, or calcium hypochlorite treatment [41]. Thus, it
appears that GFP, which is considered as a stable fluorochrome in living cells,
is lost rapidly upon cell death in bacteria. However, because of the small
percentage of fluorescent GFP cells for which the cell status is unclear, it is
preferable to confirm cell viability with an additional method when inferring
specifically on the viability of GFP-tagged cells.
26.3.3.4 Other Fluorescent Indicators of Bacterial
Physiology
Although many studies have investigated the detection of metabolically active
bacteria in aquatic environments with fluorogenic substrates as indicators of
bacterial respiratory or enzymatic activity [42,43], few of these dyes have been
used so far to investigate bacterial activity on plants. Similarly, the assessment
608 Microbiology of Fruits and Vegetables
of intracellular pH has been performed at the single bacterial cell level by
fluorescence ratio imaging with fluorescent pH indicators (e.g., 5- (and 6-)
carboxyfluorescein, BCECF, SNAFL, SNARF) [42,43], and with GFP [44],
but their utility for in situ probing of bacterial pH on plants has not been
explored. With the increasing interest in bacterial survival to acid stress in
fresh-cut fruits and vegetables, such an approach may prove to be almost
essential. Additionally, the use of fluorescent probes in combination with
flow cytometry in antimicrobial research has been widely reported. Fluorescent
probe technology and microscopy may be applied successfully to quantify
the effect of decontamination agents on human or plant pathogens on
agricultural plants.
26.3.4 Bacterial Gene Expression In Situ
on Plants
26.3.4.1 GFP as a Reporter of Gene Expression
The combination of fluorescent markers with reporter gene technology has
proven to be a powerful tool to study the behavior of bacteria on plants. The
fusion of fluorescent reporter genes to bacterial genes of interest allows for
the measurement of the transcriptional activity of that gene at the single
bacterial cell level under the microscope, rather than at the population level.
Thus, the distribution of transcriptional activity of a gene, and, potentially, the
role of its phenotype, can be assessed in particular environments.
Unless the experiments are performed in a gnotobiotic system, an addi-
tional marker is required to distinguish the bacterial cells under study from
those belonging to the indigenous microflora. This ensures that the bacterial
cells in which transcriptional activity of the gene of interest is low or off, and
therefore in which the reporter signal is low, will be detected. In the first study
of this type on plants, Brandl et al. used a transcriptional gfp fusion to an auxin
(IAA) biosynthetic gene of E. herbicola in combination with FISH to assess
the distribution of IAA synthesis in this bacterial species on bean leaves [12]
(Figure 26.7). Plants were inoculated with a strain of E. herbicola transformed
with the gfp fusion and incubated to allow for colonization to occur. Then,
the bacterial cells were washed off the leaves and subjected to FISH on micro-
scope slides with tetramethylrhodamine-labeled 16S rRNA probe specific to
E. herbicola. The green fluorescence intensity of bacterial cells that were
stained red by FISH was measured by analysis of digital images acquired under
the epifluorescence microscope. The frequency distribution of GFP fluores-
cence intensity per cell revealed that a small proportion of the E. herbicola cells
on the leaves expressed the IAA gene at very high levels, suggesting that there
were microsites on the leaf that were conducive to high production of IAA.
With the same approach, subsequent studies demonstrated the heterogeneous
distribution of the availability of sucrose [15] and fructose [14] to E. herbicola,
and of iron to Pseudomonas syringae [13], on plant surfaces. In all of the above
studies, FISH enabled the specific labeling of bacterial cells at the strain level
Methods in Microscopy
609
Inoculation with E.herbicola [ipdC-gfp]
Fixation
Wash off bacteria
N*
-o
FISH through
agar mount
f -<»^
.
FISH
Confocal microscopy
Epifluorescence
microscopy
Digital image analysis
Spatial distribution of ipdC-gfp expression
in situ on the leaf
Distribution frequency of
ipdC-gfp expression
FIGURE 26.7 Schematic diagram of fluorescence microscopy strategies to investigate
the distribution of gene expression at the bacterial cell level on plants. The protocols
make use of dual labeling with GFP as a reporter of transcriptional activity, and with
rhodamine as a marker for FISH to identify specific bacterial cells among the natural
plant microflora. Spatial distribution of ipdC-gfp expression is assessed by visualiza-
tion under CLSM of the GFP fluorescence in bacterial cells that were identified by
FISH, performed directly on plant samples (A). Frequency distribution data of the
activity of transcriptional fusions to gfp are acquired by measuring the fluorescence of
individual bacterial cells that were washed off the plant surface and identified by FISH
(B). (From Brandl, M.T., Quinones, B., and Lindow, S.E., Proc. Natl. Acad. Sci. USA,
98, 3454,2001.)
within a given species. Additionally, the rhodamine label of the 16S rRNA
probe provided a fluorescent signal with an emission spectrum sufficiently dis-
tinct from that of GFP to prevent misinterpretation of the fluorescent signals
in each microscope filter set. Optical crosstalk is a major issue in multilabeling
experiments.
Frequency distribution analysis of bacterial gene expression has been per-
formed mostly by epifluorescence microscopy to allow for fluorescence meas-
urements of a large number of bacterial cells that were recovered from plant
tissue. Brandl et al. developed a method for the assessment of bacterial GFP
fluorescence in situ on leaves under the CLSM (Figure 26.7) [12]. The method
consisted in performing FISH on leaf disks that were fixed in paraformalde-
hyde and then covered with a thin film of low-percentage agar to prevent
disruption of the spatial distribution of the bacterial cells during hybridization
procedures. The transcriptional activity of GFP reporter fusions was assessed
subsequently in 16S rRNA-labeled E. herbicola cells, through the agar, by
confocal microscopy (Figure 26.8). In this manner, spatial patterns of gene
expression in a specific bacterial population could be established.
610 Microbiology of Fruits and Vegetables
FIGURE 26.8 (Color insert follows page 594) CLSM micrograph of the spatial
distribution of E. herbicola cells harboring an ipdC-gfp fusion in the vicinity of a
glandular trichome on the surface of bean leaves. The same field of view was imaged
sequentially with a rhodamine (A) and a GFP (B) emission filter. E. herbicola cells were
detected by FISH with a rhodamine-labeled 16S rRNA probe and performed on leaf
disks mounted in agar (A). Large variations in the expression of ipdC-gfp were
detected among the population of rhodamine-labeled cells (B). White and yellow arrows
show E. herbicola cells with high and low levels of ipdC-gfp expression, respectively.
Width of white square, 5 urn. (From Brandl, M.T., Quinones, B., and Lindow, S.E.,
Proc. Natl. Acad. Sci. USA, 98, 3454, 2001. Copyright 2001 National Academy of
Sciences, USA.)
The discovery of the fluorescent protein DsRed caused much excitement
because of its potential to be used as an intrinsic bacterial label along with a
transcriptional fusion to GFP in gene expression studies. Despite reports that
GFP and DsRed can be detected in a single cell upon simultaneous excitation
[45], it appears that DsRed is also a good acceptor molecule, with GFP or CFP
as a donor, in fluorescence resonance energy transfer (FRET) imaging [46].
The interaction between these fluorescent proteins may confound the quanti-
tative interpretation of the fluorescent signals, and therefore the usefulness of
DsRed for dual-labeling with GFP or its variants still needs to be ascertained.
