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8COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 1, 2005 © 2005 Institute of Food Technologists
Comprehensive
Review of
Patulin Control
Methods in
Foods
Matthew M. Moake, Olga I. Padilla-Zakour,
and Randy W. Worobo
ABSTRAABSTRA
ABSTRAABSTRA
ABSTRACTCT
CTCT
CT: :
: :
:
The myThe my
The myThe my
The mycotocoto
cotocoto
cotoxin, patulin (4-hyxin, patulin (4-hy
xin, patulin (4-hyxin, patulin (4-hy
xin, patulin (4-hydrdr
drdr
droo
oo
oxyxy
xyxy
xy-4H-fur-4H-fur
-4H-fur-4H-fur
-4H-furo [3,2c] po [3,2c] p
o [3,2c] po [3,2c] p
o [3,2c] pyryr
yryr
yran-2[6H]-one), is pran-2[6H]-one), is pr
an-2[6H]-one), is pran-2[6H]-one), is pr
an-2[6H]-one), is produced boduced b
oduced boduced b
oduced by a number of fungi com-y a number of fungi com-
y a number of fungi com-y a number of fungi com-
y a number of fungi com-
mon to frmon to fr
mon to frmon to fr
mon to fruit- and vuit- and v
uit- and vuit- and v
uit- and vegetable-based pregetable-based pr
egetable-based pregetable-based pr
egetable-based productsoducts
oductsoducts
oducts, most notably apples, most notably apples
, most notably apples, most notably apples
, most notably apples. D. D
. D. D
. Despite patulinespite patulin
espite patulinespite patulin
espite patulin’’
’’
’s ors or
s ors or
s original discoiginal disco
iginal discoiginal disco
iginal discovv
vv
verer
erer
ery as an antibiotic, ity as an antibiotic, it
y as an antibiotic, ity as an antibiotic, it
y as an antibiotic, it
has come under heavy scrutiny for its potential negative health effects. Studies investigating these health effects havehas come under heavy scrutiny for its potential negative health effects. Studies investigating these health effects have
has come under heavy scrutiny for its potential negative health effects. Studies investigating these health effects havehas come under heavy scrutiny for its potential negative health effects. Studies investigating these health effects have
has come under heavy scrutiny for its potential negative health effects. Studies investigating these health effects have
proved inconclusive, but there is little doubt as to the potential danger inherent in the contamination of food products byproved inconclusive, but there is little doubt as to the potential danger inherent in the contamination of food products by
proved inconclusive, but there is little doubt as to the potential danger inherent in the contamination of food products byproved inconclusive, but there is little doubt as to the potential danger inherent in the contamination of food products by
proved inconclusive, but there is little doubt as to the potential danger inherent in the contamination of food products by
patulin. The danger posed by patulin necessitates its control and removal from food products, creating a demand forpatulin. The danger posed by patulin necessitates its control and removal from food products, creating a demand for
patulin. The danger posed by patulin necessitates its control and removal from food products, creating a demand forpatulin. The danger posed by patulin necessitates its control and removal from food products, creating a demand for
patulin. The danger posed by patulin necessitates its control and removal from food products, creating a demand for
handling and prhandling and pr
handling and prhandling and pr
handling and processing techniques capable of doing soocessing techniques capable of doing so
ocessing techniques capable of doing soocessing techniques capable of doing so
ocessing techniques capable of doing so, pr, pr
, pr, pr
, preferefer
eferefer
eferably at loably at lo
ably at loably at lo
ably at low cost to industrw cost to industr
w cost to industrw cost to industr
w cost to industryy
yy
y. .
. .
.
WW
WW
With this being the caseith this being the case
ith this being the caseith this being the case
ith this being the case, much, much
, much, much
, much
research has been devoted to understanding the basic chemical and biological nature of patulin, as well as its interactionresearch has been devoted to understanding the basic chemical and biological nature of patulin, as well as its interaction
research has been devoted to understanding the basic chemical and biological nature of patulin, as well as its interactionresearch has been devoted to understanding the basic chemical and biological nature of patulin, as well as its interaction
research has been devoted to understanding the basic chemical and biological nature of patulin, as well as its interaction
within foods and food prwithin foods and food pr
within foods and food prwithin foods and food pr
within foods and food production. oduction.
oduction. oduction.
oduction.
While past rWhile past r
While past rWhile past r
While past researesear
esearesear
esearch has elucidated a grch has elucidated a gr
ch has elucidated a grch has elucidated a gr
ch has elucidated a great deal, patulin contamination continues to beeat deal, patulin contamination continues to be
eat deal, patulin contamination continues to beeat deal, patulin contamination continues to be
eat deal, patulin contamination continues to be
a challenge for the food industra challenge for the food industr
a challenge for the food industra challenge for the food industr
a challenge for the food industryy
yy
y. H. H
. H. H
. Herer
erer
eree
ee
e, w, w
, w, w
, we re r
e re r
e review in depth the past review in depth the past r
eview in depth the past review in depth the past r
eview in depth the past researesear
esearesear
esearch on patulin with an emphasis upon its influencech on patulin with an emphasis upon its influence
ch on patulin with an emphasis upon its influencech on patulin with an emphasis upon its influence
ch on patulin with an emphasis upon its influence
within the food industrwithin the food industr
within the food industrwithin the food industr
within the food industry
y
yy
y, including its r, including its r
, including its r, including its r
, including its regulation, health effectsegulation, health effects
egulation, health effectsegulation, health effects
egulation, health effects, biosynthesis, biosynthesis
, biosynthesis, biosynthesis
, biosynthesis, detection, quantification, distr, detection, quantification, distr
, detection, quantification, distr, detection, quantification, distr
, detection, quantification, distribution withinibution within
ibution withinibution within
ibution within
foodsfoods
foodsfoods
foods, and contr, and contr
, and contr, and contr
, and control, durol, dur
ol, durol, dur
ol, during the ving the v
ing the ving the v
ing the varar
arar
arious stages of apple juice prious stages of apple juice pr
ious stages of apple juice prious stages of apple juice pr
ious stages of apple juice production. Foduction. F
oduction. Foduction. F
oduction. Finallyinally
inallyinally
inally, key ar, key ar
, key ar, key ar
, key areas whereas wher
eas whereas wher
eas where future futur
e future futur
e future patulin re patulin r
e patulin re patulin r
e patulin researesear
esearesear
esearchch
chch
ch
should focus to best control the patulin contamination problem within the food industry are addressed.should focus to best control the patulin contamination problem within the food industry are addressed.
should focus to best control the patulin contamination problem within the food industry are addressed.should focus to best control the patulin contamination problem within the food industry are addressed.
should focus to best control the patulin contamination problem within the food industry are addressed.
Introduction
Patulin is a mycotoxin produced by a number of fungi common
to fruit- and vegetable-based products, most notably apples. Ap-
ples are the 3rd most important fruit crop in the United States after
citrus fruits and grapes, with 40% of apples being used for juice
and other processed products (USDA 2002). Patulin contamina-
tion within apple products poses a serious health risk to consum-
ers, particularly children whom have been shown by a USDA sur-
vey to consume increased levels of apple products during the 1st
y of life (6.4 g/kg body weight/d), compared with adults (1 g/kg
bw/d) (Plunkett and others 1992), placing them at increased risk
for patulin toxicity. The health risks posed by patulin necessitate
its control and removal from apple products, creating a demand
for food-processing techniques capable of doing so, preferably at
low cost to industry. Here, we review past research devoted to the
understanding and control of patulin, with an emphasis upon pat-
ulin’s influence within the food industry.
History and Regulation
Patulin (4-hydroxy-4H-furo [3,2c] pyran-2[6H]-one), Figure 1,
is a water-soluble lactone 1st isolated as an antibiotic during the
1940s (Stott and Bullerman 1975). Owing to co-discovery of the
compound by various groups, it has historically been known by
names such as clavacin (Anslow and others 1943), expansine
(Van Luijk 1938), claviformin (Chain and others 1942), clavatin
(Bergel and others 1943), gigantic acid (Philpot 1943), and myo-
cin C (DeRosnay and others 1952). Initially isolated as a broad-
spectrum antifungal antibiotic (Korzybski and others 1976), it was
later found to inhibit more than 75 different bacterial species in-
cluding both Gram-positive and Gram-negative bacteria (Ciegler
and others 1971). Continued studies on the application of the
compound (Raistrick and others 1943), later found to be unsup-
ported by clinical studies (Medical Research Council 1944; Stans-
field and others 1944), suggested patulin to have applications in
treatment of nasal congestion and the common cold. Not long af-
ter, various studies suggested patulin to be not only toxic to fungi
and bacteria but also to animals (see below, Health Effects) and
higher plants, including cucumber, wheat, peas, corn, and flax (Iy-
engar and Starky 1953; Norstadt and McCalla 1963, 1968;
Berestets’kyi and Synyts’kyi 1973)
As continued evidence for the negative health effects of patulin
surfaced, many regulatory agencies began placing caps on patu-
lin content within foods. European countries were among the 1st
to address the concern, and today many countries across the
world, particularly those within the EU, limit allowable patulin
content within foods at 50 g/L (Moller and Josefsson 1980; van
Egmond 1989; AIJN 1990; Stoloff and others 1991; Rovira and
others 1993; Forbito and Babsky 1996; Gokmen and Akar 1998).
