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Patulin: Surveillance in Michigan Apple Cider and
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presented by
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PATULIN: SURVEILLANCE IN MICHIGAN APPLE CIDER AND JUICE, AND
FACTORS INFLUENCING ITS PRODUCTION AND CONCENTRATION IN APPLE
PRODUCTS
BY

Kerri Latrice Harris

A DISSERTATION
Submitted to
Michigan State University
In partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
Department of Food Science and Human Nutrition

2007

Abstract
PATULIN: SURVEILLANCE IN MICHIGAN APPLE CIDER AND JUICE, AND
FACTORS INF LUNENCING ITS PRODUCTION AND CONCENTRATION IN
APPLE PRODUCTS
BY

Kerri Latrice Harris

Patulin is produced by mold species such as Penicillium expansum and is the most
abundant mycotoxin in apples and apple juice. Because of its toxicity, the FDA has
established that patulin concentrations in apple juice products should not exceed 50
ug/kg. The objectives of this research were: 1) determine the concentrations of patulin in
a) apple cider produced and marketed by Michigan cider mills in 2002-2004 and b)
different brands of apple juice and cider, including shelf-stable products, marketed in
retail grocery stores in Michigan in 2005-2006, 2) determine the production of ethylene
by P. expansum and the relationship between the presence of endogenous and exogenous
ethylene and patulin production by P. expansum on liquid and solid media, 3) determine
whether fruit-produced ethylene or exogenous ethylene (100 uL/L) affects the production
of patulin in apples treated with l-methylcyclopropene (l—MCP) and inoculated with P.
expansum, and 4) determine the effect of trimming and culling of apples inoculated with
P. expansum on patulin levels in intact and decayed apple tissue and in juice produced
from two varieties of apples. To achieve these objectives, we 1) surveyed cider and juice
samples from Michigan mills (N=493) retail grocery stores (N=159) for patulin
concentrations, 2) analyzed the effects of ethylene and l-MCP on patulin production in

Red Delicious apples and liquid and solid media inoculated with P. expansum, and 3)

evaluated the effects of trimming and culling on patulin concentrations in juice from
naturally-infected Red Delicious apples and manually inoculated (P. expansum) J onagold
and Red Delicious apples stored in normal atmosphere for up to 15 days.

The survey confirmed detectable patulin (Z4 jig/L) in 18.7% of all cider mill
samples with 2.2% of samples above the legal limit of 50 ug/L. In retail grocery stores,
23% contained detectable patulin, with 11.3% of samples having patulin concentrations
above the legal limit. Exogenous ethylene administration significantly promoted patulin
production by P. expansum when inoculated on solid Potato Dextrose Agar media (P 5
0.0206) and tended to promote (P 5 0.065) patulin production by P. expansum when
inoculated on apples. l-MCP treatment of apples throughout P. expansum grth did not
influence patulin production in either medium. Juice extracted from rotten tissue of
apples inoculated with P. expansum in the laboratory typically contained >1000 pg
patulin/L by six days after inoculation. Juice prepared fiom normal appearing tissue of
both J onagold and Red Delicious apple varieties always contained detectable patulin by
nine days after inoculation, but patulin concentrations in this normal-appearing tissue
never exceeded 100 jig/L.

These experiments demonstrated that 1) apple cider and juice processors need to improve
patulin control procedures, 2) exogenous ethylene treatment does not inhibit, and may
actually enhance, patulin production by P. expansum, and 3) that careful trimming and
culling of fruit used for juice production is highly effective in controlling patulin
concentrations in apple juice, provided that removal of decayed apple flesh is essentially

complete.

Copyright by
KERRI LATRICE HARRIS

2007

DEDICATION
This work is dedicated to all those who loved me enough to pray for me, and
those who came before me, especially my parents, Willie and Emma Harris;
my sister, Kara J. Harris; my grandparents, Mary E. Hudson, Mimic C.

Adams, and Theophilus Rembert.

ACKNOWLEDGMENTS

A project indepth, and time consuming could not be completed without the
unselfish help, love, and patience of a village. Anyone and everyone who contributed in
any way are greatly appreciated. I will apologize, if I failed to recognize anyone in these
acknowledgements. It is an account that should be charged to my mind and not my heart.

I would like to thank my advisor, Dr. Leslie D. Bourquin, for his financial and
academic support, and physical labor throughout this endeavor. His advice and
dedication to the completion of this product is unmatched, especially at the end when we
were having daily marathon meetings.

My committee, Drs. Randy Beaudry, John E. Linz, and Gale Strasburg, were
supportive and encouraging. They helped me whenever called upon, even at the last
minute, without reservation. Dr. Beaudry was always available for big and small matters
pertaining to this research, including the regular use of his laboratory, even during apple
harvest season. His graduate and undergraduate students were also exceptional in sharing
equipment and helpful tips. Dr. Linz was always encouraging and thorough in
explanations. His laboratory group, too, was instrumental in giving advice, sharing
laboratory space, and helping when needed. Dr. Strasburg’s professionalism, patience,
and willingness to listen, offer advice, and loan equipment was also greatly appreciated.

I am sincerely grateful for the aid given to me by Drs. Ludmilla Roze and Gerd
Bobe. Without Ludmilla’s detailed notes, tolerance, and sincere willingness to help, the
ethylene data may not have been completed so expediciously. I could always count on
Gerd to help in any way necessary, whether traipsing around the state of Michigan to

collect cider samples, or to work half the night analyzing data.

vi

Heartfelt appreciation goes to my fellow graduate students. They encouraged me
and helped me throughout the pursuit of this degree. We worked hard, but reminded one
another to play a little, also. The friendship and comradeship we shared will always be
remembered and treasured.

I would also like to thank everyone in the Department of Food Science and
Human Nutrition and the College of Agriculture and Natural Resources for their random
acts of kindness, words of wisdom, encouragement, and humor, and for doing their jobs
so well and without hesitation.

Words alone cannot express the gratitude and love I have for my family. Without
their love, prayers, support, financial assistance, and encouragement, this phase of life
would have been intolerable. I knew this degree was a part of the Lord’s Will for me
because of them. I never felt alone and abandoned during this process. God was always
with me, and they let Him use them when I needed that reassurance. My family never let
me give up, and they were always just a phone call or email away. My parents and my
sister went well above and beyond the call of duty whenever necessary without my
having to ask. They braved the year-round weather conditions of Michigan, to help,
physically, mentally, and morally, whenever possible. My grandma talked to me for
countless hours on the phone during the dark, morning hours of all-nighters. Many
family vacations and holiday traditions were altered throughout this time, and everyone,
extended family (The Conner/Rose Families) and friends included, took it in stride.

I would also like to thank my friends, whom I also consider members of my
extended family. Mr. and Mrs. Hugh and Gladys Scott were like surrogate parents to me

in Lansing. They kept me fed, made me laugh, and made me feel like one of their

vii

children. I always felt welcome in their home, even when I appeared unaccounced and
asking for something. Mr. and Mrs. Karl and Connie Wilson always made themselves
available when I needed help in any manner. Not only Shay, but the entire Davis Family

kept me in their prayers and were only a phone call away when needed.

viii

Table of Contents

List of Tables ............................................................................................ xii
List of Figures ....................................................................................... xiv
Introduction ........................................................................................................... 1
1 Patulin: Review of the Literature .................................................................... 4
1.1 Rationale and Public Health Significance ............................................... 4
1.2 Incidence in Food and Beverage Products ............................................. 6
1.3 Patulin Biosynthesis ............................................................................... 7
1.4 Patulin Production and Stability .............................................................. 8
1.5 Patulin Toxicity ..................................................................................... 10
1.5.1 Humans ......................................................................................... 10
1 ..5 2 Animals .......................................................................................... 10
1 ..5 3 Plants ............................................................................................ 12
1.6 Detection .............................................................................................. 13
1.7 Patulin Surveillance in Apple Juice ....................................................... 16
1.8 Patulin Development in Stored Apples ................................................. 23
1.8. 1 Recommended Typical Apple Storage Conditions ........................ 23
1.8.2 Controlled and Modified Atmosphere Storage and Patulin
Production .................................................................................................... 23
1.8.3 Ethylene and Patulin Production ................................................... 24
1.8.3.1 Ethylene Effects on Fungal Metabolism ................................. 26
1.9 Methods to Reduce Patulin Concentrations .......................................... 33
1.9.1 Culling and Trimming ..................................................................... 33
1.9.2 Chemical Additives ........................................................................ 33
1.9.3 Fermentation and Clarification ....................................................... 35
1 9 4 Biocontrol and Irradiation ............................................................... 36
1.9 5 Thermal Processing ....................................................................... 36
1.10 Patulin Regulations ............................................................................... 39
1.10.1 Juice HACCP ................................................................................ 40
1.11 Objectives and Specific Aims ............................................................... 41
1 .12 References ........................................................................................... 43

2 Patulin Surveillance in apple cider produced and sold by Michigan cider mills
in 2002-2004 and apple juice and cider sold in retail grocery stores in Michigan in

2005-2006 ........................................................................................................... 56
2.1 Abstract ................................................................................................ 56
2.2 Introduction ........................................................................................... 58
2.3 Materials and Methods ......................................................................... 59

2.3.1 Solid-Phase Extraction .................................................................. 60
2.3.2 High Performance Liquid Chromatography (HPLC) ...................... 61
2.3.3 Statistical Analyses ........................................................................ 61
2.4 Results .................................................................................................. 63
2.4.1 Cider Mill Samples ......................................................................... 63

2.4.2 Retail Samples .............................................................................. 66

2.5 Discussion ............................................................................................ 70
2.5.1 Cider Mill Samples ......................................................................... 71
2.5.2 Retail Samples .............................................................................. 73

2.6 References ........................................................................................... 75

3 Ethylene production by Penicillium expansum and its endogenous and
exogenous effects on patulin production in inoculated solid and liquid media ....78

3.1 Abstract ................................................................................................ 78
3.2 Introduction ........................................................................................... 79
3.3 Materials and Methods ......................................................................... 80
3.3.1 Preparation of Commercial Mold Cultures ..................................... 80
3.3.2 Experiment 1: Ethylene and Patulin Production by P. expansum
ATCC 1117 and ATCC 28883 ..................................................................... 81
3.3.2.1 Extraction of Patulin from Media ............................................. 82
3.4 Gas chromatography (GC) Quantification of Ethylene .......................... 83
3.4.1 Experiment 2: Patulin production in relationship to the action of
endogenous and exogenous ethylene on solid and liquid* media plates ..... 83
3.4.2 Solid-Phase Extraction .................................................................. 85
3.4.3 High Performance Liquid Chromatography (HPLC) ...................... 86
3.4.4 Statistical Analyses ........................................................................ 86
3.5 Results .................................................................................................. 87
3.5.1 Experiment 1: Production of Ethylene and by P. expansum 1117
and ATCC 28883 when Grown on Solid, Semi-Solid, and Liquid Media ...... 87
3.5.1.1 P. expansum 28883 on Solid Media ....................................... 87
3.5.1.2 P. expansum 28883 on Semi-Solid Media ............................. 89
3.5.1.3 P. expansum 28883 on Liquid Media ..................................... 91
3.5.1.4 Patulin and Ethylene Production by P. expansum 1117 on
Solid PDA Media ...................................................................................... 92
3.5.2 Experiment 2: Patulin produced in relationship to the presence of
endogenous and exogenous ethylene on solid and liquid* media ............... 94
3.5.2.1 Solid Media ............................................................................ 94
3.5.2.2 Liquid Media ........................................................................... 97
3.6 Discussion ............................................................................................ 97

3.6.1 Experiment 1: Production of Ethylene by P. expansum ATCC
28883 on Solid, Semi-Solid, and Liquid Media and P. expansum ATCC 1117

on Solid Media ............................................................................................. 98

3.6.2 Experiment 2: Patulin produced in relationship to the presence of

endogenous and exogenous ethylene on solid media ................................. 99
3.7 References ......................................................................................... 102

4 Effect of fruit-produced ethylene and exogenous ethylene (100 pL/L) on the
production of patulin in apples treated with 1-MCP and inoculated with

Penicillium expansum ....................................................................................... 104
4.1 Abstract .............................................................................................. 104
4.2 Introduction ......................................................................................... 105

4.3 Materials and Methods ....................................................................... 107

4.3.1 Experiment 1 ............................................................................... 107
4.3.2 Experiment 2 ............................................................................... 109
4.3.3 Preparation of Commercial Mold Cultures ................................... 110
4.3.4 Solid-Phase Extraction ................................................................ 110
4.3.5 High Performance Liquid Chromatography (HPLC) .................... 111
4.3.6 Gas Chromatography (GC) ......................................................... 111
4.3.7 Statistical Analyses ...................................................................... 112
4.4 Results ................................................................................................ 1 13
4.4.1 Experiment 1: Ethylene, Air ......................................................... 113
4.4.2 Experiment 2: Inoculation, Ethylene, Air, 1-MCP ......................... 114
4.5 Discussion .......................................................................................... 1 18
4.6 References ......................................................................................... 121

5 Effect of trimming and culling on patulin concentrations in intact versus
decayed apple tissues ...................................................................................... 123
5.1 Abstract .............................................................................................. 123
5.2 Introduction ......................................................................................... 125
5.3 Materials and Methods ....................................................................... 126
5.3.1 Experiment 1: Naturally Infected Apples ...................................... 127
5.3.2 Experiment 2: Apples Inoculated with Penicillium expansum ...... 129
5.3.3 Preparation of Commercial Mold Cultures ................................... 131
5.3.4 Solid-Phase Extraction ................................................................ 131
5.3.5 High Performance Liquid Chromatography (HPLC) .................... 132
5.3.6 Statistical Analyses ...................................................................... 132
5.4 Results ................................................................................................ 134
5.5 Discussion .......................................................................................... 140
6 Summary, Conclusions and Future Research ........................................... 146
7 Appendices ................................................................................................ 149
7.1 General Cider Information .................................................................. 151
7.1.1.1 Processing Steps ................................................................. 151
7.1.1.2 HACCP ................................................................................. 152
7.1.2 Patulin produced in relationship to the presence of endogenous and
exogenous ethylene on solid and liquid media .......................................... 154

xi

List of Tables

Table 1. Summary of Patulin L050 values in animals (from Lindroth 1980) ........ 12
Table 2. Synopsis of Different Methods of Detection of Patulin ......................... 15

Table 3. Worldwide Patulin Surveillance in Apple Juice, Concentrates and
Processed Products ..................................................................................... 20

Table 4. Summaries of Apple Controlled Storage Studies and Development of
Patulin .......................................................................................................... 28

Table 5. Summaries of Studies and on Methods to Reduce or Eliminate Patulin
..................................................................................................................... 38

Table 6. Present Regulations for Patulin in EU Member States and the United
States .......................................................................................................... 41

Table 7. Patulin Concentrations (Log1o ug/L) in Michigan Apple Cider Samples,
2002-2004 - Overall and Stratified by Pathogen Reduction Measures and
Year of Processing ...................................................................................... 65

Table 8. Patulin Concentrations in Retail Samples Sold in the State of Michigan
2005—2006 — Overall and Stratified by Beverage Type, Growth Treatment,
Handling, Ingredients ................................................................................... 69

Table 9. Patulin Concentrations in Retail Samples Sold in the State of Michigan
2005-2006 — Overall and Stratified by Time of Processing, and Season ..... 70

Table 10. Experiment 2 Treatment Design ........................................................ 85

Table 11. Patulin and Ethylene Production by P. expansum 28883 grown on
Solid PDA Media .......................................................................................... 88

Table 12. Patulin and Ethylene Production by P. expansum ATCC 28883 When
Grown on PDA Semi-Solid Media ................................................................ 90

Table 13. Patulin and Ethylene Production by P. expansum ATCC 28883 When
Grown in Liquid PDB Media ......................................................................... 91

Table 14. Patulin and Ethylene Production by P. expansum ATCC 1117 When
Grown on Solid PDA Media ......................................................................... 93

Table 15. Patulin Concentrations (pg/L) of P. expansum 28883 Grown on Solid
PDA Media and Treated with Ethylene and 1-MCP ..................................... 96

xii

Table 16. Mycelial Weights (kg) of P. expansum 28883 Grown on Solid PDA
Media and Treated with Ethylene and 1-MCP ............................................. 96

Table 17. Treatment Design Used in Experiment 2. ................... 112

Table 18. Experiment 1- Patulin Concentrations in Juice Prepared from Apples
Treated with Ethylene (100 pL/L) and Air .................................................. 114

Table 19. Patulin Concentrations (Log1o pg/L) in Juice Pressed from Apples
Treated with Ethylene and 1-MCP + ReTain ............................................. 116

Table 20. Patulin Concentrations in Juice Prepared from Intact Apples, and
Normal-Appearing and Rotten Tissue Fractionated from Apples ............... 136

Table 21. Mean and Range of Patulin Concentrations in Juice Prepared from
Normal-Appearing and Rotten Tissue of P. expansum Inoculated Jonagold
Apples ........................................................................................................ 1 37

Table 22. Mean and Range of Patulin Concentrations in Juice Prepared from
Normal-Appearing and Rotten Tissue of P. expansum Inoculated Red
Delicious Apples ........................................................................................ 138

Table 23. Patulin Concentrations (Log1o pg/L) of P. expansum 28883 in Liquid
Media Treated with Ethylene and 1-MCP .................................................. 155

Table 24. Overall Mycelial Weights (g) and Diameters (cm) in Liquid Media
Inoculated with P. expansum 28883 Treated with Ethylene and 1-MCP 1 55

Table 25. Ethylene production (outflow - inflow in closed vessels) of Red

Delicious Apples variously treated with P. expansum, ethylene, and 1-MCP.
................................................................................................................... 1 57

xiii

List of Figures

Figure 1. Patulin and Ethylene Production by P. expansum 28883 When Grown
on Solid Media ............................................................................................. 88

Figure 2. Patulin and Ethylene Production by P. expansum ATCC 28883 When
Grown on Semi-Solid PDA Media ................................................................ 90

Figure 3. Patulin and Ethylene Production by P. expansum ATCC 28883 When
Grown in Liquid PDB Media ......................................................................... 92

Figure 4. Patulin and Ethylene Production by P. expansum ATCC 1117 When
Grown on Solid PDA Media ......................................................................... 93

Figure 5. Mycelia Areas (cmz) in Solid Media Inoculated with P. expansum
28883 Treated with Ethylene and 1-MCP — Least Squares Means Averaged
Across All Treatments .................................................................................. 97

Figure 6. Effect of Ethylene on Patulin Concentrations in Juices Prepared from

Apple infected with P. expansum 28883 .................................................... 117
Figure 7. Effect of 1-MCP + ReTain on Patulin Concentrations in Juices
Prepared from Apples infected with P. expansum 28883 .......................... 117
Figure 8. Experiment 1: Treatment of Apple Samples Obtained from Commercial
Storage Facility .......................................................................................... 128
Figure 9. Experiment 2: Treatment of samples for Trim/Cull Study .................. 130
Figure 10. Relationship between Percent Rot to Patulin Concentration (pg/L) in
rotten tissue (Experiment 1). ...................................................................... 136
Figure 11. Comparison of Patulin in Rotten Tissue by Apple Variety ............... 139

Figure 12. Comparison of Patulin in Normal Appearing Tissue by Apple Variety
................................................................................................................... 139

xiv

Introduction

Patulin (4-hydroxy-4H-furo(3,20)pyran-2(6H)-one) has a molecular weight of 154.12
and an empirical formula of C7H604 (Woodard and Singh 1949, Woodard and Singh
1950). This crystalline, colorless compound has a melting point of 110-112°C and
sublimes in high vacuum at 70-100°C (Chain et al. 1942, Katzman et al. 1944). Patulin is
a secondary fungal metabolite, is the most common mycotoxin found in apples and apple
juices, and is produced by several species of Penicillium, Aspergillus, and Byssochlamys
fungi (Wilson 1974, Lindroth 1980, Martins 2002). Penicillium expansum is the primary
source of patulin in fruit (Askar 1999, Jackson et al. 2003).

Following its discovery in 1941 , patulin was initially widely studied due to its
antibiotic activity against some gram-positive and gram-negative bacteria, as a possible
cure for the common cold, and some skin infections, but this research was abandoned
because of its toxic side effects (Raistrick et al. 1943, Martins 2002, Sorenson et al.
1985). Studies continued on the phytotoxicity and mutagenic properties of patulin
(Dickens and Jones 1961, Pohland and Allen 1970, Lindroth 1980). Patulin causes
gastrointestinal toxicity in rodents and also has mutagenic, neurotoxic, genotoxic and
carcinogenic effects in rodents (Dickens and Jones 1961, Osswald 1978, Ciegler 1976,
Reddy 1978, Reddy 1979, Lindroth 1980). Furthermore, patulin causes dermal and
gastric irritation in humans and also suppresses the immune system of rodents, producing
ulceration, congestion, and hemorrhagic lesions, especially in the gastrointestinal tract
(Dickens and Jones 1961, McKinley et a1. 1980ab, DeRosnay 1952, Dalton 1952).

Based upon this toxicity profile, an action level for patulin was established at 50

jig/kg in apple juice and in beverages containing apple juice ingredients (FDA 2001 ,

Codex Alimentarius Commission 2003). In recent years, patulin surveillance in apples
and apple juices by regulatory authorities in the US. and other countries has resulted in
significant product recalls due to patulin concentrations in excess of regulatory limits.
Thus, patulin contamination of food products has significant economic implications for
food processors in addition to its detrimental effects on public health.

The overall objectives of this research were to determine the prevalence and levels
of patulin contamination in apple cider and juice in Michigan and to assess factors
influencing patulin production in apples and its resulting concentration in apple juice
products. In order to meet these overall objectives, experiments were conducted to
achieve the following specific aims:

1. Determine the concentrations of patulin in a) apple cider produced and marketed
by Michigan cider mills in 2002, 2003, and 2004; and b) different brands of apple
juice and cider, including shelf-stable products, marketed in retail grocery stores
in Michigan in 2005 and 2006.

2. Determine the production of ethylene by Penicillium expansum and the
relationship between the presence of endogenous and exogenous ethylene and
patulin production by Penicillium expansum on liquid and solid media.

3. Determine whether fruit-produced ethylene or exogenous ethylene (100 uL/L)
affects the production of patulin in apples treated with l-Methylcyclopropene (1-
MCP) and inoculated with Penicillium expansum.

4. Determine the effect of trimming and culling of apples inoculated with
Penicillium expansum on patulin levels in intact and decayed apple tissue and in

juice produced from two varieties of apples.

The expected outcomes of this project are that the apple juice and cider industry will
have greater awareness of potential for patulin contamination in their products and will
have improved methods to reduce or prevent patulin contamination in apple and apple
juice products. We hope that increased awareness of patulin as a hazard in apples and
processed apple products will enable the industry to take preventative actions which will

lead to cost savings and a decreased risk of product recalls and adverse publicity.

1 Patulin: Review of the Literature

1.1 Rationale and Public Health Significance

Patulin is the most common mycotoxin found in apples and apple juices (Essa and
Ayesh 2000) and is produced by several species of Penicillium, Aspergillus, and
Byssochlamys fungi. Penicillium expansum is the main cause of apple rot and the
primary source of patulin formation in fruit (Ruggieri 1982, Askar 1999, Jackson 2003).
Due to concerns over human safety, the World Health Organization (WHO)
recommended that the maximum allowable concentration of patulin in apple juice be 50
jig/L (Van Egmond 1989, Stoloff, et. al. 1991). The Codex Alimentarius Commission
(2003) standard recommends an action limit for patulin of 50 ug/kg in apple juice and in
beverages containing apple juice ingredients. The regulatory limit in the United States
for apple products is no more than 50 ug/kg of patulin in apple juice, apple juice
concentrate (when diluted to single strength), and applesauce (FDA 2001). Patulin
regulations were needed because small amounts of P. expansum contamination of apples
can lead to high patulin concentrations in processed apple products depending upon the
isolate, temperature, storage time, and apple variety (McCallum et al. 2002), and because
patulin is a chemical hazard to human health.