26.3.4.2 Practical Note on the Use of GFP for Gene
Expression Measurements
Because of the great stability of GFP, unstable GFP variants with a short
half-life have been constructed to measure transient bacterial gene expression
in time-course experiments on plants [47-49]. These destabilized variants
prevent the accumulation of GFP under basal or noninduced gene expression
conditions and are more accurate reporters of transcriptional activity at a given
time point. Also, GFP fluoresces poorly under low oxygen conditions, and
some variants like EGFP have decreased fluorescence at a pH between 7.0 and
Methods in Microscopy 611
4.5, with only 50% of its fluorescence at pH 6.0; other factors affecting GFP
chromophore formation include temperature, chemical denaturants, and
certain solvents [50]. In addition, an effect of bacterial growth rate on GFP
fluorescence intensity of individual cells has been reported [49]. Therefore,
the effect of experimental conditions on the accumulation and the function of
GFP as a chromophore per se should be tested with a constitutively expressed
gfp to avoid misinterpretation of GFP fluorescence data.
26.3.4.3 FISH for the Detection of Bacterial mRNA
Although not reported in plant studies at the present, bacterial mRNA
detection and quantification by FISH has been successfully performed in single
bacterial cells in environmental samples. With as many as five fluorescently
labeled oligodeoxynucleotide probes (depending on abundance of the tran-
script), the mRNA of two enterobacterial genes that are induced at different
stages of growth was targeted to profile the physiological activity of
Enterobacteriaceae in a waste water microbial community [51]. The mRNA
profile was obtained in conjunction with rRNA FISH for the identification of
the bacterial cells at the taxon level within the community. In other cases where
abundance of target mRNA is low, signal amplification is necessary to detect
fluorescence in single cells. Pernthaler and Amann have developed a FISH
protocol for the sensitive detection of low-abundance mRNAs at the single
bacterial cell level by enzymatic amplification of the fluorescence signal emitted
from long oligonucleotide probes that were labeled at high density [52]. This
enabled the detection of the expression of a single gene in methanotrophic
bacteria present in sediment samples. The application of such powerful
reporter systems to the detection of mRNA in single bacterial cells may offer a
means of probing specific bacterial functions in natural microbial consortia in
the plant environment. It remains to be determined, however, whether detec-
tion of the hybridization signal can be achieved against the often auto-
fluorescent background on plants.
26.3.4.4 Immunolabeling of Gene Products
Immunofluorescence labeling represents an alternative method to FISH to
investigate the localization of specific bacteria on plants, but it is suitable also
for quantitative analysis of proteins or their enzymatic products. For example,
patterns of regulation of a Ralstonia solanacearum virulence gene (eps) were
determined by quantifying the amount of (3-galactosidase protein present in
single cells of a transformant of this plant pathogen that carried an eps-lacZ
reporter fusion [53]. Quantitative measurements were performed by digital
analysis of the fluorescence of single R. solanacearum cells recovered during
infection of tomato plants, and then immunolabeled against (3-galactosidase.
Immunofluorescence microscopy with antibodies against the R. solanacearum
exopoly saccharide EPS 154 and a specific Xanthomonas axonopodis lipopoly-
saccharide [55] proved useful also to determine the spatiotemporal production
612 Microbiology of Fruits and Vegetables
of these virulence factors during progression of disease in their respective
plant host. Such an approach may have great potential in the investigation
of the surface components of plant-associated or human bacterial cells while
they grow or survive in the plant environment. The increasing availability of
very bright fluorescent antibody conjugates that allow for the detection of even
small amounts of molecules at the single cell level makes this method worthy
of consideration.
Recent breakthroughs in the development of fluorescent bioconjugates
that are bright and span the visible range have contributed to the arsenal of
strategies that microbiologists can employ to investigate the molecular biology
of bacteria on plants. However, both FISH and immunofluorescence labeling
in situ on plant surfaces require great attention to avoid the perturbation of
the bacterial cells during the many washes involved in these procedures.
26.4 OTHER TYPES OF MICROSCOPY
26.4.1 Multiphoton Excitation Fluorescence
Microscopy
In multiphoton excitation microscopy, a fluorophore is excited simultaneously
by two or more photons of longer wavelength than that of the emitted light.
Because excitation occurs only at the focal point of the microscope, there
is little out-of-focus absorption, and therefore more excitation light reaches
the focal plane. In principle, there are three main advantages to this type
of microscopy: greater penetration through thick samples, minimized photo-
damage of living cells by excitation with infrared light, and decreased photo-
bleaching outside the focal plane [56].
Multiphoton microscopy may have potential for improved imaging of
microbial cells embedded deep in plant tissue. In addition, since UV illumi-
nation in other types of microscopy causes considerable photodamage of plant
cells, multiphoton microscopy may be useful for visualization of bacterial cells
labeled with UV-absorbing fluorophores on plant surfaces. This may broaden
the range of fluorophores available to microbial ecologists to investigate the
behavior of bacteria in the plant environment. However, there is evidence that
photobleaching is actually more acute within the focal volume than with one-
photon excitation, particularly with thin samples [57]. This was confirmed even
with GFP, a fluorescent molecule considered relatively stable [58]. However,
perhaps more limiting to its application to plant microbiology is the fact that
multiphoton microscopy provides inherently less resolution than single-photon
microscopy such as CLSM [59]. Thus, although this technology has improved
greatly three-dimensional imaging of plant tissue, it actually may be suited only
for the effective visualization of its larger microbial inhabitants and not for
bacteria. To our knowledge, multiphoton microscopy has been applied to
microbiology so far mostly for the study of biofilms [60,61]. As less expensive
multiphoton systems become available, time will tell if this technology is
advantageous for the imaging of bacterial cells in or on plant tissue.
Methods in Microscopy 613
26.4.2 Fluorescence Stereomicroscopy
The fluorescence stereomicroscope provides little resolution of single bacterial
cells, and thus is not widely used in microbiology. However, it may be useful
for preliminary observations of microbial assemblages on plant surfaces, such
as biofilms and fungi, or to select tissue samples for further high-resolution
microscopy. For example, GFP-labeled S. enterica and Listeria monocytogenes
cells were observed by stereomicroscopy as large aggregates on the seed coat
edge and the root hairs of alfalfa sprouts [62,63]. In contrast, GFP-labeled
E. coli 0157:H7 cells were located in these areas at significantly lower densities
[62]. These observations under the fluorescence stereomicroscope were possible
because of the intense green fluorescence emitted by large aggregates of
brightly fluorescent bacterial cells and on portions of the sprouts that have
relatively little autofluorescence in the green range. When inoculated onto
the roots of growing lettuce plants at low cell concentrations in irrigation
water, GFP-labeled E. coli 0157:H7 was present as single cells or small
colonies scattered at distant locations on the root, and detectable by confocal
microscopy only [64].
26.4.3 Immunoelectron Microscopy
Despite the important role of fluorescence microscopy in cellular imaging, its
resolution is still well below that attained with electron microscopy (EM).
However, with the exception of biofilms on plant surfaces, which were revealed
by scanning EM (SEM) as complex assemblages of diverse microbes embedded
in organic material [26,65,66], most bacterial cells that are imaged on plants
under the EM remain disappointingly anonymous to the investigator.
