Mycotoxins in Fruits, Fruit Juices, and Dried Fruits

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1 1514 Journal of Food Protection, Vol. 66, No. 8, 2003, Pages Copyright q, International Association for Food Protection Review Mycotoxins in Fruits, Fruit Juices, and Dried Fruits S. DRUSCH 1 * AND W. RAGAB 2 1 Arbeitsgruppe für Lebensmittelqualität und -sicherheit, University of Kiel, Heinrich-Hecht-Platz 10, Kiel, Germany; and 2 Department of Food Science and Technology, Assiut University, Egypt MS : Received 5 November 2002/Accepted 13 February 2003 ABSTRACT This review gives an overview of the presence of mycotoxins in fruits. Although several mycotoxins occur in nature, very few (a atoxins, ochratoxin A, patulin, Alternaria toxins) are regularly found in fruits. It has been shown that the presence of fungi on fruits is not necessarily associated with mycotoxin contamination. The formation of mycotoxins depends more on endogenous and environmental factors than fungal growth does. Mycotoxins may remain in fruits even when the fungal mycelium has been removed. Depending on the fruit and the mycotoxin, the diffusion of mycotoxins into the sound tissues of fruits may occur. The in uence of the selection and storage of fruits and the in uence of different processing steps involved in the production of fruit juices and dried fruits on possible mycotoxin contamination is described. It is shown that the careful selection, washing, and sorting of fruits is the most important factor in the reduction of mycotoxin contamination during the production of fruit juices. The processing of fruits does not result in the complete removal of mycotoxins. Mycotoxins are metabolites of lamentous fungi that have deleterious effects in animals and, in some circumstances, in humans (100). They include a very large, heterogeneous group of substances, and correspondingly mycotoxigenic species can be found in all major taxonomic groups. Mycotoxins exhibit properties of acute, subacute, and chronic toxicity in animals and/or humans, with some mycotoxins also being carcinogenic, mutagenic, and teratogenic. Moreover, mycotoxins cause a loss of viability in plant seeds, reduce the quality and acceptability of all types of products, and limit the storability and decrease the nutritional quality of foods. Therefore, mycotoxins are considered an important worldwide problem in terms of public health, agriculture, and economics (10, 12, 32, 51). It is unavoidable that human and animal foods and feedstuffs will be universally exposed to fungal invasion from the planting of the crop through harvesting, transportation, and storage, and even into the grocery store, restaurant, and home, where the product will await nal use by the consumer (31). A characteristic shared by most fruits is high acidity. The phs of fruits range from 5.0 to,2.5, and this ph range is considered the single most important factor in determining the types of microorganisms that can spoil this class of food. While most species of bacteria are inhibited by such high hydrogen ion concentrations, yeasts and molds are more aciduric, and for many fungal genera or species these ph values are tolerable, if not optimum for growth. It is because of acidity, therefore, that fungi are the principal spoilage microorganisms for fruits and fruit products (106). Fruits become increasingly susceptible to fungal * Author for correspondence. Tel: ; Fax: ; sdrusch@foodtech.uni-kiel.de. invasion during ripening, as the ph of the tissue increases, skin layers soften, soluble carbohydrates build up, and defense barriers weaken (87). However, the growth of fungi is not necessarily associated with the formation of mycotoxins. Within a species, the mycotoxigenic potential of a fungus largely depends on the strain of the fungus. Apart from the species and strain of the fungus, the physical and chemical composition of the matrix and environmental factors play major roles in mycotoxin formation. The presence of fungi provides no assurance of mycotoxin contamination, and because of the stability of mycotoxins, they may be present in food when fungi are no longer present. Furthermore, a fungus may produce different mycotoxins, and a mycotoxin may be produced by several different fungi. The contamination of fruits with mycotoxins has not only caused health hazards but also resulted in economic losses, especially to exporting countries (98). This review represents an attempt to encompass and incorporate the large body of work on mycotoxins in fruits, fruit juices, and dried fruits for producers and manufacturers of this important category of foods. RELEVANT MYCOTOXINS AND MYCOTOXIGENIC FUNGI Although a large number of different mycotoxins exist, only a few of them are regularly found in foods. The mycotoxins most commonly found in foods are a atoxins, ochratoxin A, patulin, and mycotoxins produced by Fusarium species. The latter group of mycotoxins are not covered in this review, since their occurrence is limited to grains and seeds. Table 1 gives an overview of the mycotoxins covered in this review and the relevant mycotoxigenic fungi producing them.