MS 20040335 Submitted 5/21/04, Revised 8/17/04, Accepted 10/18/04.
Authors are with Dept. of Food Science and Technology, New York State
Agricultural Experiment Station, Cornell Univ., Geneva, NY 14456-0462.
Direct inquiries to author Worobo (E-mail: rww8@cornell.edu).
Vol. 1, 2005—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 9
Patulin control in foods . . .
While 50 g/L is now the norm for patulin regulation, several
countries have set even lower limits for patulin at 25 to 35 g/L
(van Egmond 1989). In conjunction with these maximum content
standards, a joint Food and Agriculture Organization-World
Health Organization (WHO) expert committee established a provi-
sional maximum daily intake of 0.4 g/kg body weight for patulin
(WHO 1995). The United States has been much slower to set reg-
ulation on patulin, but today the U.S. Food and Drug Administra-
tion limits patulin to 50 g/L in single-strength and reconstituted
apple juices (USFDA 2004). The limitation of these regulations to
apple juice and apple juice concentrate was likely based upon
the fact that, at the time, only apple juice and cider had been
found to be naturally contaminated by patulin (WHO 1990).
While this fact has since been disproved (see below, Patulin With-
in Foods), apple juice and cider remain the major source of hu-
man patulin consumption.
Health Effects
Assessment of the health risks posed by patulin to humans is
based upon a wide number of studies during the past 50-plus
years that implicate a number of acute, chronic, and cellular level
health effects as summarized in Table 1. Results of these studies
are, as a whole, inconclusive, but suggest that acute symptoms of
patulin consumption can include agitation, convulsions, dyspo-
nea, pulmonary congestion, edema, ulceration, hyperemia, GI
tract distension, intestinal hemorrhage, epithelial cell degenera-
tion, intestinal inflammation, vomiting, and other gastrointestinal
and kidney damage (Walker and Wiesner 1944; Escoula and oth-
ers 1977; Hayes and others 1979; McKinley and Carlton 1980a,
1980b; McKinley and others 1982; Mahfoud and others 2002).
Chronic health risks of patulin consumption can include neuro-
toxic, immunotoxic, immunosuppressive, genotoxic, teratogenic,
and carcinogenic effects (Dickens and Jones 1961; Mayer and
Legaror 1969; Ciegler and others 1976; Oswald and others 1978;
Korte 1980; Thust and others 1982; Lee and Röschenthaler 1986;
Roll and others 1990; Hopkins 1993; Pfeiffer and others 1998;
Wichmann and others 2002).
The basis for these various toxic effects appears to be mixed.
On a cellular level, patulin has been shown to have effects includ-
ing plasma membrane disruption (Riley and Showker 1991; Mah-
foud and others 2002), inhibition of protein synthesis (Hatez and
Gaye 1978; Miura and others 1993; Arafat and Musa 1995), inhi-
bition of Na+-coupled amino acid transport (Ueno and others
Figure 1—The structure of
patulin (4-hydroxy-4H-furo
[3,2c] pyran-2[6H]-one)
Table 1—The health effects of patulin
Acute symptom Source
Agitation, convulsions, dysponea, pulmonary congestion, Escoula and others 1977; Hayes and others 1979
edema, hyperemia, GI tract distension
Nausea Walker and Wiesner 1944
Epithelial cell degeneration, intestinal hemorrhage Mahfoud and others 2002
Intestinal inflammation McKinley and Carlton 1980a, 1980b; McKinley and others
1982; Mahfoud and others 2002
Ulceration Escoula and others 1977; Hayes and others 1979; McKinley and Carlton 1980a,
1980b; McKinley and others 1982; Mahfoud and others 2002
Chronic symptom Source
Genotoxic Mayer and Legaror 1969; Korte 1980; Thust and others 1982; Lee and Roschenthaler
1986; Roll and others 1990; Hopkins 1993; Pfeiffer and others 1998
Neurotoxic Hopkins 1993
Immunotoxic Hopkins 1993; Wichmann and others 2002
Immunosuppressive Wichmann and others 2002
Teratogeneic Ciegler and others 1976; Roll and others 1990
Carcinogenic Dickens and Jones 1961; Oswald and others 1978
Cellular level effect Source
Plasma membrane disruption Riley and Showker 1991; Mahfoud and others 2002
Protein synthesis inhibition Hatez and Gaye 1978; Miura and others 1993; Arafat and Musa 1995
Transcription disruption, translation disruption Moule and Hatey 1977; Arafat and others 1985; Lee and Roschenthaler 1987
DNA synthesis inhibition Cooray and others 1982
Na-coupled amino acid transport inhibition Ueno and others 1976
Interferon-␥ production inhibition Wichmann and others 2002
RNA polymerase inhibition Moule and Hatey 1977
Aminoacyl-tRNA synthetases inhibition Arafat and others 1985
Na-K ATPase inhibition Phillips and Hayes 1977, 1978; Riley and Showker 1991
Muscle aldolase inhibition Ashoor and Chu 1973
Urease inhibition Reiss 1977
Loss of free glutathione Burghardt and others 1992; Barhoumi and Burghardt 1996
Protein crosslink formation Fliege and Metzler 1999
Protein prenylation inhibition Miura and others 1993
10 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 1, 2005
CRFSFS: Comprehensive Reviews in Food Science and Food Safety
1976), disruption of transcription and translation (Moule and
Hatey 1977; Arafat and others 1985; Lee and Roschenthaler
1987), inhibition of DNA synthesis (Cooray and others 1982),
and inhibition of interferon-␥–producing T-helper type 1 cells
(Wichmann and others 2002). Patulin is believed to cause cell
damage by forming adducts with thiol-containing cellular compo-
nents such as glutathione and cysteine-containing proteins (Har-
wig and others 1973a; Barhoumi and Burghardt 1996). Indeed,
many enzymes with a sulfhydryl group in their active site are sen-
sitive to patulin. Na+-K+–dependent ATPase (Phillips and Hayes
1977, 1978; Riley and Showker 1991), RNA polymerase (Moule
and Hatey 1977), aminoacyl-tRNA synthetase (Arafat and others
1985), and muscle aldolase (Ashoor and Chu 1973) have all
been shown to be inhibited by patulin. Furthermore, loss of free
glutathione in living cells is associated with patulin exposure
(Burghardt and others 1992; Barhoumi and Burghardt 1996), and
treatment with exogenous cysteine and glutathione prevented pat-
ulin toxicity within intestinal epithelium (Mahfoud and others
2002). Patulin has also been shown to induce intra- and intermo-
lecular protein cross-links. This reaction is preferential with the
thiol group of cysteine, but also occurs with the side chains of
lysine and histidine, and ␣-amino groups (Fliege and Metzler
1999). Other observations have also noted patulin’s reactivity
with NH2 groups (Lee and Roschenthaler 1986). Similarly, patulin
has been shown to inhibit protein prenylation, a necessary post-
translational protein modification involved in the activation of
many proteins, including numerous oncogenes, such as Ras, that
must be prenylated for proper function (Miura and others 1993).
However, some enzymes, such as urease, which lack a sulfhydryl
group in their active site, are also sensitive to patulin (Reiss 1977).
Finally, patulin’s inhibition of transcription and translation ap-
pears to be through direct interaction with RNA and DNA (Moule
and Hatey 1977; Arafat and others 1985; Lee and Roschenthaler
1987). Thus, while patulin toxicity may result from thiol-related in-
teraction in a number of cases, there appear to be exceptions to
this rule.
Biosynthesis
Patulin is produced by 60-plus species of mold encompassing
over 30 genera (Lai and others 2002). Included within these are
Penicillium
expansum
(
P.
leucopus
),
P.
patulum
(
P.
urticae
,
P.
griseofulvum
),
P.
crustosum
,
P.
roqueforti
,
P.
claviforme,
Paecilo-
myces
spp.
