Wageningen Academic

World Mycotoxin Journal, November 2008; 1(4): 385-396 P u b l i s h e r s

Mouldy fruits and vegetables as a source of mycotoxins: part 2

N. Paster1 and R. Barkai-Golan2

1Department of Food Science, the Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel; 2Department
https://www.wageningenacademic.com/doi/pdf/10.3920/WMJ2008.x044 - Sunday, April 28, 2024 8:48:34 AM - IP Address:45.160.36.189

of Postharvest Science of Fresh Produce, the Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel;
golaneli@bezeqint.net

Received: 15 June 2008 / Accepted: 17 October 2008

© 2008 Wageningen Academic Publishers

Abstract

Although chemical control is still the main way to reduce the preharvest and postharvest incidence of mycotoxigenic
fungi, the worldwide tendency to reduce chemical use, and the emergence of resistant strains, have accelerated the search
for non-chemical strategies. Those applied at the postharvest stage include heat treatments, biological control, and
modified-or controlled-atmosphere storage. It is now evident that combinations of treatments are more efficient than
individual treatments applied alone. Most of the studies on mycotoxins in fruits are focused on patulin (produced mainly
by Penicillium expansum) in apple products, and ochratoxin A (produced mainly by Aspergillus carbonarius) in grapes
and in wines. Patulin levels can be significantly reduced by washing and sorting apples, and trimming away rotten tissues.
Other treatments for patulin reduction have been associated with its ability to react readily with compounds containing
sulphydryl groups. Other additives that reduce patulin levels are ascorbic acid and B-complex vitamins. The rate of
ochratoxin A reduction during fermentation depends, among others, on the yeast used for fermentation and the type of
wine produced. During vinification, ochratoxin A is also reduced by binding to the solid residues of grapes. Alternaria
mycotoxins may be produced naturally in a variety of fruits and vegetables and their processed products. At least two
of these mycotoxins, alternariol and alternariol monomethyl, were shown to be stable in heated apple juice. In some
apple cultivars, Alternaria occurs in the core and the damage is hidden. Contamination with mycotoxins is recognized
as an unavoidable risk, but three major components are involved in the attempt to minimize the risk: Good Agricultural
Practices, Good Manufacturing Practices, and Hazard Analysis Critical Control Points. In many countries, regulatory
measures have been taken to limit the presence of mycotoxins in fruits and vegetables. Several factors may influence the
establishment of national and regional mycotoxin limits and regulations.

Keywords: Aspergillus, Penicillium, Alternaria, mycotoxin prevention, mycotoxin control, regulations

1. Introduction

Fungi play a substantial role in the spoilage of fruits
and vegetables, because of their pathogenicity to the
harvested products. However, during the various stages
of pathogenesis, some of these fungi may generate
different mycotoxins, toxic to humans and animals that
consume them. During recent decades a variety of fruits
and vegetables that form part of our daily diet have
been added to the list of products exposed to mycotoxin
contamination.
Since Aspergillus, Penicillium and Alternaria species are
the major mycotoxigenic fungi that attack harvested fruits
and vegetables, a variety of mycotoxins produced by these
fungal genera during pathogenesis may contaminate the
fruit and vegetable tissues. Some of these mycotoxins
are highly stable during processing, therefore, although
consumers will reject a visibly rotten fruit, processed
fruit products may still form a significant source of these
mycotoxins.

ISSN 1875-0710 print, ISSN 1875-0796 online, DOI 10.3920/WMJ2008.x044 385

 N. Paster and R. Barkai-Golan
Research has recently been increasingly focused on basic
aspects of mycotoxins in fruits and vegetables, and a
book, ‘Mycotoxins in Fruits and Vegetables’, dedicated
to these aspects has recently been published (Barkai-
Golan and Paster, 2008a). This multi-author book covers
the risk assessment and safety evaluation of fruit and
vegetable mycotoxins, their economic aspects, factors
affecting their production, and their ability to diffuse into
adjacent fruit tissues. The accumulated information on
the major mycotoxins produced in fruits and vegetables
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during pathogenesis, including their toxicity, their
sources and their occurrence, and methods for detection
and determination of the mycotoxigenic moulds and the
mycotoxins produced by them, are presented. Limits set
on the contents of mycotoxins in fruits and vegetables, and
the corresponding regulations that have been established
in many countries, are discussed. Special attention has
been given to chemical, physical and biological methods
for suppressing the growth of mycotoxigenic moulds, as
well as to direct decontamination and detoxification of
fruits, vegetables, and their derived products, with respect
to mycotoxins.
The present review, which is based on these book chapters,
is divided into two parts. The first deals with moulds and
mycotoxin formation in fruits and vegetables, diffusion
of mycotoxins, risk assessment and safety evaluation,
and methods for determination of mycotoxigenic fungi
and their toxins (Barkai-Golan and Paster, 2008b). The
present part deals with: (1) control of mycotoxigenic fungi
and mycotoxins; (2) means to prevent contamination with
patulin, ochratoxin A and Alternaria mycotoxins, and
the effects of processing on these mycotoxins; and (3)
regulations, limits and economic aspects of mycotoxins
in fruits and vegetables.
2. Control of mycotoxigenic fungi and
mycotoxins
The problem of mycotoxins in fresh produce and processed
products is of continuing concern and has received a great
deal of attention during the last four decades. Preharvest
contamination can be reduced by Good Agricultural
Practices that limit the growth of mycotoxigenic fungi,
including biocontrol (Tamm, 2001). Contamination can be
minimised by means of appropriate postharvest practices
involving chemical, physical and biological treatments.
These various approaches are discussed in the following
sections.
Chemical control of mycotoxigenic fungi
Harvested fruits and vegetables are commonly treated
with fungicides to retard postharvest disease development.
However, there is a greater likelihood of direct human
exposure to such fungicides than to chemicals that are
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applied solely to protect foliage (Tripathi and Dubey, 2004).