Patulin has mutagenic, neurotoxic, genotoxic, carcinogenic and gastrointestinal
toxicity effects in rodents. It also causes dermal and gastric irritation in humans (Dickens
and Jones 1961, Sydenham et. al. 1997, Moodley 2002). Patulin has antifungal activity
and is extremely toxic to plant and animal cells (Sorenson et. al. 1985, Martins 2002).

Patulin also suppresses the immune system of rodents and produces ulceration,

congestion, and hemorrhagic lesions, especially in the gastrointestinal tract (McKinley et
al. l9803b, Martins et al. 2002).

Michigan is one of the leading states in production of apples and apple products in
the United States, producing and processing an average of 762 million pounds of apples
in 2001-2005 (NASS 2004-2005). An average of 172 million pounds (23%) of all apples
produced in Michigan were processed to juice and cider in 2001-2005 (NASS 2004-
2005)

Mold grth in fruits results in significant economic losses worldwide (Ryu and
Holt 1993). Blue mold, the disease caused by Penicillium Sp. in apples, leads to soft,
watery, brown spots that quickly enlarge, especially when fruit is stored at moderate
temperatures. These spots generally appear after some sort of damage or bruising has
occurred to the fruit, thereby allowing a point for fungal invasion and growth (Moodley
2002). Among the molds that commonly colonize apples, P. expansum is the major
source of patulin (Harwig et al. 1973, Brian et al. 1956).

Patulin surveillance in apple juices produced by Michigan cider mills is important
to determine the incidence and levels of patulin contamination in apple cider and juice
throughout the state. These data can be used as an indicator of the quality of apples used
to make these products and to predict the prevalence of human consumption of products
containing patulin concentrations above the legal limit of 50 jig/kg (pg/L).

The effects of ethylene on patulin production in stored, l-MCP treated apples have
not been investigated. Although ethylene promotes ripening of fruit, at it is also possible
that ethylene may prevent the development of patulin in apple products that are

eventually pressed and used for juices and ciders.

Although previous studies on the effects of trimming and culling on patulin
concentrations in apples have been conducted, no systematic and quantitative trimming
studies have been determined. Trimming may be as effective as culling to reduce patulin

contamination, depending on the initial amount of patulin in the product.

1.2 Incidence in Food and Beverage Products

Patulin is the most common mycotoxin found in apples and apple juices (Essa and
Ayesh 2000). In fact, it has been found in at least 50% of apples and pears with brown
rot (Frank 1977). Patulin is formed from many mold species and found in several food
items - bread, pears, peaches, plums, apricots, bananas, cherries, pineapples, grapes,
moldy silage, and was temporarily found in fermented sausage during the ripening
process (Reiss 1972, Tyllinen et al. 1977, Anderson et a1. 1978, Frank et a1. 1977,
Escoula 1975). Furthermore, patulin has been found in processed fi'uit products such as
juices, sauces, purees, jellies, diced apples, and apple pulps (Burda 1992). Homemade
apple juice and jam have been found to be at great risk for patulin contamination due to
poor raw material selection and storage conditions (Lindroth and Niskanen 1978).

Inoculation studies with P. expansum, B. nivea, and P. urticae show that patulin
can be produced in greengages, strawberries, honeydew melons, tomatoes, cucumbers,
carrots, and red and green paprika (Frank 1977). However, this inoculated growth failed
in celeriac, kohlrabi, cauliflower, red cabbage, radish, horseradish, onions, zucchini
squash, potatoes, and egg plants (Frank 1977).

It should be noted, however, that low-carbohydrate and high-protein foods
generally do not support the production of significant amounts of patulin even though

such foods may have extensive mold growth (Olivigni and Bullerman 1977, Stott and

Bullerman 1975). The presence of and reaction with sulthydryl groups present in hi gh-
protein foods makes patulin chemically undetectable and less toxic (Olivigni and

Bullerman 1977, Stott and Bullerman 1975).

1.3 Patulin Biosynthesis

Patulin (4-hydroxy-4H-furo(3,20)pyran-2(6H)-one) has a molecular weight of
154.12 and an empirical formula of C7H6O4 (Woodard and Singh 1949, Woodard and
Singh 1950). This secondary metabolite is produced by several species of Penicillium,
Aspergillus, and Byssochlamys fungi, namely, P. expansum (P. leucopus), P. urticae (P.
patlum/P. griseofulvum), P. claviforme, P. cyclopium, P. equinum, P. granulatum (P.
divergens), P. lanosum, P. lapidosum, P. melinii, P. novae-zeelandiae, P. roqueforti, A.
clavatus, A. giganteus, A. terreus, B. fulva and B. nivea (Lindroth 1980, Paster 1995,
Brause 1996, Martins 2002, Sydenham et. al 1997, Harrison 1989, Northolt 1978,
Berretta et a1. 2000, Davis and Diener 1978, Burda 1992, Golcmen 2001, Taniwaki et al.
1989, Roland 1984, Essa and Ayesh 2000, Harwig 1978, Rice et al. 1977, Wilson 1974).

Like most mycotoxins, little is known about the biosynthesis of patulin.
Mycotoxins are secondary metabolites of fungi that are formed at the end of the
exponential growth phase of the fungus (Maggon 1977). The most agreed upon theory is
that these secondary metabolites are formed when there is an accumulation of primary
metabolic precursors, such as acetate, malonate, pyruvate, and amino acids, due to
slowed organism growth and reduced energy requirements. The fungus tries to
efficiently rid itself of these precursors by diverting them to secondary metabolites
(Maggon 1977). Many of these metabolites are precursors in fatty acid synthesis that

alternately follow a polyketide pathway since the additional energy will not be used

 

(Scott et al. 1973, Tanenbaum and Bassett 1959). This polyketide pathway leads to the
biosynthesis of 6-methylsalicyclic acid (6-MSA), the first precursor to patulin and many
other mycotoxins (Scott et al. 1973, Tanenbaum and Bassett 1959, Zamir 1980). It is
thought acetyl-CoA and Malonyl-CoA combine and the enzyme 6-MSA synthase is
introduced to create 6-MSA (Scott et al. 1973, Zamir 1980). After several more steps,

gentisaldehyde is formed, followed by patulin (Scott et a1. 1973, Zamir 1980).

1.4 Patulin Production and Stability

Numerous studies have been conducted on factors affecting patulin production by
Penicillium species. Penicillium expansum is the most common species studied since it
has been found in a wide variety of foods such as fruits, vegetables, bread, and meat
products. This wide distribution of P. expansum in food products is probably due to its
tolerance of low pH and its stability over a wide range of temperatures including normal
pasteurization times and temperatures (Rychlik 2001, McCallum et al. 2002).
Penicillium can grow and produce patulin in apples at temperatures below 5°C in normal
atmospheric storage (Northolt et al. 1978, Northolt and Bullerman 1982, Sydenham et al.
1997). P. expansum is especially noted for the production of patulin in apples and apple
products (Pohland et al. 1970, Davis and Diener 1978, Sydenham et a1 1997). The
presence or absence of patulin cannot be determined based solely upon the visible
presence or absence of mold, since toxins may remain long after molds disappear
(Lindroth 1980, Sydenham et a1. 1997 , Jackson 2003). Sound apples inoculated with P.
expansum showed that the initial firmness of the apple and the cultivar of apple might be
better determinants of the amount of patulin produced, rather than visibility of rot (Beer

and Amand 1974).

Patulin production is highly dependent upon several factors including water
activity (Aw), preharvest treatment, the type of food product and the type of fungal
species and strains present (Northolt et al. 1978). Because of the narrow AW range (0.95-
0.99) for patulin production, water activity is an important determinant of patulin
production by P. expansum and P. patulum (Northolt et al. 1978, Martins et al. 2002).
Preharvest handling and treatment of apples can influence patulin production in apples
(Watkins et al. 1990, Acar et a1. 1998, Jackson 2003). Dropped or windfall apples are
more likely to be contaminated with patulin than tree-picked apples. Unculled tree-
picked fi'uit is more likely to have patulin contamination than culled fi'uit (Jackson 2003).

Little or no patulin usually penetrates healthy tissue surrounding the disease
lesions unless fruits are overripe or had senescent breakdown (Sydenham et al. 1997,
Jackson 2003). However, patulin may be present in visibly healthy fruit (Sydenham et al.
1997, Jackson 2003, Lindroth 1980). Patulin has been known to be present up to 1-2 cm
from the source of visible rot in apples (Taniwaki et al. 1989, Sydenham et al. 1997,
Rychlik 2001, Moodley et al. 2002). However, patulin can penetrate an entire tomato
(Rychlik 2001). Juices with pulp are more susceptible to patulin contamination than
other plain juice products and fruit by—products (Beretta 2000, Buchanan et al. 1974).

Once formed, patulin is completely stable in apple juice, grape juice and dry corn.
Traditional apple processing methods will not eliminate patulin in the final products
(Pohland and Allen 1970, Scott and Somers 1970). Patulin is unstable in alkaline pH
(Chain et al. 1942, Raistrick 1943, Karow 1944, Heatley et al. 1947). In fact, it is slowly

degraded at pH 6.8 (Brackett and Marth 1980).

1.5 Patulin Toxicity
1.5.1 Humans

Initial studies that claimed patulin may be a cure for the common cold could not be
validated (Medical Research Council 1944, Stoloff 1976). Patulin is of utmost concem to
humans not only due to its acute toxicity, but also because of its suspected carcinogenic
and teratogenic properties (Dickens and Jones 1961, Martins et al. 1982, Martins 2002).
Patulin can cause extensive tissue swelling as well as additional epidermal and dermal
injury when applied directly to the skin of humans (Dalton 1952). Patulin also causes
acute toxicity when ingested (Dickens 1965, Martins 2002.) Patulin was detected in
frozen blueberries and was suspected of causing diarrhea in the children who ate them
(Lindroth 1980b). In humans, oral administration of patulin (100 mg over 10 days)
results in stomach irritation, nausea, vomiting, and diarrhea (Walker and Wiesner 1944).
However, intravenous perfirsion of 100 mg of patulin into a human caused no acute

toxicity (DeRosnay 1952).

1.5.2 Animals

Acute toxicity and death caused by patulin has been assessed using a variety of
animal models. Table 1 summarizes the LD50 for patulin observed in several studies.
The LDso by daily oral administration of patulin to mice for two weeks is 29 to 48 mg/kg
body weight, and 125 mg/kg body weight was always fatal (Broom et al. 1944, Lindroth
and von Wright 1978, McKinley and Carlton 1980ab). Seventy-five mg/kg body weight
of patulin was always fatal in mice and rats when injected intravenously (Broom et al.
1944, Chain et al. 1942, Raistrick et al. 1943, Yamamoto 1954). Comparable results

have been produced in rats, cats, dogs, hamsters, cows, and rabbits following oral

1O

administration of patulin (Broom et al. 1944, Lindroth and von Wright 1978, McKinley
and Carlton 1980ab). Subcutaneous, intravenous and intraperitoneal injection of patulin
in mice, rats, hamsters, and dogs all resulted in generally similar LDlo values, ranging
from 5.7 - 50 mg patulin/kg body weight in these species. Chickens and their embryos,
quail, rabbit skin, guppies, and zebra fish larvae also were found to be sensitive to patulin
(Broom et al. 1944, McKinley and Carlton 1980ab, Ciegler et al. 1977). Animals that did
not die during these acute toxicity studies demonstrated severe pathological changes,
such as hemorrhaging in the lungs, capillary damage in the liver, spleen and kidney, and
edema of the brain (Broom et al. 1944, Lindroth and von Wright 1978, McKinley and
Carlton 1980).

In a long-term rodent toxicity study by Dickens and Jones (1961), it was found
that patulin should be considered a carcinogen. Male rats were subcutaneously injected
with 0.2 mg of patulin in arachis oil two times per week for 61-64 weeks (Dickens and
Jones 1961). Local sarcomas were observed in six out of eight surviving rats (Dickens
and Jones 1961). However, no carcinogenic activity attributable to patulin was observed
in an experiment where patulin was administered orally by a stomach tube to female
Sprague-Dawley rats two times per week for 64 weeks with a total dosage of 358 mg of
patulin/kg body weight (Osswald et al. 1978). Becci et al. (1981) also conducted a long-
term study of 109 weeks. Male and female rats were administered 0.0, 0.1, 0.5, and 1.5
mg/kg body weight of patulin by gastric intubation three days a week. It was found that
repeated oral administration of 1.5 mg/kg body weight of patulin caused premature death

in rats (Becci et al. 1981). No adverse effects were noted at 0.1 mg/kg body weight of

11

patulin. The information from this study was used by FDA to develop the maximum

residue limits (MRL) for patulin (FDA 2001).

Table 1. Summary of Patulin LDso values in animals (from Lindroth 1980)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Animal Administration“ LDso (mgkg) Reference
Mouse Sc 8-10 Katzman et al. 1944
Sc 10.0 McKinley and Carlton
1980b
Sc 15 Broom et al. 1944
Iv 15.6 Yamamoto 1954
Iv 25 Broom et a1. 1944
Ip 5.7 Ciegler et al. 1976
Ip 7.5 McKinley and Carlton
1980b
1p 15 Hofmann et al. 1971
Ip 15 Broom et al. 1944
Ip 30 Andraud et al. 1964
Po 29 Lindroth and von
Wright 1978
P0 35 Broom et al. 1944
P0 48 McKinley and Carlton
1980b
Rat Sc 15 Broom et al. 1944
Sc 25 Katzman et al. 1944
Iv 25-50 Broom et al. 1944
Hamster Sc 23 McKinley and Carlton
19803
Ip 10 McKinley and Carlton
1980a
P0 31.5 McKinley and Carlton
1980a
Dog Iv 10.4 Reddy et al. 1979
Chick P0 170 Lovett 1972
Chicken embryo (4-day- 2.35 jig/embryo Ciegler et al. 1976
old)
Zebra fish larvae 18.0 jig/ml Abedi and Scott 1969
*sc subcutaneous injection
iv intravenous injection
ip intraperitoneal injection
po per os

1.5.3 Plants

Patulin inhibits, wilts, or stunts the growth of the seeds of cucumbers, corn, peas,

flax, safflower seedlings, and sugar beets (Berestets’kyi and Synyts’kyi 1973, Gattani,

ML 1957, Wallen and Skolko 1951, Lindroth 1980). Furthermore, patulin inhibits

12

 

germination of apple pollen and the growth of cultured soybean cell suspensions, tomato
seedlings, and wheat shoot (Polacco and Sands 1977, Norstadt and McCalla 1963,
Miescher 1950, Lindroth 1980). Patulin reduces wheat straw and grain yields in
greenhouse and growth chamber cultivation conditions (Ellis et a1. 1977, Norstadt and
McCalla 1971, Lindroth 1980). Field studies confirmed that patulin decreases
germination, plant growth, winter survival and tillering of wheat seedlings and reduces

grain yield (Ellis et al. 1973)

I. 6 Detection

Several methods, including thin-layer chromatography, gas chromatography, gas
chromatography/mass spectrometry, fluorodensitometric assay, and liquid
chromatography/mass fragrnentography, have been successfully used in the
determination of patulin (Salem and Swanson 1976, MacDonald et al. 2000, Lindroth
1980). A summary of these methods and their limits of detection for patulin is presented
in Table 2. High performance liquid chromatography is presently acknowledged as the
standard method by the Association of Official Analytical Chemists (AOAC; MacDonald
et al. 2000). The AOAC method involves extraction and isolation of patulin followed by
its separation by reverse phase liquid chromatography and detection by ultraviolet light
absorbance (MacDonald et a1. 2000).

Several methods of patulin extraction prior to liquid chromatography have been
developed. The two most common methods are extraction into ethyl acetate followed by
back-extraction into sodium carbonate, or extraction into ethyl acetate with cleanup on a
silica gel column (MacDonald et al. 2000). Another method utilizes mixtures of

acetonitrile with water or 4% aqueous KCl (9: 1) to extract patulin from apples and pears

13

and their by-products (Gimeno and Martins 1983). The detection limit of patulin in foods
using HPLC with UV detection is approximately 5 jig/kg (Woodward and Singh 1949,
Pohland and Allen 197 0). Patulin’s single ultraviolet (UV) absorption maximum is at
approximately 275 nm (Pohland and Allen 1970, Woodward and Singh 1949). Eisle and
Gibson (2003) developed a syringe-cartridge solid phase extraction method for patulin in

apple juice that has a sample recovery average greater than 92%.

14

Table 2. Synopsis of Different Methods of Detection of Patulin

 

 

 

 

 

 

 

 

Type of Instrument Limit of Type of Product Authors
Detection
HPLC l-ll pg/kg Apple Juices and Ware 1975, Ware et
Butter al. 1974, Scott and
Kennedy 1973,
Tanner and Zanier
1976, Stray 1978
5 jig/kg Breakfast cereals, Hunt et al. 1978
pork, baked
beans, cheese,
milk powder
5 jig/kg Fruit Juices Leuenberger et al.
1978
2.7 jig/kg Apple Juice MacDonald et al.
2000
TLC 40 jig/kg Corn Pohland and Allen
1970, Scott 1974,
Scott and Kennedy
1973
With 3-methyl-2- 20 ug/l Apple Juice AOAC 1975, Abedi
benzothiazolinone and Scott 1969
hydrazone hydrochloride
(MBTH)
Reflectance measurement 40 jig/kg Apples, pears, Polzhofer 1977
at 273 nm tomatoes,
cucumbers, plums
With Diethylamine 20 jig/kg Cheese Siriqadana and
Lafont 1979
GLC 20 jig/kg Grains Fujimoto et al. 1975,
Suzuki et al. 1975
2.5 jig/kg Apple Juice Josefsson and
Anderssonl976,
Fujimoto et al. 1975,
Anderson et al. 1978
Mass Fragmentography 5 ug/kg Apple Juice Rosen and Pareles
1974
Fluorodensitometric 10 jig/kg Fruits and Fruit Fritz et al. 1979
Assay Products
0.1 pg of Pure Patulin Salem and Swanson
1976
1.0 mg/l Apple Juice Salem and Swanson
1976
Rapid Thin Layer 120-130 jig/kg Apples, Pears, Gimeno and Martins
Chromatography Apple Juice and 1983
Jam, Pear Juice
and Jam

 

 

 

 

15

 

1. 7 Patulin Surveillance in Apple Juice

The presence of patulin in commercial apple products is of great concern to the
apple industry. Until recently, patulin surveillance in Michigan apple juice and cider has
been negligible due to a lack of industry awareness of the problem and the high costs and
poor availability of patulin testing by commercial laboratories. Only recently has patulin
received regulatory attention in the United States, which has increased the demand for
patulin analyses. Few food laboratories offer patulin analysis and those who do may
charge $100-$300 per sample. This regulatory attention, plus additional awareness and
surveillance, should ultimately result in lower patulin incidence as was observed in a
1998 United Kingdom study (MAFF 1998). This study showed that the percentage of
apple juice samples that contained patulin in excess of 50 ug/L decreased from 26% to
2% over a six-year period following the establishment of the action level in the UK
(MAFF 1998).

Although patulin surveillance had not been previously reported for Michigan apple
juice or cider, several surveillance studies have been reported for apple juice, apple juice
concentrate, and related products in other regions. Table 3 summarizes several of these
patulin surveillance studies.

Collectively, these studies confirm that patulin contamination of apple juice
products is a significant concern worldwide. Patulin levels as high as 1,000 ug/L were
present in samples of Canadian apple cider (Scott et al. 1972). The US. Food and Drug
Administration surveyed patulin in apple juice in the US. market in 1973, and patulin
was detected in 37% of 136 samples with an average contamination level of 69 jig/L (40-

440 jig/L range) (Stoloff 1976). In Washington, DC. area stores, 8 out of 13 apple juices

16

contained patulin with concentrations of 44-309 p g/L (Ware et al. 1975). On roadside
stands in Wisconsin, patulin was found in 29 out of 66 samples of apple juice (Brackett
and Marth 1979). Twenty-nine percent of apple juice concentrates examined in Finland
contained detectable patulin with an average patulin concentration of 196 pg/L (Lindroth
1980). The same study reported that 26% of commercial apple juices contained patulin,
with concentrations averaging 15 pg/L, and 12 out of 39 homemade apple juice samples
contained patulin. Of these 12 patulin positive homemade apple juice samples, 9 had 68
p g/L or less and the remaining 3 samples had thousands of micrograms of patulin per
liter (Lindroth 1980).

In a recent Japanese survey including domestic and imported apple juice products,
3 of 143 domestic juice products (6-10 pg/L) and 6 of 45 imported juice products (6-15
pg/L) tested positive for patulin (Watanabe and Shimizu 2005). However, all positive
samples contained less than 50 pg/L patulin (Watanabe and Shimizu 2005). A survey of
patulin in Iranian apple juice (N=42) and apple juice concentrates (N=23) reported 69%
and 78% incidence of patulin contamination, respectively (Cheraghali et al. 2005). Sixty-
nine percent of apple juice samples contained detectable (>15 pg/L) levels of patulin,
while 78% of apple juice concentrates contained detectable (>15 pg/L) levels of patulin.
Thirty-three percent of the apple juice samples and 56% of the apple juice concentrates
were above 50 pg/L with maximum levels of 285 pg/L and 148 p g/L, respectively
(Cheraghali et a1. 2005). A survey of 754 retail apple juice products in Poland revealed
166 were contaminated with patulin, 8 of which exceeded 50 pg patulin/L (Szymczyk et
al. 2004). 'A survey of patulin in 113 samples of Australian apple juice showed patulin

detected in 65% of the samples (N=75), with 33 of the positive samples had levels

17

exceeding 50 pg/L (Watkins et al. 1990). Among these 33 samples, 8 had patulin levels
greater than 300 pg/L with a maximum of 629 pg/L (Watkins et al. 1990). A survey of
patulin in 100 commercial apple juices and 12 children’s apple foods in Madrid, Spain
showed patulin detected in 82% of the apple juices; 75% of which had levels below 10
pg/L with a maximum concentration of 170 pg/L (Prieta et al. 1994). The children’s
apple foods had undetectable patulin levels (Prieta et a1 1994). Another South African
survey of 143 apple juice samples found detectable in 24%; 5% of which were above 50
pg/L (Odhav et al. 2001).

A long-term survey was conducted on patulin in 482 Turkish apple juice
concentrates, diluted to single strength, collected during 1996-1999 (Gokmen and Acar
2000). Average patulin contamination levels were 63, 43, 19, and 31 pg/L in successive
years (Gokmen and Acar 2000). The percentage of concentrates above 50 pg/L
decreased each respective year (48%, 34%, 8% and 8%) (Gokmen and Acar 2000). In a
separate survey of 215 products in 1996-1997, Gokrnan and Acar (1998) found that 46%
of commercial apple juice concentrates that contained patulin were above 50 pg/L. The
patulin in this study ranged from 7-367 pg/L (Gokrnan and Acar 1998). This decrease in
succeeding years may have been due to changing climatic conditions through the years
and the increased use of activated charcoal during clarification of heavily decayed apples
used to manufacture concentrate. A survey of patulin in 105 apple juice samples in
Taipei, Taiwan found 12 samples contained patulin, all of which were below 50 pg/L
(Lai et al. 2000). A survey of 20 New Zealand apple juice samples revealed 3

contaminated samples with patulin levels of 106, 133 and 216 pg/L (Wilson 1981).

