Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Review of
Patulin Control
Methods in
Foods
Matthew M. Moake, Olga I. Padilla-Zakour,
and Randy W. Worobo
ABSTRA
ABSTRACT CT
CT:: The my coto
mycoto xin, patulin (4-hy
cotoxin, (4-hydrdr
drooxy -4H-fur
xy-4H-fur
-4H-furoo [3,2c] p yr
pyran-2[6H]-one), is pr
yran-2[6H]-one), oduced b
produced y a number of fungi com-
by
mon to fr uit- and vvegetable-based
fruit- egetable-based pr oducts
oducts,, most notably apples
products apples.. D espite patulin
Despite patulin’’s or iginal disco
original discovver
eryy as an antibiotic, it
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 by
patulin. The danger posed by patulin necessitates its control and removal from food products, creating a demand for
handling and pr ocessing techniques capable of doing so
processing so,, prefer
prefer ably at lo
eferably w cost to industr
low industryy. With this being the case
case,, much
research has been devoted to understanding the basic chemical and biological nature of patulin, as well as its interaction
within foods and food pr oduction. While past rresear
production. esear ch has elucidated a gr
esearch eat deal, patulin contamination continues to be
great
a challenge for the food industr
industryy. H er
Her e, w
ere wee rreview
eview in depth the past rresear
esear ch on patulin with an emphasis upon its influence
esearch
within the food industr
industry y, including its rregulation,
egulation, health effects
effects,, biosynthesis
biosynthesis,, detection, quantification, distr ibution within
distribution
foods
foods,, and contr ol, dur
control, ing the vvar
during ar ious stages of apple juice pr
arious oduction. F
production. inally
inally,, key ar
Finally eas wher
areas wheree futur
future e patulin rresear
esearch
esearch
should focus to best control the patulin contamination problem within the food industry are addressed.
8 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 1, 2005 © 2005 Institute of Food Technologists
Patulin control in foods . . .
While 50 g/L is now the norm for patulin regulation, several found to be naturally contaminated by patulin (WHO 1990).
countries have set even lower limits for patulin at 25 to 35 g/L While this fact has since been disproved (see below, Patulin With-
(van Egmond 1989). In conjunction with these maximum content in Foods), apple juice and cider remain the major source of hu-
standards, a joint Food and Agriculture Organization-World man patulin consumption.
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- Health Effects
ulation on patulin, but today the U.S. Food and Drug Administra- Assessment of the health risks posed by patulin to humans is
tion limits patulin to 50 g/L in single-strength and reconstituted based upon a wide number of studies during the past 50-plus
apple juices (USFDA 2004). The limitation of these regulations to years that implicate a number of acute, chronic, and cellular level
apple juice and apple juice concentrate was likely based upon health effects as summarized in Table 1. Results of these studies
the fact that, at the time, only apple juice and cider had been 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
Figure 1—The structure of and kidney damage (Walker and Wiesner 1944; Escoula and oth-
patulin (4-hydroxy-4H-furo ers 1977; Hayes and others 1979; McKinley and Carlton 1980a,
[3,2c] pyran-2[6H]-one) 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
4 by addition of acetic acid). This prepared extract is ready for ed as its trimethyl silyl derivative (TMS-patulin) with electron ion-
HPLC analysis. The recommended liquid chromatography (LC) ization (Roach and others 2002). In addition, GC-MS with nega-
systems include analytical reversed-phase LC columns such as tive ion chemical ionization allows detection of underivatized pat-
octadecylsilane fully end-capped with 5 m particle stationary ulin in apple juice extracts. A variety of capillary columns in qua-
phase, 12 to 25 nm pore size, carbon loading of 12% to 17%, drupole, ion trap, and magnetic sector instruments can be used
and a UV detector set at 276 nm, although a photo diode array for patulin confirmation when present in apple juice at 68 to
detector is preferred to aid in the presumptive identification of the 3700 g/L (Roach and others 2000). Underivatized patulin was
patulin peak. The system can be run isocratically at 1 mL/min us- successfully analyzed via GC with electronic pressure control, on-
ing 3% to 10% acetonitrile in acidified water (0.095 parts per vol- column injection, and selected ion monitoring allowing detection
ume perchloric acid 60%) as long as patulin separates from 5-hy- limits of 4 g/L (Llovera and others 1999). In another study, a
droxymethylfurfural (HMF), a common compound found in apple combination of diphasic dialysis extraction, in-situ acylation, and
juice that elutes just before patulin. For cloudy apple juice and ap- GC-MS permitted the quantification of patulin from 10 g to 250
ple puree, a collaborative study of 12 participants from European g/L (Sheu and Shyu 1999).