Fortunately, the discovery of colloidal gold as a label in immunoelectron
microscopy has provided new opportunities to detect specific bacteria and to
unravel the more complex biology of bacterial cells in their natural envi-
ronment. This approach has the advantage of combining the highly specific
localization of molecules in situ with the high resolution of EM, and has been
used in many studies in plant pathology. For example, some elegant experi-
ments were performed under EM to propose a model for the role of the Hrp
pilus and effector proteins for type III secretion during the interaction of
Erwinia amylovora and Pseudomonas syringae with plant cells. These studies
involved single and double labeling with gold particles of different sizes to
detect two types of protein, and observations under transmission EM in plants
[67,68]. Using SEM in combination with somatic and flagellar gold-labeled
monoclonal antibodies specific to Salmonella enterica serovar Thompson,
this pathogen was visualized at high resolution on leaf surfaces after its inocu-
lation onto cilantro plants; more importantly, the immunodetection of flagellar
components allowed for the observation that S. Thompson cells produced
flagella that appeared anchored to the leaf surface, suggesting that they may
serve as attachment factor to plants (Figure 26.9) [69]. Besides immuno-
cytochemistry, other cytochemical approaches have been developed for gold
614 Microbiology of Fruits and Vegetables
FIGURE 26.9 Backscattered electron image of S. enterica serovar Thompson cells on a
cilantro leaf after their inoculation onto cilantro plants. Gold-labeled flagellar
antibodies that are specific to this serovar are shown binding to the flagellum, which
appears to be anchored to the plant surface (arrows). Bright dots are lOnm gold
particles. Bar, 500 nm. (Micrograph courtesy of Delilah F. Wood.)
labeling in EM based on the binding affinity of lectins, enzymes, or proteins
to specific molecules. For a discussion and description of methodology
regarding the use of gold labeling and EM to investigate plant-microbe
interactions, the reader is referred to an excellent review by Benhamou and
Belanger [70].
26.5 CONCLUDING REMARKS
Plant microbial ecologists face the challenge of investigating the behavior
of their subjects at the relevant spatial scale, that of the bacterial cell. Although
the discovery of fluorescent proteins and confocal microscopy has propelled
the use of cell imaging to study bacteria in the plant environment, it is evi-
dent that this approach remains in its infancy compared to the recent advances
in fluorescence imaging of protein dynamics in living eukaryotic cells, such as
FRET, FRAP (fluorescence recovery after photobleaching), FLIP (fluores-
cence loss in photobleaching), and FCS (fluorescence correlation spectroscopy)
[71]. The intense fluorescent signal required in this type of imaging due to the
small size of bacterial cells, combined with the difficulty of performing time-
lapse studies on plants under the fluorescence microscope without altering the
physicochemical microenvironment of the bacterial cells, may have limited the
application of these technologies to plant microbiology research.
Other types of microscopy are emerging that can be applied to fully
hydrated cells, and thus have great potential to impact our ability to probe
the behavior of bacteria in their natural habitat. Atomic force microscopy
(AFM), which measures the force between a sharp tip and the surface of a
sample, has extended our capability to view the minuscule. AFM has been used
Methods in Microscopy 615
already to map the surface of individual bacterial cells and various bacterial
attachment factors at unparalleled resolution, to map and quantify the adhe-
sion force of microbes to a substratum, and to measure bacterial cell wall
elasticity [72]. Scanning transmission X-ray microscopy (STXM), which uses
soft X-ray absorption spectra to provide detailed quantitative chemical infor-
mation about a sample at high resolution, is another new technology that has
been used recently to investigate the distribution of proteins, lipids, saccha-
rides, and nucleic acids in a biofilm [10]. STXM may be useful to map the
biochemistry of bacteria and their chemical environment on plant surfaces,
and thereby gain a better understanding of their physiology in this habitat.
Thus, developments in microscopy keep providing powerful tools to explore
fundamental questions regarding the biology of bacteria on plants, and their
interactions with other plant microflora and with their plant host.
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ndex
Acetic acid vapor, 424-425
Acid
organic, 322-327
resistance, 325-327
stress, 104-105
Acidified vegetables, 319-322
Adaptation, stress, 96-97, 106-107
Aeromonas, 314, 440, 452-453
Aerosols and internalization, 82
Agents, washing, 376-383
Agrobacterium tumefaciens, 41-43
Alley clobacillus acldoterrestris
alicyclic fatty acids and, 165
confirmation and identification,
178-179
D- and Z- values, 165-166, 167-169
detection and identification of, 174-179
enrichment of, 178
enumeration of, 177-178
future research on, 179-180
heat shock conditions and, 177
media used for isolation of, 175-177
physiological and phenotypic characteristics
of, 164-165
sanitation and, 173-174
significance of detection/isolation from
foods, 179
taxonomic history of, 160-164
thermal resistance characteristics of,
165-172
types of spoilage related to, 172-173
Alkaline washing agents, 388-389
Allyl isothiocyanate gas, 422-423
Alternarla alternata, 366, 465
Alternatives to chlorine, 378-383
Amendments, soil, 14, 26-27
American Society for Quality (ASQ), 355
Anisakis simplex, 505
Antagonistic interactions between soft-rot and
human pathogens, 126-127
Apples
chemical, heat, and biological control
treatments for, 297-299
cider, 21^216, 368-369
controls for processed products made
from, 300
culling, sorting, and trimming of, 297
good agricultural practices (GAPs) and, 367
harvest practices and, 295
hot water treatment of, 466, 468-469
irradiation treatments of, 297-299
patulin in, 282-300
physical, chemical, and microbial properties
of, 291-292
postproduction handling of, 295-300
preharvest practices and, 294-295
storage of, 299-300
washing treatments for, 296
Aqueous cell suspensions and infiltration of
plant surfaces, 84—85
Arabidopsis thaliana, 58, 601
Aspergillus flavus, 440, 527
Assessment studies, HACCP, 345-347
ATP, 579-581
Attachment
of human enteric pathogens to plants and
other interactions, 52-59
of microbes to melons, 236-237
by plant nitrogen fixing, epiphytic, and
pathogenic bacteria to plants, 38-48
potential factors of enteric bacterial
pathogens for plants, 48-52
Azospir ilium, 47-48
B
Bacillus, 102, 190, 199, 316, 317, 415
biocontrol of, 527
superatmospheric 2 and, 440
surface pasteurization and, 488
BacT/Alert Microbial Detection System, 582
Bacteria
in acidified vegetables, 321-322
attachment to plants, 41-48
cell viability, 605-606, 607
gene expression in situ on plants, 608-612
high hydrostatic pressure processing (HPP)
of, 501-504
irradiation effect on, 506-508
lactic acid, 314, 316-318
621
622
Index
Bacteria (continued)
in low pH foods, 160