18

An Italian survey of 40 commercial apple products, juice, vinegar, sauce, and
purees, found 11 positive samples with patulin concentrations ranging from 1.4 to 74.2
pg/l (Ritieni 2003). Ten of the 40 samples were baby foods, two of which tested positive
for patulin (Ritieni 2003). Both were labeled as “organic food” (Ritieni 2003). A survey
of patulin in 28 local and imported apple fi'uits in Cairo showed that patulin levels were
within the legal limits of 50 pg/L (Ayesh and Foaad 2001). A survey of 31 apple
samples in South Africa revealed 8 patulin positive samples, with a range of 5 to 45 pg/L
(Leggott and Shephard 2001). Six of 10 baby fruit juices and purees contained detectable
patulin levels that ranged from 5 to 20 pg/L (Leggott and Shephard 2001).

The majority of commercial products contained less than 10 pg/kg of patulin;
however patulin levels as high as 42 pg/kg have also been measured in these products
(Geipel et al. 1981). Homemade apple juice and jam are more at risk for patulin
contamination possibly due to poorer raw material selection and storage conditions

(Lindroth and Niskane 1978).

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22

1.8 Patulin Development in Stored Apples
1.8.1 Recommended Typical Apple Storage Conditions

Controlled or modified atmosphere refrigerated storage of apples is an effective
method to maintain high fruit quality even afier several months of storage. Apples are
typically stored under the following conditions, depending on cultivar: 90-95% relative
humidity, 0.5 — 8% carbon dioxide and 1 — 3% oxygen at temperatures ranging from -1 -
4°C for 1-12 months (Reed 2003). Red Delicious apples may be stored 7 to 9 months in
2.5% 02, 2.5% C02, and 0°C (DeEll 2003). Empire apples may be stored 6 to 8 months
in 1.5% 02, 1.5% C02, and 1-2°C (DeEll 2003). Controlled atmosphere storage and
more effective fruit management have increased the potential to store apples for periods
up to 13 months (Doores 1983-1984, Saftner 2002). Ethylene production by apples in
refrigerated storage is reduced more by controlled-atmosphere than in regular atmosphere

storage (Safier 2002).

1.8.2 Controlled and Modified Atmosphere Storage and Patulin Production

Controlled or modified atmosphere storage has been shown to reduce the amount
of patulin produced in stored apples. A summary of these studies is provided in Table 4.
Paster et al. (1995) observed that the ability of three strains of P. expansum (NRRL 2034,
NRRL 6069, and CBS 481.84) to grow and produce patulin in pears and apples differed
under varying temperatures (0, 3, 6, 17, and 25°C). However, the maximum patulin
production in apples occurred at 17°C. They also found that patulin production was
inhibited when the fungi were grown in apples stored under 3%C02/2%Oz (25°C).

Patulin production is inhibited by storage in 3% COz/2% 02. Although these conditions

23

are typical for pear and apple storage to delay senescence and to curb postharvest decay,
P. expansum may still grow and produce patulin after 3-6 months under controlled
atmosphere conditions (Lovett et al. 1975). P. expansum 1071 produced detectable
patulin (0.4 to 0.6 ug/ml) under atmospheric storage conditions of 1% C02, 3% 02 and
96% N and a temperature of 33°F when stored for 3 to 6 months (Lovett et al. 1975).
Frank (1977) found that any increase in oxygen concentration may lead to the formation
of patulin by some fungal strains in apples inoculated with P. expansum and stored for 5
months at 4°C, followed by being held for 2-3 days at room temperature (Frank 1977).

In a study conducted by Jackson et al. (2003), MacIntosh, Red Delicious, Golden
Delicious, Gala, Fuji, Red Rome, and Granny Smith apple cultivars were stored in
controlled atmosphere for 4-6 weeks at 0-2°C. In this study, patulin was not detected in
cider produced from culled (removed), tree-picked apples, but patulin was detected in
cider produced from unculled, tree-picked apples (concentrations ranging from 0.97 to 64
ug/L) (Jackson et al. 2003). Cider made from tree-picked, controlled-atmosphere stored
apples that were culled before pressing contained 0 to 15 pg patulin/L, while cider made
from unculled fi'uit contained 59.9 to 120.5 pg patulin/1L (Jackson et a1. 2003). Taniwaki
et al. (1989) observed patulin production ranges of 150-311 ug/L and 100 to 300 pg/L in
apples stored at 25°C and 4°C, respectively one month after inoculation with P.

expansum.

1.8.3 Ethylene and Patulin Production

Ethylene (C2H4) is a natural, gaseous compound that functions as a plant hormone
(Zeringue et al. 1990). It has been approved for use in agriculture for seventy-five years

and is ideal for use in food systems since it is relatively safe, inexpensive, and has no

24

deleterious effects on the nutritional value of food and feed (Roze et al. 2004).
Furthermore, ethylene is a regulatory agent in the formation of secondary metabolites
(Sharma et al. 1985).

Fruit produce ethylene as a natural part of the ripening process (Doores 1983-
1984). The effects of ethylene on sensory and quality aspects of fruit, particularly
firmness, have been studied. Liu and Long-Jun (1986) showed that apple softening was
caused by 500 ul/L ethylene in apples stored in 3% 02 plus 3% or 5% C02. However,
reducing 02 concentrations during apple storage diminished this effect (Liu and Long—Jun
1986). Ethylene had little or no effect on fruit firmness at 1% 02 and 3% C02 (Liu and
Long-Jun 1986). With low levels of ethylene (<1 pI_./L), reduced 02 concentrations did
not influence fruit firmness (Liu and Long-Jun 1986). The rate of ethylene production by
the fruit, however, was reduced by lowering 02 concentrations (Liu and Long-Jun 1986).

Fruit was found to be firmer when exposed to lower levels (<0.1 uL/L) of ethylene

versus high levels (>160 pL/L) (Johnson et a1. 1993). Although no effect was found on
rotting of uninoculated fruit, inoculated fruit in a low ethylene environment had higher
rotting incidence compared to the higher ethylene environment (Johnson et al. 1993).

Red Delicious, Golden Delicious, Idared, and “orchard” apples showed little difference in
firmness and ripeness when stored at ethylene concentrations of 1-3.8 ppm, 10 ppm, and
500 ppm (Liu 1977). McIntosh apples were higher in acid, had firmer texture, and were
less ripe when stored at low ethylene concentrations than at 10 ppm and 500 ppm (Liu
1977). Delicious apples were stored 7 months and had severe rot in controlled
atmosphere with 10 and 500 ppm ethylene, but no rot was observed with low ethylene

(Liu 1977).

25

Ethylene has been shown to accelerate the softening of apples that can lead to
rotting (Johnston et al. 2002). Non-treated Granny Smith apples sofiened slower at 20°C
than those treated with ethylene or cold treatment, while Pacific Rose softening did not
speed up after ethylene or cold treatment (Johnston et a1. 2002). Ripening was increased
by a two- to three-fold increase in ethylene concentration when compared to non-treated
fruit at 20°C (Johnston et al. 2002). Preclimacteric McIntosh apples were studied under
low (~6 ppm) and high (~1570 ppm) ethylene levels at 3.3°C for 189 days (Forsyth et al.
1969). Apples stored under low ethylene concentrations had higher firmness compared
to apples stored at high ethylene levels, and this higher firmness continued for more than
7 days at room temperature (F orsyth et al. 1969). However, pH was lowered and soluble
solids content was slightly increased in apples stored at low ethylene concentrations
(F orsyth et al. 1969). Low ethylene treatment causes lower ethylene emission by fruit

and reduced incidence of core browning (Forsyth et al. 1969).

1.8.3.1 Ethylene Effects on Fungal Metabolism

Aspergillus parasiticus and Aspergillus nidulans produce ethylene during the early
grth phase on media, and the onset of aflatoxin biosynthesis is marked by the absence
of ethylene evolution (Sharma et al. 1985). Sharma et a1. (1985) found that ethylene
inhibited Aspergillus development and toxin biosynthesis by adding 2-chloroethyl
phosphonic acid (CEPA), an ethylene-generating compound, directly to growth medium.
These experiments reported a tight correlation between ethylene presence and aflatoxin
suppression in A. parasiticus, as well as between ethylene concentration and stimulation
or inhibition of growth (Shanna et a1. 1985). It is thought that the mechanism whereby

ethylene reduces aflatoxin biosynthesis could be a receptor/sensor-mediated signaling

26

pathway since 1-MCP, which has a structure similar to ethylene, blocks the ethylene
receptors of higher plants, and promotes aflatoxin production (Sharma et al. 1985, Serek
et a1. 1995, Roze 2004). Additional atmospheric ethylene prevents aflatoxin
accumulation and sexual development in A. parasiticus and A. nidulans, respectively
(Roze 2004). This reduction is further enhanced in A. parasiticus by the addition of C02
(Roze 2004). It is likely that the fungi may have different responses to ethylene due to

adaptive survival mechanisms within the two Aspergillus species (Roze 2004).

27

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32

1.9 Methods to Reduce Patulin Concentrations

Table 5 outlines various studies which have examined methods to eliminate or
reduce the concentration of patulin. This section will briefly review studies examining
the effects of apple trimming and culling, the use of chemical additives, fermentation,
clarification and filtration, thermal processing, and storage time as means to control

patulin concentrations in apple products.

1.9.1 Culling and Trimming

Trimming and culling of incoming fruit is the most effective method to reduce
patulin in the resulting juice (Lovett et al. 1975). Trimming and culling has been shown
to remove 93-99% of total patulin in fungus-rotted apples (Lovett et al. 1975; FDA
2001). However, culling and trimming of fruit may not be a completely reliable means to
control patulin concentrations, as a lack of visible rot does not necessarily indicate the
absence of patulin in the fruit (Taniwaki 1992). Adequate culling of apples prior to juice
preparation takes a lot of diligence and time and is often disregarded or poorly executed.
Although over-ripened or rotting fruit can be removed, sorting based on visual
observation is sometimes inadequate, resulting in patulin-contaminated finished products
(Harwi g 1978). Washing reduces patulin concentrations in apples by 10—99%,
depending on initial patulin levels and the type of wash solution used (Acar et al. 1998,

Jackson 2003).

1.9.2 Chemical Additives

The inhibition of P. expansum by chemical additives has been studied (Ryu and

Holt 1993). Patulin production was impeded when punctured apples were dipped into

33

solutions containing either 0.3% cinnamon oil or 0.5% potassium sorbate after
inoculation with P. expansum (Ryu and Holt 1993). Patulin production was impeded
when punctured apples were dipped into solution of 0.3% cinnamon oil or 0.5%
potassium sorbate after inoculation with P. expansum (Ryu and Holt 1993). The authors
recommended that fresh juice and cider products be surface treated with cinnamon oil or
potassium sorbate like postharvest apples (Ryu and Holt 1993).

Patulin may be destroyed when sulfur dioxide is used as an antioxidant or
antimicrobial agent because of its strong oxidizing capacity (Pohland and Allen 1970).
SO; and patulin interact by the opening of the hemiacetal ring of patulin and binding of
SO; to its double bond. However, the practical efficacy of SO; to reduce patulin
concentrations in juice has been questioned, as Burroughs (1977) noted that the affinity
of patulin to SC; is of little significance at the SO; concentrations (less than 200 ppm)
normally utilized in apple juice and cider processing (Burroughs 1977). Patulin is
virtually eliminated by treatment with 2,000 ppm SO; (Burroughs 1977).

The biological activity of patulin is diminished by binding with molecules
containing a sulfliydryl group (Lindroth 1980, Martins 2002). Adducts formed when
patulin reacts with sulfhydryl compounds, such as cysteine, glutathione and proteins,
have diminished toxicity compared to the parent toxin. The bacteriocidal effect of patulin
was partially inactivated after its reaction with SH compounds (Geiger and Conn 1945,
Rinderknecht et al. 1947). Administration of patulin-glutathione at a concentration equal
to the LDso for free patulin had little or no toxic effect to mice, chicken embryos, and

rabbit skin (Hofmann et al. 1971). Patulin-cysteine mixtures were not toxic when

34

injected intraperitoneally in mice at levels corresponding to 4 times the LD50 or when
injected into chicken embryos at 50 times the LD50 (Ciegler et al. 1976a).

Adding ascorbate was observed to increase the rate of disappearance of patulin
fiom apple juice and buffer solution (Brackett and Marth 1980). The mechanism of this
disappearance is unknown (Lindroth 1980). However, it is assumed that a metal-
catalyzed oxidation of ascorbate would somehow change the structure of patulin and
account for a decrease in the detection of patulin. A clarification process is
recommended before apple juice is treated with ascorbate and/or ascorbic acid to reduce
patulin levels to reduce the possible regeneration of the toxin, and the possible toxicity of
the reaction products. Sensory tests should be conducted to determine the acceptability

of this further processing by consumers.

1.9.3 Fermentation and Clarification

Patulin is practically undetected in the presence of Saccharomyces cerevisiae after
3 - 14 days of fermentation (Harwig et al. 1973, Burroughs 1977). However, it should be
noted that patulin has been found in some fermented products (Leggott and Shephard
2001). While clarification procedures, as well as pressing followed by centrifugation
have also been found to significantly decrease the presence of patulin in juice, this
increases the patulin content of pulp which may be used in animal feed (Martins 2002,
Bissessur et al. 2001). Activated charcoal has been used to effectively to remove patulin
from apple cider (Sands et a1. 1976). It should be noted that drastic color losses have
been observed with the use of some of these clarification methods (Sands et al. 1976).

One study showed that patulin is undetectable from apple juice fermented by

Saccharomyces spp (Harwig et al. 1973). This patulin reduction by fermentation was

35

confirmed in studies by Burroughs (1977) and Stinson et al. (1979). This converted toxin
was studied with l4C-patulin (Stinson et al. 1979). The products were mostly nonvolatile
and water soluble. Little, if any, patulin was shown to be metabolized to C02, and at
least 58% of the patulin was converted to substances other than adducts of cysteine,

peptides, and proteins (Lindroth 1980).

1.9.4 Biocontrol and Irradiation

The use of biocontrol and irradiation to control patulin was also studied. Castoria
et al. (2005) found that Rhodotorula glutinis strain LSll decreased the recovery of
patulin when plated together in 96-well microtiter plates and co-inoculated into apples for
10 days. Patulin production from irradiated (100-200 Krads) spores was significantly
lower than that from untreated spores (Bullerman and Hartung 1975). When mycelia
were irradiated, patulin production was even lower than that from irradiated spores
(Bullerman and Hartung 1975). Patulin production was not completely inhibited at either

level of irradiation (Bullerman and Hartung 1975).

1.9.5 Thermal Processing

Patulin is relatively stable to heat treatment, although some decrease is noted. One
study found that 50-60% of patulin was destroyed when canned apple juice was heated at
80°C for 20 minutes (Scott and Somers 1968). Another study reported when that apple
juice was pasteurized at 87-89°C, the patulin content was reduced by 20% (Stray and
Nossen 1978). In another study, patulin concentration in apple juice and sauce was
unchanged after 30 minutes at 100°C (Andersson and J osefsson 1979). Thirty minutes of

boiling did destroy 23-48% of patulin in blackcurrants, blueberries, and strawberries, and

36

10-20% of patulin in corresponding berry jams (Andersson and J osefsson 1979).
Thermal destruction (105-125 °C) of patulin was nil in a citric acid-phosphate buffer with

pH values ranging from 3.5-5.5 (Lovett and Peeler 1973).

37

Table 5. Summaries of Studies and on Methods to Reduce or Eliminate Patulin

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Type of Intervention Level of Type of Product References
Reduction
TWQlling 93-99% Apples Lovett et al. 1975
Activated Charcoal Highly Efficient Apple Cider Sands et al. 1976
Ascorbate and Ascorbic Highly Efficient Apple Juice Brackett and
Acid Marth 1980
Irradiation
100 Krad Complete Penicillium (Bullerman and
200 Krad Complete patulum NRRL Hartung 1975)
989
100 Krad 0.2 ug/mg Penicillium (Bullerman and
Detected patulum M108 Hartung 1975)
200 Krad Complete
Ultrafiltration 25% Apple Juice Acar 1998
Clarification and 39% Apple Juice Acar 1998
Filtration
Fermentation (Yeast, Complete Apple Juice Burroughs 1977,
Saccharorglces spp.) 57, 142
Sulthydryl Compounds 4—100 times less Chicken Embryo Ciegler et al.
(Adducts formed with toxic 1976, Lindroth
cysteine, glutathione, and von Wright
etc.) 1978, von Wright
and Lindroth
1978
Sulfur Dioxide Highly Efficient Apple Juice and Pohland and
Cider Allen 1970,
Burroughs 1977
3% Cinnamon Oil Highly Efficient Apples Ryu and Holt
1993
0.5% Potassium Sorbate Highly Efficient Apples Ryu and Holt
1993
Cooking/Temperature:
10 min 90% Apple Puree Frank et al. 1976
30 min No change Apple Juice and Andersson and
Sauce Josefsson 1979
Boiling 23-48% Blackcurrants, Lindroth 1980ab
Blueberries,
Strawberries
Boiling 10-20% Corresponding Lindroth 1980ab
Berry Jams
Canning (80°C for 20 50-60% Apple Juice Scott and Somers
min) 1968
Pasteurization (87-89°C) 20% Apple Juice Stray and Nossen
1978
High Pressure Washing 54% Apples for Apple Acar et a1. 1998
Juice

 

 

 

 

38

 

1. 10 Patulin Regulations

Patulin regulations were needed because small amounts of P. expansum
contamination of apples can lead to high patulin concentrations in processed apple
products (McCallum et a1. 2002). Regulation was especially important because of the
high prevalence of patulin-contaminated products in commerce (Essa and Ayesh 2000).
The World Health Organization (1995) has determined the tolerable daily human intake
of patulin should not exceed 50 ug/kg body weight/day, using a 100-fold safety factor.
The current Codex Alimentarius Commission (2003) recommended maximum residue
limits (MRLs) for patulin is 50 ug/kg in apple juice and in beverages containing apple
juice ingredients.

Several countries worldwide have regulations for the control of patulin in fi'uit juices
and some fruit products (Majerus 2002). Table 6 outlines individual countries’ patulin
MRLs for specific products. Although most of the regulations only pertain to patulin
concentrations in clear juices, the need for patulin control in cloudy juices and apple
puree has been sought by the European Union since damaged and moldy apples may be
used in the production of these products (MacDonald et al. 2000).

The US. FDA also adopted the SOug/L MRL for patulin in apple juice, apple juice
concentrate and apple sauce (FDA 2001). This was based upon “no observed adverse
effect level” (NOAEL), provisional tolerable daily intake (PTDI), and provisional
tolerable weekly intake (PTWI) estimates developed based on animal toxicity studies
(Becci et a1. 1981, FDA 2000). The PTDI for patulin was calculated to be 0.43 jig/kg

body weight based upon 90th percentile patulin exposure for all ages (FDA 2000).

39

1.10.1 Juice HACCP

There are an estimated 16,000 to 48,000 juice-associated illnesses per year in the
United States (FDA 2001). Most of these juice-associated illnesses are suspected to be
caused by pathogenic microorganisms, especially in unpasteurized fruit juices (Besser et
al. 1993, FDA 2001). These facts and several recalls led the FDA to address foodborne
illness and hazards related to juice products (FDA 1997). A warning statement on juice
product labels was required in 1998 if they were not processed using Hazard Analysis
Critical Control Point (HACCP) or a lethality step that would lead to a 5-log reduction of
the pertinent pathogens (FDA 1998). Furthermore, in 1998 FDA stated an intent to
require juice processors to develop and implement HACCP systems (FDA 1998). To
heighten juice manufacturers’ knowledge, educational programs on juice safety and
HACCP were established (Thede 2004).

The FDA required HACCP procedures to be implemented by all the juice
processors by 2004 (FDA 2001), but the effects of FDA HACCP requirements on patulin
contamination in juice products have not been reported. In 2003, two recalls of 1,500 and
38,000 bottles of apple juice were issued by FDA for noncompliant patulin levels (ODH
2003). This shows further need for patulin surveillance and improved patulin controls in

apple juice products.

40

Table 6*. Present Regulations for Patulin in EU Member States and the United
States

 

 

 

 

 

 

 

 

 

 

 

Country Commodities Maximum limit (31kg)
Austria Fruit juice 50
Finland All foods 50
France Apple juice and derived 50
products
Germany Apple juice 50
Italy Fruit juice 50
Norway General 50
Sweden Berries and products of berries 50
United Kingdom Apple juice 50
United States Apple juice, Apple juice 50
concentrate and Apple sauce

 

 

 

 

*From Majerus et al. 2002

1. 1] Objectives and Specific Aims

Based upon this review of the literature, objectives and specific aims were
developed. The overall objectives of this research were to determine the prevalence and
levels of patulin contamination in apple cider and juice in Michigan and to assess factors
influencing patulin production in apples and its resulting concentration in apple juice
products. In order to meet these overall objectives, experiments were conducted to
achieve the following specific aims:

1. Determine the concentrations of patulin in a) apple cider produced and marketed
by Michigan cider mills in 2002, 2003, and 2004 and b) different brands of apple
juice and cider, including shelf-stable products, marketed in retail grocery stores
in Michigan in 2005 and 2006.

2. Determine the production of ethylene by Penicillium expansum and the
relationship between the presence of endogenous and exogenous ethylene and

patulin production by Penicillium expansum on liquid and solid media.

41

3. Determine whether fruit-produced ethylene or exogenous ethylene (100 uL/L)

affects the production of patulin in apples treated with 1-methylcyclopropene (1-

MCP) and inoculated with Penicillium expansum.
4. Determine the effect of trimming and culling of apples inoculated with

Penicillium expansum on patulin levels in intact and decayed apple tissue and in

juice produced from two varieties of apples.

The overall outcome of this study will be to increase the awareness of the apple
industry, allowing them to take preventative action, which will lead to cost savings and a

decrease in potential recalls and adverse publicity.

42

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2 Patulin Surveillance in apple cider produced and sold by Michigan cider mills
in 2002-2004 and apple juice and cider sold in retail grocery stores in Michigan
in 2005-2006

2.1 Abstract

Patulin is the most common mycotoxin found in apples and apple juices. Because of
its toxicity, the FDA has established that the patulin concentration of apple juice products
should not exceed 50 ug/L. The objective of this study was to determine the
concentrations of patulin in a) apple cider produced and marketed by Michigan apple
cider mills in 2002, 2003, and 2004 and b) apple juice and cider, including shelf-stable
products, marketed in retail grocery stores in Michigan in 2005 and 2006. End-product
samples (N = 493) of 104 Michigan apple cider mills were obtained in 2002, 2003, and
2004 and analyzed for patulin concentration using solid phase extraction followed by
high performance liquid chromatography. The majority (81.3%) of apple cider produced
by Michigan mills in 2002-2004 contained no detectable patulin. Patulin was detected
(24 ug/L) in 18.7% of all cider mill samples with 11 (2.2%) samples having patulin
concentrations above the legal limit of 50 ug/L. A greater percentage of cider samples
obtained from mills using thermal pasteurization contained detectable patulin (28.4%)
when compared to mills using ultraviolet-light irradiation (13.5%; P 5 0.006) or no
pathogen reduction treatment (17.0%; P S 0.02). Among juice and cider samples
obtained from retail grocery stores (N=159), 23% contained detectable patulin, with 18
(11.3%) samples having patulin concentrations above the legal limit of 50 ug/L. Some
apple juice samples obtained from retail grocery stores had exceptionally high patulin
concentrations, ranging up to 2700 ug/L. Collectively, these results indicate that some

members of apple cider and juice industries have inadequate controls over patulin

56

concentrations in products. The industry, overall, should focus on improved quality of
fruit used in juice production and improve culling procedures to reduce patulin

concentrations.