2 J. Food Prot., Vol. 66, No. 8 MYCOTOXINS IN FRUIT PRODUCTS 1515 TABLE 1. Mycotoxins and fungal species occurring in fruits Mycotoxin(s) Fungi producing mycotoxin References A atoxins B1, B2, G1, Aspergillus chevallieri, A. avo-furcatis, A. avus, A. avus var. columnaris, 7, 60, 104, 122 G2 A. niger, A. oryzae, A. parasiticus, A. repens, A. ruber, A. tamarii, A. wentii Alternariol, alternariol Alternaria alternata, A. tenuissima, A. solani 43, 114 methyl ether, tenuazonic acid Ochratoxin A Aspergillus alliaceus, A. melleus, A. ostianus, A. petrakii, A. sclerotiorum, A. 2, 3, 47, 49, 84, 97 sulphureus, A. fumigatus, A. versicolor, A. carbonarius, A. niger, A. ochraceus, Penicillium verrucosum Patulin Penicillium claviforme, P. expansum, P. urticae, P. patulum, Aspergillus clavatus, 17, 55, 64, 92, 93 A. giganteus, Byssoclamys fulva, B. nivea, Alternaria alternata Citrinin P. expansum 29, 124 Trichothecin Trichthecium roseum 118 A atoxins are biologically active secondary metabolites produced primarily by Aspergillus avus and Aspergillus parasiticus (57). They were rst detected and characterized in the 1960s (15) and have been found in many forms of human foods to date. A atoxin B1 and structurally related compounds are of major concern with respect to public health, mainly because of their potential as powerful hepatotoxins and carcinogens in humans and their proven toxicity to animals, birds, and sh (129). Ochratoxin A was originally isolated from Aspergillus ochraceus in Several different ochratoxins exist, but ochratoxin A is the most common. It is primarily a nephrotoxin, but teratogenic, immunosuppressive, and carcinogenic properties have also been ascribed to it. The International Agency for Research on Cancer classi ed ochratoxin A as a possible human carcinogen (1, 53). Several other Aspergillus species are also capable of producing ochratoxin A. Penicillium verrucosum is the only known and con rmed Penicillium species that is able to produce ochratoxin A (3). The contamination of foods with ochratoxin in cool climates is usually caused by P. verrucosum, whereas the occurrence of ochratoxin in foods in warmer and tropical climates is associated with A. ochraceus. Another mycotoxin with relevance to human health is patulin. Patulin has a broad spectrum of toxicities, including carcinogenicity (34) and teratogenicity (29) in animals. Symptoms of experimental cases of patulin toxicosis in animals are lung and brain edema; liver, spleen, and kidney damage; and toxicity to the immune system (62). For humans, nausea, gastrointestinal disturbances, and vomiting have been reported. Patulin is produced by approximately 60 species belonging to.30 genera of fungi (54). The most important producer of patulin is the apple-rotting fungus Penicillium expansum, and apple products may therefore contain patulin when rotten apples have been processed. These products are of toxicological concern, since they are frequently consumed by infants and young children. The World Health Organization recommends a maximum patulin level of 50 mg/liter for apple juice (128). Apart from apples, patulin is also of concern with regard to tomatoes, in which rotting is most frequently caused by Alternaria alternata. Many strains of A. alternata also produce mycotoxins such as alternariol, alternariol methylether, tenuazonic acid, and several altertoxins (108, 125). Alternaria toxins have produced a variety of adverse effects, including anorexia, emesis, gastrointestinal hemorrhages, and convulsions, in animals. Fetotoxic and teratogenic effects have also been described (121). MYCOTOXIN FORMATION IN INOCULATION EXPERIMENTS Table 2 gives an overview of the experimental evidence of the production of mycotoxins on fruits inoculated with mycotoxigenic species. A atoxins. Most papers concerning a atoxin formation on fruits refer to gs or citrus fruits. The fungus A. avus has been shown to be a reasonably vigorous pathogen on ripe g fruits. Fig fruits are very susceptible to infection by A. avus conidia on the exterior fruit surfaces as well as by conidia carried into the interior of the fruit