countries was recently conducted to validate the effectiveness of Rychlik and Schieberle (1999) developed 2 methods to quanti-
this LC procedure for patulin determination with a slight modifica- tate patulin by stable isotope dilution assays using 13C-labeled
tion. Prior to the ethyl acetate extraction, the samples were treated patulin as the internal standard. One method used LC/MS in neg-
with pectinase enzymes and held overnight at room temperature ative electrospray ionization mode without derivatization, while
or for 2 h at 40 °C and then centrifuged at 4500 × g for 5 min. the 2nd procedure utilized high resolution gas chromatography/
Based on the results, the method is recommended for patulin at high resolution mass spectrometry (HRGC/HRMS) after trimethylsi-
greater than 50 g/L in cloudy apple juice and purees (Mac- lylation of the patulin isotopomers. The HRGC/HRMS method
Donald and others 2000). showed better repeatability, higher recovery rates, and 100 times
The simultaneous quantification of patulin and HMF in apple lower detection limit (0.012 g/L) compared with the standard
juice by reversed-phase HPLC has been reported by Gokmen and HPLC/UV procedure. This highly sensitive method might be useful
Acar (1999). The method developed uses a 5-m C18 analytical for physiological studies on patulin metabolism in humans.
column (150 × 4 mm), a photodiode array detector operating at Sewram and others (2000) developed an HPLC-MS-MS method
250 to 300 nm, and a mobile phase of water-acetonitrile (99:1 v/ with selective reaction monitoring to achieve a detection limit of 4
v) at 1.0 mL/min. Sample preparation followed the official method g/L without derivatization. The MS detection was performed after
described earlier. Complete separation of HMF and patulin was atmospheric pressure chemical ionization in negative ion mode.
achieved in less than 9 min at detection limits of less than 0.01 This procedure correlated well (R = 0.99) with the standard HPLC/
g/L and less than 5 g/L respectively. UV method.
The HPLC/UV procedure is routinely used for quantitative deter- An improved LC/MS patulin determination has been reported
mination of patulin in apple products, but methods to confirm the by Takino and others (2003). They compared atmospheric pres-
presence of patulin usually include more specific detection tech- sure chemical ionization against atmospheric pressure photoion-
niques such as mass spectrometry (MS) after LC or gas chroma- ization and found that it provided lower chemical noise and sig-
tography (GC) separations. For GC-MS analysis, patulin is detect- nal suppression, thus allowing higher sensitivity. Quantification of
patulin. In Wisconsin, 23 of 40 roadside apple juices were found hibits the ability to effectively sanitize. Even if effective strategies
to contain between 10 to 350 g patulin/L (Brackett and Marth are successfully mapped out, the inherent variability in apple han-
1979a). A 2nd study showed that 8 of 13 commercial apple juices dling facilities will require customization of sanitation methods for
tested contained between 44 and 309 g patulin/L juice (Ware each operation (Rosenberger 2003), posing a considerable cost to
and others 1974). A Turkish study showed 215 of 215 apple juice producers.
concentrates examined had between 7 and 375 ppb patulin with Apple storage poses yet a 3rd means of fungal control and
43% being above the 50-ppb international standard (Gokmen contamination. Conflicting evidence exists as to whether stan-
and Acar 1998). Finally, a 1996 to 1998 study in South Africa dard controlled-atmosphere, refrigerated storage is sufficient to
showed that 5 of 22 juices sampled contained between 10 and prevent apple soft rot (Sommer and others 1974; Lovett and oth-
45 ppb patulin, and 29% of infant apple products showed 5 to ers 1975a; Paster and others 1995). Classically associated with
20 ppb (Brown and Shephard 1999). A summary of patulin con- wounded fruit, in the mid-1990s, nonwounded, stem-end in-
tamination within foods is given in Table 2. 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
Control During Apple Harvest, Processing, and Storage apples (Rosenberger 2003). Furthermore, these facilities are not
In North America, apple juice is typically a byproduct pro- available to all producers, forcing many to use deck-storage,
duced from culled apples unfit for other, higher quality and higher which can drastically increase patulin production. Alternatives
profit, purposes (Rosenberger 2003). Apples are typically harvest- to room storage are the use of packaging materials such as poly-
ed and processed, and those lower quality apples unfit for 1st ethylene, which through their own atmospheric control, have
quality edible retail apples are stored in controlled atmosphere, been shown to reduce patulin production in apples (Moodley
refrigerated storage for anywhere up to around 12 mo when the and others 2002). In the same study mentioned previously (Jack-
next harvest comes in. son and others 2003), patulin was not detected in cider from
Current methods for preventing mycotoxin contamination of culled, tree-picked apples stored 4 to 6 wk at 0 to 2 °C, but was
foods include (1) strict adherence to moisture control, (2) imple- detected at levels between 0.97 and 64 g/L in stored, tree-