,
Saccharomyces
vesicarium,
Alternaria
alternata,
Bys-
sochlamys
nivea
,
B.
fulva
,
Aspergillus
giganteus
,
A.
terreus
, and
A.
clavatus
(Lovett and others 1974; Rice and others 1977; Harwig
and others 1978; Northolt and others 1978; Draughon and Ayres
1980; Ough and Corison 1980; Roland and Beuchat 1984a; Ay-
tac and Acar 1994; Laidou and others 2001; Moss and Long
2002). Patulin production in culture has been shown to occur
when growth rate diminishes because of limitations on cell
growth such as nitrogen consumption (Grootwassink and Gauch-
er 1980).
Patulin biosynthesis is well understood and involves a series of
condensation and redox reactions, many, if not all, of which are
enzyme catalyzed. Studies based primarily upon kinetic pulse-ra-
diolabeling and cell-free extract systems have elucidated the patu-
lin metabolic pathway in great detail; however ongoing studies on
the subject continue to produce new intermediaries in the path-
way. Patulin is a polyacetate-derived secondary metabolite or
polyketide (Turner 1976). Its synthesis is initiated with acetyl co-
enzyme A (CoA) and 3 units of malonyl CoA, making it a tetra-
ketide (Ciegler and others 1971; Turner 1976; Steyn 1992). Acetyl
CoA and 3 malonyl-CoA are condensed into 6-methylsalicylic
acid (6-MSA) by a 760000-Dalton homotetramer enzyme called
6-methylsalicylic acid synthetase (6-MSA synthetase)(Gaucher
1975; Lynen and others 1978). The gene encoding for 6-MSA has
been cloned and characterized from
P.
patulum
and
P.
urticae
(Beck and others 1990; Wang and others 1991). Inactivation of 6-
MSA synthetase has been shown to be the 1st limitation on patu-
lin production (Neway and Gaucher 1981). 6-MSA synthetase
loss is a selective process because the highly similar fatty acid
synthetase of
P.
urticae
(Lynen and others 1978) is stable under
the same reaction conditions that inactivate 6-MSA synthetase
(Lam and others 1988). This finding is further verified by studies in
which treatment of 6-MSA synthetase reaction mixtures, contain-
ing Nicotanimide Adenine Dinucleotide Phosphate (NADPH) co-
factor, acetyl-CoA, and malonyl CoA, with the reducing agent,
dithiothreitol, and proteinase inhibitor, phenylmethylsulfonyl fluo-
ride, stabilized 6-MSA synthetase. This suggests proteolysis and
conformational integrity play a role in the regulation of 6-MSA
synthetase (Lam and others 1988).
The next stage of patulin biosynthesis involves the conversion
of 6-MSA into m-cresol via the activity of 6-MSA decarboxylase
(Lam and others 1988). M-cresol is then converted into m-hy-
droxybenzyl alcohol by m-cresol 2-hydoxylase (Murphy and
Lynen 1975). The next step in patulin’s biosynthetic pathway is
debated among 2 main mechanisms. Both agree that m-hydroxy-
benzyl alcohol is eventually converted to gentisaldehyde (Forrest-
er and Gaucher 1972; Zamir 1980), however the intermediary be-
tween these 2 compounds is believed to be either gentisyl alcohol
(Sekiguchi and others 1983; Iijima and others 1986) or m-hydrox-
ybenzaldehyde (Sekiguchi and others 1983). Some studies have
suggested that both are possible, with m-hydroxybenzaldehyde
being favorable (Gaucher 1975), whereas others believe that m-
hydroxybenzaldehyde is not converted to gentisaldehyde but
rather to m-hydroxybenzoic acid (Murphy and Lynen 1975). In
the 2nd case, m-hydroxybenzyl alcohol dehydrogenase converts
m-hydroxybenzyl alcohol into m-hydroxybenzaldehyde (Gauch-
er 1975; Murphy and Lynen 1975). Both this enzyme and m-
cresol 2-hydroxylase have been shown to require oxygen and
NADPH to function (Murphy and Lynen 1975).
Once gentisaldehyde has been formed, it is then converted to
isoepoxydon, phyllostine, neopatulin, E-ascladiol, and finally to
patulin (Sekiguchi and Gaucher 1977, 1978; Sekiguchi and Gau-
cher 1979; Sekiguchi and others 1979, 1983). The conversion of
isoepoxydon to phyllostine is accomplished via an NADP-depen-
dent isoepoxydon dehydrogenase (Sekiguchi and Gaucher
1979). Conversion of neopatulin to E-ascladiol is accomplished
through a reduction by NADPH. The product of this reaction, E-
ascladiol, is then either oxidized to patulin or nonenzymatically
transformed to its isomer Z-ascladiol (Sekiguchi and others 1983).
The biosynthetic pathway of patulin is summarized in Figure 2.
Detection and Quantification
The established limit of 50 g/L in the United States as the maxi-
mum patulin level allowed in fruit products has provided an in-
centive for the development of faster and more specific analytical
methods with lower detection limits. A comprehensive review of
the development of thin-layer gas and liquid chromatographic
methods for the detection and confirmation of identity of patulin
has been previously published by Shephard and Leggott (2000).
The most common method currently used to quantify patulin in
fruit products is high-performance liquid chromatography (HPLC)
with ultraviolet (UV) detection. This is the official method adopted
by AOAC Intl. for apple juice (method 995.10) with a detection
limit of 5 g/L. The juice is extracted 3 times with ethyl acetate and
cleaned up by liquid-liquid extraction with a 1.5% sodium car-
bonate solution. The ethyl acetate extract is dried with anhydrous
sodium sulfate; the solvent is then evaporated, normally under ni-
trogen, and the dried residue is dissolved with acidified water (pH
Vol. 1, 2005—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 11
Patulin control in foods . . .
4 by addition of acetic acid). This prepared extract is ready for
HPLC analysis. The recommended liquid chromatography (LC)
systems include analytical reversed-phase LC columns such as
octadecylsilane fully end-capped with 5 m particle stationary
phase, 12 to 25 nm pore size, carbon loading of 12% to 17%,
and a UV detector set at 276 nm, although a photo diode array
detector is preferred to aid in the presumptive identification of the
patulin peak. The system can be run isocratically at 1 mL/min us-
ing 3% to 10% acetonitrile in acidified water (0.095 parts per vol-
ume perchloric acid 60%) as long as patulin separates from 5-hy-
droxymethylfurfural (HMF), a common compound found in apple
juice that elutes just before patulin. For cloudy apple juice and ap-
ple puree, a collaborative study of 12 participants from European
countries was recently conducted to validate the effectiveness of
this LC procedure for patulin determination with a slight modifica-
tion. Prior to the ethyl acetate extraction, the samples were treated
with pectinase enzymes and held overnight at room temperature
or for 2 h at 40 °C and then centrifuged at 4500 ×
g
for 5 min.
Based on the results, the method is recommended for patulin at
greater than 50 g/L in cloudy apple juice and purees (Mac-
Donald and others 2000).
The simultaneous quantification of patulin and HMF in apple
juice by reversed-phase HPLC has been reported by Gokmen and
Acar (1999). The method developed uses a 5-m C18 analytical
column (150 × 4 mm), a photodiode array detector operating at
250 to 300 nm, and a mobile phase of water-acetonitrile (99:1 v/
v) at 1.0 mL/min. Sample preparation followed the official method
described earlier. Complete separation of HMF and patulin was
achieved in less than 9 min at detection limits of less than 0.01
g/L and less than 5 g/L respectively.
The HPLC/UV procedure is routinely used for quantitative deter-
mination of patulin in apple products, but methods to confirm the
presence of patulin usually include more specific detection tech-
niques such as mass spectrometry (MS) after LC or gas chroma-
tography (GC) separations. For GC-MS analysis, patulin is detect-
ed as its trimethyl silyl derivative (TMS-patulin) with electron ion-
ization (Roach and others 2002). In addition, GC-MS with nega-
tive ion chemical ionization allows detection of underivatized pat-
ulin in apple juice extracts. A variety of capillary columns in qua-
drupole, ion trap, and magnetic sector instruments can be used
for patulin confirmation when present in apple juice at 68 to
3700 g/L (Roach and others 2000). Underivatized patulin was
successfully analyzed via GC with electronic pressure control, on-
column injection, and selected ion monitoring allowing detection
limits of 4 g/L (Llovera and others 1999). In another study, a
combination of diphasic dialysis extraction, in-situ acylation, and
GC-MS permitted the quantification of patulin from 10 g to 250
g/L (Sheu and Shyu 1999).
Rychlik and Schieberle (1999) developed 2 methods to quanti-
tate patulin by stable isotope dilution assays using 13C-labeled
patulin as the internal standard. One method used LC/MS in neg-
ative electrospray ionization mode without derivatization, while
the 2nd procedure utilized high resolution gas chromatography/
high resolution mass spectrometry (HRGC/HRMS) after trimethylsi-
lylation of the patulin isotopomers. The HRGC/HRMS method
showed better repeatability, higher recovery rates, and 100 times
lower detection limit (0.012 g/L) compared with the standard
HPLC/UV procedure. This highly sensitive method might be useful
for physiological studies on patulin metabolism in humans.