by insects. Buchanan et al. (25) discussed the question whether A. a- vus is a fruit pathogen on gs (with infection, colonization, and a atoxin production occurring in the orchard) or a saprophyte colonizing on the dried product. These authors found that green g fruits are resistant to invasion by A. avus but that as the fruits become rm and ripe and soften they lose their resistance. Le Bars (56) reported that g fruits are a suitable medium for a atoxin production because they contain high carbohydrate levels, and this property is more favorable for toxin formation than for mold growth. In another study on the a atoxigenic potential of raw dried fruits, gs were found to have a higher potential than apricots, pineapples, or raisins (71). The experiments of Buchanan et al. (25) indicate that the conidia of A. avus are capable of penetrating fruit skin. Spores on the surfaces of ripe g fruits were able to infect and colonize the fruits, suggesting that the conidia of A. avus are capable of penetrating the fruit skin rather than entering the fruit through a wound. However, there might have been several alternative ways for a wound pathogen like A. avus to enter fruit readily. Minute wounds might have been present in the fruit skin. The foraging of microscopic animals, such as insects or mites, might have caused wounds that were not readily visible to the human eye. Another possibility is that suf cient fruit juice may have

3 1516 DRUSCH AND RAGAB J. Food Prot., Vol. 66, No. 8 TABLE 2. Mycotoxin production on experimentally infected fruits Commodity Fungus Mycotoxin(s) Reference(s) Oranges Aspergillus parasiticus A atoxins B1, B2, G1, G2 90 Oranges A. avus A atoxin B1 123 Grapefruit juice, grapefruit peel A. avus, A. parasiticus A atoxins B1, B2, G1, G2 7 Orange juice A. avus A atoxin B1 18 Figs A. avus A atoxins B1, B2, G1, G2 25 Tomatoes Alternaria alternata Alternariol, alternariol methyl ether 82 altenuene, altertoxin I, altertoxin II Apples, strawberries, blueberries, A. alternata Alternariol, alternariol methyl ether 63, 121 grapes, mandarines Apples, tomatoes, blueberries A. alternata, A. tenuissima, A. solani Tenuazonic acid, alternariol, alternariol 114 methyl ether (altenuene, al- tertoxin) Oranges, lemons A. citrii Tenuazonic acid, alternariol, alternariol 115 methyl ether (altenuene, alter- toxin I) Apples, peaches, strawberries, greengage tomatoes, bananas, Patulin 40 melons Cherries Pears Aspergillus clavatus, Penicillium claviforme, P. expansum, P. patulum A. alternata, P. expansum, Saccharomyces vesicarium Patulin 64 Patulin 55 been present on the fruit surface to permit some fungal colonization, an event often facilitating penetration. Rapid fungal colonization and a atoxin accumulation presumably continued until fungal growth was stopped by a lack of moisture in the dried fruits (25). In any case, a atoxins constitute a problem that is already present in the orchard. Firm, ripe fruits show little contamination when they are dried immediately (112). In contrast to the situation for gs, A. avus grows on oranges only after the peel has been partially deteriorated by other microorganisms, partly because of the antifungal action of citrus oil against A. avus (123). Furthermore, studies involving the inoculation of citrus fruits with Aspergillus con rm the importance of the carbohydrate content of the substrate but also indicate that other endogenous factors in uence mold growth and a atoxin production. Because of its higher content of nitrogenous compounds and carbohydrates, the grapefruit peel was found to be a better Aspergillus substrate than grapefruit juice (7). A atoxin concentrations produced by the same strains of A. avus or A. parasiticus were 5 to 10 times as high in grapefruit peel as in grapefruit juice. Citric acid, a major constituent of grapefruit juice, does not support toxin formation by A. parasiticus (31). A decrease in a atoxin production over time was observed for A. avus grown on oranges and in orange juice (18, 123). A atoxins are degraded by the mycelium of A. avus itself. The production of a atoxins is at its maximum when