mentation of good manufacturing practices, (3) good quality as- picked, unculled fruit. Cider from apples stored in controlled at-
surance efforts, and (4) adherence to Hazard Analysis Critical mosphere and culled showed 0 to 15.1 g/L patulin while un-
Control Points principles (Lopez-Garcia and Park 1998; Park and culled apple fruit showed 59.9 to 120.5 g/L patulin. Thus, stor-
others 1999). Existing and developmental preventive measures age can exacerbate any flaws in prior handling steps, such as
during preproduction are based upon fruit quality and facility harvesting. The longer the storage of the apples, the greater the
sanitation measures. The quality of fruit resulting from harvesting risk of increased patulin content. This is particularly evident to
is the 1st step in controlling patulin levels. With the highest juice producers within the U.K. who see dramatic rises of patulin
quality hand-picked fruit being used for direct-for-retail sale, content within juice produced in June, July, and August—the
processed apple products usually are produced from mechani- months just prior to the new harvest season where source ap-
cal harvest, windfalls, insect-damaged, or culled fruit. Bruises, ples have been stored for almost a year (Corbett 2003). Further-
skin breaks, and other physical damage within these apples pro- more, even if apples are perfectly processed prior to storage, ad-
vide a perfect entry for P. expansum and other patulin-producing dition of diphenylamine, which is used to treat ‘storage scald’ in
species into the fruit. Studies have examined the effect of fruit stored apples, can provide an inoculum of patulin-producing
quality and harvest method on the patulin content of the result- fungi in the fruit (Rosenberger 2003).
ant juices. In 1 study, patulin was undetectable in 7 cultivars of Various treatments have been investigated for the ability to re-
tree-picked cider, whereas it was detected between 40.2 and duce the apple rot and patulin production seen within the har-
374 g/L in 4 cultivars of ground-harvest cider (Jackson and vest, processing, and storage steps. Postharvest treatment of ap-
others 2003). Many of the patulin control measures suggested ples with benzimidazole fungicides was used from the 1970s
by the Joint FAO/WHO Food Standards Programme are based through the early 1990s but has since been largely abandoned
upon the careful selection of fruits as based upon good agricul- due to fungal resistance (Rosenberger 2003). Insecticide treatment
tural practice (CODEX 2002). However, as indicated by a New of apples can reduce growth of patulin-producing molds and
York State Hudson Valley Lab study that showed around 20% of thereby patulin production between 85% and 100% (Draughon
bagged, in-store, retail apples to contain consumer-visible blue- and Ayres 1980), but the treatment raises questions about the
rot (Rosenberger 2003), even the highest level of fruit selection safety of organophosphate insecticides and is thus nonpreferen-
and good agricultural practices cannot eliminate apple-rot and tial. Combinations of heat treatment, calcium infiltration, and bio-
associated patulin contamination. logical control with an antagonistic bacteria, Pseudomonas syrin-
Standard postharvest processing, including washing, sorting, gae, were investigated alone or in combination to reduce inci-
and packaging, poses a 2nd means of both fungal control and dence of postharvest apple decay. Apples inoculated with P. ex-
contamination alike. Washing with high-pressure water has been pansum and heated at 38 °C for 4 d showed no decay lesions af-
shown to reduce patulin levels within apple juice by 21% to 54% ter storage at 1 °C for 6 mo. After heat-treatment inoculation with
(Acar and others 1998). A 2nd study showed that washing of P. expansum followed by infusion of 2% calcium chloride alone
ground-harvested apples resulted in a 10% to 100% patulin re- and Ps. syringae treatment alone reduced decay incidence by
duction, depending on initial patulin level and washing treatment 25% each. Calcium treatment plus Ps. syringae treatment without
(Jackson and others 2003). However, these same washes can also and with subsequent heat treatment reduced decay by 89% and
serve as a source of contamination. Contaminated bins, storage 91%, respectively (Conway and others 1999). Finally, other meth-
rooms, drencher washes, drying brushes after apple wash, and ods used for the control of human pathogens like Escherichia
other steps within the processing cycle can all provide a source of coli, such as washing with solutions of peroxyacetic acid, chlo-
fungal inoculum cycling in poorly sanitized setups. Prevention rine dioxide, and chlorine (Sapers and others 1999; Wisniewsky
methods aimed at cleaning and sterilizing storage and processing and others 2000; Annous and others 2001), may also provide
facilities routinely and in between seasons are being mapped out, some benefit toward preventing postharvest apple decay. Howev-
but have not yet been fully developed. Plus, the older, complicat- er, these methods have yet to be analyzed for their effectiveness
ed design of many packing-house and processing equipment in- against fungal spores.