Sewram and others (2000) developed an HPLC-MS-MS method
with selective reaction monitoring to achieve a detection limit of 4
g/L without derivatization. The MS detection was performed after
atmospheric pressure chemical ionization in negative ion mode.
This procedure correlated well (
R
= 0.99) with the standard HPLC/
UV method.
An improved LC/MS patulin determination has been reported
by Takino and others (2003). They compared atmospheric pres-
sure chemical ionization against atmospheric pressure photoion-
ization and found that it provided lower chemical noise and sig-
nal suppression, thus allowing higher sensitivity. Quantification of
Figure 2—Patulin bio-
synthetic pathway.
Shown here are the
metabolic intermediar-
ies and relevant en-
zymes involved in the
biosynthesis of patulin
(Gaucher 1975; Murphy
and Lynen 1975;
Sekiguchi and Gaucher
1979; Sekiguchi and
others 1983; Iijma and
others 1986; Priest and
Light 1989).
12 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 1, 2005
CRFSFS: Comprehensive Reviews in Food Science and Food Safety
patulin was linear in the 0.2 to 100 g/L range with detection lim-
its of 1 g/L in apple juice samples. Analysis time was 10 min us-
ing an on-line extraction method based on column switching that
eliminated the need for time-consuming off-line sample prepara-
tion. Another technique to perform rapid analysis of patulin with
simpler sample preparation using capillary electrophoresis was
reported by Tsao and Zhou (2000). In this study, a micellar elec-
trokinetic capillary chromatography mode was selected with a
photodiode array detector set at 273 nm. The procedure requires
a small sample volume (2 mL) and minimal usage of solvents as
the separation is achieved by migration of charged particles in the
run buffer. Detection limits were reported as 3.8 g/L.
Rapid clean-up and faster analytical methods based on immu-
nochemical technology are currently being investigated. The low
molecular weight of patulin presents a challenge for this area of
research and development (McElroy and Weiss 1993).
In addition to traditional isolation methods for potential patulin-
producing molds, numerous rapid methods utilizing polymerase
chain reaction (PCR)–based methodologies have been developed.
Marek and others (2003) have developed a PCR-based method
using primers specific to the polygalacturonase gene to identify
patulin-producing fungi (
P.
expansum
) in 4 to 5 h, a much faster
time compared with culture methods that require 48 h. The lowest
level of detection with spore extracts was the DNA equivalent
from 25 spores of
P.
expansum
. Further work will be required to
apply this method to food systems. A gene probe for the patulin
metabolic pathway has also been described by Paterson and oth-
ers (2000). The probe for this PCR detection method was based
on the unique iso-epoxy dehydrogenase gene to allow testing for
potential patulin production directly from food and environmen-
tal samples without any need for pre-enrichment of patulin-pro-
ducing molds.
Patulin Within Foods
Patulin-producing strains have been isolated from a variety of
fruits and vegetables including apples, grapes, cherries, crabap-
ples, pears, apricots, persimmons, strawberries, nectarines, rasp-
berries, black mulberries, white mulberries, lingon berries, peach-
es, plums, tomatoes, greengages, bananas, blueberries, black cur-
rants, almonds, pecans, peanuts, and hazelnuts (Pierson and oth-
ers 1971; Harvey and others 1972; Buchanan and others 1974;
Lovett and others 1974; Sommer and others 1974; Akerstrand
and others 1976; Andersson and others 1977; Frank and others
1977; Harwig and others 1978; Brackett and Marth 1979a; Je-
linek and others 1989; Jiminez and others 1991; Prieta and others
1994; Leggott and Shephard 2001; Demirci and others 2003; Ri-
tieni 2003).
The presence of patulin-producing fungi does not necessarily
guarantee patulin production, however. Patulin production is usu-
ally, but not exclusively, associated with apple soft rot and blue
mold rot, most commonly caused by
P.
expansum
(Brian and oth-
ers 1956; Pierson and others 1971; Harwig and others 1973b;
Sommer and others 1974). This fungi, which is the most common
cause of apple rot in the apple industry (Rosenberger 2003), has
been shown to naturally cause blue-rot in apples, cherries, plums,
apricots, peaches, nectarines, pears, and quince (Pierson and oth-
ers 1971; Harvey and others 1972; Buchanan and others 1974).
Further, apples, peaches, strawberries, bananas, greengages, to-
matoes, cherries, and pears have all been shown experimentally
to support blue-rot (Lovett and others 1974; Laidou and others
2001).
Patulin-production within fruits, vegetables, and their products
has been investigated and often appears to be dependent on such
factors as water activity (aw), temperature, pH, and other chemical
characteristics intrinsic to fruits (Sommer and others 1974;
Northolt and others 1978; McCallum and others 2002). pH and
patulin production have been shown to be inversely related, with
patulin being unstable at high pH (McCallum and others 2002).
Temperature has been shown to affect pathogen growth and, to a
greater extent, the production of patulin (McCallum and others
2002). Patulin production has been observed at all temperatures
permitting
P.
expansum
growth, encompassing an approximate
range of 0 to 30 °C (Sommer and others 1974).
B.
nivea
has been
shown to grow faster at temperatures of 30 and 37 °C while patu-
lin production was highest at 21 °C (Roland and Beuchat 1984b).
As testament to the variability seen in patulin production, even
cultivar differences among apples affect the patulin production of
P.
expansum
(McCallum and others 2002), with Jonathan, Gou-
dreinetter, Cox’s Orange Pippin, and Bramley being particularly
susceptible, whereas Golden Delicious appears to be particularly
resistant (Northolt and others 1978; Corbett 2003).
Apple juice and cider were once thought to be the only prod-
ucts to naturally contain patulin (WHO 1995). However, compil-
ing research has now shown patulin to occur naturally in such
fruit and vegetable products as apple, apple-acerola, grape, pear,
sour cherry, blackcurrant, orange, pineapple, and passion fruit
juices, pasteurized and unpasteurized apple cider, apple puree,
corn, strawberry, blackcurrant, and blueberry jams, and some
types of baby food (Lovett and others 1974; Sommer and others
1974; Frank and others 1977; Scott and others 1972; Harwig and
others 1978; Brackett and Marth 1979a; Ehlers 1986; Wheeler
and others 1987; Jelinek and others 1989; Lin and others 1993;
Prieta and others 1994; Rychlik and Schieberle 1999; Ake and
others 2001; Leggott and Shephard 2001; Ritieni 2003). Further-
more, evidence suggests that other juices, including blueberry,
red raspberry, boysenberry, pear, cranberry, strawberry, black
cherry, and peach can support growth and associated patulin
production of
B.
fulva
and/or
P.
expansum
(Rice and others 1977;
Larsen and others 1998).
Besides fruit and vegetable products, patulin has also been iso-
lated from cheddar cheese (Bullerman and Olivigni 1974) and
grain products such as barley and wheat malts, feed silages, cere-
al stubbles (Escoula 1974, 1977; Lopez-Diaz and Flannigan
1997; Pittet 1998), bread, and related flour/dough products (Reiss
1972, 1976). In 1 study, 23 strains of
Aspergillus
and
Penicillium
isolated from moldy bread were tested for patulin production.
Twenty-one of 23 moldy bread samples were found positive for
27 to 160 g patulin/kg (Tyllinen and others 1977). Finally, patu-
lin has been attributed to the death of 100 cows fed malt (Ciegler
1977) and to hemorrhagic syndrome and death in silage-fed cat-
tle (Syrett 1979).
Within the food industry, apples and their respective products
are of greatest concern for patulin contamination. While a variety
of other food sources and products have demonstrated patulin
and/or patulin-producer contamination, the frequency of these
events is much less than that of the apple industry. Patulin has
been identified in apples from Canada (Harwig and others 1973b;
Sommer and others 1974), England (Brian and others 1956; Som-
mer and others 1974), New Zealand (Walker 1969), United States
(Sommer and others 1974; Ware and others 1974), Australia
(Sommer and others 1974), and South Africa (Leggott and Shep-
hard 2001), and has been found in apple juices from Canada
(Scott and others 1972), United States (Ware and others 1974),
Sweden (Josefsson and Andersson 1976), South Africa (Brown
and Shephard 1999; Leggott and Shephard 2001), Turkey (Gok-
men and Acar 1998), Brazil (de Sylos and Rodriquez-Amaya
1999), Austria (Steiner and others 1999a), Italy (Ritieni 2003), Bel-
gium (Tangi and others 2003), France, and Australia (Sommer and
others 1974).