the biomass reaches its optimal value, and a atoxin production rapidly declines after the mycelium starts to autolyze (123). Patulin. Apples are commonly used for inoculation experiments undertaken to investigate the formation of patulin on fruits. Early ndings indicated that the presence of a patulin-producing species does not necessarily imply patulin production. Factors like incubation temperature, lesion size, and substrate play important roles (105). Hasan (44) studied mycelial growth and patulin production over time in apples inoculated with P. expansum. Patulin production increased when the mycelial growth reached the late phase, in which the energy source in the medium is nearly depleted and suf cient intermediates have accumulated. The degradation of patulin over time has been observed, and this degradation has been attributed to the action of chemicals like organic acids leaching from the vacuole and to the mycelium of P. expansum itself (44). Other fungi, e.g., Penicillium patulum, have also been shown to produce patulin when cultured on apples (64). Peaches, apricots, strawberries, greengage, and pears are also suitable substrates for patulin production (40, 55). With regard to the safety of fruit products, e.g., when apples are to be cleaned and trimmed for juice production, the diffusion behavior of patulin in the fruit is of interest. In apples, the diffusion of patulin is limited to a depth of up to 1 to 2 cm from the infected area (96, 119). Similar results have been reported for sound pear tissues, in which patulin diffusion at up to 0.6 cm was observed (55). In contrast, for tomatoes, the mycotoxin penetrates the entire fruit (96). For tomatoes, Rychlik and Schieberle (96) found 450 mg of patulin per kg at a distance of 4 cm from the infected tissue with an inoculum concentration of 52.9 mg of patulin per kg. On the basis of the higher water content and the lower content of structure-forming polysaccharides in to-

4 J. Food Prot., Vol. 66, No. 8 MYCOTOXINS IN FRUIT PRODUCTS 1517 matoes than in apples, these authors deduced a higher diffusion coef cient for patulin in tomatoes than for patulin in apples. The mycotoxin may therefore also penetrate through other low-viscous foods like blueberries, grapes, and melons. Because of health concerns about synthetic fungicides, the use of natural substances to control the growth of Penicillium species and subsequent patulin production has recently been investigated. Patulin production was found to be inhibited by 0.2% lemon oil, and the use of 0.05% lemon and 0.2% orange oil was found to result in a reduction of 90% (44). Research has also focused on microorganisms that may be used as biofungicides. The antifungal activity of Bacillus subtilis against the growth of P. expansum has recently been demonstrated (39). Alternaria toxins. Alternaria molds are commonly involved in the fungal spoilage of stored fruits. A key characteristic of Alternaria spp. is their ability to grow at low temperatures. For this reason, they are particularly involved in the spoilage of fruits during refrigerated storage. The growth of A. alternata and the production of alternariol and alternariol methyl ether in grapes were demonstrated by Tournas and Stack (121). Fruits generally affected by Alternaria rot are apples, tomatoes, grapes, blueberries, peaches, cherries, and citrus fruits (115). Some species of Alternaria are nonspeci c, while the occurrence of others, such as Alternaria citrii and Alternaria solanaceous, is limited to certain fruits. Stinson et al. (114) reported the formation of tenuazonic acid, alternariol, alternariol monomethylether altenuene, and altertoxin I on tomatoes and apples after inoculation with known toxigenic Alternaria strains. All species (Alternaria tenuis, A. alternata, Alternaria tenuissima, and Alternaria solani) and strains produced tenuazonic acid, alternariol, and alternariol methyl ether when cultivated on apple and tomato tissue, except for A. alternata 938 and A. solani, which did not produce any mycotoxins when cultivated on tomato tissue. Concentrations of tenuazonic acid were