57

2.2 Introduction

Patulin, a secondary fungal metabolite produced by several species of Penicillium,
Aspergillus, and Byssochlamys, is the most common mycotoxin found in apples and apple
juices (Wilson 1974, Lindroth 1980, Martins 2002). Penicillium expansum, which is
commonly found in soil, is the primary source of patulin in fi'uit (Askar 1999, Jackson et
al. 2003). Patulin causes gastrointestinal toxicity in rodents and also has mutagenic,
neurotoxic, genotoxic and carcinogenic effects in rodents (Dickens and Jones 1961,
Osswald 1978, Ciegler 1976, Reddy 1978, Reddy 1979, Lindroth 1980). Furthermore,
patulin causes dermal and gastric irritation in humans and also suppresses the immune
system of rodents, producing ulceration, congestion, and hemorrhagic lesions, especially
in the gastrointestinal tract (Dickens and Jones 1961, McKinley et al. 1980ab, DeRosnay
1952, Dalton 1952). Based upon its toxicity profile, an action level of 50 ug/kg was
established for patulin in apple juice, apple juice concentrate (at single strength dilution),
and apple sauce (FDA 2001).

Patulin surveillance in apples and apple juices by regulatory authorities in the US.
and other countries has resulted in a significant number of product recalls due to patulin
concentrations in excess of regulatory limits. In fact, several recent recalls of juice
products due to excessive patulin concentrations have occurred in the US. and UK.
Thus, patulin contamination of food products has significant economic implications for
food processors in addition to its detrimental effects on public health. Establishment and
enforcement of maximum residue levels (MRLs) for patulin in apple juice and cider in
other countries has resulted in its reduced prevalence in products. For example, a 1998

United Kingdom study reported that the percentage of UK. apple juice samples that

58

contained patulin concentrations greater than 50 ug/L decreased from 26% to 2% over a
six-year period following the establishment of the action level in the UK (MAFF 1998).
Previous studies have surveyed patulin concentrations in apple cider and juices in
the US. For example, the US. Food and Drug Administration surveyed patulin in apple
juice in the US. market in 1973, and patulin was detected in 37% of 136 samples with an
average contamination level of 69 ug/L (40-440 ug/L range) (Stoloff 1976). In
Washington, DC. area stores, 8 out of 13 apple juices contained patulin with
concentrations of 44—309 jug/L (Ware et al. 1975). On roadside stands in Wisconsin,
patulin was detected in 29 out of 66 samples of apple juice (Brackett and Marth 1980).
Statewide surveillance of patulin concentrations in apple cider produced by
Michigan cider mills has not been previously conducted. Furthermore, recent data on
patulin concentrations in US. apple juice products are not available. We were
particularly interested in assessing patulin contamination of apple cider and juice
products in the years during and immediately subsequent to implementation of F DA’s
juice HACCP regulation (1998, FDA 2001) and establishment of the US. action level for
patulin in apple juice, apple juice concentrate, and apple sauce (FDA 1998; FDA 2001).
Therefore, the objectives of this research were to determine the concentrations of patulin
in apple juice and cider a) produced and marketed by apple cider mills in Michigan in
2002, 2003, and 2004 and b) in different brands of apple juice and cider, including shelf-

stable products, marketed in retail grocery stores in Michigan in 2005 and 2006.

2.3 Materials and Methods

Samples of Michigan apple cider were purchased at 59, 104, and 74 cider mills

and retail establishments throughout the state of Michigan in 2002, 2003, and 2004,

59

respectively. All samples (0.473 — 3.875 L) were obtained within 14 days of manufacture
during cider season and prior to any “sell by” or “use by” dates on product packages.

The samples were stored on ice or refrigerated and immediately delivered to the
Michigan Department of Agriculture (MDA) Region Six Laboratory in East Lansing, M1
for microbial analyses. Results of these microbial analyses are published elsewhere
(Bobe et a1. 2007). The MDA saved aliquots (at least 50 ml) of the samples, which were
frozen and returned to MSU for patulin testing in our laboratory.

We also obtained samples of apple juice and cider (including shelf-stable
products) produced by large juice processing facilities (including many national brands)
and sold in retail grocery stores in Michigan at four times during 2005 and 2006. Apple
juice and cider products (31, 24, 37, and 32 samples obtained in Summer 2005, Fall 2005,
Spring 2006, and Summer 2006, respectively) were purchased and aliquots were placed
into 50 ml centrifuge tubes and frozen until patulin analysis. To determine patulin
concentrations, all samples were subjected to solid phase extraction and patulin was
quantified by high performance liquid chromatography. Samples were analyzed in
triplicate and analyses were repeated if the standard deviation of the three samples was

more than 10% of the mean.

2.3.1 Solid-Phase Extraction

Solid-phase extraction (SPE) of patulin from apple juice was carried out using a
syringe-cartridge method (Eisele and Gibson 2003). The SPE cartridge (Waters Oasis®
HLB 60 mg, Milford, MA) was activated by applying 2-3 ml of water, followed by 2 ml
of methanol and a final wash of 2-3 ml of water. Each sample (2.5 mL volume) was then

applied to cartridges under vacuum. The cartridges were rinsed with 2.0 ml of 1.0%

60

sodium bicarbonate solution (pH=8.3) followed by 2 m1 of 1% acetic acid solution. One
milliliter of 10% ethyl acetate in ethyl ether solution was then added to each cartridge to
elute patulin. The eluates were collected in 15x45 mm vials, evaporated to dryness under
a stream of nitrogen and then reconstituted in 0.5 m1 of 0.1% acetic acid. The vials were

capped, vigorously mixed, and fi'ozen for future analysis by HPLC.

2.3.2 High Performance Liquid Chromatography (HPLC)

Patulin was quantified using a Waters Corporation (Milford, MA) high performance
liquid chromatograph (HPLC). The Waters system included two model 510 pumps, a
model 717-plus auto-sampler, and a model 996 Photodiode Array detector. An Alltech
C13, 5 pm column (4.6 x 250 mm) was used to separate compounds. Each sample (50 uL
injection volume) was subjected to isocratic elution for 30 minutes with 10% aqueous
acetonitrile (ACN), followed by a 30-minute clean with 70% of 100% ACN and 30% of
10% ACN. The photodiode array detector collected the UV and visible light spectrum
from 190 to 800 nm to allow comparison of sample UV spectra to that of patulin
standards. Chromatograms were analyzed using Waters’ Milleniurn software. Patulin
concentration was quantified by comparing UV absorbance at 276 nm to known patulin

standards.

2.3.3 Statistical Analyses

For statistical analysis, SAS Version 9.1 was used (SAS Institute Inc., Cary, NC).
The proportion of samples containing detectable patulin and the proportion of samples
containing patulin concentrations above the legal limits were compiled for cider mills

who used thermal processing, UV-light treatment, or no pathogen reduction technology.

61

These same data also were stratified based on year of production (2002, 2003, 2004). In
addition, the relationship between the presence of microorganisms and pathogen
reduction measures (none, thermal pasteurization, UV light irradiation) and patulin
concentration was examined in the cider mill samples obtained in 2002-2004. The
average proportion of samples that tested positive for patulin was compared among the
groups by using chi-square tests in PROC GENMOD in SAS. The same procedure was
used to compare the average proportion of samples that exceeded the legal limit of 50
ug/L to counts for generic E. coli, total colifonns, or aerobic microorganisms. We
evaluated the data by comparing the average logm—transformed counts of samples that
tested positive for E. coli 0157:H7, total coliforrns, or aerobic microorganisms, as well as
the patulin concentration, respectively, between the groups using two-sided t-tests in
PROC MIXED in SAS. The same statistical procedures were used to evaluate the impact
of year of processing (2002, 2003, and 2004) on microbiological criteria in Michigan
apple cider. In Table 7, means 3: standard error of means (SEM) of proportions of
samples testing positive and loglo-transformed counts are presented and significant
differences between groups at P S 0.05 and tendency for differences between groups at P
S 0.10 are designated by different superscripts, a,b,c and d,e,f, respectively. The same
statistical procedures were used for analyzing patulin concentrations in samples obtained
from the retail grocery stores, but this information was further explored based on
ingredients used, beverage type (juice vs. cider), handling (refrigerated vs. shelf stable),

production system (organic vs. conventional), and time of sampling.

62

2.4 Results
2.4.] Cider Mill Samples

Results of patulin surveillance conducted on juice and cider samples obtained from
Michigan cider mills are presented in Table 7. The majority of apple cider and juice
produced in Michigan in 2002-2004 contained no detectable patulin. When all 2002-
2004 Michigan cider mill patulin surveillance data are combined (N=493), patulin was
detected (24 [lg/L) in 18.7% of all cider samples with 11 (2.2%) samples having patulin
concentrations above the legal limit of 50 ug/L. The average concentration of patulin in
samples that contained detectable patulin was 36.9 ug/L, with a range of 4.6 to 467.4
its/L-

We also examined the effect of pathogen control measures and year of processing
on patulin concentrations (Table 7). Among samples in which no pathogen control
measure was used (n=324), 17.0% of all samples had detectable patulin and 2.8% (9
samples) were above the legal limit. The average patulin concentration of the samples
containing detectable patulin was 42.1 ug/L with a range of 4.8 to 329.8 jig/L. Among
samples that were thermally pasteurized (n=95), 28.4% contained detectable patulin with
1.1% (1 sample) above the legal limit. The average concentration of the samples
containing detectable patulin was 33.4 ug/L with a range of 4.6 to 467 .4 jig/L. Among
samples treated with UV-light irradiation, 13.5% contained detectable patulin with 1.4%
(1 sample) above the legal limit. The average concentration of samples containing
detectable patulin was 17.3 ug/L, with a range of 5.5 to 59.1 jig/L. A significantly
greater percentage of samples obtained from facilities using thermal pasteurization

contained detectable patulin (28.4%) compared to facilities using ultraviolet-light

63

irradiation (13.5%; P S 0.02) or no pathogen reduction technology (17.0%; P S 0.01).
However, there was no difference among these treatments in likelihood of samples
containing patulin concentrations 250 ug/L.

Bacterial counts detected in these apple cider samples and the influence of
processing procedures (e.g. thermal processing, ultraviolet light processing) used by cider
mills on bacterial counts were reported by Thede et al. (2004) and Bobe et al. (in press).
Detectable bacterial counts (total aerobic plate count, total coliforrns, and generic E. coli)
were not associated (P 2 0.10) with logic transformed patulin concentrations in these
samples (data not shown).

The influence of cider production year on patulin concentrations is reported in
Table 7. Among samples obtained in 2002 (n=71), 21.1% contained detectable patulin,
with 4.2% (3 samples) above the legal limit. The average of patulin concentrations and
range were 55.2 ug/L and 4.9 to 329.8 ug/L, respectively. Among the 2003 samples (n=
241), 12.9% contained detectable patulin, with 0.8% (2 samples) above the legal limit.
The average and range of patulin concentrations were 23.9 ug/L and 4.8 to 96.4 ug/L,
respectively. Among 2004 samples (n= 181), 25.4% contained detectable patulin, with
3.3% (5 samples) above the legal limit. The average of patulin concentrations and range
were 39.7 ug/L and 4.6 to 467.4 ug/L, respectively.

Apple cider samples obtained in 2003 were significantly (P S 0.05) less likely to
contain patulin than those obtained in 2004, and tended (P S 0.10) to be less likely to
contain patulin when compared to 2002 samples. There was a tendency (P S 0.10) for
more samples obtained in 2002 and 2004 to contain 2 50 ug/L patulin when compared to

samples obtained in 2003.

64

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65

2.4.2 Retail Samples

Data on patulin concentrations in apple juice samples obtained from Michigan
retail grocery stores are summarized in Tables 8 and 9. The majority of apple juice and
apple cider samples (N=159) obtained from Michigan retail establishments in 2005 and
2006 had non-detectable patulin concentrations (Table 8). However, 23.3% of all cider
samples contained detectable (Z4 ug/L) patulin, with 18 (11.3%) samples having patulin
concentrations above the legal limit of 50 ug/L. The average patulin concentration (i
standard error) of patulin-positive retail samples was 226 i 85 ug/L, respectively. The
range of patulin concentrations in positive samples was 8.8 to 2700 ug/L.

These samples were also categorized and statistically analyzed to compare
ingredients used (apples, concentrate, or both), beverage type (juice or cider), production
system (organic vs. conventional), handling (shelf-stable or refrigerated), time of
sampling (Summer 2005, Fall 2005, Spring 2006, or Summer 2006), and whether apples
were used for juice processing in-season or off-season. Among juice samples
manufactured from apples (n=49), 40.8% contained detectable patulin, 9 (18.4%) of
which were above the legal limit. Among juice samples made from concentrate (n=99),
13.4% contained detectable patulin, 7 (7.1%) of which were above the legal limit.
According to the statement of ingredients on the product labels, 11 samples used both
apples and concentrate as ingredients. Among these samples, 36.4% contained detectable
patulin, 2 (18.2%) ofwhich were above the legal limit. Juices made from apples were
more likely to contain detectable patulin (P = 0.0003) than juices made from concentrate.
Juices made using both apples and concentrate tended to be more likely to contain

detectable patulin (P = 0.0553) than samples made from concentrate. Similarly, juices

66

made from apples were more likely (P = 0.0440) to have patulin levels above 50 ug/L
when compared to juices manufactured from concentrate.

Among the cider samples that had not been clarified (n=18), 27.8% contained
detectable patulin, with none above the legal limit of 50 jig/L (Table 8). Among the juice
samples (n=141), 22.7% contained detectable patulin, with 12.8% (18 samples) above the
legal limit. Compared to products designated as ciders (all of which were manufactured
using apples as an ingredient), juice products were significantly more likely to contain
patulin concentrations exceeding the legal limit. The average concentration of patulin in
juice samples also tended (P < 0.10) to be greater than that observed in ciders.

Of the juices labeled as organic (n=14), 42.9% had detectable patulin with 21.4%
(3 samples) above the legal limit. Of the samples produced using conventionally grown
ingredients (n=145), 21 .4% had detectable patulin with 10.3% above the legal limit.
There was a tendency (P=0.08) for organic juices to be more likely to contain detectable
patulin when compared to conventional juices.

Among refrigerated retail juices (n=29), 24.1% contained detectable patulin, 1
(3.4%) of which was above the legal limit (Table 8). Among shelf-stable retail juices
(n=130), 23.1% contained detectable patulin, 17 (13.1%) of which were above the legal
limit. Shelf-stable samples showed a tendency (P = 0.0711) to be more likely to contain
illegal levels of patulin when compared to refrigerated samples.

Comparison of patulin concentrations of juices obtained at retail outlets at
different times are summarized in Table 9. No differences were detected in the
proportion of samples containing detectable patulin, the proportion of samples containing

> 50 ug/L patulin, or the mean patulin concentration when comparing the four sampling

67

periods. Similarly, no differences in these parameters were detected when samples were
categorized as being obtained during apple harvest season (Fall 2005 juices only) or in
the off-season (combined results for Summer 2005, Spring 2006, and Summer 2005
sampling periods). However, off-season-procured juices made from apples were
significantly more likely to contain detectable patulin (P S 0.02) when compared to juices
produced from apples during apple harvest season. When broken down by ingredients
and time, of those juices produced from apples during the harvest season, 9.1% (1
sample) contained detectable patulin. This juice sample also happened to exceed the
legal limit. Of those juices produced from apples during the off-season, 50.0% contained
detectable patulin, 8 (21.1%) of which were above the legal limit. Among those juice
samples produced from concentrate, obtaining samples during apple harvest season or in
the off-season did not influence patulin detection frequency, the proportion exceeding the

legal limit, nor the mean patulin concentration.

68

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69

Table 9. Patulin Concentrations in Retail Samples Sold in the State of Michigan
2005-2006 - Overall and Stratified by Time of Processing, and Season

 

 

 

 

 

 

 

 

 

 

 

Time of No. of Samples Samples Average Range of
Sampling/ samples containing containing concentrations of concentration of
Season/ detectablel patulin 2510 samples with samples with
Ingredients patulin (0/0) lig/L (O/o) detectable l detectable platulm
Patulin (Pg/L) (Hg/L)
Time of
Sampling:
Summer 35 28.6 i 7.7 5.7x 4.0 53.8 i 24.4 15.3 — 264.8
2005
Fall 2005 39 23.1 :1: 6.8 12.821: 5.4 338.1 i 186.6 13.4 — 1722.6
Spring 2006 44 20.5 i 6.2 13.6:1: 5.2 126.4 0 47.5 13.6 — 417.6
Summer 41 22.0 i 6.5 12.2:t 5.2 404.8 :1: 293.3 8.8 - 2700.4
2006
Season:
In-season 39 23.1 i 6.8 12.821: 5.4 338.1 i 186.6 13.4 — 1722.6
Off-season 120 23.3 i 3.9 10.8 i: 2.8 190.0 i 96.5 8.8 — 2700.4
For apples
only:
III-season 11 9_1b i 9.1 9.1 :t 9.1 114.5 114.5
Off-season 38 5003 :t 8.2 21.1 i 6.7 83.6 d: 26.9 8.8 - 417.6
For
concentrate
only:
In-season 24 20.8 i 8.5 8.3 i 5.8 485.8 :t 331.9 13.4 — 1722.6
Off-season 75 10.7 d: 3.6 6.7i 2.9 464.5 i 326.0 26.8 — 2700.4

 

I
Least square means i standard errors are shown.

a,b,c

year of processing significantly differ at P S 0.05.

Numbers with different superscripts (a,b,c) within a column and within pathogen reduction measures or

,e . . . . . . . .
Numbers With different superscripts (d,e) Within a column and Within pathogen reduction measures or

year of processing tend to differ at P S 0.10.

2.5 Discussion

Public and governmental concern over the safe production of j uice increased in

1996 in light of foodbome illness outbreaks associated with the consumption of

unpasteurized apple juice and cider products. To increase juice safety and enhance

7O

 

pathogen control, in 2001 FDA issued juice HACCP regulations to be implemented in all
juice processing plants by January 2004. Also in 2001, FDA implemented additional
regulations by establishing a maximum limit for patulin concentrations in apple juice and
cider, apple juice concentrate (at single strength), and apple sauce at 50 ug/L. We were
especially interested in determining the prevalence of patulin in apple juice and cider
produced in mills and sold in grocery stores in the state of Michigan subsequent to FDA
rulemaking on juice HACCP and patulin maximum residue requirements.

To conduct this study, we collected cider produced and sold in Michigan cider
mills and juice and cider sold in retail grocery stores in Michigan, including local and
national brands. To our knowledge, this is the first large-scale, multi-year surveillance
study that has been conducted to estimate the frequency of patulin presence in

commercial apple cider and juice samples in Michigan.

2.5.1 Cider Mill Samples

Bacterial counts in the samples obtained from cider mills and the processing
procedures (e. g. thermal processing, ultraviolet light processing) used to produce these
samples were reported by Thede et al. (2004) and Bobe et al. (Journal of Food Protection
in press). Bacterial counts (total plate count, coliforms, and E. coli 0157:H7) were not
associated with logo transformed patulin concentrations (P 2 0.10) in these samples.
However, illegal concentrations of patulin were found in 2.2% of the cider mill samples.
It should be noted that samples containing patulin concentrations above 50 [Ag/L are
legally adulterated, just as would be the case if they were found to contain pathogenic

microorganisms.

71

A significantly greater percentage of samples obtained from facilities using thermal
pasteurization contained detectable patulin (28.4%) compared to facilities using
ultraviolet-light irradiation (13.5%; P S 0.02) or no pathogen reduction technology
(17.0%; P S 0.01) (Table 7). This difference likely was due to the use of a greater
proportion of unsound fi'uit by thermal processors knowing that adequate thermal
processing will effectively control potential microbial hazards associated with this lower-
quality fruit.

We observed significant differences in patulin contamination in the three years of
sampling apple cider (Table 7). We hypothesize that differences in patulin contamination
could be due to a late frost and poor apple production in 2002 and more puncture wounds
in apples due to hail storms in 2004, thereby limiting fi'uit availability and perhaps
diverting more unsound fruit into juice production in 2002 and 2004. The noticeably
lower patulin incidence observed in 2003 samples might be explained by a considerably
greater availability of fresh apples that year according to the Michigan Agricultural
Statistics (2005-2006). Michigan apple production in 2003 (930 million pounds) was
significantly greater than in 2002 (520 million pounds) and 2004 (730 million pounds).
Apple processing was also higher in 2003 (580 million pounds) than in 2002 (365 million
pounds) and 2004 (490 million pounds). The patulin incidence in 2002 may be slightly
lower than 2004 due to fewer apples being held in cold and controlled atmosphere storage
during that year (134,627,000 pounds vs. 223,445,000 pounds). Collectively, this
information leads us to hypothesize that processors may compensate for lower apple
production during a year by culling and trimming less, or by simply using whatever fruit

is available.

72

2.5.2 Retail Samples

A considerably larger percentage (11.3%) of apple juice samples obtained at retail
grocery stores in 2005-2006 contained patulin concentrations above the legal limit than
the cider mill samples collected in 2002-2004. This observation suggests that processors
supplying juice year-round to retail chains have greater problems with patulin
contamination than the small cider mills. This could be due to longer storage times for
fruit before juice manufacture and also due to inadequate controls of patulin
concentrations in apple juice concentrates used for juice production by larger processors.

Shelf-stable samples showed a tendency to have a greater frequency of patulin
contamination when compared to refrigerated samples (Table 8). This difference may be
due to faster rotation of refrigerated products, and because they tend to be from more
localized sources.

Juices made from apples were more likely to have detectable patulin (P = 0.0003)
than juices made from concentrate (Table 8). Juices made using both apples and
concentrate tended to be more likely to contain detectable patulin (P = 0.0553) than juices
made from concentrate alone. Furthermore, juices made from apples were more likely (P
= 0.0440) to have samples with patulin levels above 50 jig/L when compared to those
manufactured from concentrates. However, juices manufactured using concentrates
contained exceptionally high levels of patulin contamination on several occasions,
indicating that some apple juice concentrate suppliers are using fi'uit of very low quality.

There was a tendency (P=0.08) for organic juices to have a greater proportion
containing detectable patulin in comparison to conventionally produced juices (Table 8).

Organic juices are considered more wholesome by the average consumer, but our results

73

(based on a relatively small number of observations) indicate that organic apple juices
might be more likely to contain patulin when compared to their conventional
counterparts. This is not particularly surprising since it has been found that some
insecticides reduce and even inhibit patulin production (Draughon and Ayres 1979).
2.2% and 11.3% of randomly obtained apple juice samples from Michigan cider
mills and retail establishments, respectively, contained >50 pg patulin/liter. This is an
excessive proportion of adulterated apple juice in the marketplace two years after juice
HACCP implementation. Juice processors must be encouraged to use appropriate apple
storage and culling practices. It is hoped that these surveillance data will encourage
processors to take further steps to reduce the likelihood of patulin contamination in juice
products. We hope that increased awareness of patulin as a hazard in apples and

processed apple products will enable the industry to take preventative actions which will

result in decreased risk of product recalls and adverse publicity.

74

 

 

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food problems. Advances in chemistry series 149, ed. By JV Rodricks. Washington, DC,
American Chemical Society, 90-109.

 

 

77

 

3 Ethylene production by Penicillium expansum and its endogenous and
exogenous effects on patulin production in inoculated solid and liquid media

3. 1 Abstract

Patulin is produced by mold species such as Penicillium expansum and is the most
abundant mycotoxin in apples and apple juice. Production of patulin in infected apples
can be reduced by modified atmosphere fruit storage conditions. The objectives of this
research were to determine the production of ethylene and patulin by P. expansum and
the influence of exogenous ethylene and the ethylene action inhibitor, l-MCP, on patulin
production by P. expansum when cultured on liquid or solid media. In experiment 1,
spores of P. expansum ATCC 28883 and/or ATCC 1117 were cultured in tubes
containing PDA or PDB. P. expansum ATCC 28 883 produced much larger
concentrations of patulin and ethylene than P. expansum ATCC 1117. Timing of patulin
and ethylene production by P. expansum was inversely related for both strains. The
second experiment was conducted on plates containing PDA or PDB and treating P.
expansum ATCC 28883 in a flow through system with combinations of ethylene and 1-
MCP using a factorial design. Patulin production in Petri plates containing PDA
inoculated with P. expansum ATCC 28883 were significantly increased by the presence
of exogenous ethylene (P = 0.0206). There was no overall l-MCP effect (P = 0.72) on
patulin production. These results confirm that P. expansum produces ethylene and

suggests an inverse relationship between endogenous ethylene and patulin production.