The side effects of synthetic fungicides raise the need to
develop alternative strategies for reducing losses due to
postharvest decay (Marin et al., 2008).
Chemical control remains the principal way to reduce the
incidence of blue mould in apples that are cold-stored for
long periods. Intensive and exclusive use of benzimidazoles
has led to the emergence of benzimidazole-resistant
pathogens in most packing houses. Since the discovery of
thiabendazole (TBZ)-resistant P. expansum isolates in apple
packing houses, the search for alternative chemical control
strategies has greatly intensified. Fungicides are applied
to fruits as prestorage drenches or line sprays or, in some
countries, in the field, several days before harvest. Up to
now a few trials of chemical controls have regarded patulin
as a variable, and have considered only fruit decay, in order
to evaluate the effectiveness of chemical treatments.
Currently, the fungicide most commonly used for postharvest
application is imazalil, as well as its mixtures with other
fungicidal products such as iprodione, TBZ, etc. Imazalil
is an imidazole-type systemic fungicide, which inhibits the
biosynthesis of ergosterol. It has a broad-spectrum activity,
wider than that of TBZ, and is active against isolates that
show resistance to this type of product.
Fludioxonil belongs to phenylpyrroles – a new class of
non-systemic fungicides and it is effective in preventing
apple decay by TBZ-sensitive and TBZ-resistant isolates
of P. expansum. The protective effects of three fungicides,
fludioxonil, cyprodinil and a mixture of the two, against
blue mould caused by TBZ-sensitive and TBZ-resistant
isolates of P. expansum, were evaluated, and all three
were found to be equally effective against both groups of
isolates. A recent in vitro study showed that carbendazim,
captan, and bupirimate inhibited patulin production by
P. expansum, but less effectively than they inhibited its
growth (Paterson, 2007).
In vivo testing of ammonium molybdate as a potential
fungicide to protect apples against blue mould showed
significant reduction in P. expansum lesion diameters
(Nunes et al., 2001). Pyrimethanil was also very effective
as a postharvest treatment, in controlling decay in wounds
inoculated with P. expansum conidia; no deleterious effects
on fruit quality were reported (Sholberg et al., 2005a).
Replacement of synthetic fungicides with natural products,
particularly those of plant origin, that are non-toxic and
specific in their action, is gaining considerable attention,
and the use of natural products in the management of fruit
decay caused by plant pathogenic fungi was reviewed by
Tripathi and Dubey (2004). Chitosan, a deacetylated form
of chitin, has been used as an alternative control agent
against blue mould in harvested ‘Red Delicious’ apple fruits,
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  Mouldy fruits and vegetables as a source of mycotoxins: part 2
and has been shown to induce resistance in the fruit rather
than merely inhibiting the pathogen directly (Capdeville
et al., 2002).
Several in vitro and field trials have been carried out
on the influence of fungicides on ochratoxin A (OTA)-
producing fungi and on the subsequent OTA accumulation
in grapes and/or wine, but at present there is no systematic
application of fungicides specifically to control black
aspergilli in these products. Bellí et al. (2006) found that
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fungicides that prevent fungal growth obviously prevent
OTA production, but not all the fungicides that reduced
growth reduced OTA synthesis. In general, fungicides that
contain copper or strobilurins reduced both growth and
OTA production, unlike sulphur fungicides. Natamycin,
a fungicide produced by Streptomyces natalensis, was
shown to control Aspergillus carbonarius growth and OTA
production in a red grape extract medium, in a way that
depended on water activity and temperature (Medina et
al., 2005).
Production of Alternaria mycotoxins in whole tomatoes,
apples, oranges, and lemons infected with indigenous
Alternaria was demonstrated for the first time by Stinson et
al. (1981). Several chemicals such as triazoles, strobilurins
and chlortanolil are recommended for controlling
Alternaria in tomatoes (Marin et al., 2008).
The natural compounds tested for controlling Alternaria
include chitosan, some essential oils, and isothiocyanates.
Chitosan retarded the appearance and growth of visible
black lesions, and the synthesis of mycotoxins such as
alternariol (AOH) and alternariol monomethyl ether (AME).
Chitosan also induced the production of the phytoalexin
rishitin in tomato tissue. Another alternative treatment
could involve the use of essential oils: Feng and Zheng
(2007) found that cassia and thyme oils exhibited antifungal
activity against Alternaria alternata on tomatoes, but that
cassia oil impaired the flavour. Isothiocyanates were tested
against Alternaria by Troncoso-Rojas et al. (2005). These
compounds completely inhibited Alternaria growth in
vitro, and when used to control fungal rot on packed bell
peppers and packed tomatoes, they yielded better results
than the synthetic fungicide, with no adverse effects on
fruit quality.
Physical control of mycotoxigenic fungi
Several physical practices can be applied to prevent
postharvest contamination of fruits and vegetables
with toxigenic fungi. However, mycotoxins have high
decomposition temperatures and are therefore very resistant
to heat inactivation. Nevertheless, use of heat treatments
to destroy the spores and mycelia of the mycotoxigenic
fungi, in combination with other means, might reduce or
eliminate fungal proliferation and, thereby, production of
World Mycotoxin Journal 1 (4)
the toxin. To maximise control of fruit decay by postharvest
application of alternatives to synthetic fungicides, various
control strategies can be integrated. The inhibition of
physiological and/or pathological deterioration of freshly
harvested produce by combinations of all types of physical,
chemical, and environmentally friendly chemical methods,
known as ‘physicochemical’ treatments, has been studied
(Fallik, 2008).