Vol. 1, 2005—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 13
CRFSFS: Comprehensive Reviews in Food Science and Food Safety
Control during Production new toxic substances, (3) retain nutritive value/acceptability of the
For both removal of patulin during production and postproduc- product, (4) not significantly alter the technological processes as-
tion, effective decontamination/detoxification procedures must (1) sociated with the product, and (5) if possible, destroy fungal
inactivate, destroy, or remove the toxin, (2) not produce or leave spores (Park and others 1988). Methods of patulin control within
standard apple juice production steps are centered on 3 areas. levels is the pasteurization process. Of the 3 processes mentioned
The 1st of these involves the quality of the fruit and processing of thus far, this is by far the least effective. Repeated studies have
this fruit, prior to pressing. As previously mentioned, processed shown that, while unstable at high pH (McCallum and others
apple products often utilize lower quality fruit that is unsuitable 2002), patulin is relatively stable to thermal degradation in the pH
for direct market retail. Removal of decayed/ damaged fruit or range of 3.5 to 5.5, with lower pH leading to greater stability
trimming of moldy portions can significantly reduce patulin levels (Heatley and Philpot 1947; Lovett and Peeler 1973). The half-life
in apple products (Lovett and others 1975b; Taniwaki and others of patulin held at 25 °C at pH 6.0 and 8.0 has been shown to be
1992; Sydenham and others 1995, 1997; Beretta and others 55 and 2.6 d, respectively (Brackett and Marth 1979c). In 1 study,
2000). Trimming of rotten sections of apple has been shown to re- no patulin reduction occurred during concentration and pasteur-
move up to 99% of patulin contamination (Lovett and others ization (Aytac and Acar 1994), while a 2nd study did show a 50%
1975b). However, this process is expensive and labor intensive. reduction of patulin in apple juice treated for 20 min at 80 °C
Furthermore, patulin can be detected in visibly sound fruit (Jack- (Scott and Somers 1968)—a much longer treatment than used in
son and others 2003) and can spread from rotten areas of apples standard pasteurization procedures. A 3rd study showed pasteur-
into sound areas (Beretta and others 2000). Two studies showed ization treatments between 60 and 90 °C for 10 s were only able
that patulin could diffuse 1 to 2 cm from the rotten core in apples to reduce patulin levels by 18.8% (Wheeler and others 1987). Ev-
(Taniwaki and others 1992; Rychlik and Schieberle 2001), 0.6 cm idence also shows that patulin is nonvolatile and, upon distilla-
in pears (Laidou and others 2001), and throughout the entire fruit tion production of apple aroma, patulin remains within juice con-
in tomatoes (Rychlik and Schieberle 2001). Producers’ tests on centrate (Kryger 2001). Finally, not only is the pasteurization pro-
apples have shown that patulin level is often not related to the cess unable to significantly reduce patulin levels, it often fails to
physical quality of the fruit, with both high-rot fruit and top-quali- fully remove heat resistant patulin-producing fungi, such as B.
ty eating fruit often having high levels of patulin in tests (Corbett nivea and B. fulva (Ough and Corison 1980), allowing for poten-
2003), as further demonstrated by the previously mentioned Hud- tial continued production of patulin within the finished juice.
son Valley study on retail apples. While often beneficial, the sort- As a final stage of the production process, studies have exam-
ing of decayed apples to the level of being effective is difficult to ined the effects of storage on patulin content. Mixed studies show
impossible, often necessitating the need to reject entire juice either a decrease (Scott and Somers 1968; Harwig and others
loads of apples. If reliant on this method, small-scale producers 1973a) or no change (Pohland and Allen 1970; Zegota and others
who cannot afford these losses will be forced to sort by hand, in- 1988) of patulin levels in apple juice with refrigerated storage.
creasing costs to the point that many may cease operating (Rosen- Similar studies within grape juice have shown stability (Ough and
berger 2003). Corison 1980) or an approximate 50% decrease (Scott and Som-
The 2nd area of standard juice production capable of reducing ers 1968) of spiked patulin levels in grape juice after 5 wk.