Numerous studies around the world have examined the extent
and degree to which apple products have been contaminated by
Vol. 1, 2005—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 13
Patulin control in foods . . .
patulin. In Wisconsin, 23 of 40 roadside apple juices were found
to contain between 10 to 350 g patulin/L (Brackett and Marth
1979a). A 2nd study showed that 8 of 13 commercial apple juices
tested contained between 44 and 309 g patulin/L juice (Ware
and others 1974). A Turkish study showed 215 of 215 apple juice
concentrates examined had between 7 and 375 ppb patulin with
43% being above the 50-ppb international standard (Gokmen
and Acar 1998). Finally, a 1996 to 1998 study in South Africa
showed that 5 of 22 juices sampled contained between 10 and
45 ppb patulin, and 29% of infant apple products showed 5 to
20 ppb (Brown and Shephard 1999). A summary of patulin con-
tamination within foods is given in Table 2.
Control During Apple Harvest, Processing, and Storage
In North America, apple juice is typically a byproduct pro-
duced from culled apples unfit for other, higher quality and higher
profit, purposes (Rosenberger 2003). Apples are typically harvest-
ed and processed, and those lower quality apples unfit for 1st
quality edible retail apples are stored in controlled atmosphere,
refrigerated storage for anywhere up to around 12 mo when the
next harvest comes in.
Current methods for preventing mycotoxin contamination of
foods include (1) strict adherence to moisture control, (2) imple-
mentation of good manufacturing practices, (3) good quality as-
surance efforts, and (4) adherence to Hazard Analysis Critical
Control Points principles (Lopez-Garcia and Park 1998; Park and
others 1999). Existing and developmental preventive measures
during preproduction are based upon fruit quality and facility
sanitation measures. The quality of fruit resulting from harvesting
is the 1st step in controlling patulin levels. With the highest
quality hand-picked fruit being used for direct-for-retail sale,
processed apple products usually are produced from mechani-
cal harvest, windfalls, insect-damaged, or culled fruit. Bruises,
skin breaks, and other physical damage within these apples pro-
vide a perfect entry for
P.
expansum
and other patulin-producing
species into the fruit. Studies have examined the effect of fruit
quality and harvest method on the patulin content of the result-
ant juices. In 1 study, patulin was undetectable in 7 cultivars of
tree-picked cider, whereas it was detected between 40.2 and
374 g/L in 4 cultivars of ground-harvest cider (Jackson and
others 2003). Many of the patulin control measures suggested
by the Joint FAO/WHO Food Standards Programme are based
upon the careful selection of fruits as based upon good agricul-
tural practice (CODEX 2002). However, as indicated by a New
York State Hudson Valley Lab study that showed around 20% of
bagged, in-store, retail apples to contain consumer-visible blue-
rot (Rosenberger 2003), even the highest level of fruit selection
and good agricultural practices cannot eliminate apple-rot and
associated patulin contamination.
Standard postharvest processing, including washing, sorting,
and packaging, poses a 2nd means of both fungal control and
contamination alike. Washing with high-pressure water has been
shown to reduce patulin levels within apple juice by 21% to 54%
(Acar and others 1998). A 2nd study showed that washing of
ground-harvested apples resulted in a 10% to 100% patulin re-
duction, depending on initial patulin level and washing treatment
(Jackson and others 2003). However, these same washes can also
serve as a source of contamination. Contaminated bins, storage
rooms, drencher washes, drying brushes after apple wash, and
other steps within the processing cycle can all provide a source of
fungal inoculum cycling in poorly sanitized setups. Prevention
methods aimed at cleaning and sterilizing storage and processing
facilities routinely and in between seasons are being mapped out,
but have not yet been fully developed. Plus, the older, complicat-
ed design of many packing-house and processing equipment in-
hibits the ability to effectively sanitize. Even if effective strategies
are successfully mapped out, the inherent variability in apple han-
dling facilities will require customization of sanitation methods for
each operation (Rosenberger 2003), posing a considerable cost to
producers.
Apple storage poses yet a 3rd means of fungal control and
contamination. Conflicting evidence exists as to whether stan-
dard controlled-atmosphere, refrigerated storage is sufficient to
prevent apple soft rot (Sommer and others 1974; Lovett and oth-
ers 1975a; Paster and others 1995). Classically associated with
wounded fruit, in the mid-1990s, nonwounded, stem-end in-
fected blue-rot began to appear with increasing frequency.
Long-term, controlled atmosphere storage has now been shown
to allow the slow growth and stem-based invasion of fungi into
apples (Rosenberger 2003). Furthermore, these facilities are not
available to all producers, forcing many to use deck-storage,
which can drastically increase patulin production. Alternatives
to room storage are the use of packaging materials such as poly-
ethylene, which through their own atmospheric control, have
been shown to reduce patulin production in apples (Moodley
and others 2002). In the same study mentioned previously (Jack-
son and others 2003), patulin was not detected in cider from
culled, tree-picked apples stored 4 to 6 wk at 0 to 2 °C, but was
detected at levels between 0.97 and 64 g/L in stored, tree-
picked, unculled fruit. Cider from apples stored in controlled at-
mosphere and culled showed 0 to 15.1 g/L patulin while un-
culled apple fruit showed 59.9 to 120.5 g/L patulin. Thus, stor-
age can exacerbate any flaws in prior handling steps, such as
harvesting. The longer the storage of the apples, the greater the
risk of increased patulin content. This is particularly evident to
juice producers within the U.K. who see dramatic rises of patulin
content within juice produced in June, July, and August—the
months just prior to the new harvest season where source ap-
ples have been stored for almost a year (Corbett 2003). Further-
more, even if apples are perfectly processed prior to storage, ad-
dition of diphenylamine, which is used to treat ‘storage scald’ in
stored apples, can provide an inoculum of patulin-producing
fungi in the fruit (Rosenberger 2003).
Various treatments have been investigated for the ability to re-
duce the apple rot and patulin production seen within the har-
vest, processing, and storage steps. Postharvest treatment of ap-
ples with benzimidazole fungicides was used from the 1970s
through the early 1990s but has since been largely abandoned
due to fungal resistance (Rosenberger 2003). Insecticide treatment
of apples can reduce growth of patulin-producing molds and
thereby patulin production between 85% and 100% (Draughon
and Ayres 1980), but the treatment raises questions about the
safety of organophosphate insecticides and is thus nonpreferen-
tial. Combinations of heat treatment, calcium infiltration, and bio-
logical control with an antagonistic bacteria,
Pseudomonas
syrin-
gae,
were investigated alone or in combination to reduce inci-
dence of postharvest apple decay. Apples inoculated with
P.
ex-
pansum
and heated at 38 °C for 4 d showed no decay lesions af-
ter storage at 1 °C for 6 mo. After heat-treatment inoculation with
P.
expansum
followed by infusion of 2% calcium chloride alone
and
Ps.
syringae
treatment alone reduced decay incidence by
25% each. Calcium treatment plus
Ps.
syringae
treatment without
and with subsequent heat treatment reduced decay by 89% and
91%, respectively (Conway and others 1999). Finally, other meth-
ods used for the control of human pathogens like
Escherichia
coli
, such as washing with solutions of peroxyacetic acid, chlo-
rine dioxide, and chlorine (Sapers and others 1999; Wisniewsky
and others 2000; Annous and others 2001), may also provide
some benefit toward preventing postharvest apple decay. Howev-
er, these methods have yet to be analyzed for their effectiveness
against fungal spores.