Utilisation of antagonistic microorganisms in combination
with physical treatments appears to be a promising
technology. Some antagonist-based products are
commercially available and others are currently in various
stages of development (De Costa and Erabadupitiya,
2005). A combination of heat and yeast antagonists was
more effective than either treatment alone in controlling
P. expansum decay development on apples (Leverentz
et al., 2003), and microwave power combined with an
antagonistic yeast reduced the percentage of infected
wounds and diameters of lesions, without impairing the
quality parameters of fruit. Saftner et al. (2003) reported
that prestorage application of 1-methylcyclopropene (1-
MCP), heat (38 °C for 4 days), 1-MCP plus heat treatments,
and controlled atmosphere storage decreased the severity
of decay caused in wound-inoculated ‘Golden Delicious’
apples by P. expansum. Heat treatment and sodium
bicarbonate significantly improved the efficacy of the
biocontrol agent – the BIO126 strain of Metchinkowia
pulcherrima – against blue mould on apple fruits stored
at 23 °C (Spadaro et al., 2004).
Several combinations of physicochemical treatments
were studied in order to improve the efficacy of Hot
Water Rinsing and Brushing (HWRB) in controlling decay
development. A combination of HWRB and heat treatment
was highly effective in controlling Alternaria rot in mango
fruits, and enabled significant reduction of prochloraz use
against Penicillium spp. decay. An integrated approach
that was studied for the control of postharvest diseases of
peaches included application of a yeast antagonist (Candida
oleophila), hot water treatment at 55 °C for 10 seconds, and
storage in a modified atmosphere at 0 °C (Karabulut and
Baykal, 2004). Each of the treatments significantly reduced
the decay incidence caused by natural infection, but the
highest efficacy was achieved by the combination of all
three strategies.
It is now evident that combinations of treatments are more
efficient in controlling postharvest pathogens than each
of the treatments applied alone. Heat treatments, such
as hot-water dips, vapour heat, dry heat or curing, and
HWRB may reduce decay when applied alone, but their
application in combination with modified or controlled
atmosphere, biocontrol agents, ethanol, bicarbonate salts,
and even with low dosages of fungicide, can significantly
387
 N. Paster and R. Barkai-Golan
enhance their efficacy in controlling the development of
postharvest fungal decay.
Biological control of mycotoxigenic fungi in fruits
In the postharvest environment, parameters such as
temperature and humidity can be rigidly controlled
and can be taken into account when selecting a suitable
biocontrol agent. Also, harvested commodities present a
more concentrated target for the application of biocontrol
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agents (Castoria et al., 2008). However, in spite of all
the fundamental and applied research on postharvest
biocontrol that has been published, the commercial use
of these products has been and still is limited, and covers
only a very small fraction of the potential market.
The main obstacle to the use of postharvest biocontrol
products has been inconsistency in performance, especially
when they are used in a stand-alone mode, in place of
synthetic fungicides. A second problem with the currently
available products is their inability to control previously
established and latent infections. Biological control has
been tested within Integrated Pest Management (IPM)
programmes, against bunch rots caused by Aspergillus and
Penicillium in grapes, both in vineyards and after harvest.
The efficacy of microbial antagonists, in combination with
reduced-rate fungicide applications has been evaluated.
Several antagonistic microbes have been successfully used
for preventing infections by postharvest fungal pathogens
of pome fruits, including P. expansum (Janisiewicz and
Korsten, 2002). Postharvest biocontrol agents act through
a variety of complex mechanisms (Castoria et al., 2003;
Droby and Chalutz 1994; Janisiewicz and Korsten 2002),
which makes the emergence, through selection, of resistant
strains of the pathogen unlikely. In contrast, such selection
takes place when the few authorised postharvest fungicides
are used, especially those acting on fungal targets coded by
one or a few genes, as in the cases of benzimidazoles and
β-tubulin (Sholberg et al., 2005b). Castoria et al. (2005)
found that in apples pretreated with the biocontrol agent
Rhodotorula glutinis strain LS11, the overall accumulation
of patulin in the small percentage of fruits that were infected
by P. expansum was significantly lower than in infected,
non-pretreated apples. Furthermore, the biocontrol yeast
was able to resist and degrade a high concentration of
patulin, both in vitro and in a model system consisting of
browning apple homogenate, which mimicked decaying
fruit tissue.
The activity of biocontrol yeasts does not appear to rely on
production of undesirable antibiotics, since competition
for space and nutrients is still considered to be a major
factor in making many biocontrol agents effective on
pome fruits. Studies at a biochemical and genetic level also
suggested that yeast extracellular enzymes (β-glucanases
and chitinases) that depolymerise fungal cell walls fill a role
388
in postharvest biocontrol of fungi (Janisiewicz and Korsten,
2002). Induction of resistance has also been hypothesised
to be a mechanism in the activity of postharvest biocontrol
agents (Droby and Chalutz, 1994); it has been described
for apples and citrus fruits, and it involves the production
of glucanases and chitinases, structural barriers such as
wall appositions, phytoalexins, and ethylene, and enhanced
activity of the enzyme phenylalanine ammonia lyase (Droby
et al., 2002; El Ghaouth et al., 2003). Studies of induced
resistance suggest that a deeper understanding of the
tritrophic interaction – plant tissue-pathogen-biocontrol
agent could also help in achieving enhanced antagonistic
activity and crop protection.
In general, yeasts and bacteria appear to be the predominant
colonisers early in the season; filamentous fungi tend to
appear later in the season and during storage. Microbial
populations with diverse habitats change over time,
and this also holds true for the epiphytic microflora on
fruits (Spurr, 1994). Many of these yeast genera harbour
biocontrol agents, and the composition of the mycoflora
will also depend on the host cultivar and on the climate.
The role of mycotoxins in the microbial ecology of the
fruit surface and fruit wounds is not clear, since research
on their activity and the definition of these compounds
usually address their toxicity to humans and animals.
However, it is possible that mycotoxins may play a direct
role in intermicrobial competition and survival, as had
been speculated with regard to the ecological role of
the phytotoxins (microbial toxins that inhibit plants)
that also exhibit antimicrobial activity (Castoria et al.,
2008). Biocontrol yeasts and bacteria that are to be used
to control mycotoxigenic fungi must be able to resist the
antimicrobial activity of a mycotoxin such as patulin.
Indeed, if a mycotoxin exhibits antimicrobial activity
and if its production continues from the time that the
mycotoxigenic fungus appears on the surfaces of leaves and
fruits in the field until and during the postharvest period,
then the biocontrol agent needs to overcome its toxicity
in order to establish itself.