much higher when Alternaria spp. were grown on tomatoes, while concentrations of the dibenzo-a-pyrone toxins alternariol and alternariol methyl ether were higher when Alternaria spp. were cultured on apples. The formation of altenuene and altertoxin I was limited to A. alternata strains. These results clearly show that the extent of toxin formation depends on the substrate on which the fungus is cultivated. The inoculation of homogenized blueberry and tomato tissue with wild strains isolated from those fruits resulted in the formation of considerable amounts of tenuazonic acid, alternariol, and alternariol methyl ether (114). In contrast, Tournas and Stack (121) reported the failure of Alternaria strains to produce alternariol and alternariol methyl ether on blueberries. Two major differences between the two studies concern the Alternaria strains involved and the pretreatments used for the fruits. In the rst study, by Stinson et al. (114), the fruits were mechanically broken and steam disinfected, and this process causes substantial changes in the texture and composition of the fruit. However, the fruit s integrity does not necessarily protect it against mycotoxin formation. The production of Alternaria toxins was demonstrated for naturally infected intact apples and tomatoes and for inoculated intact oranges and lemons (115). Tenuazonic acid was the mycotoxin that was produced most abundantly by Alternaria species on lemons (A. citrii) and on oranges and tomatoes (Alternaria spp.), while alternariol was the predominant mycotoxin for apples. Overall, mycotoxin contents in lemons were lower than those in oranges. Another factor affecting the growth of Alternaria species is competition between fungal genera during growth. For example, it was shown that on strawberries the growth of Alternaria is inhibited by the presence of fast-growing molds like Botrytis and Rhizopus. Therefore, Alternaria species as mycotoxin-producing molds on strawberries are not of great concern (121). NATURAL OCCURRENCE OF MYCOTOXINS ON FRUITS Mycotoxin formation subsequent to the experimental cultivation of a fungus on fruit tissue does not necessarily indicate that a mycotoxin naturally occurs on a fruit. The natural occurrence of mycotoxins also depends on environmental factors that determine the growth of fungi, the fungal spectrum on a fruit, and endogenous factors. According to the review by Pitt and Hocking (87), defense mechanisms in fruit appear to be highly effective against a variety of fungi; relatively few genera and species are able to invade fruits. Some fungi are highly specialized pathogens, attacking only one or two kinds of fruits, and others have a more general ability to invade fruit tissue. A atoxins and ochratoxin A. Reports concerning aflatoxins on fruits are limited to fruits from regions with relatively high temperatures, which favor the growth of aflatoxin-producing species like gs, dates, and citrus fruits. Natural contamination with a atoxins has been demonstrated for gs and oranges as well as apples, but in some studies no contamination could be observed for gs, peaches, or dates. Concentrations of up to 78 mg of a atoxin G1 per kg and up to 63 mg of a atoxin B1 per kg have been analyzed in gs at different production stages (81). Özay et al. (79) concluded from the results of studies on gs at different harvesting times that contamination with a atoxins occurs only if environmental conditions like temperature and humidity are favorable. A similar conclusion can be drawn for ochratoxin A. For example, of six peaches with cleaved moldy stones, only one was found to contain ochratoxin A (37). Engelhardt et al. (37) analyzed different fruits after the removal of rotten tissue and found up to 2.71 mg of ochratoxin A per kg in cherries and up to 1.44 mg/kg in tomatoes and strawberries. Peaches and apples were also contaminated with ochratoxin A, but to a lesser degree. The results obtained by these authors show that damaged or moldy fruits can be contaminated with ochratoxin A to a certain degree, even after the removal of the rotten parts. Patulin. The occurrence patulin on bananas, pineapples, grapes, peaches, apricots, plums, and tomatoes was