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 Control Postproduction
and authenticity qualities. Some sources suggest that standard
fruit juice production processes remove only about 20% of patu- Filtering and adsorption
lin (Stray 1978; Harrison 1989). One study showed that the tradi- A number of studies have been devoted to the removal of patu-
tional apple juice production processes of depectinization, clarifi- lin from juice through the use of adsorption filters, columns, and
cation, and filtration through a rotary vacuum precoat filter could agitation treatments using carbon-based material. Agitation with
reduce patulin levels by 39%. In the same study, use of depectini- 20 mg/mL activated charcoal followed by filtration through a 40-
zation, clarification, mixing with gelatin/bentonite, and ultrafiltra- or 60-mesh charcoal column reduced a 30-g/mL patulin solu-
tion resulted in a 25% decrease in patulin (Acar and others 1998). tion to below detectable levels. Further, use of 5 mg/mL charcoal
A 2nd study examined the effects of gelatin/bentonite flocculation, in agitation was able to reduce patulin to below detectable levels
filtration, activated charcoal, ultrafiltration, polyvinylpolypyrroli- in naturally contaminated cider. However, color loss was marked-
done, and polystyrene-Divinyl Benzene (DVB)–based macro po- ly present in this resulting juice (Sands and others 1976). In a 2nd
rous resin on apple juice color, clarity, phenolic content, organic study, 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3 g/L activated charcoal was
acid content, and patulin reduction. Activated charcoal treatment added to naturally contaminated apple juice containing 62.3 ppb
had the highest patulin reduction with 40.9% but also significant- patulin. Samples were mixed for 0, 5, 10, 20, and 30 min. Three
ly reduced color and phenolic content, 2 characteristics associat- grams/liter activated charcoal was found to be most effective with
ed with juice authenticity. Polystyrene-DVB–based macro porous a time period of 5 min. Clearness of juice increased, color of juice
resin was the 2nd most effective, reducing patulin by 11%. None decreased, and small decreases in fumaric acid, pH, and °Brix
of the treatments altered organic acid content significantly (Artik were also seen (Kadakal and Nas 2002). In another study, ultrafine
and others 2001). Centrifugation and fining of juice pulp have activated carbon was bound to granular quartz producing a com-
been shown to reduce patulin levels by 89% and 77%, respec- posite carbon adsorbent (CCA). Columns with varying amounts of
tively. However, these methods make the removed cake and/or fil- CCA were prepared and 10 g/mL patulin were filtered through at
ter potentially highly toxic and unfit for any further use, such as 1 mL/min. Fifty percent breakthrough values for columns with 1.0,
often is done with juice sediments in animal feed (Bissessur and 0.5, and 0.25 g CCA were 137.5, 38.5, and 19.9 g, respectively
others 2001). This could represent a loss of income for many juice (Huebner and others 2000).
producers. Batch absorption with synthetic polymers has also In a 4th study designed to compare the effects of different car-
been investigated (Canas and Aranda 1996), as has enzyme treat- bon activation methods, steam activated carbon NORIT SA 4 and
ment with pectinase enzymes used to break down the pectin coat NORIT SX 4 performed equally, removing 80% and 70%, respec-
surrounding protein particles, allowing aggregation and sedimen- tively, of an initial 1 g/L patulin solution in 12 °Brix juice at 55 °C.
tation of protein particles and, in the case of patulin contamina- Chemically activated carbon NORIT CA 1 removed only 45% of
tion, their associated patulin adducts. This method has resulted in the same solution. This study also showed that increased °Brix
a 73% decrease in patulin content within juice (Bissessur and oth- levels of juice led to decreased patulin removal efficiency, with
ers 2002). NORIT SA 4 removing only 20% of patulin from 20 °Brix juice.