The major challenges facing the postharvest implementation
of biological control based on antagonistic microorganisms
are of economic feasibility and the achievement of levels of
efficacy and reliability comparable with those provided by
chemical control. The integrated approach that combines
the biocontrol agents with reduced dosages of chemical
fungicides can actually contribute to meeting these
challenges.
To develop more efficient methods for controlling
postharvest mould contamination it is essential to elucidate
the relationship between infection of various plant parts in
the field, on the one hand, and the incidence of disease or
toxic contaminants, on the other hand. Control strategies
World Mycotoxin Journal 1 (4)
  Mouldy fruits and vegetables as a source of mycotoxins: part 2
should be developed based on a holistic approach that
includes modelling and prediction systems, early detection
techniques, biological and physical methods, and cultural
practices.
3. Means to prevent contamination with patulin,
ochratoxin A and Alternaria mycotoxins and
the effects of processing on these mycotoxins
The main factors affecting fungal growth and mycotoxin
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production in vivo include: environmental conditions,
e.g. water availability, temperature and photoperiod; the
composition and condition of the substrate, (i.e. in the case
of OTA in grapes, stage of maturation; competition among
the microorganisms present on the substrate; and fungal
factors, e.g. ability to colonise the substrate and to produce
the toxin). Controlling one or more of these factors may
contribute to the prevention of toxin formation.
Prevention of patulin contamination in fruit juice
The fungus P. expansum Link is the main organism
responsible for the production of patulin in apples, and
the toxin is considered also to be a natural contaminant
of apple juice. The optimum growth temperature for P.
expansum is near 25 °C and the optimum temperature for
patulin production is reported in the range of 23-25 °C.
However, there are reports on fungal growth at -3 °C
and patulin can be produced at 0-4 °C. Minimum water
activity (aw) for spore germination is 0.82-0.83. The Codex
Committee on Food Additives and Contaminants issued
a ‘Code of Practice for the Prevention and Reduction of
Patulin Contamination in Apple Juice and Apple Juice
Ingredients in other Beverages’ (CAC, 2003). Fruits received
by a factory during peak harvesting time are generally
stored for later processing and, therefore, measures to
reduce fungal growth and to delay postharvest decay during
storage are frequently applied. These objectives are best
achieved at low temperatures (<4 °C) and under modified
atmospheres, typically containing 3% CO2 and 2% O2.
However, even under these conditions, P. expansum can
grow and produce patulin to a limited extent, depending
on the fungal strain and on the apple cultivar involved.
Although organic vapours such as trans-2-hexenal have
been tested as fungus-control agents, the production
of off-flavours in some apple cultivars can restrict their
application. Trans-2-hexenal has similarly been shown to
be effective against P. expansum and patulin production in
‘Conference’ pears, although off-odours and off-flavours
are also a concern (Neri et al., 2006).
Patulin levels can rise during preprocessing storage if
apples are retained in deck storage for extended periods
of time, and the levels produced depend on uncontrollable
extraneous variables such as the initial presence of fungal
spores and the weather conditions over the storage period.
World Mycotoxin Journal 1 (4)
A portable drencher, capable of treating apples in single
storage bins, has been described and used in studies of
the possibility of controlling fungal decay by drenching
the fruits with 2% solutions of sodium bicarbonate, the
yeast Metschnikowia pulcherrima, or a combination of
both. The combination treatment was found to reduce
the incidence of fungal decay significantly. Natural food
ingredients that showed potential to control P. expansum
and/or reduce patulin levels included lemon and orange
oils, and hydrogen peroxide. Trimming of the rotten
tissue could not remove all of the toxin from the apple
since patulin can diffuse into whole apple tissue, but the
patulin concentration rapidly decreased to negligible levels
over a distance of 1 cm from the rot. Manual sorting and
trimming may assist in removing significant amounts of
patulin prior to its entry into the factory juice stream.
Although washing, sorting and trimming can significantly
reduce the levels of patulin that enter the later stages of
processing, they cannot alone achieve adequate removal of
patulin on an industrial scale. The basic factory operations
required to produce apple juice concentrate from washed
apples include: juicing and pressing, pasteurisation,
depectinisation, filtration, clarification and concentration
(Shephard and Vismer, 2008). Patulin levels are not
decreased during the first pressing of the mash that leaves
the milling stage, and a small decrease that was observed
at the second pressing could be related to dilution by the
water added to the pressing waste from the first stage.
The most significant decrease in patulin levels during
these trials occurred during rotary vacuum filtration and
clarification with gelatine and bentonite. Leggott et al.
(2000) showed that only the depectinisation/activated
charcoal/ultrafiltration step achieved a significant reduction
in patulin. The decrease was attributed to the action of the
activated charcoal. A carbon-based composite material has
been developed, in which activated carbon was bound to
granular quartz to produce a fixed-bed adsorption column
with good bed porosity, and its effectiveness in patulin
removal was demonstrated by Huebner et al. (2000).
The reactions of patulin with a number of naturally
occurring or added juice components have been extensively
documented. Patulin reacts readily with compounds
such as cysteine, thioglycolic acid and glutathione,
which contain sulphydryl groups. Indeed, the use of
sulphydryl-containing reducing agents such as cysteine
as a possible additive to cattle feed has been mooted as
a means to reduce the toxicity of contaminated hay or
silage to ruminants (Morgavi et al., 2003). Another well-
documented additive that reduces patulin levels is ascorbic
acid. Other compounds that have been shown to reduce
patulin levels in juices include the B vitamins, thiamine,
pyridoxine and calcium pantothenate (Yazici and Velioglu,
2002) and sulphur dioxide, which may be used as a juice
preservative (Steiner et al., 1999). The effectiveness of all
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 N. Paster and R. Barkai-Golan
these additives depends on their concentration, the storage
time, and the type of apple juice.