78

 

3.2 Introduction

Patulin, a secondary fungal metabolite produced by several species of Penicillium,
Aspergillus, and Byssochlamys fungi, is the most common mycotoxin found in apples and
apple juices (Wilson 1974, Lindroth 1980, Martins 2002). Penicillium expansum is the
primary source of patulin in fruit (Askar 1999, Jackson et al. 2003). Patulin causes
gastrointestinal toxicity in rodents and also has mutagenic, neurotoxic, genotoxic and
carcinogenic effects in rodents. Furthermore, patulin causes dermal and gastric irritation
in humans and also suppresses the immune system of rodents, producing ulceration,
congestion, and hemorrhagic lesions, especially in the gastrointestinal tract (Dickens and
Jones 1961, Moodley 2002, McKinley et al. 1980ab, Martins et al. 2002). Based upon
this toxicity profile, the US Food and Drug Administration (FDA) established an action
level for patulin at 50 jig/kg in apple juice and in beverages containing apple juice, apple
juice concentrate (at single strength) and apple sauce (FDA 2001). In recent years,
patulin surveillance in apples and apple juices by regulatory authorities in the US. and
other countries has resulted in a significant number of product recalls due to patulin
concentrations in excess of regulatory limits. Thus, patulin contamination of food
products has significant economic implications for food processors in addition to its
detrimental effects on public health.

It is well established that patulin is formed predominantly by P. expansum in apples
after harvest and that patulin is commonly present in juice or cider produced from these
moldy apples. Several strains of P. expansum have been reported to produce ethylene in
non-peer-reviewed reports (Taiz and Zeiger 2002). However, the relationship between

ethylene production and onset of toxin by P. expansum production has not been

79

 

 

examined. Suppression of ethylene production by Aspergillus parasiticus and
Aspergillusflavus is strongly associated with the onset of aflatoxin production by these
fungi (Sharma et al. 1985). Aflatoxin production by Aspergillus parasiticus also was
found to be significantly inhibited by exposure to 100 pI/L exogenous ethylene (Roze et
a1. 2004). Although the biosynthesis pathways of patulin and aflatoxin by these fungi
differ, they are both polyketides, and the relationship between the presence of ethylene
and toxin production may be similar.

The overall objective of this research was to investigate possible associations
between ethylene and patulin production by P. expansum. A series of experiments was
conducted to answer three main questions concerning the potential relationship between
ethylene and patulin: 1) do P. expansum strains ATCC 1117 and ATCC 28883 produce
ethylene, and if so, how much? 2) when is patulin produced by P. expansum relative to
the presence of endogenous and exogenous ethylene when grown on solid media? and 3)
what is the influence of endogenous and exogenous ethylene on patulin production by P.

expansum when cultured in liquid media?

3.3 Materials and Methods

3.3.1 Preparation of Commercial Mold Cultures

Pure cultures of Penicillium expansum ATCC 28883 and ATCC 1117 were
obtained as freeze-dried cultures from the American Type Culture Collection (ATCC;
Manassas, VA). After each capsule was aseptically opened, the freeze-dried cultures
were rehydrated according to ATCC instructions. In brief, one-half ml of sterile water

was added to each of two test tubes and the contents of each capsule were added to each

80

tube using a sterile pipet tip. The contents were thoroughly mixed and another 5 m1 of
sterile water was added to each test tube and mixed. These mixtures were allowed to
incubate overnight at room temperature. The next day, the mixtures were transferred to
Petri dishes containing Potato Dextrose Agar (PDA) (Becton, Dickinson and Company,
Sparks, MD). Three plates for each strain were center inoculated and allowed to incubate
in the dark at 30°C for two to three weeks. The spores were loosened by adding 2-3 ml of
sterile water (repeated three times) and by brushing the mycelia with a sterile, bent glass
rod. Spores of P. expansum 28883 and P. expansum 1117, (2.09x107 CFU/ml spore
suspension and 2.69x107 CFU/ml spore suspension, respectively) were collected in a 15-
ml centrifuge tube and counted using a hemacytometer. The spore samples were stored
at —80°C in 25% glycerol. Prior to use, spores of P. expansum 28883 and P. expansum
1117 were thawed at room temperature in a water bath and immediately transferred to the

appropriate growth medium and incubated at 30°C.

3.3.2 Experiment 1: Ethylene and Patulin Production by P. expansum ATCC 1117
and ATCC 28883

To facilitate headspace gas sampling in the slant tubes used for this experiment,
holes were drilled into the caps of SO-ml centrifuge tubes and Alltech, F -145 septum
plugs were inserted. Twenty-five ml of horizontally aligned PDA or PDB was added to
each tube. Spores of P. expansum ATCC 28883 and ATCC 1117 were added to 0.7%
agar or additional broth to achieve a 105 CFU/ml spore suspension. The agar or broth in
each SO-ml centrifiige tube was top inoculated with 4 ml of this mixture. Tubes were
incubated at room temperature in darkness with the caps loosely threaded to allow gas

exchange. Measurements of headspace gas for ethylene concentrations were conducted

81

every 20 to 28 hours for 10 days by gas chromatography. In order to measure headspace
gas, tube caps were closed tight for 10-15 minutes to allow ethylene to accumulate.
Headspace gas of each tube was then sampled by inserting a 26-gauge needle (attached to
a 1 mL syringe) through the septum to obtain a 1 ml sample of headspace gas for
ethylene measurement by gas chromatography (GC). Ethylene was determined in each
tube in triplicate. This experiment was conducted to determine if P. expansum ATCC
1117 and ATCC 28883 produce ethylene when grown on solid PDA (1 .5% agar), semi-
solid PDA (0.7% agar), or liquid PDB (0% agar) media and, if so, to develop an

approximate timeline for the ethylene synthesis by these fiingi.

3.3.2.1 Extraction of Patulin from Media

The PDA samples were analyzed for patulin concentration by a combination of
methods described by MacDonald, et a1. (2000), Eisle and Gibson (2003), and Roze et al.
(2004). Triplicate samples were obtained immediately afier inoculation (day 0), daily on
days 1-10, and finally on day 30 after inoculation and used for patulin quantification.
First, the agar from each tube (including fungal mycelia) was cut into small pieces with a
sterile knife and transferred to.a 100-ml Erlenmeyer flask (Roze et al. 2005). Thrice, 25
milliliters of ethyl acetate were added to each flask and shaken for 1 minute (Roze et al.
2004). This mixture was allowed to sit for 8 to 12 hours and, again, shaken for 1 minute.
Each time, the solvent was quickly removed using a Pasteur pipette and combined in a
glass test tube (MacDonald, et al. 2000 and Roze et a1. 2004). This solvent was
evaporated to dryness under a gentle stream of nitrogen and reconstituted in 30 ml of pH

4 water (Prieta et al. 1993 and Rovira et al. 1993). Sample clean-up was conducted using

82

the solid phase extraction technique described by Eisle and Gibson (2003) and HPLC
analysis was conducted.

Triplicate samples of each fungal strain grown in PDB also were analyzed for
patulin concentrations immediately after inoculation (day 0), daily through day 10, and
on day 25. First, the broth and mycelia from each tube was filtered through a Gooch
crucible to collect the mycelia. The mycelia remaining in each Gooch crucible was thrice
extracted with 25 milliliters of ethyl acetate added to the tube and shaken for 1 minute
(Roze et al. 2004). This mixture was allowed to sit for 8 to 12 hours and, again, shaken
for 1 minute. Each time, the solvent was quickly removed using a Pasteur pipette and
combined in a glass container (MacDonald, et al. 2000 and Roze et al. 2005). This
solvent was evaporated to dryness under a gentle stream of nitrogen and reconstituted in
30 ml of pH 4 water (Prieta et a1. 1993 and Rovira et al. 1993). Samples were then

subjected to solid-phase extraction of patulin followed by HPLC analysis.

3.4 Gas chromatography (GC) Quantification of Ethylene

Gas chromatography (GC) was performed using a 400 Series model gas
chromatograph (Carle AGC, HACH Carle Chromatography, Loveland, CO). Ethylene
concentrations in samples were calculated by comparison to known ethylene standards.
These readings were performed in triplicate by taking l-ml samples through septums

placed in the cap of each tube using a 1-ml syringe.

3.4.1 Experiment 2: Patulin production in relationship to the action of endogenous
and exogenous ethylene on solid and liquid* media plates

This experiment was conducted to determine the influence of endogenous and

exogenous ethylene on patulin production by P. expansum in solid and liquid media,

83

respectively. The treatment design is presented in Table 10. This experiment was
performed using a flow-through system consisting of eighteen 10-liter dessicators, each
containing 10 Petri plates (100 mm x 20 mm) containing either PDA or PDB and center
inoculated with 1 ml of 105 P. expansum spores. The flow-through system involved
either 100 uL/L ethylene or air (containing no ethylene) being continuously flushed
through the appropriate dessicator. The appropriate plates were treated with 1- MCP, the
one time treatment at day 5, before they were placed in the dessicator (to compare
directly with a concurrent study using apples), and others were continuously flushed with
a 0.3 to 0.4 uL/L concentration of l-MCP to control ethylene action of the fungi as it
grew. The continuous l-MCP treatment was dosed using a l-MCP-releasing chemical
mix [3000 pL/L, made fresh every other day; [stock of SmartFresh, Inc., Rohm and Hoas,
Philadelphia, PA; 6.5 g plus 100 ml of water in a 980-ml jar (Roze et al. 2004)]. 1-MCP
was delivered continuously using a syringe pump (KD Scientific, model 220, New Hope,
PA). Appropriate dessicators contained granulated potassium permanganate (KMnO4)
and a carbon dioxide (C02) scrubber to absorb residual ethylene and residual C02,
respectively. Cultures were grown in complete darkness. Ethylene concentrations in
headspace gas were measured daily and plates were removed every 2 days for 10 days for
patulin assays.

Ethylene measurements of head space gas in each dessicator were conducted daily
by gas chromatography and the plates were analyzed for patulin presence using sample
preparation techniques described by Prieta et al. 1993, Rovira, et al. (1993), MacDonald,
et a1. (2000), Eisle and Gibson (2003), and Roze et al. (2004). Patulin was extracted from

the Petri plates containing potato dextrose broth using procedures similar to those

84

described for experiment 1. The mycelia] diameter of fungal colonies on each plate was
measured in centimeters using a ruler and recorded. The tubes containing solid media
were heated for 30-45 minutes in a 90°C water bath. These samples were then filtered
through Gooch crucibles to harvest mycelia. The mycelia and Gooch crucibles were
dried in a 70°C oven for 12 hours, cooled in a dessicator, and weighed. They were then
placed in the oven for two, subsequent, 30 minute periods, cooled, and weighed again.

All three weights were recorded and averaged.

Table 10. Experiment 2 Treatment Design

 

 

 

 

 

 

 

 

Treatment Inoculation Ethylene l-MCP (1- l-MCP Replicates
time (Continuous)
injection)
1 + + + - 3
2 + + - - 3
3 + - + - 3
4 + - - - 3
5 + + + + 3
6 + + + 3

 

 

 

 

 

 

 

3.4.2 Solid-Phase Extraction

Solid-phase extraction (SPE) of patulin from apple juice was carried out using a
syringe-cartridge method (Eisele and Gibson 2003). The SPE cartridge (Waters Oasis®
HLB 60 mg, Milford, MA) was activated by applying 2-3 ml of water, followed by 2 m1
of methanol and a final wash of 2-3 ml of water. Each sample (2.5 mL volume) was then
applied to cartridges under vacuum. The cartridges were rinsed with 2.0 m1 of 1.0%
sodium bicarbonate solution (pH=8.3) followed by 2 ml of 1% acetic acid solution. One
milliliter of 10% ethyl acetate in ethyl ether solution was then added to each cartridge to

elute patulin. The eluates were collected in 15x45 mm vials, evaporated to dryness under

85

a stream of nitrogen and then reconstituted in 0.5 ml of 0.1% acetic acid. The vials were

capped, vigorously mixed, and fi'ozen for future analysis by HPLC.

3.4.3 High Performance Liquid Chromatography (HPLC)

Patulin was quantified using a Waters Corporation (Milford, MA) high
performance liquid chromatograph (HPLC). The Waters system included two model 510
pumps, a model 717-plus auto-sampler, and a Photodiode Array detector (model 996,
Milford, MA). An Alltech C13, 5pm column (4.6 x 250 mm) was used to separate
compounds. Each sample (50 11L injection volume) was subjected to isocratic elution for
30 minutes with 10% aqueous acetonitrile (ACN), followed by a 30 minute clean with
70% of 100% ACN and 30% of 10% ACN. The photodiode array detector collected the
UV and visible light spectra from 190 to 800 nm to allow comparison of sample UV
spectra to that of patulin standards. Chromatograms were analyzed using Waters’
Millenium software. Patulin concentration was quantified by comparing UV absorbance

at 27 6 nm to known patulin standards.

3.4.4 Statistical Analyses

For statistical analyses, SAS Version 9.1 was used (SAS Institute Inc., Cary, NC).
In Experiment 1 (Tables 11-14), means and standard deviations were conducted for
ethylene and patulin production by fungi grown on different media, but no statistical
comparisons among media types or fimgal strains were conducted due to individual sub-
experiments being completed at different times.
In Experiment 2, data on logo patulin concentrations were analyzed using PROC

MIXED. Independent variables used in the model were the effects of ethylene

86

(exogenous or endogenous only), l-MCP (none, once, continuous), the ethylene by 1-
MCP interaction, time, and interactions between time with ethylene and l-MCP.
Experimental vessel (dessicator) was used as a repeated variable. An unstructured
covariance procedure was used in the MIXED procedure. We evaluated the impact of
ethylene and l-MCP on the concentrations of patulin by comparing the appropriate
means using two-sided t-tests in PROC MIXED in SAS. The value of l was assumed for
samples having undetectable patulin (<4 ug/L patulin before log transformations to
prevent undefined logIo values. Mycelia weights and mycelia diameters for Experiment 2

were analyzed using PROC MIXED and an autoregressive covariance matrix.

3.5 Results

3.5.1 Experiment 1: Production of Ethylene and by P. expansum 1117 and ATCC
28883 when Grown on Solid, Semi-Solid, and Liquid Media

3.5.1.1 P. expansum 28883 on Solid Media

Data on ethylene and patulin productions by P. expansum 28883 grown on PDA
(solid media) are presented in Table 11 and Figure 1. Patulin was first detectable 2 days
after inoculation and approached its plateau by day 4. Ethylene production was zero or
minimal until day 4, and reached peak production (77.5 $18.5 uL/L) by day 6. Ethylene
production was maintained at high levels through day 10 and still was being produced in

significant concentrations on day 30 following inoculation.

87

Table 11. Patulin and Ethylene Production by P. expansum 28883 grown on Solid
PDA Media

 

Figure l. Patulin and Ethylene Production by P. expansum 28883 When Grown on
Solid Media

 

 

 

 

 

 

 

 

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I —I—Dally Ethylene Average —0— Log 10 Daily Patulin Average

 

88

3.5.1.2 P. expansum 28883 on Semi-Solid Media

Data on ethylene and patulin productions by P. expansum 28883 grown on PDA
(0.7% agar; semi-solid media) are presented in Table 12 and Figure 2. Since patulin was
detectable immediately following inoculation, those samples on days 0 and 1 were treated
as false positives, since no patulin should have been present at day 0 and at all times the
patulin levels in semi-solid PDA was higher than that in solid PDA. As was observed
when this strain was cultured on solid PDA, patulin production occurred before ethylene
and peaked at day 4 (Figure 2). Ethylene production peaked at day 9. Relative patulin
concentrations recovered from P. expansum 28883 grown on semi-solid PDA were
slightly greater than for solid PDA. However, this appearance of increased recovery
could be due to increased efficiency of patulin recovery from semi-solid versus solid

PDA or due to an increase recovery of byproducts that coelute with patulin.

89

Table 12. Patulin and Ethylene Production by P. expansum ATCC 28883 When
Grown on PDA Semi-Solid Media

 

Figure 2. Patulin and Ethylene Production by P. expansum ATCC 28883 When
Grown on Semi-Solid PDA Media

I, 300 6

 

 

 

 

 

 

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—l—Dally Ethylene Average —0— Log 10 Daily Patulin Average

Log 10 Patulin (ppb)

 

 

90

3.5.1.3 P. expansum 28883 on Liquid Media

Data on ethylene and patulin productions by P. expansum ATCC 28883 grown on
PDB (liquid media) are presented in Table 13 and Figure 3. Patulin was first detectable 4
days afier inoculation and approached its plateau by day 5. Ethylene production was zero
or minimal until day 6, and reached a peak production (14.8 $12.04 uL/L) by day 8.
Production of both patulin and ethylene by P. expansum ATCC 28883 in liquid media
were very attenuated relative to their productions when this organism was cultured on

solid or semi-solid PDA.

Table 13. Patulin and Ethylene Production by P. expansum ATCC 28883 When
Grown in Liquid PDB Media

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Daily Patulin Loglo Daily Patulin Daily Ethylene
Day AveragteL/L) Average (pL/L) Average (pL/L)
0 < 4 < 4 0.055 $0.0020
1 < 4 < 4 0.042 $0.0039
2 < 4 < 4 0.065 $1.32
3 < 4 < 4 0.060 $0.006]
4 539 $228 2.70 $0.22 0.053 $0.013
5 879 $741 2.80 $0.48 0.069 $0.016
6 118 $167 1.38 $1.27 1.50$l.61
7 45 $71.2 1.00 $1.06 1.09 $0.51
8 321 $522 1.52 $1.48 14.8 $012.04
9 751 $1202 1.80 $1.68 1.62 $0.77
10 243 $302 1.95 $0.92 5.75 $9.04
25 2573 $4018 2.19 $1.98 0.57 $0.41

 

 

 

91

 

Figure 3. Patulin and Ethylene Production by P. expansum ATCC 28883 When
Grown in Liquid PDB Media

 

Log 10 Patulin Production vs. Ethylene Production of
P. expansum 28883 in Liquid Media

 

 

 

 

 

 

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3.5.1.4 Patulin and Ethylene Production by P. expansum 1117 on Solid PDA Media
Data on ethylene and patulin production by P. expansum ATCC 28883 grown on
PDB (liquid media) are presented in Table 14 and Figure 4. Patulin was first detectable 6
days after inoculation and reached its plateau by day 9. Ethylene production was zero or
minimal until day 4, and reached a peak production (1.21 $0.20 uL/L) by day 5. Unlike
the results observed with P. expansum 28883, in this strain ethylene production occurred
before patulin was detectable (Figure 4). Production of both ethylene and patulin by P.
expansum ATCC 1117 were about 2 logs lower than were observed when P. expansum

28883 was grown on the same media.

92

Table 14. Patulin and Ethylene Production by P. expansum ATCC 1117 When
Grown on Solid PDA Media

 

Figure 4. Patulin and Ethylene Production by P. expansum ATCC 1117 When
Grown on Solid PDA Media

1.6

1.4

0.8 V" i

0.6

Ethylene (ppm)

Log10 Patulln (ppb)

 

 

 

 

 

-0.2

 

 

 

 

‘ (+03in Ethylene Average — + — Log 10 Daily Patulin Axeragel

93

3.5.2 Experiment 2: Patulin produced in relationship to the presence of
endogenous and exogenous ethylene on solid and liquid* media

3.5.2.1 Solid Media

Since P. expansum 28883 was the strain to consistently produce high levels of
patulin and ethylene in Experiment 1, it was the only strain used in Experiment 2. The
effects of exogenous ethylene and l-MCP treatment of patulin production by P.
expansum when cultured on solid PDA media are presented in Table 15. Since no
interaction between ethylene and l-MCP on patulin production was detected, main effect
least square means are presented. Averaged across all days and treatment levels, there
was a significant (P = 0.02) effect of ethylene treatment on patulin production by P.
expansum 28883 when grown on solid PDA media (Table 15). Plates exposed to 100
uL/L exogenous ethylene had significantly greater patulin production when compared to
plates that were not exposed to exogenous ethylene. Treatment with l-MCP, whether
single exposure or continuous administration, did not influence patulin production by P.
expansum when grown on solid PDA media.

Mycelia of P. expansum 28883 were collected by filtration of media through
filtered glass crucibles and the mean mycelia weights on days 2, 6, and 10 following
inoculation are presented in Table 16. As expected, mycelia] weights increased with time
after plate inoculation. Mycelial weights were not influenced by treatment with ethylene
or l-MCP (data not shown).

Mycelial colony areas on plates were measured on days 2, 4, 6, and 8 after

inoculation and overall means averaged across treatments are presented in Figure 5.

94

Mycelia areas increased significantly (P < 0.0001) with time, but were not significantly
influenced by ethylene or l—MCP treatment (data not shown). Mycelia area was highly
correlated with patulin production by P. expansum 28883 when grown on solid PDA

media (r = 0.80; P < 0.0001)

95

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Figure 5. Mycelia Areas (cmz) in Solid Media Inoculated with P. expansum 28883
Treated with Ethylene and l-MCP — Least Squares Means Averaged Across All
Treatments

 

 

 

18- O

Diam eter Area (cm A:2)

 

 

 

0 1 2 3 4 5 6 7 8 9
oArea (cm"2) Day

 

 

 

 

 

 

 

3.5.2.2 Liquid Media

When this experiment was conducted using PDB, too many plates contained
undetectable patulin to allow for reliable statistical analysis. Therefore, these results are
tabulated in the appendix to this dissertation (Appendix B), but are not discussed in this

chapter.

3. 6 Discussion

To improve safety of j uice beverage products, in 2001, the US. FDA promulgated
juice HACCP regulations to be implemented in all juice processing plants by January
2004 (FDA 2001). Also in 2001, FDA implemented additional regulations by limiting
patulin concentrations in apple juice and cider, apple juice concentrate (at single

strength), and apple sauce to 50 ng/L (FDA 2001). Thus, regulators are closely

97

scrutinizing patulin as a hazard of concern in apple juice products. The juice processing
industry needs practical, useful methods to prevent or minimize the occurrence of patulin.
We hypothesized that ethylene regulation/manipulation could be a practical and
economical solution to controlling patulin production in stored apples and, subsequently,
in apple cider and juice produced from stored apples. Since patulin production generally
occurs while apples are being stored, prevention in storage would likely be most
beneficial to the juice and cider industry. Ethylene is a natural, gaseous plant hormone
that has been found to inhibit aflatoxin biosynthesis in media and raw peanuts (Roze et al.
2004). Several strains of P. expansum have been reported, in non-peer-reviewed
publications, to naturally produce large amounts of ethylene (Taiz and Zeiger 2002). It is
not known how these ethylene levels related to the production of patulin by the mold.
We conducted these experiments to determine if ethylene regulated patulin production by
P. expansum. We also wanted to compare the effects of exogenous and endogenous

ethylene on patulin production.