14 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 1, 2005
CRFSFS: Comprehensive Reviews in Food Science and Food Safety
Control during Production
For both removal of patulin during production and postproduc-
tion, effective decontamination/detoxification procedures must (1)
inactivate, destroy, or remove the toxin, (2) not produce or leave
new toxic substances, (3) retain nutritive value/acceptability of the
product, (4) not significantly alter the technological processes as-
sociated with the product, and (5) if possible, destroy fungal
spores (Park and others 1988). Methods of patulin control within
Table 2—Food industry distribution of patulin and patulin-producing fungi
Foods contaminated with
patulin-producing fungal strains Source
Apples Pierson and others 1971; Sommer and others 1974; Frank and others 1977; Brackett and Marth
1979a; Prieta and others 1994; Leggott and Shephard 2001; Ritieni 2003
Grapes Sommer and others 1974; Harwig and others 1978
Cherries Harvey and others 1972; Buchanan and others 1974; Lovett and others 1974; Andersson and others
1977; Arici and others 2002
Crabapples Sommer and others 1974
Pears Pierson and others 1971; Buchanan and others 1974; Sommer and others 1974
Apricots Harvey and others 1972; Buchanan and others 1974; Sommer and others 1974
Persimmons Sommer and others 1974
Strawberries Andersson and others 1977; Frank and others 1977; Arici and others 2002
Nectarines Harvey and others 1972
Raspberries Andersson and others 1977; Arici and others 2002
Black mulberries Arici and others 2002
White mulberries
Lingon berries Andersson and others 1977
Peaches Harvey and others 1972; Buchanan and others 1974; Andersson and others 1977; Frank and others 1977
Plums Harvey and others 1972; Buchanan and others 1974; Andersson and others 1977
Tomatoes
Greengages Frank and others 1977
Bananas
Blueberries Akerstrand and others 1976; Andersson and others 1977
Black currants Andersson and others 1977
Almonds
Pecans Jiminez and others 1991
Peanuts
Hazelnuts
Foods contaminated with patulin Source
Apple juice Lovett and others 1974; Sommer and others 1974; Frank and others 1977; Scott and others 1977;
Brackett and Marth 1979a; Prieta and others 1994; Rychlik and Schieberle 1999;
Leggott and Shephard 2001; Ritieni 2003
Apple-acerola juice Rychlik and Schieberle 1999
Pear juice Ehlers 1986
Grape juice Harwig and others 1978; Rychlik and Schieberle 1999
Sour cherry juice
Blackcurrant juice Rychlik and Schieberle 1999
Orange juice
Pineapple juice Ake and others 2001
Passion fruit juice
Apple cider Brackett and Marth 1979; Wheeler and others 1987; Leggott and Shephard 2001
Apple puree Leggott and Shephard 2001; Ritieni 2003
Corn Lin and others 1993
Strawberry jam
Blackcurrant jam Jelinek and others 1989
Blueberry jam
Baby food Prieta and others 1994; Leggott and Shephard 2001; Ritieni 2003
Cheddar cheese Bullerman and Olivigni 1974
Barley Malt Lopez-Diaz and Flannigan 1997
Wheat Malt
Bread Reiss 1972, 1976
Countries with apple and
juice contamination Source
Canada Scott and others 1972; Harwig and others 1973b; Sommer and others 1974
England Brian and others 1956; Sommer and others 1974
New Zealand Walker 1969
United States Sommer and others 1974; Ware and others 1974
South Africa Leggott and Shephard 2001
Sweden Josefsson and Andersson 1976
Turkey Gokmen and Acar 1998
Brazil de Sylos and Rodriguez-Amaya 1999
Austria Steiner and others 1999a
Belgium Tangi and others 2003
Australia Sommer and others 1974
France
Vol. 1, 2005—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 15
Patulin control in foods . . .
standard apple juice production steps are centered on 3 areas.
The 1st of these involves the quality of the fruit and processing of
this fruit, prior to pressing. As previously mentioned, processed
apple products often utilize lower quality fruit that is unsuitable
for direct market retail. Removal of decayed/ damaged fruit or
trimming of moldy portions can significantly reduce patulin levels
in apple products (Lovett and others 1975b; Taniwaki and others
1992; Sydenham and others 1995, 1997; Beretta and others
2000). Trimming of rotten sections of apple has been shown to re-
move up to 99% of patulin contamination (Lovett and others
1975b). However, this process is expensive and labor intensive.
Furthermore, patulin can be detected in visibly sound fruit (Jack-
son and others 2003) and can spread from rotten areas of apples
into sound areas (Beretta and others 2000). Two studies showed
that patulin could diffuse 1 to 2 cm from the rotten core in apples
(Taniwaki and others 1992; Rychlik and Schieberle 2001), 0.6 cm
in pears (Laidou and others 2001), and throughout the entire fruit
in tomatoes (Rychlik and Schieberle 2001). Producers’ tests on
apples have shown that patulin level is often not related to the
physical quality of the fruit, with both high-rot fruit and top-quali-
ty eating fruit often having high levels of patulin in tests (Corbett
2003), as further demonstrated by the previously mentioned Hud-
son Valley study on retail apples. While often beneficial, the sort-
ing of decayed apples to the level of being effective is difficult to
impossible, often necessitating the need to reject entire juice
loads of apples. If reliant on this method, small-scale producers
who cannot afford these losses will be forced to sort by hand, in-
creasing costs to the point that many may cease operating (Rosen-
berger 2003).
The 2nd area of standard juice production capable of reducing
patulin levels involves the juice clarification process. Results from
this process have been mixed, and those methods most success-
ful at removing patulin often do so at the expense of juice sensory
and authenticity qualities. Some sources suggest that standard
fruit juice production processes remove only about 20% of patu-
lin (Stray 1978; Harrison 1989). One study showed that the tradi-
tional apple juice production processes of depectinization, clarifi-
cation, and filtration through a rotary vacuum precoat filter could
reduce patulin levels by 39%. In the same study, use of depectini-
zation, clarification, mixing with gelatin/bentonite, and ultrafiltra-
tion resulted in a 25% decrease in patulin (Acar and others 1998).
A 2nd study examined the effects of gelatin/bentonite flocculation,
filtration, activated charcoal, ultrafiltration, polyvinylpolypyrroli-
done, and polystyrene-Divinyl Benzene (DVB)–based macro po-
rous resin on apple juice color, clarity, phenolic content, organic
acid content, and patulin reduction. Activated charcoal treatment
had the highest patulin reduction with 40.9% but also significant-
ly reduced color and phenolic content, 2 characteristics associat-
ed with juice authenticity. Polystyrene-DVB–based macro porous
resin was the 2nd most effective, reducing patulin by 11%. None
of the treatments altered organic acid content significantly (Artik
and others 2001). Centrifugation and fining of juice pulp have
been shown to reduce patulin levels by 89% and 77%, respec-
tively. However, these methods make the removed cake and/or fil-
ter potentially highly toxic and unfit for any further use, such as
often is done with juice sediments in animal feed (Bissessur and
others 2001). This could represent a loss of income for many juice
producers. Batch absorption with synthetic polymers has also
been investigated (Canas and Aranda 1996), as has enzyme treat-
ment with pectinase enzymes used to break down the pectin coat
surrounding protein particles, allowing aggregation and sedimen-
tation of protein particles and, in the case of patulin contamina-
tion, their associated patulin adducts. This method has resulted in
a 73% decrease in patulin content within juice (Bissessur and oth-
ers 2002).
The 3rd juice production process capable of reducing patulin
levels is the pasteurization process. Of the 3 processes mentioned
thus far, this is by far the least effective. Repeated studies have
shown that, while unstable at high pH (McCallum and others
2002), patulin is relatively stable to thermal degradation in the pH
range of 3.5 to 5.5, with lower pH leading to greater stability
(Heatley and Philpot 1947; Lovett and Peeler 1973). The half-life
of patulin held at 25 °C at pH 6.0 and 8.0 has been shown to be
55 and 2.6 d, respectively (Brackett and Marth 1979c). In 1 study,
no patulin reduction occurred during concentration and pasteur-
ization (Aytac and Acar 1994), while a 2nd study did show a 50%
reduction of patulin in apple juice treated for 20 min at 80 °C
(Scott and Somers 1968)—a much longer treatment than used in
standard pasteurization procedures. A 3rd study showed pasteur-
ization treatments between 60 and 90 °C for 10 s were only able
to reduce patulin levels by 18.8% (Wheeler and others 1987). Ev-
idence also shows that patulin is nonvolatile and, upon distilla-
tion production of apple aroma, patulin remains within juice con-
centrate (Kryger 2001). Finally, not only is the pasteurization pro-
cess unable to significantly reduce patulin levels, it often fails to
fully remove heat resistant patulin-producing fungi, such as
B.
nivea
and
B.
fulva
(Ough and Corison 1980), allowing for poten-
tial continued production of patulin within the finished juice.
As a final stage of the production process, studies have exam-
ined the effects of storage on patulin content. Mixed studies show
either a decrease (Scott and Somers 1968; Harwig and others
1973a) or no change (Pohland and Allen 1970; Zegota and others
1988) of patulin levels in apple juice with refrigerated storage.
Similar studies within grape juice have shown stability (Ough and
Corison 1980) or an approximate 50% decrease (Scott and Som-
ers 1968) of spiked patulin levels in grape juice after 5 wk.