The chemical processes responsible for the disappearance
of patulin are complex and not fully understood, although
recent studies using 14C-labelled patulin have shown the
presence of two major metabolites, one of which was
identified as a reduction product, E-ascladiol, which is
presumed to be further degraded during fermentation
(Moss and Long, 2002). Although patulin is removed
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during fermentation, commercial ciders have been found to
contain patulin (Leggott and Shephard, 2001). The reasons
for this could lie in the use of extremely contaminated
fruit juice, in the use of alcohol-intolerant yeast strains
that cause arrested fermentation, or in the practice of
adding unfermented juice to adjust the sugar content and
flavour of the cider after fermentation. Patulin is a heat-
stable compound in acidic aqueous solutions (pH 3.5-5.5)
and, depending on temperature and storage time, it has
been shown to have considerable stability in apple juice.
The extent to which patulin loss is due to reaction with
the naturally present ascorbic acid or with compounds
containing sulphydryl groups is unknown.
Prevention of ochratoxin A contamination in wines
A. carbonarius has been proven to be the main producer
of OTA in grapes, and it is considered to be the main
source of OTA in wines and vine fruits, especially in
regions with hot and dry climates. In colder countries,
Penicillium species are considered to be the source of OTA
contamination. In vitro studies found that the optimum
growth of A. carbonarius occurred at 30-35 °C and aw of
0.98-0.99 whereas the most favourable ranges for OTA
production were 15-25 °C and 0.93-0.95, respectively.
However, differences among the optimal growth conditions
of different isolates were reported, and the conditions for
rapid or maximal growth are not necessarily the same
as those for maximal OTA production. The presence of
OTA in wine has been reported worldwide during the last
decade; red wines – especially European ones – have been
found to contain greater amounts of OTA than white wine
(Blesa et al., 2006; Zimmereli and Dick, 1996).
For OTA in grapes, the period between early veraison
and harvesting can be regarded as the critical period for
OTA accumulation, which varies according to the weather
during that period (Battilani et al., 2006). It is quite evident
that OTA contents in wines from the warmer, southern
regions are almost always higher than those in wines from
the cooler, northern growing areas. Production of OTA by
A. carbonarius was correlated positively with total acidity
and negatively with reducing sugars content. Differences
in fungal invasion of the grapes are related not only to the
climatic conditions, the agricultural practices and the stage
of maturation of the berries, but also to the condition of
390
the grapes and the aggressiveness of the fungi. Since OTA
may be a particularly severe problem in wine prepared
from poor-quality grapes, efforts should be devoted to
minimising injuries caused in the field, especially in the
period between veraison and harvest, and to trimming
the poor-quality or infested berries. Also, OTA may be
transferred from infested to healthy grapes, and may be
present in undamaged berries.
Biodegradation or removal of OTA by several bacteria,
yeasts and fungi has been reported and was attributed, in
several cases, to enzymatic activity. The microorganism
population present in grapes comprises a variety of fungal
species, including yeasts. Therefore, the interaction between
the components of that population is of interest, and studies
may throw light on the inhibition of ochratoxigenic fungi
and of OTA production by competition, and also on the
biodegradation of OTA. Xiao et al. (1996) reported that in
an Aspergillus ochraceus culture, OTA was biotransformed
into various metabolites, such as ochratoxin α, which lacks
the phenylalanine moiety and is less toxic than OTA.
Isolates of Aspergillus fumigatus and black Aspergilli could
eliminate the toxin from the medium (Varga et al., 2000).
The fate of OTA during the various steps of the wine-
making processes has been studied, especially with respect
to the effects of the yeasts used for fermentation and of the
materials applied to improve the wine quality. It was found
that during crushing and maceration, which are the initial
stages of the wine-making process, OTA was released to the
product. The rate of OTA reduction during fermentation
depends on the yeast used for fermentation, the yeast
concentration and the type of wine produced, (Bejaoui et
al., 2004). Since the level of OTA in the finished wine is
markedly lower than that found in the grapes, as a result
of toxin removal throughout the vinification processes,
and especially during the fermentation in which binding
to yeast cells occurs, monitoring the OTA level is of the
utmost importance in manufacturing unfermented grape
products.
Reduction of OTA during vinification is also attributed to
its binding to the solid parts of the grapes. Reduction of the
toxin occurs at each of the solid-liquid separation stages,
the toxin being discarded with the solid phase.
The efficiency of various clarifying technologies in reducing
OTA levels has been studied; microfiltration of red wine
through a 0.45-µm, but not through a 0.10-µm membrane,
decreased OTA concentration by about 80%. With regard
to OTA removal during wine fining, Leong et al. (2006)
noted that the differences between the wine matrix after
racking from the gross lees and the juice may affect the
efficacy of OTA removal by fining agents. Hydrophobic and
positively charged fining agents, and especially potassium
caseinate and activated carbon, were found to be the most
World Mycotoxin Journal 1 (4)
  Mouldy fruits and vegetables as a source of mycotoxins: part 2
efficient in removing the toxin from red wines. Treatment
with decolourising carbon significantly decreased OTA
concentrations. However, the use of carbon was associated
with decreases in some important aroma compounds of
wine. One major factor that limits the efficacy of OTA
binding to the added materials is the competition between
the toxin and other wine constituents, e.g. polyphenols
and proteins, for the binding sites present in the agent;
competition that might interfere with the removal of OTA
(Paster, 2008).
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The competitive interactions between OTA, fining agents,
and wine proteins and polyphenols are still not completely
clear and could be attributed, inter alia, to negative charge
in the cell walls and to the acidic nature of OTA. The
cell wall components include polysaccharides, proteins
and lipids, which provide sites for adsorption through a
hydrogen-bonding hydrophobic interaction.
It can be concluded that OTA contamination of grapes and
wines can be regarded as a worldwide problem. Differing
findings regarding the presence and amount of the toxin in
the produce arriving from a given area in different seasons
can be attributed to, among other factors, changes in
fungal populations, weather conditions and agricultural
management, which could affect fungal growth and toxin
production.