3.6.1 Experiment 1: Production of Ethylene by P. expansum ATCC 28883 on Solid,
Semi-Solid, and Liquid Media and P. expansum ATCC 1117 on Solid Media

While it appears to be common knowledge that several species of Penicillium
produce large amounts of ethylene (Taiz and Zeiger 2002), no studies were found that
quantified these concentrations. When P. expansum ATCC 28883 was plated on solid,
semi-solid, and liquid media, patulin production preceded ethylene production by the
mold. Regardless of the media used, patulin consistently peaked at day 4, typically at
least two days before ethylene production peaked. In fact, patulin production had

declined somewhat during the times when ethylene production peaked and began to

98

decline. Patulin and ethylene concentrations when P. expansum ATCC 28883 was grown
in liquid PDB media were much lower than those observed in solid and semi-solid media,
which indicates that patulin production is significantly affected by host matrix.
Conversely, when P. expansum ATCC 1117 was cultured on solid PDA media,
ethylene production preceded patulin production. Furthermore, P. expansum ATCC 1117
produced significantly less patulin and ethylene than P. expansum ATCC 28883,
suggesting that considerable strain differences exist. Although this experiment proved
that P. expansum is capable of producing large amounts of ethylene (albeit in a strain
dependent manner) it did not give us a clear indication as to how patulin production is
affected by endogenous ethylene. We observed in both strains of P. expansum that
maximal production of ethylene and patulin occurred at different times. However, no
clear hypothesis could be drawn about the influence of endogenous ethylene production

on patulin synthesis.

3.6.2 Experiment 2: Patulin produced in relationship to the presence of
endogenous and exogenous ethylene on solid media

Although there was no overall day effect on patulin production in liquid media, there
appeared to be a tendency (P = 0.06) for exogenous ethylene exposure to accelerate
patulin production by P. expansum 28883. Unlike experiments conducted with aflatoxin,
ethylene did not inhibit or reduce patulin production in solid media inoculated with P.
expansum (Sharma et al. 1985, Roze et al. 2004). In fact, patulin concentrations in Petri
plates containing solid media inoculated with P. expansum ATCC 28883 were
significantly greater in the presence of exogenous ethylene (P = 0.0206). We expected 1-

MCP to promote patulin production (by blocking ethylene) in media in a manner similar

99

to that which occurred with aflatoxin studies (Roze et al. 2004). However, in this study
we found no overall l-MCP effect (P = 0.72) on patulin production nor on mycelia
weight or mycelia area. It is possible that this lack of effect could be explained by the
absorption of the l-MCP by the KMnO4 added to the containers to absorb ethylene, as it
absorbs l-MCP as well as ethylene.

Plates exposed to exogenous ethylene had high patulin concentrations at all times,
which may be the main reason why no overall day effect was detected. Our finding that
ethylene enhanced patulin concentrations was completely unexpected since exposure to
exogenous ethylene in media and raw peanuts inhibited and/or reduced aflatoxin
production by Aspergillus parasiticus (Roze et al. 2004, Sharma et al. 1985). This
suggests that there is, in fact, a relationship between patulin production and the presence
of ethylene.

Two differing conclusions could be drawn based on these results, 1) ethylene and
patulin production by P. expansum might be unrelated or 2) ethylene promotes patulin
biosynthesis by P. expansum. Since ethylene is a natural promoter of fruit softening and
ripening, exposure to exogenous ethylene may simply increase fungal growth by
facilitating mycelia penetration and, hence, indirectly increase patulin production.
However, we consider the second explanation to be more likely. Although endogenous
ethylene appears to be inversely related to patulin production in P. expansum ATCC
28883 and 1117, exogenous ethylene marginally promotes patulin production by P.
expansum in solid media, despite the presence of continuous l-MCP. Further research on

potential mechanistic relationships between patulin and ethylene are warranted and it

100

would be useful to study the influence of l-MCP and exogenous ethylene on patulin

production in inoculated apples.

101

3. 7 References

Askar A. 1999. Patulin in apple juice and children’s apple food. I. Toxicological and
legal aspects. Fruit Processing, 9(3): 74-77.

Dickens F and Jones HEH. 1961. Carcinogenic activity of a series of reactive lactones
and related substances. British Journal of Cancer, 15: 85-100.

Doores S. 1983—1984. The Microbiology of Apples and Apple Products. Critical Reviews
in Food Science and Nutrition, 19(2): 133-149.

Eisele, TA and Gibson MZ. 2003. Syringe-Cartridge Solid-Phase Extraction Method for
Patulin in Apple Juice. Journal of the Association of Official Analytical Chemists,
86(6):1160-1163.

Jackson LS, Beacham-Bowden T, Keller UW, and Adhikari C. 2003. Apple quality,
storage, and washing treatments affect patulin levels in apple cider. Journal of Food
Protection, 66(4): 618-624.

Johnston JW, Hewett EW, Maarten LAT, Hertog M, and Harker FR. 2002. Temperature
and ethylene affect induction of rapid softening in ‘Granny Smith’ and ‘Pacific RoseTM
apple cultivars. Postharvest Biology and Technology, 25(3): 257-264.

Lindroth, S. 1980. Occurrence, Formation and Detoxification of Patulin Mycotoxin.
Technical Research Centre of Finland: Materials and Processing Technology. 24: 4-46.

Liu FW and Long-Jun C. 1986. Responses of Daminozide-sprayed ‘McIntosh’ Apples to
Various Concentrations of Oxygen and Ethylene in Simulated CA Storage. Journal of the
American Society for Horticultural Science, 111(3): 400-403.

Lovett J, Thompson Jr RG, and Boutin BK. 1975. Trimming as a means of removing
patulin from fungus-rotted apples. Journal of the Association of Official Analytical
Chemists, 58(5):909-91 1.

MacDonald S, Long M, and Gilbert J. 2000. Liquid chromatographic method for
determination of patulin in clear and cloudy apple juices and apple puree: Collaborative.
Journal of the Association of Official Analytical Chemists, 83(6): 1387-1394.

Martins ML, Gimeno A, Martins HM, and Bernardo F. 2002. Co-occurrence of patulin
and citrinin in Portuguese apples with rotten spots. Food Additives and Contaminants,

19(6): 568-574.

McKinley ER and Carlton WW. 1980a. Patulin mycotoxicosis in the Syrian hamster.
Food Cosmetics Toxicology 18(2): 173-179.

102

McKinley ER and Carlton WW. 1980b. Patulin mycotoxicosis in Swiss ICR mice. Food
Cosmetics Toxicology 18(2): 181-187.

Moodley RS, Govinden R and Odhav B. 2002. The effect of modified atmospheres and
packaging on patulin production in apples. J oumal of Food Protection, 65(5): 867-871.

Prieta J, Moreno MA, Diaz S, Suarez G, and Dominguez L. 1994. Survey of patulin in
apple juice and children’s apple food by the diphasic dialysis membrane procedure. J.
Agric Food Chem, 42(8): 1701-1703.

Rovira R, Ribera F, Sanchis V, and Canela R. 1993. Improvements in the quantitation of
patulin in apple juice by high-performance liquid chromatography. Journal of
Agricultural and Food Chemistry, 41(2): 214-216.

Roze LV, Calvo AM, Gunterus A, Beaudry R, Kall M, and Linz J E. 2004. Ethylene
modulates development and toxin biosynthesis in Aspergillus possibly via an ethylene
sensor-mediated signaling pathway. Journal of Food Protection, 67(3): 43 8-447.

Saf’tner RA, Abbot J A, Conway WS, Barden CL, and Vinyard, BT. 2002. Instrumental
and Sensory Quality Characteristics of ‘Gala’ Apples in Response to Prestorage Heat,
Controlled Atmosphere, and Air Storage. J oumal of the American Society of
Horticultural Science, 127(6): 1006-1012.

Sharma A, Padwal-Desai SR, and Nadkarni, GB. 1985. Possible implication of
reciprocity between ethylene and aflatoxin biogenesis in Aspergillusflavus and
Aspergillus parasiticus. Applied Environmental Microbiology 49(1): 79-82.

Taiz L and Zeiger E. 2002. Plant Physiology. Sinauer Associates, Sunderland.

United States Food and Drug Administration (FDA). 2001. 21 CFR Part 120. Hazard
analysis and critical control point (HACCP); procedures for the safe and

sanitaryprocessing and importing of juice; final rule. US Federal Register, 66(13): 6138-
6202.

Wilson DM. 1974. Patulin and penicillic acid. In Mycotoxins and other fungal related

food problems. Advances in chemistry series 149, ed. By JV Rodricks. Washington, DC,
American Chemical Society, 90-109.

103

4 Effect of fruit-produced ethylene and exogenous ethylene (100 pL/L) on the
production of patulin in apples treated with l-MCP and inoculated with
Penicillium expansum

4.1 Abstract

Patulin is produced by molds including Penicillium expansum and is the most
abundant mycotoxin in apples and apple juice. Production of patulin can be reduced
under modified atmospheric storage conditions. Recent research shows that ethylene
reduces aflatoxin production. The objective of this study was to determine whether fruit-
produced ethylene or exogenous ethylene (100 pL/L) reduces the production of patulin in
apples treated with 1-Methylcyclopropene (l-MCP), an ethylene receptor blocker, and
inoculated with Penicillium expansum. Two studies were conducted to meet this
objective. Both used freshly picked Red Delicious apples that were treated with l-MCP,
ReTain (an ethylene biosynthesis inhibitor), and then inoculated with P. expansum. The
second experiment was expanded by the addition of sample vessels containing
uninoculated apples to control for ethylene potentially produced by P. expansum. The
apples were randomly assigned to lO-liter dessicators and then stored at ambient
temperature either under 100 uL/L ethylene or 100% air. Apples from each container
were obtained every two days after treatment and pressed to apple juice. Patulin in apple
juice was quantified using solid phase extraction followed by high performance liquid
chromatography. In the first experiment, concentrations of patulin in apple juice of P.
expansum-inoculated apples increased exponentially from undetectable (< 4 jig/L)
concentrations at day 0 to 3.44$0.10 rig/L after 16 days of storage, respectively (P S

0.0012). Ethylene treatment did not influence patulin concentrations in juice prepared

104

from apples in this experiment. In the second experiment juice from uninoculated fi'uit
contained no detectable patulin. Concentrations of patulin in apple juice of P. expansum
inoculated apples increased exponentially from undetectable (< 4 ug/L) concentrations at
day 0 to 3.16$0.10 pg/L after 16 days of storage, respectively (P 5 0.0182). Averaged
across all time points, exposure of apples to 100 uL/L ethylene tended (P S 0.065) to
increase the production of patulin in P. expansum inoculated apples. Neither ethylene
nor 1-MCP and ReTain treatment affected patulin accumulation. We conclude that,
contrary to our original hypothesis, exogenous ethylene treatment does not inhibit (and

may actually promote) patulin synthesis by P. expansum.

4.2 Introduction

Patulin, a secondary fungal metabolite produced by several species of Penicillium,
Aspergillus, and Byssochlamys, is the most common mycotoxin found in apples and apple
juices (Wilson 1974, Lindroth 1980, Martins 2002). Penicillium expansum, which is
commonly found in soil, is the primary source of patulin in fi'uit (Askar 1999, Jackson et
al. 2003). Based upon its proven toxicity to animals and humans, an action level of 50
jig/kg was established by the US. FDA for patulin in apple juice, apple juice concentrate
(at single strength), and apple sauce (FDA 2001).

Ethylene (C2H4) is a natural, gaseous compound that functions a plant hormone
(Zeringue et al. 1990, Abeles et al. 1992, Savaldi-Goldstein and Fluhr 2000). The effects
of ethylene on fruit ripening are well-characterized. Treatment causes lower ethylene
emission by fruit and reduced incidence of core browning (Forsyth et al. 1969).

However, in recent years it has become apparent that ethylene also functions as a

105

regulatory agent in the formation of secondary metabolites by certain species of fungi
(Sharma et a1. 1985).

For example, ethylene exposure has been shown to accelerate the softening of
apples that can lead to rotting (Johnston et al. 2002). Preclimacteric McIntosh apples
were studied under low (~6 ppm) and high (~1570 ppm) ethylene levels at 33°C for 189
days (Forsyth et al. 1969). Apples stored under low ethylene concentrations had higher
firmness compared to apples stored at high ethylene levels, and this higher firmness
continued for more than 7 days at room temperature (Forsyth et al. 1969). However, pH
was lowered and soluble solids content was slightly increased in apples stored at low
ethylene concentrations (Forsyth et al. 1969).

Aspergillus parasiticus and Aspergillus nidulans produce ethylene during the early
growth phase on media, and the onset of aflatoxin biosynthesis is marked by the absence
of ethylene evolution (Sharma et al. 1985). Shanna et al. ( 1985) demonstrated that
ethylene may inhibit Aspergillus development and toxin biosynthesis by adding 2-
chloroethyl phosphonic acid (CEPA), an ethylene-generating compound, directly to
growth medium. These experiments demonstrate a tight relationship between ethylene
presence and aflatoxin suppression in A. parasiticus, as well as between ethylene
concentration and stimulation or inhibition of growth (Sharma et al. 1985). It is thought
that the mechanism whereby ethylene reduces aflatoxin biosynthesis could be a
receptor/sensor-mediated signaling pathway since l-MCP, which has a structure similar
to ethylene and blocks the ethylene receptors of higher plants, also promotes aflatoxin
production by Aspergillus (Sharma et al. 1985, Serek et a1. 1995, Roze 2004).

Exogenous ethylene treatment prevents aflatoxin accumulation and sexual development

106

in A. parasiticus and A. nidulans, respectively (Roze 2004). The overall objective of this
research was to determine the impact of ethylene exposure on patulin development by P.
expansum inoculated on apples. We have previously demonstrated that patulin and
endogenous ethylene production by P. expansum is inversely related, but that exogenous
administration of ethylene to P. expansum 28883 stimulated patulin production.

The specific aims of this research were 1) to determine the effects of exogenous
ethylene administration on patulin formation by P. expansum on Red Delicous apples,
and 2) to assess the impact of l-MCP on patulin production by P. expansum while

growing on apples.

4.3 Materials and Methods
4.3.] Experiment 1

The first experiment was conducted to determine the effect of exogenous ethylene
treatment on patulin production by P. expansum on apples. Freshly picked Red Delicious
apples (n=108) were treated with the ethylene inhibitor l-Methylcyclopropene (l-MCP)
overnight in three 5-gallon plastic containers. The apples were then dipped in ethanol
and dual—hole punctured, using a wooden block containing two 3 mm metal pins that
were spaced 1.8 cm apart. Before being used to inoculate fruit, the P. expansum used for
this experiment was collected from decaying apples at MSU and propagated by
vigorously mixing one moldy Honeycrisp apple in 200 mL of water. This solution was
fiirther diluted, serially, and plated. Pure ATCC cultures were not used due to time
constraints and the desire to conduct immediate preliminary experiments. After culturing
for 5—7 days, colonies were isolated and individually plated. The Penicillium

classification was determined with the aid of a professional mycologist using a mycology

107

key (Barnett and Hunter 1998). Next, the apples were inoculated with Penicillium
expansum using a 200 pl pipette tip. The apples were then randomly assigned to six
dessicators (sixteen apples/dessicator) and stored at ambient temperature with a normal
atmosphere or normal atmosphere plus 100 uL/L ethylene. The ethylene was produced
by creating a vacuum in a gas cylinder, adding 540 mL of ethylene to the cylinder, and
then adding compressed air to the cylinder until the final ethylene concentration was 100
uL/L. Rolling the tank mixed this combination and ethylene concentration was measured
by gas chromatography. The gas phase of containers was maintained by the constant
flow of gas from either the 100 uL/L ethylene or air tanks at a flow rate of 30 ml/min.
Three dessicators were used for ethylene treatments and the other three dessicators were
used for air treatments. Ethylene concentrations in the dessicators and the room were
measured daily. Two apples fiom each container were randomly selected and removed
after 8, 10, 12, 14, and 16 days of treatment, respectively, and pressed to obtain apple
juice.

After sampling, apples were placed in freezer bags and frozen in a walk-in cooler (-
20°C) until juicing. All equipment and utensils used for juicing were washed with water
and ethanol and allowed to air dry between each sample. The apple samples were
defrosted and cut in half on a cutting board using a sharp knife. The halves were then
ground using a General Electric 4—speed food processor with a 450-Watt motor and then
pressed using a potato ricer lined with cheesecloth into a beaker. The juice was poured
into plastic culture tubes and capped and frozen until patulin was quantified using solid
phase extraction (Eisele and Gibson 2003) followed by high performance liquid

chromatography (MacDonald et al. 2000).

108

4.3.2 Experiment 2

Since preliminary results from Experiment 1 indicated that ethylene levels in the
dessicators were not completely controlled in that study, a second eXperiment was
designed. The treatment design used in this experiment (Table 17) included four
treatments using apples inoculated with Penicillium expansum 28883 using a 2X2
factorial design of treatments having two levels of exogenous ethylene (100 uL/L
ethylene; no ethylene) and two levels of l-MCP and ReTain (treated; untreated). In
addition, two treatments were included to test the effects of l-MCP and ReTain on
patulin and ethylene production of apples that were not inoculated with P. expansum and
not treated with exogenous ethylene. Ethylene was prepared and dosed as described in
Experiment 1. Apples were treated with ReTain (50 g/acre) four weeks before harvest.
l-MCP was also applied to apples as described in Experiment 1, with the exception that
only half of the apples were treated. Inoculation with mold was the same as in
Experiment 1, except that a commercial mold, P. expansum ATCC 28883 American
Type Culture Collection (AT CC) (Manassas, VA), was used. After inoculation, the
apples were then randomly assigned to 18 dessicators (sixteen apples/dessicator). Two
apples from each container were randomly selected and removed after 3, 5, 7, 10, 12, 14,
and 16 days of treatment, respectively, and pressed to obtain apple juice. Ethylene
measurements in exit gas streams and patulin concentrations in juice produced from the
treated apples were reported using gas chromatography and solid phase extraction and

high performance liquid chromatography, respectively.

109

4.3.3 Preparation of Commercial Mold Cultures

A pure strain of Penicillium expansum ATCC 28883 mold culture was obtained as
a freeze-dried culture from the American Type Culture Collection (ATCC; Manassas,
VA). After the capsule was aseptically opened, the freeze-dried culture was rehydrated
according to ATCC instructions. In brief, one-half ml of sterile water was added to each
of two test tubes and the contents of each capsule were added to each tube using a sterile
pipet tip. The contents were thoroughly mixed and another 5 ml of sterile water was
added to each test tube and mixed. These mixtures were allowed to sit overnight in
darkness at room temperature. The next day, the mixtures were transferred to Petri dishes
containing Potato Dextrose Agar (PDA) (Becton, Dickinson and Company, Sparks, MD).
Three plates for each test tube were center inoculated and allowed to incubate at 30°C for
two to three weeks. The spores were loosened by adding 2-3 ml of sterile water (repeated
three times) and by brushing the mycelia with a sterile, bent glass rod. Spores (2.9x107
CFU/ml spore suspension) were collected in a 15-ml centrifuge tube and counted using a
hemacytometer. The spore samples were stored at —80°C in 25% glycerol. Prior to use,
spores were thawed in a room temperature water bath and immediately transferred to the

appropriate growth medium and incubated at 30°C.

4.3.4 Solid-Phase Extraction

Solid-phase extraction (SPE) of patulin from apple juice was carried out using a
syringe-cartridge method (Eisele and Gibson 2003). The SPE cartridge (Waters Oasis®
HLB 60 mg, Milford, MA) was activated by applying 2-3 ml of water, followed by 2 ml
of methanol and a final wash of 2-3 ml of water. Each sample (2.5 mL volume) was then

applied to cartridges under vacuum. The cartridges were rinsed with 2.0 ml of 1.0%

110

sodium bicarbonate solution (pH=8.3) followed by 2 m1 of 1% acetic acid solution. One
milliliter of 10% ethyl acetate in ethyl ether solution was then added to each cartridge to
elute patulin. The eluates were collected in 15x45 mm vials, evaporated to dryness under
a stream of nitrogen and then reconstituted in 0.5 ml of 0.1% acetic acid. The vials were

capped, vigorously mixed, and frozen for future analysis by HPLC.

4.3.5 High Performance Liquid Chromatography (HPLC)

Patulin was quantified using a Waters Corporation (Milford, MA) high
performance liquid chromatograph (HPLC). The Waters system included two model 510
pumps, a model 717-plus auto-sampler, and a model 996 Photodiode Array detector. An
Alltech C13, 5 mm column (4.6 x 250 mm) was used to separate compounds. Each sample
(50 uL injection volume) was subjected to isocratic elution for 30 minutes with 10%
aqueous acetonitrile (ACN), followed by a 30 minute clean with 70% of 100% ACN and
30% of 10% ACN. The photodiode array detector collected the UV and visible light
spectrum from 190 to 800 nm to allow comparison of sample UV spectra to that of
patulin standards. Chromatograms were analyzed using Waters’ Millenium software.
Patulin concentration was quantified by comparing UV absorbance at 276 nm to known

patulin standards.

4.3.6 Gas Chromatography (GC)

Gas chromatography (GC) was performed using a 400 Series model gas
chromatograph (Carle AGC, HACH Carle Chromatography, Loveland, CO). Ethylene

concentrations in samples were calculated by comparison to known ethylene standards.

111

 

These readings were performed in triplicate by taking l-ml samples through septums

placed in the cap of each tube using a 1-ml syringe.

Table 17. Treatment Design Used in Experiment 2.

 

 

 

 

 

 

 

 

Treatment P. expansum 100 pL/L l-MCP + Replicates
Inoculation Ethylene ReTain
1 + + + 3
2 + + - 3
3 + - + 3
4 + - - 3
5 - - + 3
6 - - - 3

 

 

 

 

 

 

4.3.7 Statistical Analyses

In Experiment 1, data on loglo patulin concentrations was analyzed using PROC
MIXED. Independent variables used in the model were the effects of ethylene
(exogenous or endogenous only), time, and interactions between time with ethylene.
Experimental vessel (dessicator) was used as a repeated variable. An unstructured
covariance procedure was used in the MIXED procedure. We evaluated the impact of
ethylene on the concentrations of patulin by comparing the appropriate means using two-
sided t-tests in PROC MIXED in SAS. The value of 1 was assumed for samples having
undetectable patulin (<4 ug/L patulin before log transformations to prevent undefined
logm values.

In Experiment 2, data on loglo patulin concentrations was analyzed using PROC
MIXED. Independent variables used in the model were the effects of ethylene
(exogenous or endogenous only), l-MCP (none, once), inoculation (inoculated, not
inoculated), the ethylene by 1-MCP interaction, time, and interactions between time with
ethylene, inoculation, and l-MCP. Experimental vessel (dessicator) was used as a

repeated variable. An unstructured covariance procedure was used in the MIXED

112

 

procedure. We evaluated the impact of ethylene and l—MCP on the concentrations of
patulin by comparing the appropriate means using two-sided t-tests in PROC MDCED in
SAS. The value of 1 was assumed for samples having undetectable patulin (<4 [lg/L

patulin before log transformations to prevent undefined loglo values.

4.4 Results
4.4.1 Experiment 1: Ethylene, Air

Data on patulin concentrations in juice from Penicillum-infected apples treated with
ethylene (100 uL/L) and air are presented in Table 18.

Concentrations of patulin in apple juice of Penicillium-inoculated apples increased
exponentially from undetectable (< 4 ug/L) concentrations at day 0 to 3.44$0.10 ug/L
after 16 days of storage, respectively (P 5 0.0012). Patulin concentrations were not
significantly influenced by exogenous ethylene treatment (P = 0.40), indicating that
storage of apples in 100 ppm ethylene did not decrease the production of patulin in
Penicillium inoculated apples.

Ethylene analyses of gases exiting the dessicators indicated that ethylene was being
produced in the dessicators (data not shown). We could not attribute this ethylene
production to 1) inadequate l-MCP treatment of apples, or 2) ethylene production by
Penicillium. Thus, Experiment 2 was designed to more carefully control for potential

ethylene production by fruit or P. expansum.