5 1518 DRUSCH AND RAGAB J. Food Prot., Vol. 66, No. 8 TABLE 3. Natural occurrence of mycotoxins on fruits Reference Toxin(s) Commodity No. of samples positive/ no. of samples analyzed Maximum concn. 90 A atoxin B1 A atoxins B1, B2, G1, G2 Oranges 8/25 52 mg/kg 120 m/kg 44 A atoxins B1, B2, G1, G2 Apples, brown lesions Apples, remainders 30/30 0/ mg/kg 81 A atoxin B1 A atoxin B2 A atoxin G1 A atoxin G2 Figs, different production stages before processing 23/ mg/kg 37.7 mg/kg 78.3 mg/kg 15.0 mg/kg 79 A atoxins B1, B2, G1, G2 Figs 0/12 89 A atoxins B1, B2, G1, G2 Dates 0/5 66 Citrinin Apples 14/ mg/kg 37 Ochratoxin A Tomato a Cherry a Strawberry a Apple a Apricot a Peach a Nectarine a 6/11 6/6 4/10 2/4 0/2 1/9 0/ mg/kg 2.71 mg/kg 1.44 mg/kg 0.41 mg/kg 0.59 mg/kg 81 Ochratoxin A Figs 1/ mg/kg 79 Ochratoxin A Figs 0/12 37 Ochratoxin A Peaches 21/ mg/kg 21 Patulin Rotten areas of apples 21/ mg/kg 44 Patulin Apples, rotten areas Apples, remainders 30/30 30/30 1,000 mg/kg 300 mg/kg 120 Patulin Apples 5 mg/kg 9 Patulin Blueberries Raspberries Strawberries Cherries Lingon berries Peaches Plums Black currants 14 Patulin Cherries Strawberries Raspberries Black mulberries White mulberries 115 Alternariol Tomatoes Apples 1/12 0/10 0/10 0/10 1/2 1/8 1/6 0/6 9/10 8/10 3/5 7/10 4/6 6/19 7/8 21 mg/kg 265 mg/kg 6 mg/kg 4 mg/kg 113 mg/kg 145 mg/kg 746 mg/kg 157 mg/kg 426 mg/kg 5.3 mg/kg 59 mg/kg 127 Alternariol Apples 1/ mg/kg 115 Alternariol methyl ether Tomatoes Apples 6/19 8/8 0.8 mg/kg 2.3 mg/kg 227 Alternariol methyl ether Apples 1/ mg/kg 115 Tenuazonic acid Tomatoes Apples 11/19 8/8 139 mg/kg 0.5 mg/kg 107 Tenuazonic acid Tomatoes, moldy 73/ mg/kg a After removal of rotten area from fruit. reported in early studies, but the authors of these reports did not present data on the incidence and concentration of contamination (40, 120). Table 3 shows that patulin has been quanti ed in different stone fruits, berries, and strawberries in other studies; for example, during storage at 158C for 1 week, patulin was found to be detectable in blueberries at a maximum concentration of 1,000 mg/kg (9). Patulin occurs most frequently by far in brown rot lesions of apples. Up to 130 mg of patulin per kg has been detected in the lesions of apples (21), and no correlation between the size of the lesion and the patulin concentration was found. Martins et al. (68) recently showed that there are differences in incidences and levels of patulin contamination for different apple varieties. Lovett et al. (65) reported that in naturally rotted apples, P. expansum is the predominant patulin-producing fungus. Other fungi also produce the mycotoxin but are not as rapidly invasive. They may be identi ed as patulin producers only when they gain a

6 J. Food Prot., Vol. 66, No. 8 MYCOTOXINS IN FRUIT PRODUCTS 1519 competitive advantage. For grapes, a strong in uence of vine variety on patulin occurrence was observed (8). Most samples of the variety Müller Thurgau were more frequently contaminated with patulin than most samples of the variety Riesling. It therefore seems likely that vine variety also in uences the occurrence of patulin in apples. High levels of patulin contamination in apples have recently been reported in Turkey (14). Alternaria toxins. In addition to patulin, Alternaria toxins have also been detected in apples. According to the review by Scott (101), alternariol has also been detected in red currants, raspberries, strawberries, gooseberries, and blueberries. The storage of fruits may lead to a signi cant increase in mycotoxin concentration. MYCOTOXINS IN FRUIT JUICE For fruit juices, contamination with mycotoxins depends largely on the fruit from which the juice is produced. The contamination of juice with mycotoxins usually results from the use of poor-quality fruit. The spectrum of mycotoxins occurring depends on the type of fruit. Table 4 gives an overview of types of juices, mycotoxins detected, and the maximum concentrations of mycotoxins. In several countries, including Switzerland, Sweden, Belgium, Norway, and