The 3rd juice production process capable of reducing patulin Within this study, patulin removal was independent of juice tem-
Vol. 1, 2005—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 15
CRFSFS: Comprehensive Reviews in Food Science and Food Safety
perature between 30 and 65 °C (Leggott and others 2001), while of the double bonds (Burroughs 1977). A 4th study found the sul-
in another study, patulin binding to activated carbon compounds fur dioxide inactivation of patulin to be reversible or irreversible de-
was shown to be endothermic, with increased temperatures re- pending on pH (Steiner and others 1999b).
sulting in improved patulin removal (Mutlu and others 1997). Sulfur compounds common to biological systems and fre-
Despite these initial studies where activated carbon was shown quently associated with patulin toxicity, such as glutathione, cys-
to reduce patulin, little work has been done to optimize carbon teine, and thioglycolate, are believed by many to produce biologi-
for this process. Furthermore, the use of activated carbon poses a cally inactive products when reacted with patulin (Cavallito and
substantial cost to the juice industry (Leggott and others 2001), Bailey 1944; Geiger and Conn 1945; Reinderknecht and others
being both time consuming and expensive (Bissessur and others 1947; Singh 1967; Hoffman and others 1971; Ciegler and others
2001). Outside of the cost of carbon material itself, activated char- 1976). In 1 study, treatment with cysteine and glutathione pre-
coal treatment creates excess waste that must be dealt with eco- vented the negative effects of patulin infection on rumen fermen-
logically (Artik and others 2001). Also, as mentioned within sever- tation, while non-sulfhydryl–containing ascorbic acid and ferulic
al of the studies, negative affects on color, fumaric acid content, acid did not protect against patulin toxicity (Morgavi and others
pH, and °Brix have been observed with carbon adsorption. These 2003). Patulin has been shown to form a number of adducts with
and other modifications made by carbon absorption treatments N-acetylcysteine, glutathione, and cysteine (Fliege and Metzler
can negatively alter the taste perception and quality of the juice 2000). While patulin-cysteine adduct mixtures are slightly bacteri-
(Huebner and others 2000). Finally, the use of clays can pose a cidal to Gram-positive and Gram-negative species (Geiger and
risk due to removal of essential nutrients from juice (Park and Conn 1945; Reinderknecht and others 1947), they have been
Troxell 2002). Similar analysis with activated carbon compounds shown to reduce the bactericidal effects of patulin by more than
has not been performed, but could very likely have the same neg- 100 fold. Furthermore, treatment of mice with a cysteine-patulin
ative result. adduct mix, 85 times the equivalent lethal dose50 concentration
of patulin alone, resulted in no deaths or macroscopic pathologi-
Chemical modification cal effects (Lindroth and von Wright 1990).
Chemical decontamination methods have also been shown to Another chemical method examined for the decontamination
be effective and are likely the most easily suitable for industry. of patulin has been treatment with a variety of organic acids and
Many of the chemicals examined thus far are already considered vitamins, many of which are considered to be food-grade addi-
food grade additives/treatments, and therefore, their use would be tives. In one study, addition of ascorbate and ascorbic acid in-
preferential to many other forms of treatment. Plus, because addi- creased the rate at which patulin was reduced from solution in a
tion of chemical agents to human foods solely for reducing myc- concentration-dependent manner. These reduction rates were
otoxin levels is currently not permitted in the U.S. (Park and Trox- found to be slower in juice, but still present. A mechanism was
ell 2002), treatments already accepted for other purposes would proposed in which a reaction of ascorbate or ascorbic acid with
allow more rapid integration within industry. Furthermore, many metal ions produced singlet oxygen molecules that attacked and
of these same treatments have been shown to be effective in in- oxidized patulin. However, no evidence for any such reaction
hibiting patulin-producing molds (see below, Patulin Production products was given (Brackett and Marth 1979b). A 2nd study uti-
in the Final Product), making them a double-edged sword in patu- lizing ascorbic acid found that treatment with 500 mg/kg juice
lin control. could produce a 50% reduction of patulin (Aytac and Acar 1994).
Numerous chemical treatments have been utilized to detoxify A 3rd study, however, found that degradation by ascorbic acid re-
patulin. Included within these treatments are chemicals designed sulted in only 5% degradation in 3 h and 36% degradation after
to oxidize and reduce patulin to hopefully less toxic compounds, 44 h (Fremy and others 1995). Besides ascorbic acid, treatment of
as well as treatments to bind up patulin in the form of less toxic apple juice with the B vitamins, thiamine hydrochloride, pyridox-
thiol-based adducts. Two of the most potent means of chemical ine hydrochloride, and calcium-d-pantothenate, caused statisti-
detoxification of patulin are ammoniation and potassium perman- cally significant reductions in patulin content over control juices
ganate oxidation. Treatment with ammonia has been shown to re- (Yazici and Velioglu 2002).