Prevention of Alternaria toxins
Alternaria species are among the most common
postharvest pathogens of fruits and vegetables. During
pathogenesis, several species are capable of producing
several mycotoxins including AOH, AME, tenuazonic
acid, altenuene and altertoxins. Although A. alternata
has been regarded as the major mycotoxin-producing
species, several other Alternaria species may also be
involved in toxin production. The production of Alternaria
mycotoxins under natural infection conditions or following
inoculation has been studied in a variety of fruits and
vegetables including: tomatoes, apples, grapes and dried
vine fruits, oranges, lemons, mandarins and olives. The
optimal temperature range for Alternaria growth is 22-
28 °C. However, Alternaria is also capable of growing at low
temperatures (0-3 °C) and may produce toxins in tomato
and apples at a wide range of temperatures. Although
the fungus can grow and produce mycotoxins at 0.86 aw,
optimum production of the toxins is at aw above 0.97.
Prevention of Alternaria toxins may be achieved by reducing
fungal infestation at the pre harvest phase and treatments
with fungicides were useful for disease control (e.g. in
citrus fruits). More studies are needed to better understand
the effect of fungicide applications on Alternaria toxins
accumulation. Trimming away rotten or mouldy fruits may
reduce the possibility of consuming toxins already present
World Mycotoxin Journal 1 (4)
in the produce. However, in some apple cultivar, where
Alternaria rot may occur in the apple core, contaminated
fruits cannot be identified (Barkai-Golan, 2008). Methods
should be developed to detect fungal presence in the core of
fruits. The storage period and temperature may influence
the level of toxins produced. A. alternaria growth and toxin
production in tomatoes can be controlled if the fruits are
stored below 7 °C for less than 10 days. Decrease in the
concentration of AOH and AME in Alternaria-infected
tomatoes was recorded during the course of storage. It was
suggested that some degradation of toxins may occur after
prolonged storage, especially at 25 °C. Tomatoes which
were shrink-wrapped with high-density polyethylene film,
contained less toxins than unpackaged tomatoes. The
natural occurrence of Alternaria toxins have been reported
in many fruits and vegetables products (e.g. juices, nectars
and wines). Furthermore, in apple juice, AOH and AME
were shown to be stable when heated at 80 °C for 20 min
and in the presence of vitamin C. It seems that, in order to
reduce the level of Alternaria toxins in the final product,
decayed fruits (in the case where they can be identified)
should not be taken for processing (Jackson and Al-Taher,
2008).
4. Regulations, limits and economic aspects of
mycotoxins in fruits vegetables
All levels of government and food industries have been
involved in a global effort to develop and implement safe
food handling throughout the food chain. Three major
components are involved in these endeavours: Good
Agricultural Practices, Good Manufacturing Practices,
and Hazard Analysis Critical Control Points (HACCP)
(Dombrink-Kurtzman, 2008). Many foods can become
contaminated with mycotoxins before they are harvested,
as well as between harvesting and drying, and during
storage. Analysis is applied only to certain select products
likely to contain mycotoxins, such as peanuts, corn,
grapes, and dried fruits that are likely to contain aflatoxin
(Jelinek, 1987). The Rapid Alert System for Food and
Feed (RASFF), operated by the European Commission,
provides control authorities in the EU with an effective
tool for exchange of information on measures taken to
ensure food safety. The RASFF received in 2006 a total of
874 notifications on mycotoxins, of which 802 concerned
aflatoxins (most of these notifications concerned pistachio
nuts primarily originating from Iran). Within the group
of fruit and vegetables, 57 notifications (on aflatoxin
presence) concerned dried figs and derived products
primarily originating from Turkey, 77 notifications
concerned mycotoxins other than aflatoxins. The large
majority of notifications concerned OTA (mainly in
dried vine fruits) and to a much lesser extent fumonisins
(in maize products) and patulin (in concentrated apple
juice). There were 745 notifications on mycotoxins in 2007,
of which 705 concerned aflatoxins (63 concerned dried
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 N. Paster and R. Barkai-Golan
figs and derived products). In that year, 51 notifications
concerned mycotoxins other than aflatoxins, the majority
of which concerned OTA (including OTA in dried vine
fruits and figs). A large updated body of food and feed
legislation regarding food safety took effect in the EU on
1 January 2006. This legislation applies both within the EU
and in non-EU countries planning to export to Europe.
Such countries must guarantee that their products meet
the standards required for imports by the EU. For many
vegetables there is little information available on the extent
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of mycotoxin contamination that occurs. With regard to
fruits, however, patulin in apples and pears, and OTA in
grapes are the main mycotoxins currently considered to
be important in fruit products.
Regulations and limits for mycotoxins in fruits and
vegetables
Regulations of mycotoxins have been established in many
countries to protect the consumer from the harmful effects
of mycotoxins. In 2004 approximately 100 countries were
known that had specific limits for various combinations
of mycotoxins and commodities, often accompanied by
prescribed or recommended procedures for sampling
and analysis (FAO, 2004). Many of these countries had
specific regulations and limits for mycotoxins in fruits and
vegetables. Several factors may influence the establishment
of mycotoxin limits and regulations (Van Egmond and
Jonker, 2008). These are also valid for fruits and vegetables.
They include:
• availability of toxicological data on mycotoxins;
• availability of exposure data on mycotoxins;
• knowledge of the distribution of mycotoxin
concentrations within a lot;
• availability of analytical methods;
• legislation in other countries with which there are trade
contacts;
• need for a sufficient food supply.