Control Postproduction
Filtering and adsorption
A number of studies have been devoted to the removal of patu-
lin from juice through the use of adsorption filters, columns, and
agitation treatments using carbon-based material. Agitation with
20 mg/mL activated charcoal followed by filtration through a 40-
or 60-mesh charcoal column reduced a 30-g/mL patulin solu-
tion to below detectable levels. Further, use of 5 mg/mL charcoal
in agitation was able to reduce patulin to below detectable levels
in naturally contaminated cider. However, color loss was marked-
ly present in this resulting juice (Sands and others 1976). In a 2nd
study, 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3 g/L activated charcoal was
added to naturally contaminated apple juice containing 62.3 ppb
patulin. Samples were mixed for 0, 5, 10, 20, and 30 min. Three
grams/liter activated charcoal was found to be most effective with
a time period of 5 min. Clearness of juice increased, color of juice
decreased, and small decreases in fumaric acid, pH, and °Brix
were also seen (Kadakal and Nas 2002). In another study, ultrafine
activated carbon was bound to granular quartz producing a com-
posite carbon adsorbent (CCA). Columns with varying amounts of
CCA were prepared and 10 g/mL patulin were filtered through at
1 mL/min. Fifty percent breakthrough values for columns with 1.0,
0.5, and 0.25 g CCA were 137.5, 38.5, and 19.9 g, respectively
(Huebner and others 2000).
In a 4th study designed to compare the effects of different car-
bon activation methods, steam activated carbon NORIT SA 4 and
NORIT SX 4 performed equally, removing 80% and 70%, respec-
tively, of an initial 1 g/L patulin solution in 12 °Brix juice at 55 °C.
Chemically activated carbon NORIT CA 1 removed only 45% of
the same solution. This study also showed that increased °Brix
levels of juice led to decreased patulin removal efficiency, with
NORIT SA 4 removing only 20% of patulin from 20 °Brix juice.
Within this study, patulin removal was independent of juice tem-
16 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 1, 2005
CRFSFS: Comprehensive Reviews in Food Science and Food Safety
perature between 30 and 65 °C (Leggott and others 2001), while
in another study, patulin binding to activated carbon compounds
was shown to be endothermic, with increased temperatures re-
sulting in improved patulin removal (Mutlu and others 1997).
Despite these initial studies where activated carbon was shown
to reduce patulin, little work has been done to optimize carbon
for this process. Furthermore, the use of activated carbon poses a
substantial cost to the juice industry (Leggott and others 2001),
being both time consuming and expensive (Bissessur and others
2001). Outside of the cost of carbon material itself, activated char-
coal treatment creates excess waste that must be dealt with eco-
logically (Artik and others 2001). Also, as mentioned within sever-
al of the studies, negative affects on color, fumaric acid content,
pH, and °Brix have been observed with carbon adsorption. These
and other modifications made by carbon absorption treatments
can negatively alter the taste perception and quality of the juice
(Huebner and others 2000). Finally, the use of clays can pose a
risk due to removal of essential nutrients from juice (Park and
Troxell 2002). Similar analysis with activated carbon compounds
has not been performed, but could very likely have the same neg-
ative result.
Chemical modification
Chemical decontamination methods have also been shown to
be effective and are likely the most easily suitable for industry.
Many of the chemicals examined thus far are already considered
food grade additives/treatments, and therefore, their use would be
preferential to many other forms of treatment. Plus, because addi-
tion of chemical agents to human foods solely for reducing myc-
otoxin levels is currently not permitted in the U.S. (Park and Trox-
ell 2002), treatments already accepted for other purposes would
allow more rapid integration within industry. Furthermore, many
of these same treatments have been shown to be effective in in-
hibiting patulin-producing molds (see below, Patulin Production
in the Final Product), making them a double-edged sword in patu-
lin control.
Numerous chemical treatments have been utilized to detoxify
patulin. Included within these treatments are chemicals designed
to oxidize and reduce patulin to hopefully less toxic compounds,
as well as treatments to bind up patulin in the form of less toxic
thiol-based adducts. Two of the most potent means of chemical
detoxification of patulin are ammoniation and potassium perman-
ganate oxidation. Treatment with ammonia has been shown to re-
duce patulin levels by up to 99.9% in laboratory waste (Fremy
and others 1995) and 99.8% in juice. However, in the 2nd study,
the resultant product was unfit for consumption (Ellis and others
1980). Oxidation by potassium permanganate in acidic and alkali
conditions also resulted in better than 99.99% patulin reduction
in laboratory waste (Fremy and others 1995).
Treatment with various sulfur-containing compounds has been
another area of intense investigation. Studies involving the use of
sulfur dioxide have produced mixed results. Most studies agree that
patulin is unstable in the presence of sulfur dioxide (Pohland and
Allen 1970), but they vary on its degree of effectiveness. One study
showed that 100 ppm sulfur dioxide immediately reduced patulin
levels by 50% (Ough and Corison 1980). In a 2nd study, a 42% re-
duction of patulin was achieved with 100 mg sulfur dioxide/kg ap-
ple juice (Aytac and Acar 1994). However, in a 3rd study, 200 ppm
sulfur dioxide, the maximum allowable within the food industry,
caused only a 12% patulin decrease in 24 h. Sulfur dioxide (2000
ppm) showed a 90% decrease after 2 d and plateaued at that level.
Within this study, 2 reactions were shown to be involved. The 1st, a
reversible reaction, occurred presumably through the addition of
sulfur dioxide to the hemiacetal ring of patulin, forming a carbonyl
hydroxysulfonate. The 2nd reaction is thought to be irreversible
and to occur due to an opening of the lactone ring structure at one
of the double bonds (Burroughs 1977). A 4th study found the sul-
fur dioxide inactivation of patulin to be reversible or irreversible de-
pending on pH (Steiner and others 1999b).
Sulfur compounds common to biological systems and fre-
quently associated with patulin toxicity, such as glutathione, cys-
teine, and thioglycolate, are believed by many to produce biologi-
cally inactive products when reacted with patulin (Cavallito and
Bailey 1944; Geiger and Conn 1945; Reinderknecht and others
1947; Singh 1967; Hoffman and others 1971; Ciegler and others
1976). In 1 study, treatment with cysteine and glutathione pre-
vented the negative effects of patulin infection on rumen fermen-
tation, while non-sulfhydryl–containing ascorbic acid and ferulic
acid did not protect against patulin toxicity (Morgavi and others
2003). Patulin has been shown to form a number of adducts with
N-acetylcysteine, glutathione, and cysteine (Fliege and Metzler
2000). While patulin-cysteine adduct mixtures are slightly bacteri-
cidal to Gram-positive and Gram-negative species (Geiger and
Conn 1945; Reinderknecht and others 1947), they have been
shown to reduce the bactericidal effects of patulin by more than
100 fold. Furthermore, treatment of mice with a cysteine-patulin
adduct mix, 85 times the equivalent lethal dose50 concentration
of patulin alone, resulted in no deaths or macroscopic pathologi-
cal effects (Lindroth and von Wright 1990).
Another chemical method examined for the decontamination
of patulin has been treatment with a variety of organic acids and
vitamins, many of which are considered to be food-grade addi-
tives. In one study, addition of ascorbate and ascorbic acid in-
creased the rate at which patulin was reduced from solution in a
concentration-dependent manner. These reduction rates were
found to be slower in juice, but still present. A mechanism was
proposed in which a reaction of ascorbate or ascorbic acid with
metal ions produced singlet oxygen molecules that attacked and
oxidized patulin. However, no evidence for any such reaction
products was given (Brackett and Marth 1979b). A 2nd study uti-
lizing ascorbic acid found that treatment with 500 mg/kg juice
could produce a 50% reduction of patulin (Aytac and Acar 1994).
A 3rd study, however, found that degradation by ascorbic acid re-
sulted in only 5% degradation in 3 h and 36% degradation after
44 h (Fremy and others 1995). Besides ascorbic acid, treatment of
apple juice with the B vitamins, thiamine hydrochloride, pyridox-
ine hydrochloride, and calcium-d-pantothenate, caused statisti-
cally significant reductions in patulin content over control juices
(Yazici and Velioglu 2002).
Yet another chemical detoxification method examined has been
the use of ozone, which has recently been approved for use in
the treatment and processing of foods (FR 2003) and has been
shown as an effective alternative to heat pasteurization of juice. In
1 study, treatment of a 32 M patulin solution with 10% ozone
for 15 s reduced patulin to undetectable level and produced no
detectable reaction products (McKenzie and others 1997). Fur-
ther, other mycotoxins such as aflatoxins B1, G1, B2, and G2
(Maeba 1988) as well as zearalonone (Doyle and others 1982)
have been shown to be effectively degraded by ozone.
In all of these cases, preliminary evidence is promising. Howev-
er, inadequate research has been done to examine the efficacy
and full functional range of applicability for the treatment. Further-
more, the reaction mechanisms for few if any of the chemical
detoxification treatments are fully understood, and both the reac-
tion products and their respective toxicities are still unknown and
must be determined prior to use. The regulatory approval for
some of the proposed chemical treatments such as ammoniation
and potassium permanganate must be sought prior to use in the
food industry.