duce patulin levels by up to 99.9% in laboratory waste (Fremy Yet another chemical detoxification method examined has been
and others 1995) and 99.8% in juice. However, in the 2nd study, the use of ozone, which has recently been approved for use in
the resultant product was unfit for consumption (Ellis and others the treatment and processing of foods (FR 2003) and has been
1980). Oxidation by potassium permanganate in acidic and alkali shown as an effective alternative to heat pasteurization of juice. In
conditions also resulted in better than 99.99% patulin reduction 1 study, treatment of a 32 M patulin solution with 10% ozone
in laboratory waste (Fremy and others 1995). for 15 s reduced patulin to undetectable level and produced no
Treatment with various sulfur-containing compounds has been detectable reaction products (McKenzie and others 1997). Fur-
another area of intense investigation. Studies involving the use of ther, other mycotoxins such as aflatoxins B1, G1, B2, and G2
sulfur dioxide have produced mixed results. Most studies agree that (Maeba 1988) as well as zearalonone (Doyle and others 1982)
patulin is unstable in the presence of sulfur dioxide (Pohland and have been shown to be effectively degraded by ozone.
Allen 1970), but they vary on its degree of effectiveness. One study In all of these cases, preliminary evidence is promising. Howev-
showed that 100 ppm sulfur dioxide immediately reduced patulin er, inadequate research has been done to examine the efficacy
levels by 50% (Ough and Corison 1980). In a 2nd study, a 42% re- and full functional range of applicability for the treatment. Further-
duction of patulin was achieved with 100 mg sulfur dioxide/kg ap- more, the reaction mechanisms for few if any of the chemical
ple juice (Aytac and Acar 1994). However, in a 3rd study, 200 ppm detoxification treatments are fully understood, and both the reac-
sulfur dioxide, the maximum allowable within the food industry, tion products and their respective toxicities are still unknown and
caused only a 12% patulin decrease in 24 h. Sulfur dioxide (2000 must be determined prior to use. The regulatory approval for
ppm) showed a 90% decrease after 2 d and plateaued at that level. some of the proposed chemical treatments such as ammoniation
Within this study, 2 reactions were shown to be involved. The 1st, a and potassium permanganate must be sought prior to use in the
reversible reaction, occurred presumably through the addition of food industry.
sulfur dioxide to the hemiacetal ring of patulin, forming a carbonyl
hydroxysulfonate. The 2nd reaction is thought to be irreversible Biological control
and to occur due to an opening of the lactone ring structure at one Biological methods of patulin control result largely from the ob-
16 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 1, 2005
Patulin control in foods . . .
servation that patulin is almost always completely degraded dur- standard cold storage temperature down to 0°C (Sommer and
ing yeast fermentation. Besides being quite successful, this meth- others 1974). Taken together, these 2 facts suggest that finished
od is much better understood compared with other decontamina- apple juice and cider could very well support the continued pro-
tion methods. Approximately 90% of patulin can be removed duction of patulin by fungi. If this is the case, any single treatment
during yeast fermentation (Burroughs 1977). In 1 study, 6 of 8 patulin control method, such as physical adsorption, would po-
yeast strains reduced patulin levels to below detectable levels, tentially be moot due to production of patulin by active fungi
while all 8 strains resulted in a 99% or better decrease in total pat- postprocessing. Studies must be done to examine this potential
ulin content. Control juice, on the other hand, stored for an equal within juices, and, if possible, to provide correlates between mold
amount of time (2 weeks), had only a 10% reduction (Stinson and numbers and patulin levels within juice.
others 1978). In a 2nd study, yeast fermentation reduced patulin With the possibility of fungi actively producing patulin within
levels completely after 2 wk. This same study also showed that juice, control measures should aim not only at the reduction of
patulin levels failed to decrease significantly in juices that had patulin itself, but the inhibition of both fungal growth and patulin
been yeast fermented and then filter sterilized to remove yeast, production within juice. Some studies have examined the effects
suggesting that active yeast, and not their byproducts, were re- of many patulin-reducing treatments on live patulin-producing
quired for the reduction (Harwig and others 1973a). Treatments of fungi. Sulfur dioxide, sodium benzoate, and potassium sorbate
patulin along with cyclohexamide, a protein synthesis blocker of have been investigated for the affect on B. nivea growth and patu-
yeast, completely blocked protein synthesis and prevented the lin production within juice. Seventy-five ppm sulfur dioxide, 150
detoxification of patulin. Addition of cyclohexamide 3 h after pat- ppm potassium sorbate, and 500 ppm sodium benzoate all signif-
ulin addition, however, resulted in a reduced, but continued, rate icantly retarded B. nivea growth and patulin production (Roland
of patulin degradation, suggesting that the proteins synthesized and Beuchat 1984a). A 2nd study showed that treatment of B.