membrane integrity, 606-607
mRNA, 611
pathogenic, 38-48
plant surface characteristics and, 37-38
potential factors of enteric, 48-52
soft-rot, 117-128
spatial distribution of, 600-602
spores, 501-504, 506-508
taxonomic history of, 160-164
visualization using microscopy, 596-600
Bacteriological Analytical Manual (BAM), 546
Bactometer, 581-582
Basil, 270-271
Biochips, 589-590
BioControl, 576, 578
Biocontrol
advantages and limitations of, 528-530
biological fumigation and, 528
combination with other treatments, 530-531
commercial products for, 523-524
improvement in formulation, 531-532
mechanisms, 526-527
postharvest, 524-525, 528-529
preharvest, 525-526
regulatory process for, 533-534
research in, 532-533
screening and selection of microorganisms
for use in, 532
by use of mutant pathogen strains, 527
Biocontrol Network, 532-533
Biofllms, plant-microbe, 59
Biological parameters measurements, 604-605
Biolog system, 573-574
Biosensors, 588-590
BioSys, 582
Botrytis, 84, 440, 450, 464-465, 507
biocontrol of, 527
Broccoli, 467
Cabbage, 255-256
Calcium ions and soft-rot, 124-125
California Environmental Protection
Agency, 534
Campylobacter, 314, 454
Candida, 440, 450
biocontrol of, 525, 526, 527
Cantaloupe. See Melons
Capsular polysaccharide (CPS), 41
Carbon dioxide
effect on microbial growth, 439-440
/0 2 absorbers and generators, 447
superatmospheric 2 and, 440-441
Carrots, 254-255
Carvacrol, 425
Cell
-cell interactions in microorganisms,
602-604
viability, 605-606, 607
walls, plant, 81
Cellulose, 43
Cetylpyridinium chloride (CPC), 390
Channels, water, 82-83
Chemunex Scan RDI system, 572
Chlorine, 89, 376-378
Chlorine dioxide gas, 380-381
antimicrobial properties of aqueous
and, 404
effects on quality of produce, 414
efficacy in reducing microorganisms on
different produce samples, 412-413
factors influencing treatment by, 409-412
general treatment systems, 407-408
generation, 404-407
mechanisms for microbial inactivation
by, 409
physical, chemical, and safety properties,
402-404
Cilantro and Se Thompson, 56
Cinnamic aldehyde, 425
Citrus juices, 213-214, 500-501
Cloning and analysis of PL genes, 123
Clostridium, 1, 18-19, 103, 119, 316, 415
biodegradable and edible films and, 444
carbon dioxide and, 439-440
modified atmosphere packaging (MAP)
and, 451-453
in mushrooms, 148
Clostridium parvum, 222
spores, 503
superatmospheric 2 and, 440
surface pasteurization and, 488
temperature and survival of, 121
Cold storage
facilities, 17-18, 20
patulin and, 299-300
refrigerated transport, distribution and, 19
Cold stress, 102-103
Colletotrichum, 464-465, 530
Compendium of Methods for the
Microbiological Examination of
Foods, 546
Congestion, water, 82-83
Consumer handling of produce, 21
Index
623
Contamination
interventions for parasites, 273-274
prevention and intervention, 21-25
research needs in, 25-27
sources of parasitic, 269
sources of potential, 5-6, 14-21
Control of internalization, 87-90
Critical control points (CCPs), 345
determining corrective action procedures
for, 356-357
establishing SBC monitoring procedures
for, 354-356
identifying and stabilizing variability at,
348-351
Critical limits (CLs), 351-354
Crop cultivars, 89
Cryptococcus, 450, 525
infirmo-miniatus, 445
Cryptosporidium, 7, 218, 505
detection and enumeration methodologies
for, 271-273
foodborne outbreaks of, 270-271
interventions for decontamination by,
273-274
overview of, 267-268
ozone and, 415
sources of, 269
Cyclospora, 7, 505
detection and enumeration methodologies
for, 271-273
foodborne outbreaks of, 270-271
interventions for decontamination by,
273-274
overview of, 268-269
selection of samples, 550
sources of, 269
D
D- and Z- values of Alicyclobacillus,
165-169
Deoxyribonucleic acid (DNA) testing, 583-588
Department of Agriculture (USDA)
Microbiological Data Program
(MDP), 10-12
Detection of microorganisms, rapid. See also
Research
biosensors and, 588-590
future directions in, 592-593
genetic testing, 583-588
history of, 565
immunological testing and, 575-579
instrumentation and biomass measurements
for, 579-582
miniaturization and diagnostic kits for,
572-575
sample preparation and treatments for, 566
total viable cell count methodologies for,
567-572
U. S. and world market trends in,
590-592
Detergent formulations, 379-380
Diagnostic kits and miniaturization, 572-575
Direct epifluorescent filter techniques
(DFET), 572
Discoma, 597
Diversity of soft-rot bacteria, 118-120
Documentation and record keeping, HACCP,
359-361
Domestic produce survey, FDA, 9-10
Dothiorella dominicana, 464
Dyes and fluorescent conjugates, 598-600
Dynal, 578
E
EcO\51, 58
Electrochemiluminescence (ECL), 271
Electrolyzed oxidizing (EO) water, 514-516
Employee hygiene, 16
Enter obacter, 49, 314, 447, 450, 452
Enter ococcus
faecalis, 440
mundtii, 446
Enterohemorrhagic E. coli, 216-217
Enterovirus, 7
Environmental Protection Agency (EPA), 533
Enzymatic and molecular mechanism of tissue
maceration by soft-rot bacteria,
122-125
Enzyme-linked immunosorbent assay
(ELISA), 285, 575-576
Equilibrated internal atmosphere (EMA),
438-439, 449
Equipment
high hydrostatic pressure processing
(HPP), 500
packinghouse facility, 16-17
rapid methods, 579-582
washing, 383-387
Erwinia, 45-46, 84, 314, 426, 450
carotovora, 366
chlorine dioxide and, 381
hot water treatments for, 464
microscopy of, 613
pectolytic, 119
production of pectin lyase (PNL) by,
122-123, 123
624
Index
Escherichia coli, 7, 314, 426-427
acetic acid and, 424-425
in acidified vegetables, 320-322
acid stress and, 104
chlorine dioxide and, 381, 409-413
chlorine washing and, 378
detergent formulations and, 380
effects of organic acids on, 323-327
and efficacy of washers, 385-387
electrolyzed oxidizing (EO) water and,
515-516
in fresh-cut vegetables, 260, 390, 392
in fresh juices, 216-217, 222-223
general stress response and, 97
genetic regulation of acid resistance in,
325-327
GFP and, 597
good agricultural practices (GAPs) and, 367
high hydrostatic pressure processing (HPP)
of, 503-504
hot water treatment for, 240-241
in imported and domestic produce, 8, 9, 10
interactions between soft-rot and, 125-126
in lettuce, 52-53, 89-90
in melons, 58, 232, 233-234, 237-239, 240,
245, 2424
membrane integrity, 606-607
microscopy of, 613
modified atmosphere packaging of fresh
produce and, 19, 443, 444, 453-454
osmotic stress and, 102
oxidative stress and, 105
ozone and, 381-382, 415, 417, 418-421
peroxyacetic acid and, 383
preharvest stress and, 100
pressure-driven infiltration and, 84-85
pulsed electric field (PEF) processing of, 511
sample selection, 549
soft-rot and, 121
spatial distribution, 600-602
in sprouts, 54-55, 188-190, 192, 195-197,
198, 200
superatmospheric 2 and, 440
surface pasteurization and, 481, 485-486
ultraviolet radiation and, 101
USDA Microbiological Data Program
(MDP) measurements of, 11-12
Eugenol, 425
Exopolysaccharide (EPS), 40-41, 43-44
Fasciola hepatica, 505
Feces-associated pathogenic bacteria, 7
Fermentation of vegetables, 314, 318-319
Films
edible and biodegradable, 443-444
permeability, 441-442
synthetic polymer, 442-443
Flavobacterium, 450
Fluorescence in situ hybridization (FISH),
611-612
Fluorescent conjugates, 598-600
Fluorescent proteins, 596-597
Food and Drug Administration (FDA).