Regulations are primarily imposed in the light of known toxic
effects. With regard to the mycotoxins currently considered
most significant (aflatoxins, ochratoxin A, patulin,
fumonisins, zearalenone, and some of the trichothecenes,
including deoxynivalenol), the Joint Expert Committee
on Food Additives (JECFA), a scientific advisory body
of the World Health Organization (WHO) and the Food
and Agriculture Organization (FAO) (2004) of the United
Nations (UN), has devoted several sessions to evaluating
their hazards. In addition to information about toxicity,
exposure assessment is another main component of the risk
assessment. Reliable data on the occurrence of mycotoxins
in various commodities and on food intake are needed for
exposure assessment, but the quantitative evaluation of
mycotoxins intake is quite difficult. In its 56th meeting,
JECFA stressed the importance of the use of validated
analytical methods and the application of analytical quality
392
assurance to ensure that the results of surveys provide a
reliable assessment of intake (WHO, 2002).
Many countries pursue activities that contribute to the
mycotoxins risk assessment process. The distribution of the
concentration of mycotoxins in products is an important
factor to be considered in establishing regulatory sampling
criteria; if insufficient care is taken to ensure representative
sampling, the mycotoxin concentration in an inspected lot
may be wrongly estimated. Reliable analytical methods must
be available, to facilitate enforcement of the regulations
in daily practice. In addition to reliability, simplicity is
desirable with regard to minimising the amount of data
generated and enhancing the practicability of the measures
ultimately taken. A very important aspect, especially in
free-trade areas, is how enforcement bodies handle an issue
such as measurement uncertainty. European legislation
for mycotoxins requires that ‘The analytical result must
be reported as x/U whereby x is the analytical result and
U is the expanded measurement uncertainty’ (EC, 2006;
Stroka and Van Egmond, 2006). However, so far, no detailed
guidance on how to estimate measurement uncertainty
has been provided. Preferably, regulations in each country
should be brought into harmony with those in force in
other countries with which it maintains trade contacts.
Strict regulatory actions may lead importing countries to
ban or limit the import of specific commodities, which
could cause difficulties for exporting countries in finding or
maintaining markets for their products. The distortion of
the market caused by mycotoxin regulations in importing
countries could lead to export of only the less contaminated
foods and feeds, with inferior foods and feeds being left
for the local market, and the exporting countries suffering
severe financial losses (Wilson and Otsuki, 2001). Weighing
the various factors at the interface of science, food security
and regulatory activities is not a trivial matter, and
common sense is a major factor in reaching decisions. On
a worldwide basis, at least 99 countries operated mycotoxin
regulations for food and/or feed in 2003.
The category of fruits and vegetables to which mycotoxin
regulations are applied, is broadly defined; it includes nuts,
processed foodstuffs such as juices, nectars, baby food and
other fruit-derived products, as well as spices, many of
which originate from vegetables. Ninety-three countries
have formulated regulations for this category of food. It is
obvious that patulin is the most regulated mycotoxin; it is
subject to approximately 160 different regulations, in some
50 countries; it is regulated not only in apples and their
products, but also in berries, tomato paste, mushrooms,
(canned) vegetables, fermented drinks, baby food, and jams,
confitures and syrups. Within the Codex Alimentarius
Commission, which aims to facilitate world trade and to
protect the health of consumers through the development
of international standards for foods and feeds, the Codex
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  Mouldy fruits and vegetables as a source of mycotoxins: part 2
Committee on Food Additives and Contaminants (CCFAC)
established a standard for patulin in apple juice in 2003.
The CCFAC has also developed a code of practice for the
prevention and reduction of patulin in apple juice and in
apple juice-derived components in other beverages (CAC,
2003).
There are also quite numerous regulations for aflatoxins
and OTA, and several other mycotoxins form the subject
of occasional specific regulations. For OTA in fruits and
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vegetables the limits range from 10 to 50 mg/kg with
10 mg/kg occurring most frequently. The significance
of OTA contamination at levels around the legal limits
remains difficult to estimate: it was the subject of a
workshop conducted by ILSI Europe in 2005 (Hazel and
Walker, 2005), entitled ‘Ochratoxin A in food: recent
developments and significance’. At this workshop it was
concluded that attempts to characterise the risk associated
with OTA exposure were handicapped by significant
gaps in the current knowledge needed to make this risk
assessment possible. In contrast to the aflatoxins and OTA,
no carcinogenic properties have been attributed to patulin
but, nevertheless, this pre-eminent ‘fruit-mycotoxin’ is
regulated by many countries, with limits ranging from 5
to 50 mg/kg. The vast majority of the 50 countries that
impose regulations or guideline levels in relation to patulin
in fruits and vegetables apply a limit of 50 mg/kg.
Economic aspects of mycotoxins in fruits and vegetables
As the world’s largest food importer, Europe has a
strong relationship with Latin America. The EU imports
approximately 45% of the agricultural exports from Latin
America, and 15% of Latin America’s exports in 2003 were
from Central America (South Centre, 2005). To ensure
the safety of its imports and to provide these countries
with in-depth knowledge of EU import rules for fruits
and vegetables, the EU organised workshops specifically
for third-world countries, to ensure safety of EU imports.
The training focused on ensuring that fruit and vegetable
imports from third-world countries meet EU standards.
The United States of America imports more than 12%, or
$58 billion-worth, of its food (South Centre, 2005), and
approximately 20% of its fruits and vegetables come from
Central and South America. In the past several years, there
has been a fivefold increase in the number of food products
entering the US market.
In order to determine the economic effects of mycotoxins
in fruits and vegetables, it is necessary to have access
to detailed comparative information about the cost
structures of domestic and foreign industries (Roberts
and Krissoff, 2004). Unfortunately, this information is not
readily available, which makes it difficult to determine
the net effect of food safety measures on horticultural
trade patterns. Exporters in countries that do not require
World Mycotoxin Journal 1 (4)
HACCP for products in the domestic market must
either invest financially in risk-reduction technology, in
order to maintain access to the USA and EU markets,
or divert their products to countries without HACCP
requirements (Roberts and Krissoff, 2004). Contamination
with mycotoxins is recognised as an unavoidable risk, but
it is difficult to assess the economic losses associated
with mycotoxin contamination. Rather than regulating
the treatment of a commodity during processing, the
authorities usually set a tolerance level for the amount
of mycotoxin in the final product. Mycotoxin concerns
in developing countries are more commonly expressed
in relation to cereals, nuts and alcoholic beverages, and
usually from a technical or a health perspective rather than
from an economic perspective. The FAO has performed
extensive studies of mycotoxins in developing countries,
but the economic aspects are rarely examined.