113

Table 18. Experiment 1- Patulin Concentrations in Juice Prepared from Apples
Treated with Ethylene (100 uL/L) and Air

 

 

 

 

 

 

 

 

 

 

 

Day Mean Ethylenea Airb
8 1.35 a $ 0.30 1.80 $0.42 0.90 $0.42
10 2.45b $0.12 2.68 $0.17 2.23 $0.17
12 2.73"c $0.18 2.57 $0.26 2.88 $0.26
14 3.22Cd $0.16 3.03 $0.23 3.41 $0.23
16 3.44d $0.10 3.48 $0.15 3.39 $0.15

 

Least square means $ standard errors are shown.

a’b’c’dNumbers with different superscripts (a,b,c,d) within a column and row significantly differ at P S 0.05.

4.4.2 Experiment 2: Inoculation, Ethylene, Air, l-MCP

Patulin concentrations (logio ug/L) in juice from apples treated with ethylene
and/or 1-MCP + ReTain in Experiment 2 are presented in Table 19. The results
presented in Table 19 and the overall least mean square values of patulin concentrations
in juice prepared from apples in treatments 1-4 of Table 17, as well as the main effect
means for apples treated with 100 uL/L ethylene or no ethylene, and apples treated with
l-MCP + ReTain or untreated apples. Results for treatments 5 GVICP treatment, no
inoculation, no ethylene treatment) and 6 (no treatment), which were not inoculated with
P. expansum, are not presented because patulin concentrations were undetectable in the
majority of juice samples pressed from these apples (data not shown).

Concentrations of patulin in apple juice pressed from Penicillium expansum
inoculated apples (data not shown) increased exponentially (P S 0.0182) from
undetectable (< 4 ug/L) concentrations at day 0 to 3.16 $ 0.10 ug/L after 16 days of
storage. Averaged across all time points, treatment of apples with 100 ppm of exogenous

ethylene tended (P S 0.065) to increase the concentration of patulin in juice pressed from

114

P. expansum-inoculated apples (Table 19 and Figure 6). There was no overall significant
difference in patulin concentrations of juice prepared from apples treated with l-MCP +
ReTain versus those that were not (P S 0.72) (Table 19 and Figure 7). No significant
interactions between ethylene and l-MCP + ReTain treatment of apples were detected

(data not shown).

115

 

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Figure 6. Effect of Ethylene on Patulin Concentrations in Juices Prepared from
Apple infected with P. expansum 28883

 

 

 

 

 

 

 

 

 

Log10 patulin

 

 

 

 

 

5 7 10 12 14 16
i+ Ethylene (+) ----- -- Ethylene (-)J Day

 

 

 

Figure 7. Effect of l-MCP + ReTain on Patulin Concentrations in Juices Prepared
from Apples infected with P. expansum 28883

 

 

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117

4.5 Discussion

To increase juice safety, prevent foodbome illness, and enhance pathogen control,
in 2001 the US. FDA promulgated juice HACCP regulations to be implemented in all
juice processing plants by January 2004. In 2001, FDA also implemented additional
regulations by limiting patulin concentrations in apple juice and cider, apple juice
concentrate (at single strength), and apple sauce to 50 ug/L. The juice processing
industry needs practical guidance to prevent or reduce the occurrence of patulin in juice
beverages and other processed apple products. Based on its proven effectiveness in
inhibiting aflatoxin production by Aspergillus (Roze et a1. 2004 and Sharma et a. 1985),
we hypothesized that ethylene treatment could be a practical and economical method to
control patulin production in stored apples and, subsequently, in apple cider and juice
produced from stored apples. Since patulin production generally occurs while fruit is
being stored, prevention in storage would likely be most beneficial to the juice and cider
industry.

Experiment 1 was a simple, preliminary study to examine the effects of exogenous
ethylene administration (100 111/L) on patulin production when compared to samples
treated with only air. We chose 100 11le ethylene for these experiments because of its
proven efficacy to reduce aflatoxin biosynthesis at this concentration (Roze et al. 2004).
Contrary to our hypothesis, we observed no overall significant difference in patulin
concentrations in juice prepared from apples stored in air versus those stored in 100 111/L
ethylene (P = 0.40). Our preliminary results from Experiment 1 indicate that patulin
production by Penicillium is not subject to regulation by ethylene concentrations.

However, we also were unable to completely inhibit ethylene production by apples (or

118

perhaps from P. expansum) with l-MCP in this experiment. We developed the further
hypothesis that the fungus was the source of ethylene when this study was repeated twice
without completely inhibiting ethylene production, despite the use of l-MCP. Therefore,
it was concluded that these preliminary results were confounded and needed to be
repeated, beginning with the verification that the 1-MCP was working properly using
inoculation and lack of inoculation to allow calculation of endogenous ethylene
production by the fungus.

Results fi'om Experiment 2 confirmed that l-MCP + ReTain treatment did not
influence patulin concentrations in juice prepared from apples inoculated with P.
expansum. This was unexpected to us since l-MCP was found to promote aflatoxin
production in media (Roze et a1. 2004).

Our results from Experiment 2 do, however, suggest that treatment of apples with
100 111/L ethylene tended (P 5 0.065) to increase the production of patulin in P.
expansum-inoculated apples. Although this result was unexpected, the finding that
exogenous ethylene treatment may increase patulin production by P. expansum is in
agreement with findings from experiments we conducted on patulin production by P.
expansum 28883 when grown on PDA media (Chapter 3). This hypothesis was derived
based upon similarities between aflatoxin and patulin, in that they are both mycotoxins
that are synthesized via the polyketide pathway, producing 6-MSA, and have similar
structures (Maggon et al. 1977, Tanenbaum and Bassett 1958, Zamir 1980).

This study was set up to further determine the effect of inoculation, to verify the
proper operation of the l-MCP/ReTain. By checking the ethylene levels daily using gas

chromatography, it was found that the l-MCP/ReTain was effective in inhibiting ethylene

119

production by the apples (See Appendix C). We also were able to confirm that P.
expansum also was producing its own ethylene in the fruit, which agrees with our
previous observations when culturing P. expansum on PDA media (Chapter 3). The
average concentration of additional ethylene produced by apples that were not treated
with l-MCP + ReTain was 41.7 111/L ethylene, which is significantly lower than the 100
111/L exogenous ethylene used in this experiment (See Appendix C). It is unclear whether
or not the concentrations of ethylene normally produced by untreated apples would
actually promote patulin synthesis by P. expansum. This question should be addressed
by future studies.

Ethylene is a hormone whose affects may be time sensitive. Continuous exposure
to 100 uL/L ethylene tended to promote patulin production in apples inoculated with P.
expansum. This long-term, continuous exposure may have influenced the biosynthesis of

patulin in apples.

120

4. 6 References

Abeles FB, Morgan PW, and Saltveit ME. 1992. Ethylene in plant biology. Academic
Press, San Diego, CA.

Askar A. 1999. Patulin in apple juice and children’s apple food. I. Toxicological and
legal aspects. Fruit Processing, 9(3): 74-77.

Barnett HL, Hunter BB. 1998. Illustrated Genera of Imperfect Fungi, 4th ed. APS Press,
St. Paul, MN.

Eisele, TA and Gibson MZ. 2003. Syringe-Cartridge Solid—Phase Extraction Method for
Patulin in Apple Juice. Journal of the Association of Official Analytical Chemists,
86(6):1160-1163.

Forsyth FR, Eaves CA, and Li ghtfoot HJ . 1969. Storage quality of McIntosh apples as
affected by removal of ethylene from the storage atmosphere. Canadian Journal of Plant
Science, 49(5): 567-572.

Jackson LS, Beacham-Bowden T, Keller UW, and Adhikari C. 2003. Apple quality,
storage, and washing treatments affect patulin levels in apple cider. Journal of Food
Protection, 66(4): 618-624.

Johnson DS, Stow JR, Dover CJ. 1993. Prospect for the control of fungal rotting in
‘Cox’s Orange Pippin’ Apples by low oxygen and low ethylene storage. Acta
Horticulturae, 343:334-336.

Johnston JW, Hewett EW, Maarten LAT, Hertog M, and Harker FR. 2002. Temperature
and ethylene affect induction of rapid softening in ‘Granny Smith’ and ‘Pacific RoseTM
apple cultivars. Postharvest Biology and Technology, 25(3): 257-264.

Lindroth, S. 1980. Occurrence, Formation and Detoxification of Patulin Mycotoxin.
Technical Research Centre of Finland: Materials and Processing Technology. 24: 4-46.

Liu FW, Long-Jun C. 1986. Responses of Daminozide-sprayed ‘McIntosh’ Apples to
Various Concentrations of Oxygen and Ethylene in Simulated CA Storage. J. Amer. Soc.
Hort. Sci., 111(3): 400-403.

MacDonald S, Long M, and Gilbert J. 2000. Liquid chromatographic method for
determination of patulin in clear and cloudy apple juices and apple puree: Collaborative.

Journal of the Association of Official Analytical Chemists, 83(6): 1387-1394.

Maggon KK, Gupta SK, and Venkitasubramanian TA. 1977. Biosythesis of Aflatoxins.
Bacteriological Reviews, 41(4): 822-855.

121

Martins ML, Gimeno A, Martins HM, and Bernardo F. 2002. Co-occurrence of patulin
and citrinin in Portuguese apples with rotten spots. Food Additives and Contaminants,
19(6): 568-574.

Roze LV, Calvo AM, Gunterus A, Beaudry R, Kall M, and Linz J E. 2004. Ethylene
modulates development and toxin biosynthesis in Aspergillus possibly via an ethylene
sensor-mediated signaling pathway. Journal of Food Protection, 67(3): 43 8-447.

Savaldi-Goldstein S. and Fluhr R. 2000. Signal transduction of ethylene perception,
Results Probl. Cell. Differ., 27:146-161.

Serek M, Sisler EC, and Reid MS. 1995. l-Methylcyclopropene, a novel gaseous
inhibitor of ethylene action, improves the life of fruits, cut flowers and potted plants.
Acta Horticulturae, 394: 337-347.

Shanna A, Padwal-Desai SR, and Nadkarni, GB. 1985. Possible implication of
reciprocity between ethylene and aflatoxin biogenesis in Aspergillusflavus and
Aspergillus parasiticus. Applied Environmental Microbiology 49(1): 79-82.

Tanenbaum SW, Bassett EW. 1958. The biosynthesis of patulin. 1. Related aromatic
substances from Penicillium patulum Strain 2159A. Biochim. Biophys. Acta, 28221-31.

United States Food and Drug Administration (FDA). 2001. 21 CFR Part 120. Hazard
analysis and critical control point (HACCP); procedures for the safe and sanitary
processing and importing of juice; final rule. US Federal Register, 66(13): 6138-6202.

Wilson DM. 1974. Patulin and penicillic acid. In Mycotoxins and other fungal related
food problems. Advances in chemistry series 149, ed. By JV Rodricks. Washington, DC,
American Chemical Society, 90-109.

Zamir LO. 1980. The biosynthesis of patulin and penicillic acid, p.223-255. In P.S. Steyn
(ed.), The biosynthesis of mycotoxins, A study in secondary metabolism, Academic
Press, Inc., New York, NY.

Zeringue, Jr. HJ, McCormick SP. 1990. Aflatoxin production in cultures of Aspergullus

flaus incubated in atmospheres containing selected cotton leaf-derived volatiles. Toxicon
28: 445-448.

122

5 Effect of trimming and culling on patulin concentrations in intact versus
decayed apple tissues

5.] Abstract

Patulin is the most common mycotoxin found in apples and apple juices. Because
if its toxicity, the FDA has established that the patulin concentration of apple juice
products should not exceed 50 ug/L. Additional research on quantitative apple-culling
criteria necessary to produce juice meeting this criterion are required. Therefore, the
objective of this study was to determine the effect of trimming of apples infected with P.
expansum on patulin levels in normal-appearing and decayed apple tissue and in juice
produced from two varieties of apples.

Two experiments were conducted to address the objective of this study.
Experiment 1 utilized apples naturally infected. Experiment 2 utilized apples that we
inoculated with P. expansum and stored for 3, 6, 9, 12, and 15 days. Infected apples were
carefully trimmed to separate rot from normal-appearing tissue and juice obtained fiom
each fraction tested for patulin by solid phase extraction and HPLC analysis.

In Experiment 1, juice prepared from hi gh-quality apples that were free of decay
with P. expansum did not contain detectable patulin. The average patulin concentration
of juice produced from unculled naturally-infected apples was 918 [lg/L. The proportion
of rot in apples was not correlated to patulin concentrations in rot (r = 0.054). Juice
prepared from normal-appearing tissue in five out of six replicates contained no
detectable patulin, but the one replicate contained 110 ug/L of patulin.

In Experiment 2, there was no difference in patulin concentrations (loglo rig/L) in
juice prepared from rotten tissue for J onagold and Red Delicious apples when compared

over a series of 15 days. Juice prepared fiom rotten tissue typically contained >1000 pg

123

patulin/L at all times beyond 6 days after inoculation. Juice prepared from normal-
appearing tissue of both apple varieties always contained detectable patulin starting at 9
days after inoculation, but patulin concentrations in these samples never exceeded 100

[.1 g/L. Trimming and culling rotten from normal-appearing apple flesh is effective in
removing most patulin contamination fiom apples for juice production, although normal-
appearing flesh of infected apples may still contain patulin concentrations in excess of

regulatory limits.

124

5.2 Introduction

Patulin, a secondary fungal metabolite produced by several species of Penicillium,
Aspergillus, and Byssochlamys, is the most common mycotoxin found in apples and apple
juices (Wilson 1974, Lindroth 1980, Martins 2002). Penicillium expansum, which is
commonly found in soil, is the primary source of patulin in fi'uit (Askar 1999, Jackson et
a1. 2003). Patulin causes gastrointestinal toxicity in rodents and also has mutagenic,
neurotoxic, genotoxic and carcinogenic effects in rodents (Dickens and Jones 1961,
Osswald 1978, Ciegler 1976, Reddy 1978, Reddy 1979, Lindroth 1980). Furthermore,
patulin causes dermal and gastric irritation in humans and also suppresses the immune
system of rodents, producing ulceration, congestion, and hemorrhagic lesions, especially
in the gastrointestinal tract (Dickens and Jones 1961, McKinley et al. 1980ab, DeRosnay
1952, Dalton 1952). Based upon its toxicity profile, an action level of 50 jig/kg was
established for patulin in apple juice, apple juice concentrate (at single strength dilution),
and in beverages containing apple juice ingredients (FDA 2001).

Although thermal effects, charcoal treatment, ascorbic acid treatment, irradiation,
filtration, centrifirgation, and fermentation have been demonstrated to reduce
concentrations of patulin in juice, trimming and culling of incoming fi'uit is the most
effective method to reduce patulin in the resulting juice (Sands et al. 1976, Brackett and
Marth 1979, Burroughs 1977, Bullerman and Hartung et al. 1975, Bissessur et al. 2001,
Lovett et al. 1975). Trimming and culling has been shown to remove 93-99% of total
patulin in fungus-rotted apples (Lovett et al. 1975). However, culling and trimming of
fruit may not be a completely reliable means to control patulin concentrations, as a lack

of visible rot does not necessarily indicate the absence of patulin in the fruit (Taniwaki

125

1992). Adequate culling of apples prior to juice preparation requires diligence and time
and is often disregarded or poorly executed. Although over-ripened or rotting fruit can
be removed, sorting based on visual observation is sometimes inadequate, resulting in
patulin-contaminated finished products (Harwig et al. 1973).

Although previous studies on the effects of trimming and culling on patulin
concentrations in apples have been conducted, several questions remain: 1) does removal
or trimming of visible rot provide adequate control of patulin concentrations in the
resulting juice? 2) are patulin concentrations in decayed or normal-appearing apple flesh
influenced by apple variety? and 3) what is the relative patulin concentration in apple
flesh decayed by P. expansum? It is important to determine the concentrations of patulin
present in decayed and non-decayed portions of rotting apples, as well as the
concentration of patulin in juice produced from these apples. This research will help
determine apple-culling criteria necessary for processors to produce juice containing less
than 50 pg patulin/L or juice. Therefore, the objective of this study was to determine the
effect of trimming of apples infected with P. expansum on patulin levels in normal-

appearing and decayed apple tissue and in juice produced from two varieties of apples.

5.3 Materials and Methods

Two different experiments were conducted to complete this study, one of which
was comprised of three smaller trials. The first set of experiments involved Red
Delicious apples that were naturally infected with P. expansum while the second
experiment involved J onagold and Red Delicious apples that were inoculated with P.

expansum in the laboratory.

126

5.3.] Experiment 1: Naturally Infected Apples

Apples used in this experiment were obtained from a commercial controlled
atmosphere (CA) fruit storage facility in Grand Rapids, MI in July, 2005. Figure 1
outlines the steps used to fractionate apples and prepare samples for patulin analysis.
First, red delicious apples were removed from CA storage and table sorted at the facility
into intact, high quality fruit destined for the fresh market and culled fruit that included a
significant proportion of damaged, moldy and decayed fruit. Lots of the high-quality
fruit and the culled fruit were returned to the laboratory for further fractionation and
sample preparation.

Five replicates (n=5 for each replicate) of high-quality fi'uit were randomly
selected, homogenized, juice extracted, and tested for the presence of patulin by solid
phase extraction and HPLC analysis. To extract juice, the samples were ground in a
General Electric 4-speed food processor with a 450-Watt motor, and then pressed using a
potato ricer lined with cheesecloth into a beaker. The juice was poured into plastic
culture tubes, capped, and frozen until solid phase extraction and HPLC analysis was
performed.

Among the culled apples, six replicates (n=10 for each replicate) of the red
delicious apples were randomly selected, homogenized, and juice extracted as described
above. Concurrently, 6 replicates of the same apples were selected and carefully trimmed
to separate rot from normal-appearing tissue and juice was extracted from each fraction
and tested for patulin. The rotten and normal-appearing tissue fractions were weighed to

determine the percent rot for each replicate.

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5. 3.2 Experiment 2: Apples Inoculated with Penicillium expansum

This experiment was conducted using J onagold and Red Delicious apples that
were obtained from commercial sources and then inoculated with P. expansum. Figure 2
outlines the steps used to perform this study. Both varieties of apples were obtained from
MSU Food Stores in August 2006 who received them from the State of Washington. P.
expansum inoculation was conducted by wounding the apple surface using a two-hole (3
mm each) puncture wound and directly applying one drop (0.1 ml) of spore suspended
(105 ug/mL water) P. expansum ATCC 28883 (Manassas, VA) to each wound. After
inoculation, apples were stored in 6 (3 for each variety) SO-gallon plastic barrels at room
temperature and normal atmosphere. Five apples from each container were randomly
collected from each container on days 3, 6, 9, 12, and 15. A paring knife was used to
remove the rotten area affected by P. expansum inoculation and separate it from the
normal appearing tissue. These two different tissue types were placed in separate
containers, then homogenized, pressed to extract juice, and analyzed for patulin using

solid phase extraction followed by HPLC analysis.

129

 

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5.3.3 Preparation of Commercial Mold Cultures

Several steps were involved in the initial preparation of the freeze-dried capsule
ofPenicillium expansum ATCC 28883 mold culture obtained from the American Type
Culture Collection (ATCC) (Manassas, VA). After the capsule was aseptically broken,
the freeze-dried cultures were rehydrated according to American Type Culture Collection
(ATCC) (Manassas, VA) instructions. One-half ml of sterile water was added to two test
tubes and a capsule was added to each. The contents were thoroughly mixed and another
5 ml of sterile water was added to the test tube and mixed. This mixture was allowed to
sit overnight. Next, the mixture was transferred to Potato Dextrose Agar (PDA) (Becton,
Dickinson and Company, Sparks, MD). Three plates for each strain were center
inoculated and allowed to incubate at 30°C for two to three weeks. The spores were
loosened by adding 2-3 ml of sterile water (repeated three times) and by brushing the
mycelia with a sterile, bent glass rod. Spores (2.09x107 CFU/ml spore suspension) were
collected in a 15-ml centrifuge tube and counted using a hemacytometer. The spore
samples were stored at —80°C in 25% glycerol. Before use, the samples were thawed in a
room temperature water bath and immediately transferred to the appropriate growth

medium and incubated at 30°C.

5.3.4 Solid-Phase Extraction

Solid-phase extraction (SPE) of patulin from apple juice was carried out using a
syringe-cartridge method (Eisele and Gibson 2003). The SPE cartridge (Waters Oasis®
HLB 60 mg, Milford, MA) was activated by applying 2-3 ml of water, followed by 2 m1
of methanol and a final wash of 2-3 ml of water. Each sample (2.5 mL volume) was then

applied to cartridges under vacuum. The cartridges were rinsed with 2.0 ml of 1.0%

131

sodium bicarbonate solution (pH=8.3) followed by 2 ml of 1% acetic acid solution. One
milliliter of 10% ethyl acetate in ethyl ether solution was then added to each cartridge to
elute patulin. The eluates were collected in 15x45 mm vials, evaporated to dryness under
a stream of nitrogen and then reconstituted in 0.5 m1 of 0.1% acetic acid. The vials were

capped, vigorously mixed, and frozen for future analysis by HPLC.

5.3.5 High Performance Liquid Chromatography (HPLC)

Patulin was quantified using a Waters Corporation (Milford, MA) high
performance liquid chromatograph (HPLC). The Waters system included two model 510
pumps, a model 717-plus auto-sampler, and a model 996 Photodiode Array detector. An
Alltech C18, Sum column (4.6 x 250 mm) was used to separate compounds. Each sample
(50 uL injection volume) was subjected to isocratic elution for 30 minutes with 10%
aqueous acetonitrile (ACN), followed by a 30 minute clean with 70% of 100% ACN and
30% of 10% ACN. The photodiode array detector collected the UV and visible light
spectrum fiom 190 to 800 nm to allow comparison of sample UV spectra to that of
patulin standards. Chromatograms were analyzed using Waters’ Millenium software.
Patulin concentration was quantified by comparing UV absorbance at 276 nm to known

patulin standards.

5.3.6 Statistical Analyses

For statistical analysis, SAS Version 9.1 was used (SAS Institute Inc., Cary, NC).
In Experiment 11 (Table 20), means, standard deviation, and ranges of patulin
concentrations (pg/L) were calculated for each fraction, Whole Apple (High-Quality),

Whole Apple (Culled), Culled Apple Normal-Appearing Tissue, and Culled Apple Rotten

132

Tissue. Pearson Correlation Coefficients were calculated to determine the relationship
between percent rot and patulin concentration or logo patulin concentration in rotten
tissue.

In Experiment 2, least square means of loglo patulin concentrations, as well as
differences in varieties, days, containers, and tissue type, were analyzed using were
obtained using PROC MIXED of SAS 9.1. We evaluated the impact of trimming,
variety, day after inoculation, and their respective interactions on the concentration of
patulin after using a logo transformation of patulin concentrations. Container (replicate)
was used as a repeated factor in this statistical analysis. One was added to undetectable
(<4 rig/L patulin; 0.602 rig/L Loglo patulin) samples for log transformations to prevent

unreadable error messages.

133

5.4 Results

Patulin concentrations in juice prepared from intact and fractionated apples in
Experiment 1 are presented in Table 20. Juice prepared from high-quality apples culled
by the packhouse manager did not contain detectable patulin. The lowest detectable
concentration of patulin was 4 ug/L. The average patulin concentration of juice produced
from whole culled apples was 918 rig/L, with a standard deviation of 1090 rig/L, and a
range of patulin concentrations of 210 to 2522 rig/L. Normal-appearing apple tissue in
five out of six replicates contained no detectable patulin. However, the replicate in which
patulin was detected in normal-appearing tissue contained 110 ug/L of patulin. The
mean and standard deviation of the patulin concentration in rotten tissue were 3553 rig/L
and 3650 rig/L, respectively. The range of patulin concentrations in rotten tissues was
1529 to 10418 rig/L. The proportion of rot in apples (Figure 10) was not correlated to
patulin concentrations (r = 0.054).