Biological control
Biological methods of patulin control result largely from the ob-
Vol. 1, 2005—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 17
Patulin control in foods . . .
servation that patulin is almost always completely degraded dur-
ing yeast fermentation. Besides being quite successful, this meth-
od is much better understood compared with other decontamina-
tion methods. Approximately 90% of patulin can be removed
during yeast fermentation (Burroughs 1977). In 1 study, 6 of 8
yeast strains reduced patulin levels to below detectable levels,
while all 8 strains resulted in a 99% or better decrease in total pat-
ulin content. Control juice, on the other hand, stored for an equal
amount of time (2 weeks), had only a 10% reduction (Stinson and
others 1978). In a 2nd study, yeast fermentation reduced patulin
levels completely after 2 wk. This same study also showed that
patulin levels failed to decrease significantly in juices that had
been yeast fermented and then filter sterilized to remove yeast,
suggesting that active yeast, and not their byproducts, were re-
quired for the reduction (Harwig and others 1973a). Treatments of
patulin along with cyclohexamide, a protein synthesis blocker of
yeast, completely blocked protein synthesis and prevented the
detoxification of patulin. Addition of cyclohexamide 3 h after pat-
ulin addition, however, resulted in a reduced, but continued, rate
of patulin degradation, suggesting that the proteins synthesized
within the 3-h window were catalytically active against patulin
and did not just bind it up in adduct formation (Sumbu and others
1983). A later study showed that 3 strains of
Saccharomyces
cere-
visiae
reduced patulin levels during fermentive growth but not
aerobic growth. This reduction resulted in the production of 2 ma-
jor products: E-ascladiol, patulin’s immediate biosynthetic precur-
sor, and its isomer Z-ascladiol. These 2 products were also seen in
the treatment of patulin with the reducing agent sodium borohy-
drate (Moss and Long 2002). E-ascladiol is itself a mycotoxin (Su-
zuki and others 1971), which has reduced toxicity compared with
patulin and also reacts with sulfhydryl-containing compounds
(Sekiguchi and others 1983).
While effective, biological control with yeast is limited to prod-
ucts that can be fermented. Furthermore, yeast are themselves
sensitive to patulin, and at concentrations greater than 200 g/
mL, yeast have been shown to be completely inhibited, prevent-
ing fermentive detoxification (Sumbu and others 1983). No re-
search has been done to examine the potential use of other fer-
menting microbes, such as lactic acid bacteria, in decreasing pat-
ulin content within juices. Similar reducing enzymes and environ-
ments produced by these bacteria may very well be able to de-
grade patulin. Finally, no research has investigated the direct en-
zymatic degradation of patulin. Reducing enzymes such as those
involved in yeast fermentation, as well as lactone degrading en-
zymes such as -lactamase, may well be able to degrade patulin
alone.
Electromagnetic irradiation
Electromagnetic radiation has been shown in preliminary stud-
ies to reduce the content of patulin and other mycotoxins within
juice. In 1 study, treatment of juice with 0.35-kGy ionizing radia-
tion caused a 50% reduction of patulin with no acceleration of
nonenzymatic browning of the juice (Zegota and others 1988).
The UV light sensitivity of aflatoxins has been demonstrated on
several accounts, but no studies have been performed specifically
on patulin (Doyle and others 1982; Valletrisco and others 1990).
Patulin Production in the Final Product
Limited work has been done to examine the potential growth
and production of patulin by fungi in stored, finished product
juices. It is well known that many
Aspergillus
and
Byssochlamys
spp. molds, many of which produce patulin, are heat resistant
and can survive the pasteurization processes used in juice and ci-
der production. Furthermore, many of the same patulin-produc-
ing molds have also been shown to grow and produce patulin at
standard cold storage temperature down to 0°C (Sommer and
others 1974). Taken together, these 2 facts suggest that finished
apple juice and cider could very well support the continued pro-
duction of patulin by fungi. If this is the case, any single treatment
patulin control method, such as physical adsorption, would po-
tentially be moot due to production of patulin by active fungi
postprocessing. Studies must be done to examine this potential
within juices, and, if possible, to provide correlates between mold
numbers and patulin levels within juice.
With the possibility of fungi actively producing patulin within
juice, control measures should aim not only at the reduction of
patulin itself, but the inhibition of both fungal growth and patulin
production within juice. Some studies have examined the effects
of many patulin-reducing treatments on live patulin-producing
fungi. Sulfur dioxide, sodium benzoate, and potassium sorbate
have been investigated for the affect on
B.
nivea
growth and patu-
lin production within juice. Seventy-five ppm sulfur dioxide, 150
ppm potassium sorbate, and 500 ppm sodium benzoate all signif-
icantly retarded
B.
nivea
growth and patulin production (Roland
and Beuchat 1984a). A 2nd study showed that treatment of
B.
nivea
with 50 g/mL potassium sorbate completely eliminated
patulin production at 37 °C. However, higher concentrations (75
and 100 g/mL) were needed at lower temperatures (21 °C) to in-
hibit growth, and even with growth inhibition, these treatments
failed to inhibit patulin production (Roland and others 1984b). A
related study showed that 0.1% of potassium sorbate eliminated
patulin production in
P.
patulum
, but interestingly, this same treat-
ment elevated patulin production in
P.
roqueforti
(Bullerman and
Olivigni 1974). A 4th study showed that 0.1 M potassium sorbate
reduced
P.
expansum
growth by 83% and patulin production by
98%, while 0.1 M sodium propionate caused a 48% reduction in
growth and 98% reduction in patulin production (Lennox and
McElroy 1984).
A separate set of studies showed that treatment with 0.2% lem-
on oil reduced patulin production, and a mix of 0.05% lemon oil
and 0.2% orange oil resulted in a 90% reduction of patulin pro-
duction (Hasan 2000). Finally, in a series of unsuccessful studies,
addition of 0.5% and 0.75% citric and lactic acid reduced pro-
duction of aflatoxins and sterigmatocystin, but not patulin. These
same treatments also failed to inhibit
P.
expansum
growth (Reiss
1976).
Conclusions
While studies investigating the health effects of patulin have
proved inconclusive, there is little doubt as to the potential danger
inherent in the contamination of food products by patulin. Past re-
search has elucidated a great deal about the chemical and biolog-
ical nature of patulin, has made advances in methods for detect-
ing patulin, and has revealed a number of potential control mea-
sures that may provide a basis for fully effective control measures
within the future. Still, patulin contamination continues to be
present within the food industry. Thus, future research must con-
tinue to address the threat of patulin contamination within food
products, with an emphasis upon the following areas.
Patulin detection
While methods for detecting and quantifying patulin have great-
ly improved over the years, the sensitivity of these methods is still
often a limiting factor for many aspects of patulin control re-
search. Currently, no rapid analytical technique exists for the anal-
ysis of patulin, and the standard methodology for patulin quantifi-
cation requires dedicated instrumentation and trained operators.
A rapid detection method that can be used “in-house” by the
food industry without any complicated equipment would be ex-
tremely beneficial.
18 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 1, 2005
CRFSFS: Comprehensive Reviews in Food Science and Food Safety
Patulin control
Efficacy evaluation of food-processing techniques for mycotox-
in reduction must consider (1) stability of mycotoxin, (2) nature of
process, (3) mycotoxin-food interactions, and (4) mycotoxin-myc-
otoxin interactions (Park and Troxell 2002). Efforts to control the
contamination of apple products by patulin currently focus on 3
areas: prevention of patulin contamination in the harvesting, pro-
cessing, and storage steps; removal of patulin during processing;
and postproduction treatments to remove or detoxify patulin.
While many of the control measures mentioned previously have
shown promise, none are yet at the point of providing guaranteed
patulin control within food products. Also to be considered is the
increasing level of juice imports into the United States. Imports
comprise 60% of the U.S. apple juice market, with most being low
acid juices from China that are later blended with higher acid U.S.
juices for consumption. Higher U.S. demand for apple products
will increase the levels of these often quality-questioned imports
(USDA 2002). Domestic control measures based upon prepro-
duction and production-stage treatment of patulin cannot be ex-
pected to be utilized by juice exporters, and thus blending of im-
ported juices could serve as a source of patulin contamination ca-
pable of undermining any of the aforementioned control mea-
sures. With this trend in mind, postproduction patulin control
measures must almost certainly be included within any successful
control regime. Successful patulin control will most likely result,
not from a single treatment, but from a combination of control
measures throughout the production process.
Patulin prevalence
While most commonly associated with apple juice and apple
cider, patulin has been detected as a natural contaminant in a va-
riety of other apple-based and non–apple-based products. Stud-
ies should continue to investigate and monitor other varieties of
fruit and vegetable products to guarantee that patulin control
measures are applied to all products where a threat exists.
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