The most important benefits of food safety regulations
are those associated with improvements in human health,
including reductions in medical costs, productivity losses
and risks of foodborne illness, enhanced food safety, and
improved nutrition. Costs resulting from regulation include
those caused when companies change production processes
and make investments: these affect the unit cost of
production. HACCP was mandated by federal regulation in
2001, but specific HACCP plans are not mandated, because
the individual industries formulate their own programmes
(Unnevehr and Jensen, 2005). At present, both foreign and
domestic suppliers of produce to major US supermarket
chains must certify food safety practices (Calvin and Cook,
2001). The EU is the world’s largest importer of fruits and
vegetables, and currently applies marketing standards to 36
different fruits and vegetables (Elinder, 2003). In June 2004
the US Food and Drugs Administration (FDA) proposed an
Action Plan to minimise foodborne illness associated with
fresh produce consumption. This plan had four general
objectives: (1) to prevent contamination of fresh produce
with pathogens; (2) to minimise the public health impact
when contamination occurs; (3) to improve communication
about fresh produce, between the FDA and producers,
packers, processors, transporters, distributors, preparers
and consumers; and (4) to facilitate and support research
relevant to contamination in fresh produce. Fruit producers
and processors must rely on risk reduction rather than risk
elimination, because it is not currently feasible to eliminate
all potential hazards associated with fresh produce.
5. Conclusions
Mycotoxins still pose a serious threat to human and animal
health throughout the world. Since mycotoxins can be
produced during both the preharvest and the postharvest
phases, combating the problem should start in the field,
and thereby prevent the entry of contaminated produce
into the storage or processing facilities.
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 N. Paster and R. Barkai-Golan
During the last decades studies have been dedicated to the
circumstances under which mycotoxigenic fungi infect
fruits and vegetables, and produce mycotoxins, so that
conditions can be made unfavourable for toxin production.
For instance, the knowledge that surface defects on apples
are important for fruit infection by the wound pathogen
P. expansum, and that chilling injury in tomatoes stored
below 8-12 °C and sweet peppers stored below 7 °C exhibit
increased sensitivity to Alternaria infection, can help in
devising means to minimise such infections. Similarly,
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various preharvest, harvest and postharvest factors that
affect the growth of A. carbonarius will affect infection
of grapes, and the consequent production of OTA.
Among the methods intended for suppressing mycotoxin
contamination in fruits and vegetables, special prominence
should be given to those capable of reducing mould
development. Application of these methods forms the
initial stages in the control of mycotoxigenic fungi and
toxin formation.
Means to eliminate or detoxify mycotoxins that are
already present in the fruits or vegetables are not yet
widely implemented. Therefore, preventing mycotoxins
necessitates controlling the mycotoxigenic fungi in the field
or in storage, and chemical control is still the main way
to reduce the incidence of fungi. However, the worldwide
tendency to reduce the application of chemicals and,
especially, the development of fungal strains resistant to
the applied fungicides have accelerated and intensified
the search for alternative means. Physical means, use of
biological agents, and the application of natural products
are among the non-chemical strategies which have been
thoroughly studied. However, use of any one of these
means alone has achieved only limited success in reducing
fungal damage, whereas integration of several means has
been more successful. Future studies intended to lead to
the application of non-chemical means should, therefore,
focus on ‘integrated means’ strategies that seem to be
promising.
Patulin and OTA are the most studied of the mycotoxins
that appear in fruits. P. expansum has been recognised
as the main patulin producer in apples, whereas OTA in
grapes has been attributed mainly to the presence of A.
carbonarius, in hot and dry regions. Models for predicting
the appearance of A. carbonarius, based mainly on
meteorological data, have been developed. Such models are
needed for use against other mycotoxigenic fungi, and they
could undoubtedly contribute to a better understanding of
fungal behaviour, and to the identification of regions with
high risks for presence of mycotoxins in the produce.
Most mycotoxins are highly stable during industrial food
processing, and may appear in the final products. Therefore,
studies have been aimed at eliminating them before they
enter, or during the processing. Indeed, trimming away
394
damaged or rotten produce was found to significantly
reduce patulin and OTA levels in the final product.
However, as regards to Alternaria toxins, the damage
is sometimes hidden, as in apples, in which Alternaria
may occur in the core of the fruit. The development and
application of means to identify and reject rotten fruits,
even when they are not contaminated with mycotoxins,
should be accelerated. Several compounds which react with
patulin and could be used for reducing its level during apple
processing have been identified. Similarly, reduction of
OTA during vinification has been achieved through the
binding of the toxin to various constituents or additives.
More studies that lead to better understanding of the
mechanisms involved in mycotoxin elimination through
binding, destruction, etc., will elucidate the mechanisms
involved, and should assist in development of a rationale
for mycotoxin elimination during processing. However,
each of the procedures that may be adopted should
conform to some crucial criteria such as not altering the
organoleptic properties of the product, and not generating
toxic residues.
Despite the efforts to eliminate the presence of mycotoxins,
they are still recognised as an unavoidable risk. As a result,
there is a need to develop and implement working systems
which will minimise the risks presented by the presence
of mycotoxins in the produce. Examples of such systems
are: Good Agricultural Practices, Good Manufacturing
Practices, and Hazard Analysis and Critical Control
Points.
In addition to the formulation of appropriate working
systems, regulations and limits for mycotoxin concentrations
have been established, and they are strictly imposed in
many countries. These regulation are based mainly on risk
assessment, but also on other factors such as availability
of analytical methods and needs for food supplies. It
seems likely that efforts will continue at various levels,
e.g. national, food industry, to collect data on mycotoxin
accumulation in the final products, in order to create an
international database which will assist in evaluating the
systems for reducing mycotoxin levels and in assessing the
utility of the existing regulations.
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