See also Hazard Analysis Critical
Control Point (HACCP)
definition of juice, 212
domestic produce survey, 9-10
good agricultural practices (GAPs)
guidelines, 22-23, 366-367
imported produce survey, 8-9
Model Food Code, 20-21
outbreak alerts issued by, 271
washing guidelines established by, 377, 388,
391-392
Foodborne illnesses
associated with sprouts, 188-190
microorganisms of concern, 7-8
produce contamination and, 5-7
risk of contracting, 4-5, 6-7
Foodservice stores handling of produce,
20-21, 391-394
Fresh-cut vegetables
cabbage, 255-256
carrots, 254-255
interactions between microorganisms and
plant tissues in, 259-262
lettuce, 256-257
microorganisms on, 253-254
occurrence and behavior of human
pathogens in, 257-259
popularity of, 499
Fruit attachment structures, 79
Fumigation, biological, 528
Fungi and viruses on plants, 48, 288-289
high hydrostatic pressure processing (HPP)
of, 501-504
Fusarium, 450, 464-465, 465
G
Gas chromatography (GC), 284
Gas/vapor-phase sanitation. See also Washing
acetic acid, 424-425
allyl isothiocyanate, 422-423
chlorine dioxide
Index
625
antimicrobial properties of aqueous
and, 404
effects on quality of produce, 414
efficacy in reducing microorganisms,
412-413
factors influencing treatment by, 409-412
generation, 404-407
mechanisms for microbial inactivation
by, 409
physical, chemical, and safety properties
of, 402-404
general gas/vapor treatment systems for,
407-408
history of, 40
natural plant volatiles in, 425
ozone
effects on quality of produce, 421
efficacy in reducing microorganisms,
418-421
factors influencing sanitation treatment
by, 417-418
generation, 415-416
mechanisms for microbial inactivation
by, 417
potential applications of, 415
properties of, 414-415
treatment systems, 416-417
present and future applications of, 426-427
regulatory considerations, 427-428
Gene expression, bacterial, 608-612
Generally regarded as safe (GRAS) materials,
443, 444
Genetic regulation of acid resistance,
325-327
Genetic testing, 583-588
GFP, 596-597
fluorescence and cell viability, 607
as a reporter of gene expression, 608-61 1
Giardia, 415, 505
Good agricultural practices (GAPs), 6, 22-23,
343, 366-367, 376
Good manufacturing practices (GMPs),
23-24, 201, 212, 236, 343, 376
for juice, 219-221
Grade standards, 366
Growth cracks in plant surfaces, 86
Guide to Minimize Microbial Food Safety
Hazards for Fresh Fruits and
Vegetables, 22, 366, 370-371
H
Harvest operations and potential sources of
contamination, 15-16, 295
Hazard Analysis Critical Control Point
(HACCP), 24-25, 87-88, 153, 201,
236, 371
application of, 342-343
assessment studies and, 345-347
basic objectives of, 339-340
conducting process capability analyses in,
351-354
determining corrective action procedures in,
356-357
establishing documentation and recording
keeping for, 359-361
identifying and stabilizing variability at
CCPs and, 348-351, 354-356
for juice, 219-224
planning and conducting a study, 344-345
prerequisites for, 343-344
scope of, 340-342
SPC monitoring procedures in, 354-356
and using SPC to ensure control, 347-348
Heat treatments. See Hot water treatments
Hepatitis A, 7, 504
Heteractic crispa, 597
High hydrostatic pressure processing (HPP)
critical processing factors, 500-501
definition and historical perspective,
499-500
effect on parasites, 505
effect on spores and vegetative bacteria,
501-504
effect on viruses, 504-505
equipment, 500
inactivation of microorganisms
by, 501-505
Home washing of fruits and vegetables,
391-394
Hot water treatments, 240-241, 461-462.
See also Surface pasteurization
heat damage caused by, 469-470
in vitro studies, 464-465
in vivo studies, 465-469
mode of action, 470-472
rinsing and brushing system (HWRB), 463,
464
Human pathogens
attachment to plants and other interactions,
52-59
common foodborne illness causing, 7-8,
52-59
in domestic produce, 9-10
farm practices for control of soft-rot and,
127-128
in imported produce, 8-9
626
Index
Human pathogens {continued)
incidence and association with produce,
8-13
interactions between soft-rot and, 125-127
microbial ecology of, 26
occurrence and behavior in fresh-cut
vegetables, 257-259
potential sources of produce contamination
by, 13-21
produce-associated foodborne illness
traceback investigation results
on, 12-13
research on, 26, 27
USDA Microbiological Data Program
(MDP) measurements of, 10-12
Hydathodes, 79, 83
Hydrocooling water, 17, 316
Hydrogen peroxide, 239-240, 387-388
Hydrostatic pressure, 85
Hygiene, employee, 16
Immunoelectron microscopy, 613-614
Immunolabeling of gene products, 611-612
Immunological testing, 575-579
Imported produce survey, FDA, 8-9
Inoculation of produce, 556-557
Instrumentation and biomass measurements,
579-582
Internalization
aerosols and, 82
implications and control of, 87-90
infiltration of plant surface by aqueous cell
suspensions and, 84-85
internal structures of plants involved in,
80-82
location in plants, 77-78
overview of, 75-77
plant development and, 85-86
process of, 80
of soft-rot bacteria, 121
structures that enable, 78-79
types of, 82-86
water channels and water congestion and,
82-83
in wounds, 83-84
Ion-chelating agents for control of
pseudomonas rot, 125
Irradiation
of apples, 297-299
effect on spores and vegetative bacteria,
506-508
of mushrooms, 150-151
of parasites, 508
types of gamma rays used in, 505-506
of viruses, 508-509
Irrigation water as potential source of
contamination, 15, 269
Isogrid system, 569-570
Joint Food and Agriculture Organization/
World Health Organization Expert
Committee on Food Additives, 288
Journal of Quality Technology, 355
Juices and beverages
Alicyclobacillus in, 171-172, 171-180
apple, 214-216, 368-369
citrus, 213-214, 500-501
Cryptosporidium parvum in, 218
E. coli in, 216-217, 222-223
foodborne illness risk in, 211-212
HACCP rule, 219-224
intervention treatments for, 222-223
labeling of, 224
Listeria monocytogenes in, 218-219
5 log pathogen reduction standard applied
to, 221-222
pathogens associated with fresh, 216-219
physiochemical properties and endogenous
microflora of, 213-216
production of, 212-213
pulsed electric fields in processing of,
509-512
salmonella in, 217-218
sanitation standard operating procedures
(SSOPs) for, 219-221
ultraviolet (UV) radiation of, 222-223
K
Klebsiella, 48, 49, 86
Kogure assay for cell viability, 605-606
Labeling of juices, 224
Lactic acid bacteria (LAB), 314, 316-318,
445-447
Lactobacillus, 450
brevis, 446
plantarum, 318-319, 439-440
surface pasteurization and, 485-486
Lactococcus lac t is, 101, 103, 440, 446
Land use and potential contamination
sources, 14
Index
627
Latent infection of plant organs, 121
Lateral flow technology, 576-577
Lectins, Rs, 45
Lenticels, 78
Lettuce
cyclospora outbreaks in, 270-271
E. coli in, 52-53, 89-90
fresh-cut, 256-257
Leuconostoc
citrium, 446
mesenteroides, 450
Linalool, 425
Lipooligosaccharides (LOS), 41
Lipopolysaccharide (LPS), 40-41, 43-44
Liquid chromatography (LC), 284-285
Listeria monocytogenes, 7, 314, 316-318, 427
acetic acid and, 424-425
in acidified vegetables, 321-322
biodegradable and edible films and, 444
biopreservation and protective cultures and,
446-447
carbon dioxide and, 439-440
chlorine dioxide and, 381, 412-413