within the 3-h window were catalytically active against patulin nivea with 50 g/mL potassium sorbate completely eliminated
and did not just bind it up in adduct formation (Sumbu and others patulin production at 37 °C. However, higher concentrations (75
1983). A later study showed that 3 strains of Saccharomyces cere- and 100 g/mL) were needed at lower temperatures (21 °C) to in-
visiae reduced patulin levels during fermentive growth but not hibit growth, and even with growth inhibition, these treatments
aerobic growth. This reduction resulted in the production of 2 ma- failed to inhibit patulin production (Roland and others 1984b). A
jor products: E-ascladiol, patulin’s immediate biosynthetic precur- related study showed that 0.1% of potassium sorbate eliminated
sor, and its isomer Z-ascladiol. These 2 products were also seen in patulin production in P. patulum, but interestingly, this same treat-
the treatment of patulin with the reducing agent sodium borohy- ment elevated patulin production in P. roqueforti (Bullerman and
drate (Moss and Long 2002). E-ascladiol is itself a mycotoxin (Su- Olivigni 1974). A 4th study showed that 0.1 M potassium sorbate
zuki and others 1971), which has reduced toxicity compared with reduced P. expansum growth by 83% and patulin production by
patulin and also reacts with sulfhydryl-containing compounds 98%, while 0.1 M sodium propionate caused a 48% reduction in
(Sekiguchi and others 1983). growth and 98% reduction in patulin production (Lennox and
While effective, biological control with yeast is limited to prod- McElroy 1984).
ucts that can be fermented. Furthermore, yeast are themselves A separate set of studies showed that treatment with 0.2% lem-
sensitive to patulin, and at concentrations greater than 200 g/ on oil reduced patulin production, and a mix of 0.05% lemon oil
mL, yeast have been shown to be completely inhibited, prevent- and 0.2% orange oil resulted in a 90% reduction of patulin pro-
ing fermentive detoxification (Sumbu and others 1983). No re- duction (Hasan 2000). Finally, in a series of unsuccessful studies,
search has been done to examine the potential use of other fer- addition of 0.5% and 0.75% citric and lactic acid reduced pro-
menting microbes, such as lactic acid bacteria, in decreasing pat- duction of aflatoxins and sterigmatocystin, but not patulin. These
ulin content within juices. Similar reducing enzymes and environ- same treatments also failed to inhibit P. expansum growth (Reiss
ments produced by these bacteria may very well be able to de- 1976).
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- Conclusions
zymes such as -lactamase, may well be able to degrade patulin While studies investigating the health effects of patulin have
alone. proved inconclusive, there is little doubt as to the potential danger
inherent in the contamination of food products by patulin. Past re-
Electromagnetic irradiation search has elucidated a great deal about the chemical and biolog-
Electromagnetic radiation has been shown in preliminary stud- ical nature of patulin, has made advances in methods for detect-
ies to reduce the content of patulin and other mycotoxins within ing patulin, and has revealed a number of potential control mea-
juice. In 1 study, treatment of juice with 0.35-kGy ionizing radia- sures that may provide a basis for fully effective control measures
tion caused a 50% reduction of patulin with no acceleration of within the future. Still, patulin contamination continues to be
nonenzymatic browning of the juice (Zegota and others 1988). present within the food industry. Thus, future research must con-
The UV light sensitivity of aflatoxins has been demonstrated on tinue to address the threat of patulin contamination within food
several accounts, but no studies have been performed specifically products, with an emphasis upon the following areas.
on patulin (Doyle and others 1982; Valletrisco and others 1990).
Patulin detection
While methods for detecting and quantifying patulin have great-
Patulin Production in the Final Product ly improved over the years, the sensitivity of these methods is still
Limited work has been done to examine the potential growth often a limiting factor for many aspects of patulin control re-
and production of patulin by fungi in stored, finished product search. Currently, no rapid analytical technique exists for the anal-
juices. It is well known that many Aspergillus and Byssochlamys ysis of patulin, and the standard methodology for patulin quantifi-
spp. molds, many of which produce patulin, are heat resistant cation requires dedicated instrumentation and trained operators.
and can survive the pasteurization processes used in juice and ci- A rapid detection method that can be used “in-house” by the
der production. Furthermore, many of the same patulin-produc- food industry without any complicated equipment would be ex-
ing molds have also been shown to grow and produce patulin at tremely beneficial.
Vol. 1, 2005—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 17
CRFSFS: Comprehensive Reviews in Food Science and Food Safety
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