The proportion of rotten tissue in fractionated lots of apples was calculated for 5
of 6 the replicates (the sixth replicate rot yield was lost), and ranged from 25-50% rot.
The patulin concentration detected in juice prepared from this rotten tissue ranged from
1529-10418 rig/L.

Table 21 shows the mean and range of concentrations of patulin detected in
inoculated J onagold apples. Patulin was detectable in juice from normal-appearing tissue
beginning on day 9 after inoculation and patulin concentrations in juice from normal-
appearing tissue ranged up to 65 ug/L. Patulin was detectable in juice prepared from
rotten tissue at all times following fi'uit inoculation. Patulin concentrations in juice

prepared from rotten tissue of J onagold apples reached a plateau on day 9 following

134

inoculation at a mean patulin concentration of 2,951 ug/L. Trimming rotten tissue from
normal-appearing tissue removed over 95% of patulin at all time points.

Table 22 shows the mean and range of concentrations of patulin detected in
inoculated Red Delicious apples. Patulin was detectable in juice from normal-appearing
tissue beginning on day 6 after inoculation and patulin concentrations in juice from
normal-appearing tissue ranged up to 86 rig/L. Patulin was detectable in juice prepared
from rotten tissue at all times following fruit inoculation. Patulin concentrations in juice
prepared from rotten tissue of Red Delicious apples reached a plateau on day 12
following inoculation at a mean patulin concentration of 5128 rig/L. Trimming rotten
tissue from normal-appearing tissue removed over 97% of patulin at all time points.

Figures 11 and 12 show the mean patulin concentrations over time in juice
prepared from rotten and normal-appearing tissue of J onagold and Red Delicious apples.
In juice prepared from rotten tissue (Figure 11), there were not statistically significant
differences in patulin concentrations between the two varieties at any time point after
inoculation. In juice prepared from normal-appearing tissue (Figure 12), a significant
difference (P = 0.0025) in apple variety was detected on day 9 after inoculation, but not at

the other time points.

135

Table 20. Patulin Concentrations in Juice Prepared from Intact Apples, and
Normal-Appearing and Rotten Tissue Fractionated from Apples

 

 

 

 

 

 

 

 

 

 

Among samples Whole Apple Whole Apple Normal-Appearing Rotten Tissue of
containing (High- with apparent Tissue of Fractionated Cull
detectable patulin Quality) rot (Culled) Fractionated Cull Apples
Apples
Replicates 5 6 6 6
Total number of 0 6 1 6
replicates
containing
detectable patulin
(11)
Mean (pg/L) ND" 918 110 3553
Standard Deviation N/A 1090 N/A 3650
_Q§ZL)
‘ Rangegpg/L) ND 210 to 2522 <4to 110 1529 to 10418

 

 

 

*ND = Not Detected, < 4 ug/L is the detection limit for this experiment. These samples were undetectable.

Figure 10. Relationship between Percent Rot to Patulin Concentration (pg/L) in

rotten tissue (Experiment 1).

 

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25 30 35

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Rotten Tissue (%)

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Table 21. Mean and Range of Patulin Concentrations in Juice Prepared from

N ormal-Appearing and Rotten Tissue of P. expansum Inoculated Jonagold Apples

 

 

 

 

 

 

 

Mean in Normal- Range in Normal- . Range in Patulin Removed
Day . . . Mean 1n . . .
Appearing Appearmg Tissue Rotten Rotten Tissue by Trrmmmg
Tissue (rig/L) Tissue (pg/L) (rig/L) %
Lo L
( g” “g’ ) (LogroPg/L)
3 ND“ ND 1.66 :h 0.29 26 to 100 100
6 ND ND 2.84 d: 0.21 226 to 1345 100
9 1.54zt 0.12 18to 51 3.47i0.12 1566 to 5169 98.83
12 1.36 i 0.17 9 to 53 3.35 i 0.17 1177 to 3509 98.98
15 1.79:t0.15 56 to 65 3.1lt0.15 382 to 3249

 

 

 

 

 

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*ND = Not Detected, < 4 ug/L is the detection limit for this experiment. These samples were undetectable.

137

 

Table 22. Mean and Range of Patulin Concentrations in Juice Prepared from
N ormal-Appearing and Rotten Tissue of P. expansum Inoculated Red Delicious

 

 

 

 

 

 

Apples
ND = Not Detected, < 4 ug/L is the detection limit for this experiment. These samples were undetectable.
Mean in Nonnal- Range in . Range in Patulin
Day Appearing Normal- Mean m Rotten Rotten Tissue Removed by
. . Tissue (Loglo . .
Tissue Appearmg g/L) (pg/L) Trimmmg
(Log... ug/L) Tissue ” (pg/L) %
tin-fl)
3 ND“ ND 1.09 i 0.29 ND to 37 100
6 0.30 i 0.21 ND to 8 2.31 i 0.21 92 to 382 99.02
9 0.69i0.12 3 to7 3.13i0.12 1236 to 1463 99.64
12 1.50 i 0.17 24 to 40 3.71 i 0.17 3036 to 12385 99.38
15 1.80 :1: 0.15 42 to 86 3.49 :l: 0.15 2518 to 3778 97.96

 

 

 

 

 

 

 

138

 

Figure 11. Comparison of Patulin in Rotten Tissue by Apple Variety

 

 

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Figure 12. Comparison of Patulin in Normal Appearing Tissue by Apple Variety

 

 

 

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139

5. 5 Discussion

Our objectives in designing this experiment were to answer the three questions in
the introduction. First, does removal or trimming of visible rot fiom apples provide
adequate control of patulin concentrations in the resulting juice? Second, are patulin
concentrations in decayed or normal-appearing apple flesh influenced by apple variety?
Third, what is the relative patulin concentration in apple flesh decayed by P. expansum?

Previous research has addressed trimming of rot as a means to control patulin.
Taniwaki et al. (1992) found that patulin did not migrate more than 1 cm into normal-
appearing flesh of Ohio Beauty apples after all visible rot was removed. They did,
however, note that the higher the percentage of rot allowed in apples used for juice, the
higher the patulin concentrations of the resulting juice. They also noted that complete
patulin removal based upon visibility, alone, did not exclude all patulin from juice.

In this experiment, we also found that trimming is quite effective in reducing
patulin. However, apple samples containing only normal-appearing tissue still contained
patulin levels near or at the legal limit of 50 ug/L after only 9 days following inoculation
with P. expansum. While the inoculation level (105 CFU/ml) used in this experiment
may not commonly occur in nature, P. expansum is naturally present in soil and has been
found to inoculate apples in a natural environment (Harwig et a1. 1973). Lovett et al.
demonstrated a 93-99% patulin reduction in trimmed apples (197 5). Our research
demonstrated that trimming removed 97% of patulin fiom juice prepared from naturally-
decayed Red Delicious apples (Experiment 1) and <95% of patulin from Red Delicious

and J onagold apples that were artificially inoculated with P. expansum in the laboratory.

140

Lovett et al. (1975) suggested that variety of apple may play a role in patulin
production, since rotten tissue in Red Delicious apples contained less patulin (<5 jig/ml)
than rotten tissue in Golden Delicious and McIntosh apples (42.5-117.5 rig/ml) when
inoculated with P. expansum 1071 and 1172 in their experiment. No significant
differences were due to apple variety found in this study in patulin concentrations of j uice
prepared from rotten or normal-appearing tissue of Red Delicious and J onagold apples.
However, we did note that, after inoculation, J onagold apples decayed at approximately
twice the rate of Red Delicious apples. Hence, we found that apple variety may affect
rate of decay, but not patulin concentration in rot.

The majority of moldy apples in which only P. expansum is isolated will contain
patulin. Moldy apples that do not contain patulin tend to be inoculated with other molds
(Harwi g 1973). Patulin concentrations in juice prepared from culled apples in
Experiment 1 and trimmed, rotten tissue and some normal appearing tissue after only
three days of inoculation in Experment 2 were well above the F DA established maximum
residue levels (MRLs) of 50 ug/L. These data represent the importance of a trim, cull, or
patulin prevention method for all varieties of apples. The Juice HACCP Alliance (2002)
recommends that “No more than 1% by weight rot after culling” remain in apples for
juicing in order to control patulin. Based on the maximal concentrations of patulin in
apple rot determined in this research, the suggested critical limit of 1% rot allowed in
apples destined for apple juice may not be completely sufficient. Based on the maximal
patulin concentrations extracted from apple rot in these experiments, it appears that

culling to 0.4% rot may be necessary to maintain juice <50 ug/kg patulin. However,

141

further studies should be conducted to ensure that the remaining pulp used for animal
feed will also contain <50 rig/kg patulin when this percentage rot is used.

The proportion of rot in apples used in Experiment 1 was not correlated with
patulin. This lack of correlation could be due to some rot in experiment 1 being due to
molds other than P. expansum which do not produce patulin. However, rot caused by P.
expansum is likely to have a uniformly high concentration of pautlin based on our results
in Experiment 2.

Commericial facilities use different methods to prevent the entry of patulin into their
samples. Scrubbers with hard bristles, pressure washing, and culling by humans are
popular methods used in small and large cider mills and commercial, juice facilities.
Although there is no guarantee that patulin will not remain in normal-appearing tissue,
scrubbers with hard bristles to remove decayed flesh are probably effective for patulin
prevention. Human culling could be just as effective, depending on the diligence of the
individual performing the task. However, it is important to note that normal-appearing
apple tissue from carefully trimmed fi'uit in this experiment still yielded juice having >50
ug patulin/L. It is reasonable to presume that manual culling and trimming by humans in
typical juice processing facilities would be less stringent than was conducted in this
experiment. Culling must be done with extreme care and attentiveness, which makes it a
tiring job that is often either poorly conducted or entirely neglected.

When the 2002-2004 Michigan cider mill results were combined (N=493), patulin
was detected (24 rig/L) in 18.7% of all cider samples with 2.2% of samples having
patulin concentrations above the legal limit of 50 jig/L. Approximately 23.3% of all

cider and juice samples (N=159) collected fiom retail grocery stores in 2005-2006

142

contained detectable (24 rig/L) patulin, with 11.3% samples having patulin
concentrations above the legal limit of 50 jig/L. These statistics are indicative of a
serious problem in the apple juice and cider industry, not only in Michigan cider mills,
but also internationally with the analysis of national brands. The industry is in serious
need of effective, easily implemented methods of patulin control in juice from whole
apple fruit and concentrate. Although the industry does implement trim and cull
methodology, it, apparently, is not completely reliable.

Although trimming is effective, culling appears to be a more reliable method of
patulin control, since no- matter how meticulous the trimmer, normal-appearing tissue
may still contain patulin levels above 50 ug/L. Therefore, we highly recommend that the
industry cull rotten apples from samples destined for juice until patulin production can be
prevented or significantly reduced in controlled atmosphere storage. Since the industry,
due to historical and economically efficient practices, will likely continue to trim in order
utilize as much fruit as possible, we recommend that trimming be done to a maximum of

0.4% rot (weight/weight).

143

5. 6 References

Brackett RE, Marth EH. 1979. Ascorbic acid and ascorbate cause disappearance of
patulin from buffer solutions and apple juice. Journal of Food Protection, 42(11): 864-
866.

Broom WA, Biilbring E, Chapman CJ, Hampton JWF, Thomson AM, Ungar J, Wien R,
Woolfe G. 1944. The pharmacology of patulin. British Journal of Experimental
Pathology. 25: 195-207.

Bullerman LB and Hartung TE. 1975. Effect of low level gamma irradiation on growth
and patulin production by Penicillium patulum. Journal of Food Science, 40(1): 195-196.

Burroughs LF. 1977. Stability of patulin to sulfur dioxide and yeast fermentation. Journal
of Association of Official Analytical Chemists, 60(1): 100-103.

Ciegler A, Beckwith AC, Jackson LK. 1976. Teratogenicity of patulin and patulin
adducts formed with cysteine. Applied Environmental Microbiology 31(5): 664-667.

Ciegler A, Vesonder RF, Jackson LK. 1977. Production and biological activity of patulin
and citrinin from Penicillium expansum. Applied Environmental Microbiology 33(4):
1004- 1 006.

Dalton J E. 1952. Keloid resulting from a positive patch test. AMA Archives of
Dermatology Syphilology, 65: 53-56.

DeRosnay CD, Dupont M, Jensen R. 1952. An antibiotic, mycotoxin. Journal of Med.
Bordeaux Sud-Quest, 129:189-199.

Dickens F and Jones HEH. 1961. Carcinogenic activity of a series of reactive lactones
and related substances. British Journal of Cancer, 15: 85-100.

Eisele, TA and Gibson MZ. 2003. Syringe-Cartridge Solid-Phase Extraction Method for
Patulin in Apple Juice. Journal of Association of Official Analytical Chemists,
86(6):1160-1163.

Harwig J, Chen YK, Kennedy BPC, and Scott PM. 1973. Occurrence of patulin and
patulin-producing strains of Penixillium expansum in natural rots of apple in Canada.
Canadian Institute of Food Science Technology Journal, 6(1): 22-25.

Jackson LS, Beacham-Bowden T, Keller UW, Adhikari C. 2003. Apple quality, storage,
and washing treatments affect patulin levels in apple cider. Journal of Food Protection,

66(4): 618-624.

Juice HACCP Alliance. August 2002. Juice HACCP Training Curriculum, First Edition.

144

Lindroth, S. 1980. Occurrence, Formation and Detoxification of Patulin Mycotoxin.
Technical Research Centre of F inland: Materials and Processing Technology. 24: 4-46.

Lovett J, Thompson Jr RG, Boutin BK. 1975. Trimming as a means of removing patulin
from fungus—rotted apples. Journal of Association of Official Analytical Chemists,
58(5):909-911.

Martins ML, Gimeno A, Martins HM, and Bernardo F. 2002. Co-occurrence of patulin
and citrinin in Portuguese apples with rotten spots. Food Additives and Contaminants,
19(6): 568-574.

McKinley ER, Carlton WW. 1980a. Patulin mycotoxicosis in the Syrian hamster. Food
Cosmetics Toxicology 18(2): 173-179.

McKinley ER, Carlton WW. 1980b. Patulin mycotoxicosis in Swiss ICR mice. Food
Cosmetics Toxicology 18(2): 181-187.

Moodley RS, Govinden R, Odhav B. 2002. The effect of modified atmospheres and
packaging on patulin production in apples. Journal of Food Protection, 65(5): 867-871.

Osswald H, Frank HK, Komitowski D, Winter H. 1978. Long-term testing of patulin
administered orally to Sprague-Dawley rats and Swiss mice. Food Cosmetics Toxicology,
16(3): 243-247.

Reddy CS, Chan PK, and Hayes AW. 1978. Teratogenic and dominant lethal studies of
patulin in mice. Toxicology, 11: 219-223.

Reddy CS, Chan PK, and Williams WL. 1979. Acute Toxicity of pautlin and its
interaction with penicillic acid in dogs. Food and Cosmetics Toxicology, 17(6): 605-609.

Sands DC, McIntyre J L, Walton GS. 1976. Use of activated charcoal for the removal of
patulin from cider. Applied Environmental Microbiology, 32(3): 388-391.

Taniwaki MH, Hoenderboom JM,Vitali ADA, Eiroa MNU. 1992. Migration of Patuin in
Apples. Journal of Food Protection, 55(11): 902-904.

United States Food and Drug Administration. 2001. 21 CFR Part 120. Hazard analysis
and critical control point (HACCP); procedures for the safe and sanitaryprocessing and
importing of juice; final rule. US Federal Register, 66( 13): 6138-6202.

Wilson DM. 1974. Patulin and penicillic acid. In Mycotixins and other fungal related

food problems. Advances in chemistry series 149, ed. By JV Rodricks. Washington, DC,
American Chemical Society, 90-109.

145

6 Summary, Conclusions and Future Research

Patulin is produced by mold species such as Penicillium expansum and is the most
abundant mycotoxin in apples and apple juice. Because of its toxicity, the FDA has
established that patulin concentration of apple juice products should not exceed 50
jig/liter (U .S. FDA, 2001, 21 CFR Part 120, Fed. Register 66:6138-6202). Michigan
ranks as one of the top three apple producing states with approximately 30% of Michigan
apples processed into cider. However, the levels of patulin in Michigan apple cider have
not been systematically determined. Production of patulin can be reduced under
modified atmospheric storage conditions. Recent research shows that ethylene reduces
the biosynthesis of aflatoxin, another mycotoxin, by Aspergillus spp. The relationship
between ethylene production by P. expansum and the onset of toxin production has not
been examined.

Our surveillance research indicates that the majority of apple cider produced by
Michigan mills in 2002-2004 contained no detectable patulin. Patulin was detected (24
jig/L) in 18.7% of all cider mill samples with 11 (2.2%) samples having patulin
concentrations above the legal limit of 50 ug/L. A greater percentage of cider samples
obtained fiom mills using thermal pasteurization contained detectable patulin (28.4%)
when compared to mills using ultraviolet-light irradiation (13.5%) or no pathogen
reduction treatment (17.0%). Among juice and cider samples obtained from retail
grocery stores (N=159), 23% contained detectable patulin, with 18 (11.3%) samples
having patulin concentrations above the legal limit of 50 ug/L. Some apple juice samples

obtained from retail grocery stores had exceptionally high patulin concentrations, ranging

up to 2700 rig/L.

146

Timing of patulin and ethylene production by P. expansum was inversely related
for P. expansum ATCC 28883 and 1117. Patulin production in Petri plates containing
PDA inoculated with P. expansum ATCC 28883 was significantly increased by the
presence of exogenous ethylene, but there was no effect of 1-MCP on patulin production.
These results confirm that P. expansum produces ethylene and suggests an inverse
relationship between ethylene and patulin production.

Concentrations of patulin in apple juice produced from P. expansum 28883
inoculated apples increased exponentially from undetectable (< 4 ug/L) concentrations at
day 0 to 3.16i0.10 ug/L after 16 days of storage. Averaged across all time points,
exposure of apples to 100 uL/L ethylene tended to increase the production of patulin in
P. expansum inoculated apples. Therefore, exogenous ethylene treatment does not inhibit
(and may actually promote) patulin synthesis by P. expansum.

The average patulin concentration of juice produced from unculled naturally-
infected Red Delicious apples was 918 ug/L. The proportion of rot in apples was not
correlated to patulin concentrations in rot. Juice prepared fiom normal-appearing tissue
in five out of six replicates contained no detectable patulin, but the one replicate
contained 110 ug/L of patulin. Juice prepared fiom rotten tissue typically contained
>1000 ug patulin/L at all times beyond 6 days after inoculation. Juice prepared from
normal-appearing tissue of both apple varieties always contained detectable patulin
starting at 9 days after inoculation, but patulin concentrations in these samples never
exceeded 100 ug/L. We highly recommend that the industry cull rotten apples from
samples destined for juice until patulin production can be prevented or significantly

reduced in controlled atmosphere storage. Since the industry, due to historical and

147

economically efficient practices, will likely continue to trim in order utilize as much fruit
as possible, we recommend that trimming and culling be done such that no more than
0.4% rot (weight/weight) remains in apples pressed for juice.

Several important questions remain to be studied and should be addressed in
future research, 1) does “core rot,” also known as “moldy core,” contain patulin, 2) what
are optimal control atmosphere conditions to minimize patulin contamination, 3) by what
mechanism does exogenous ethylene enhance patulin production by P. expansum, 4.)
what is the natural variance in ethylene production and patulin production across different
strains of P. expansum, and 5) what is the effect of inoculum level on rot and patulin

production.

148

7 Appendices

149

Appendix A

General Cider Information

150

7.1 General Cider Information

Michigan is one of the largest fresh apple and apple products producers in the United
States, producing and processing an average of 764 million pounds of apples 2000-2003
(Doores 1983-1984, Michigan Apple Association 2004, Michigan Agricultural Statistics
2003, Thede 2004). Only the states of Washington and California produce more fresh
apples and apple products, respectively (Doores 1983-1984). An average of 207.5
million pounds (27%) of juice and cider were donated to juice in 2000-2003 (Doores
1983-1984, Michigan Apple Association 2004, Michigan Agricultural Statistics 2003,
Thede 2004). The Michigan apple industry grossed $250,457,000 pounds in 2003

(Michigan Agricultural Statistics Service 2004).

7.1.1.1 Processing Steps

Several steps are required in the processing of cider. After the apples are harvested,
they are generally placed in storage, washed, and inspected. This inspection is generally
used to separate apples to be sold wholly or as juice from dropped, decayed and damaged
apples, a process known as culling (Beaudry 2005, Thede 2004). Culling is essential
since damaged fruit is more likely to carry pathogens and are more susceptible to
chemical hazards, such as patulin. This may be followed by a grinding step (Thede
2004). A hydraulic press is then used to further crush the ground samples into juice
(Thede 2004). The juice is filtered and held in a tank where it may or may not undergo a
5-log reduction step (Thede 2004). This is followed by bottling and refrigerated storage

(Thede 2004).

151

7.1.1.2 HACCP

There are an estimated 16,000 to 48,000 juice related illnesses per year in the
United States (FDA 2001). Most of these are suspected to be caused by contaminated
fruit and pathogenic microorganisms, especially in unpasteurized juice (Besser et al.
1993, FDA 2001). These facts and several recalls led the FDA to address foodbome
illness and hazards related to juice products (1997, Thede 2004). A warning statement on
juice product labels was required in 1998 if they were not processed using HACCP or a
lethality step that would lead to a 5-log reduction of the pertinent pathogens (F DAb,
Thede 2004). Furthermore in 1998, FDA stated an intent to require juice processors to
develop and implement HACCP systems (Thede 2004). To heighten juice
manufacturers’ knowledge, educational programs on juice safety and HACCP were
established (Thede 2004). The FDA gave the juice industry three years to fully execute
HACCP programs in 100% juice beverage products and in retail establishments or
businesses that make and sell juice to other businesses (2001). Large processors were to
implement programs by January 2002, followed by small and very small processors in
January 2003 and January 2004, respectively (1998a). Although HACCP does not
directly apply to growing, harvesting, and transporting of fi'uits and vegetables, key
personnel are encouraged to implement Good Agricultural Practices (GAPS), Current
Good Manufacturing Practices (CGMPs), and Sanitation Standard Operating Practices

(SSOPs) (Dingman 1999, Codex 2003).

152

Appendix B

Patulin and Ethylene Production when Grown in P. expansum 28883 in Liquid PDB
Media

153

7.1.2 Patulin produced in relationship to the presence of endogenous and
exogenous ethylene on solid and liquid media

154

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Appendix C

Supplemental Data on Ethylene Production by Apples for Chapter 4

156

Table 25. Ethylene production (outflow — inflow in closed vessels) of Red Delicious
Apples variously treated with P. expansum, ethylene, and l-MCP.

 

 

 

 

 

 

 

Overall Difference
Treatment Ethylene Mean (uL/L)
Ethylene, l-MCP, a
Inoculated 1.50 :t 5.71
Ethylene, No l-MCP, ab
Inoculated 14.08 :t 5.7
No Ethylene, l-MCP, ab
Inoculated 2.13 i 5.71
No Ethylene, No l-MCP, b
Inoculated 19.42 i 5.71
No Ethylene, l-MCP, a
Uninoculated 0.16 i 5.71
No Treatment 41.54 i 5.71c

 

 

 

 

Least square means i: standard errors are shown.

a’b’CNumbers with different superscripts (a,b,c) within a column significantly differ at P S 0.05.

157

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