cold storage facilities and, 18, 20
cold stress and, 103
effects of organic acids on, 322-327
in fresh-cut vegetables, 258-259, 260-262,
390, 392
interactions between soft-rot and,
125-127
irradiation of, 507
in juice, 218-219
in melons, 58, 232, 233-234, 242-244,
258-259
microscopy of, 613
minimal processing and, 106
modified atmosphere packaging of fresh
produce and, 19, 452-453
in mushrooms, 146
osmotic stress and, 102
ozone and, 415
produce samples and, 56-58
in sprouts, 195, 198
superatmospheric O2 and, 440-441
surface pasteurization and, 485-486
temperature fluctuation and, 100
ultrasound and, 513-514
Location of internalized organisms, 77-78
5 log pathogen reduction standard, 221-222
M
Malthus system, 582
Mass spectrometry (MS), 284
Mathematical predictive modeling and MAP
systems, 455
Media for routine microbiological analysis,
546-548
Melons
efficacy of conventional washing of,
237-239
Escherichia coli in, 58, 232, 233-234,
237-239, 240, 242, 245
factors contributing to contamination of
postharvest condition, 235-236
preharvest and harvest condition,
234-235
hot water treatment of, 240-241, 465-466
human pathogens found in, 232, 233-234
hydrogen peroxide treatment of, 239-240
issues with fresh-cut, 242-244
laboratory-scale washing studies of, 237-239
Listeria monocytogenes in, 58, 232,
233-234, 242-244
methodology for microbiological evaluation
of, 244-246
microflora of, 232-234
mode of microbial attachment to, 236-237
novel disinfection treatments of, 239-242
outgrowth of flesh on, 243-244
popularity of, 231-232
research on, 246-247
salmonella in, 232, 233-234, 243-244, 549
spoilage organisms in, 233
steam treatment of, 241-242
transfer of bacteria from rind to flesh of, 243
washing in the packinghouse, 237
Membrane integrity, 606-607
Methyl jasmonate, 425
Micellar electrokinetic capillary
chromatography (MECC), 285
Microbiological Data Program (MDP),
USD A, 10-12
Microorganisms
attachment of human enteric pathogen,
52-59
attachment by plant nitrogen fixing,
epiphytic, and pathogenic bacterial,
38—48
cell-cell interactions in, 602-604
of concern with produce foodborne illnesses,
7-8, 34-35
fungal and viral, 48
interactions between plant tissues and,
259-262
internalized
location in plants, 77-78
628
Index
Microorganisms (continued)
overview of, 75-77
plant structure and, 80-82
process, 80
structures that enable, 78-80
types of, 82-86
media for analysis of, 546-548
plant-microbe biofilms and, 59
on plant surfaces, 37-38
potential attachment factors of enterial
bacterial, 48-52
retrieval efficiency, 557-558
structures that enable, 78-79
on vegetable products, 314-318
Micropores, 81
Microscopy
and bacterial gene expression in situ on
plants, 608-612
cell-cell interactions, 602-604
discovery of, 596
dyes and fluorescent conjugates used in,
598-600
emerging technologies in, 614-615
fluorescence stereo-, 613
fluorescent proteins used in, 596-597
immunoelecton, 613-614
Kogure assay for cell viability, 605-606
measurement of biological parameters,
604-605
membrane integrity, 606-607
multiphoton excitation fluorescence, 612
spatial distribution, 600-602
visualization of bacteria on plants using,
596-600
MicroStar system, 572
Miniaturization and diagnostic kits, 572-575
Minimal processing, 106, 316-318, 448-450
Modified atmosphere packaging (MAP),
18-19, 437-438
antimicrobial activity of MAP gases in,
439-440
biopreservation and protective cultures in,
445-447
characteristics of minimally processed
produce in, 448-450
definitions in, 438-439
equilibrated internal atmosphere
(EMA) and, 438-439
films
edible and biodegradable, 443-444
packaging and, 441-444
synthetic polymer, 442-443
food safety risk of produce in, 451-452
mathematical predictive modeling and, 455
microbial ecology of systems of, 454-455
microbiology of fruits and vegetables in,
448-455
multiple barriers and, 445-448
2 /C0 2 absorbers and generators, 447
pretreatments and miscellaneous strategies
in, 447-448
psychotrophic pathogens and, 452-453
spoilage organisms and commodity shelf life
in, 450-451
superatmospheric 2 and, 440-441
Molecular Beacon Technology, 585-586
Molecularly imprinted polymers (MIPs),
285-286
Moraxella, 439
mRNA, 611
Mucor, 450
Multiphoton excitation fluorescence micro-
scopy, 612
Multiple barriers and MAP, 445-448
Mushrooms
characteristics of, 135-136
commercial growing practices, 136-137
general composition of, 137-138
irradiation of, 150-151
microbiology of, 138
packaging of, 148-149
pulsed ultraviolet (UV) light treatment of,
151-152
quality of, 139-142, 152-153
spoilage of
cultural (growing) practices favoring, 142
cultural practices to suppress spoilage of,
142-146
postharvest conditions favoring, 146-147
postharvest practices to suppress, 147-152
quality and, 139-142
sources of microorganisms causing, 142
washing treatments for, 149-150
Mycotoxins. See Patulin
N
National Advisory Committee on the
Microbiological Criteria for Foods
(NACMCF), 2^25
National Molecular Subtyping Network
for Food borne Disease
Surveillance, 588
Natural plant volatiles, 425
Neogen, 577
Nonthermal processing methods
advantages and disadvantages of, 498-499
Index
629
electrolyzed oxidizing (EO) water,
514-516
high hydrostatic pressure processing (HPP),
499-505
irradiation, 150-151, 297-299, 505-508
popularity of, 498
pulsed electric fields in juice, 509-512
ultrasonic, 512-514
Norwalk-like viruses, 7
O
o 2
/C0 2 absorbers and generators, 447
superatmospheric, 440-441
Organic acids
and destruction of pathogens, 322-323
genetic regulation of, 325-327
specific effects of, 323-325
washing and sanitation with, 389-390
Organic foods, 13-14
Osmotic stress, 98-99, 101-102
Oxidative stress, 105-106
Ozone gas, 381-382
effects on quality of produce, 421
efficacy in reducing foodborne
microorganisms on produce
samples, 418-421
factors influencing sanitation treatment by,
417-418
generation of, 415-416
mechanisms for microbial inactivation by,
417
potential applications of, 415
properties of, 414-415
treatment systems, 416-417
Packaging materials, 18. See also Modified
atmosphere packaging (MAP)
antimicrobial film, 442-443
film permeability and, 441-442
for MAP produce systems, 441-444
mushroom, 148-149
Pantoea agglomerans, 238-239, 450, 604
Parameters, biological, 604-605
Parasites, pathogenic. See also
Cryptosporidium; Cyclospora
high hydrostatic pressure processing (HPP)
of, 505
irradiation of, 508
Pathatrix system, 578-579
Pathogenic parasites, 7
Pathogenic viruses, 7
Patulin
approaches for controlling levels of,
293-300
characteristics of, 282-283
chemical, heat, and biological control of,
297-299
cold storage and, 299-300
environmental factors affecting, 292-293
enzyme-linked immunosorbent assay
(ELISA) of, 285
factors affecting production of, 290-293
fungal species producing, 288-289
gas chromatography (GC) of, 284
good agricultural practices (GAPs) and, 367
harvest production of, 295
irradiation treatments of, 297-299
liquid chromatography (LC) and, 284-285
mechanism of toxicity of, 288
methods of analysis of, 283-286
micellar electrokinetic capillary chromato-
graphy (MECC) of, 285
molecularly imprinted polymers (MIPs) and,
285-286
natural occurrence in fruits and vegetables,
289-290
preharvest production of, 294-295
in processed apple products, 300
regulatory aspects of, 288
studies on
acute toxicity, 286
carcinogenicity, 287
genotoxicity, 287
immunotoxicity, 286-287
reproductive toxicity and teratogenicity,
287
thin-layer chromatography (TLC) of,
283-284
toxicological effects of, 286-288
washing treatments to reduce, 296
Pectate lyase (PL), 122-125
Pectic enzymes, 122-125
Pectolytic erwinia spp., 119
Pectolytic fluorescent (PF) pseudomonads,
119-120
Pediococcus acidilactici, 446
Penicillium, 146, 286, 420, 450. See also Patulin
expansum, 282-283, 289-292, 289-300,
530-531
Peppers, 466-467
Peroxyacetic acid, 382-383
Petrifilm, 570
Phylloplane, 36-37
Pichia, 525, 527
630
Index
Pili, 43
Plants. See also Produce
anatomy and biochemistry of roots and
leaves on, 35-37
attachment of pathogenic bacteria on, 38^48
bacterial gene expression in situ on, 608-612
crop cultivars, 89
development, 85-86
fungi and viruses on, 48
interactions between microorganisms and
tissues of, 259-262
-microbe biofilms, 59
microbial flora of, 37-38
potential attachment factors of enteric
bacterial pathogens for, 48-52
vegetation and survival of soft-rot bacteria,
120
Polymerase chain reaction (PCR) assays,
272-273, 584-588
Polyscyalum pustulans, 465
Postharvest produce
biocontrol applications, 524-525, 528-529
contamination of, 5-6, 16-19, 88
factors contributing to contamination of,
146-147, 234-235
patulin production in, 295-300
practices to suppress spoilage of, 147-152
stress, 102
Preharvest produce
biocontrol applications, 525-526
factors contributing to contamination in,
13-16, 234-235
patulin production in, 294-295
practices in growing mushrooms, 136-137
stress, 98-102
Pressure-driven infiltration of produce, 84-85
Prevention and intervention, contamination
current good manufacturing practices
(cGMPs), 23-24
effective management strategies, 21-22
good agricultural practices (GAPs), 6,
21-22
Hazard Analysis Critical Control Point
(HACCP), 24-25
Process capability analyses in HACCP,
351-354
Processing, minimal, 106, 316-318
Process of internalization, 80
Produce. See also Plants; Postharvest produce;
Preharvest produce; individual fruits
and vegetables
associated foodborne illness traceback
investigations, 12-13
attachment of human enteric pathogens to,
52-59
contamination of, 5-7, 13-21
domestic, 9-10
grade standards, 366
health benefit of consuming, 4
imported, 8-9
increase in production of, 117-118
inoculation of, 556-557
postproduction handling of, 5-6, 16-19, 88
probability of contracting foodborne
illnesses via, 4-5, 6-7
vegetable
biocontrol in minimally processed,
316-318
microflora, 314-318
Pseudomonas, 46-47, 49, 314, 450
biocontrol of, 525, 527
carbon dioxide and, 439
cell viability of, 605, 607
gene expression of, 608
microscopy of, 613
modified atmosphere packaging and, 455
2 /C0 2 absorbers and generators and, 447
ozone and, 382, 415
soft-rot by, 123-125
superatmospheric 2 and, 440
tolaasii, 141, 146, 148
Psychotrophic pathogens, 452-453
Pulsed electric field (PEF) processing, 509-512
Q
Quality Progress, 355
R
Ralstonia (Pseudomonas) solanacearum,
43-45, 86, 605, 611
Raspberries, 270-271
Redigel system, 570
Refrigerated transport, distribution, and cold
storage, 19
Regulations
for acid and acidified foods, 320-321
biocontrol agents, 533-534
of the general stress response, 97-98
Relative light units (RLUs), 580
Research. See also Detection of
microorganisms, rapid
agricultural water, 26
biocontrol, 532-533
contamination, 25-27
efficacy of decontamination treatment, 558
human pathogens, 26, 27
Index
631
media used in, 546-548
melons, 246-247
number of samples analyzed and reporting
results of, 559-560
procedures for detection and enumeration,
558-559
proximity risk of potential contaminant
sources, 27
selection of test strains for, 548-551
soil amendments, 26-27
sprouts, 201-202
type of produce and preparation of samples
for, 551-556
Restaurant handling of produce, 20-21
Retail food stores handling of produce, 20-21
Retrieval, microorganism, 557-558
Rhicadhesin, 40, 43
Rhizobium spp., 38-41, 601
Rhizoplane, 35-36
Rhizopus, 450
stolonifer, 382
Rhodotorula, 450
RiboPrinter microbial characterization
system, 587
Roots and leaves
phylloplane area of, 36-37
rhizoplane area of, 35-36
RpoS gene, 97-98
Saccharomyces cerevisiae, 511
Salmonella, 7, 89, 314, 390, 392, 427
acetic acid and, 424-425
acid stress and, 104-105
cell-cell interactions, 603-604
cell viability, 607
chlorine dioxide and, 412-413
detergent formulations and, 380
and efficacy of washers, 386
in fresh j uices, 2 1 7-2 1 8
general stress response and, 97
high hydrostatic pressure processing (HPP)
of, 503
in imported and domestic produce, 8, 9, 10
irradiation of, 508
in melons, 232, 233-234, 243-244, 549
membrane integrity, 606
microscopy of, 613
modified atmosphere packaging of fresh
produce and, 19, 453-454
oxidative stress, 106
ozone and, 415
preharvest stress and, 99
pulsed electric field (PEF) processing
and, 511
spatial distribution, 600-601
in sprouts, 190, 195
superatmospheric 2 and, 440
surface pasteurization and, 481, 487
in tomatoes and apples, 54
USDA Microbiological Data Program
(MDP) measurement of, 1 1
Sampling and study of microorganisms
efficacy of decontamination treatments and,
558
efficiency of retrieval in, 557-558
importance of accurate, 543-545
inoculation procedures in, 556-557
media for, 546-548
number of samples analyzed and reporting
of results in, 559-560
procedures for detection and enumeration,
558-559
for rapid detection, 566
selection of test strains for, 548-551
type of produce and preparation methods
for, 551-556
Sanitation standard operating procedures
(SSOPs), 24, 201, 343, 349
for juice, 219-221
Sclerotinia sclerotiorum, 527
Secretion system
type II, 45
type III, 44-45
Se Thompson, 56
Shelf life of MAP produce, 450-451
Shigella, 7, 314
detergent formulations and, 380
in fresh-cut vegetables, 258-259
general stress response and, 97
high hydrostatic pressure processing (HPP)
of, 504
in imported and domestic produce,
8, 9, 10
modified atmosphere packaging of fresh
produce and, 19, 453-454
Short-wave UV radiation and microbial stress,
101
SimPlate system, 570-571
Soft-rot, bacterial
diversity of, 118-120
economic impact of, 117-118
enzymatic and molecular mechanism of
tissue maceration by, 122-125
factors affecting survival of,
120-121
632
Index
Soft-rot, bacterial (continued)
farm practices for control of human patho-
gens and, 127-128
interactions between human pathogens and,
125-127
latent infection and internalization of, 121
pectate lyase (PL), 122-125
pectolytic Erwinia spp., 119
pectolytic fluorescent (PF) pseudomonad,
119-120
role of calcium ions in, 124-125
temperature and atmospheric conditions
affecting, 121
two-component regulatory gene system and,
123-124
Soil
amendments, 14, 26-27
associated pathogenic bacteria, 7
Solid phase extraction (SPE) methods, 285
Sorting, culling and trimming
apples, 297
effectiveness of, 367-370
purpose of, 365
using grade standards, 366
Sources of contamination
consumer handling of produce, 21
foodservice, restaurant, and retail food
stores produce handling, 20-21
postharvest
cold storage facility, 17-18
employee hygiene, 16
modified atmosphere packaging, 18-19
packaging material, 18
packinghouse equipment, 16-17
refrigerated transport, distribution, and
cold storage, 19
wash and hydrocooling water, 17
production
harvest operations, 15-16
irrigation water, 15
land use, 14
soil amendment, 14
wild and domestic animal, 14
Spatial distribution of bacteria, 600-602
Spiral plating method, 569
Spores, bacterial, 501-504, 506-508
Sprouts
classification of, 187-188
E. coli in, 54-55, 188-190, 192, 195-197,
198, 200
foodborne illnesses associated with, 188-190
interventions
irrigation water, 198-200
seed, 191-198
reducing risk of future outbreaks of
foodborne illness in, 200-201
research on, 201-202
seeds
biological interventions, 197-198
chemical and physical interventions,
191-197
Standard operating procedures (SOPs),
343, 349
Standard plate count method, 567-569
Staphylococcus aureus, 148-149, 314, 415,
440, 443
Statistical control charts, 348
Statistical process control (SPC)
and HACCP control, 347-348
monitoring procedures, 354-356
Steam treatments, 241-242
Stereomicroscopy, fluorescence, 613
Stomata, 81-82
Storage
apple, 299-300
cold, 17-18, 20
Stress
acid, 104-105
adaptation and associated risks assessment,
107-108
adaptation phenomenon, 95-98
cold, 102-103
definition of, 95-96
microbial adaptation on produce, 106-107
minimal processing and, 106
osmotic, 98-99, 101-102
oxidative, 105-106
postharvest, 102
preharvest, 98-102
produce microbiota as influenced by,
98-106
regulation of, 97-98
response, adaptation and the general,
96-97
temperature fluctuation and, 99-100
ultraviolet radiation and, 100-101
Structures that enable internalization,
78-79
Superatmospheric 2 , 440-441
Surface pasteurization
with hot water, 480-483
with steam, 483-485
thermosafe process, 485-486
University of Bristol process, 486-487
vacuum-steam-vacuum (VSV) process,
488-493
Index
633
ventilex continuous steam sterilizing
system, 487-488
Synergistic interactions between soft-rot and
human pathogens, 126
Synergistic treatment combinations in washing
and sanitizing, 390-391
Synthetic polymer films, 442-443
Talaromyces macrosporus, 501
TaqMan system, 585
Tecra, 577
Temperature
fluctuation and preharvest stress,
99-100
survival of soft-rot bacteria and, 121
Test strains, selection of, 548-551
Thermosafe process, 485
Thin-layer chromatography (TLC),
283-284
Thymol, 425
Tissue maceration by soft-rot bacteria,
122-125
Total viable cell count, 567-572
Toxicity of patulin, 286-288
Traceback investigations, 12-13
7ra/«-anethole, 425
Transposon mutagenesis, 123
Trichoderma harzianum, 141
Trisodium phosphate (TSP), 388-389
Two-component regulatory gene system,
123-124
Two-step model of attachment, 40
Type II secretion system (T3SS), 45
Type III secretion system (T2SS), 44-45
Types of internalization, 82-86
U
Ultrasound, microbial inactivation by,
512-514
Ultraviolet radiation, 100-101
of juice, 222-223
of mushrooms, 1 5 1-1 52
University of Bristol process for surface
pasteurization, 486-487
Vacuum-steam-vacuum (VSV) process,
488-493
Vegetables and fruits. See also individual
vegetables and fruits
acidified, 319-322
conventional washing agents for,
376-383
fermented, 318-319
microflora, 314-318, 450-451, 461-462,
479-480
minimally processed, 316-318,
448-450
Ventilex continuous steam sterilizing system,
487-488
Vibrio cholera, 199
Vibrio parahaemolyticus, 103, 104,440
VIDAS, 576
Viruses, pathogenic, 7
high hydrostatic pressure processing (HPP)
of, 504-505
irradiation of, 508-509
VITEX system, 573
W
Washing and sanitation, 17, 88-89, 316
cetylpyridinium chloride (CPC), 390
chlorine dioxide, 380-381
commercial equipment and wash
formulations for home, 393
conventional agents used in, 376-383
detergent formulations for, 379-380
equipment, 383-387
experimental technology in, 387-391
foodservice and home applications,
391-394
hot water, 240-241
hydrogen peroxide, 387-388
melon, 237-239
mushroom, 149-150
organic acids, 389-390
ozone used in, 381-382
patulin production and, 296
peroxyacetic acid, 382-383
sprout, 199-200
synergistic treatment combinations in,
390-391
trisodium phosphate (TSP), 388-389
Water
agricultural, 15, 26-27
channels and water congestion, 82-83
chlorination, 89
contaminated, 15, 269
electrolyzed oxidizing (EO), 514-516
hot, 240-241, 461-462
heat damage due to, 469-470
in vitro studies, 464-465
in vivo studies, 465-469
mode of action, 470-472
634
Index
Water (continued)
rinsing and brushing system (HWRB),
463, 464
technologies, 463
steam, 241-242
surface pasteurization using, 479-494
wash and hydrocooling, 17, 88-89, 199-200,
237-239, 296, 316
Wild and domestic animals as sources of
potential contamination, 14
World market testing trends, 590-592
Wounds, 79
infiltration by water caused by, 85
internalization in, 83-84
X
Xanthomonas, 47, 450, 611
Xylem, 77
Yersinia enter ocolitica, 314, 415, 452
Zygosaccharomyces bailii, 502, 511,
513-514
FOOD SCIENCE AND TECHNOLOGY
MICROBIOLOGY of
Fresh and fresh-cut fruits and vegetables have an excellent safety record. However,
surveillance data from the U.S. Centers for Disease Control and Prevention and
recent foodbome illness outbreaks have demonstrated that the incidence of
foodborne illnesses linked to the consumption of contaminated fresh fruit and
vegetable products may in fact be more prevalent than previously thought. U.S.
FDA and USDA microbiological surveys of domestic and imported fresh fruits and
vegetables demonstrate that human pathogens are sporadically found to be
associated with fresh produce. In addition to increased safety concerns, microbial
spoilage represents a significant source of waste for growers, packers, retailers,
and consumers.
Microbiology of Fruits and Vegetables concerns itself with the extensive research
that has been conducted on microbiological problems relating to the safety and
spoilage of fruits and vegetables in recent years. It considers incidences of human
pathogen contamination, sources of microbial contamination, microbial attachment
to produce surfaces, intractable spoilage problems, efficacy of sanitizing treatments
for fresh produce, novel interventions for produce disinfection, and methodology
for the microbiological evaluation of fruits and vegetables.
In Microbiology of Fruits and Vegetables, the editors, three leaders in the field,
have attempted to present a comprehensive examination, focusing on issues
needing coverage. They have selected chapter authors who are active researchers
in their respective fields and thus bring a working knowledge of current issues,
industry practices, and advances in technology.
Features
• Examines both pre-harvest and post-harvest practices and sources
of microbial contamination
• Considers continuing problems with post-harvest decay, bacterial soft rot,
and microbial spoilage of fresh-cuts and processed juices
Addresses important food safety issues
• Provides recommendations for good agricultural practices and improving
the efficacy of produce decontamination
► Includes recommendations for standardizing methods for
microbiological evaluation of produce 2 2 LI
ISBN D-aM^-BSbl-fl
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A CRC PRESS BOOK
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