Italy, a maximum allowable concentration of 50 mg of patulin per kg of apple juice has been established. The U.S. Food and Drug Administration has also established an action level of 50 mg/kg on a singlestrength basis for patulin in apple juice, apple juice concentrates, and apple juice products in the United States. In Germany, a maximum patulin limit of 25 mg/kg for apple juice and puree is being discussed. Most of the research on patulin has therefore focused on its behavior during the production of apple products, which will be reviewed in the following section. Selection and storage of fruits. As described by Sydenham et al. (117), ripened fruits are required for juice production, and these fruits are normally stored at low temperatures under modi ed atmospheres. Storage at low temperatures alone is not suf cient to prevent mycotoxin formation, and therefore additional measures, such as the dipping of apple fruits in 3% sodium hypochlorite, are recommended (44). The limited availability of warehouse storage facilities may also lead to the open storage of fruits. As was demonstrated by Sydenham et al. (117), patulin levels for fruits stored in the open for 15 or 33 days were signi cantly higher than those for fruits stored in the open for 7 days. The percentage of waste material (rotten and damaged fruits) removed during the sorting of the fruits was found to increase from 1.8% (on day 15) to 3.2% (on day 33). Patulin levels for the rotten fraction increased from 1,120 to up to 6,235 mg/kg over time. On the basis of the results obtained, these authors concluded that pressed juice from apples subjected to storage in the open for.7 days may contain patulin at.50 mg/kg. Processing. As shown in different studies, a key step in the prevention of the occurrence of patulin in juice is the removal of rotten fruits and fruit parts prior to further processing (44, 117). Up to 99% of patulin can be removed from the product through the trimming of the fruits (65). Another effective step in the reduction of patulin is the washing of fruits. Acar et al. (5) demonstrated that 54% of patulin could be removed from the product by high-pressure water spraying. A combination of washing and the removal of rotten and damaged apples resulted in a 10-fold decrease in the initial patulin concentration (58). After the washing and trimming of the fruits, juice is pressed and usually clari ed, heated, and lled. Some of these processing steps signi cantly contribute to a reduction in patulin levels in fruit juice, while others have little in- uence. Analysis of the juice, the lter cake, the pellet after centrifugation, and the sediment showed that nearly half of the patulin had already been removed during the pressing of the apples (22). This removal was attributed to the binding of the mycotoxin to solid substances during pressing. The second most effective step for the removal of patulin is centrifugation, which accounts for a ;20% reduction in patulin content. Centrifugation in combination with ning or enzyme treatment resulted in a smaller reduction in patulin content because of a decrease in solid particles. For the preparation of a clear juice, clari cation is required. Clari cation can be achieved by conventional methods such as ltration with gelatin and bentonite or by ultra ltration. For ultra ltration, no ltering aids are required. In terms of the removal of patulin, conventional clari cation is more effective than ultra ltration (5). Conventional clari cation and ultra ltration resulted in average patulin reductions of 39 and 25%, respectively. Similar results were observed by Gökmen et al. (42). In their study, six different clari cation processes were compared with respect to the reduction of patulin levels in the juice. The processes compared included conventional clari cation with gelatin, conventional clari cation with bentonite, conventional clari cation with bentonite in combination with activated charcoal treatment, ultra ltration, and ultra ltration in combination with pre occulation with gelatin and bentonite, with an adsorbent resin, or with polyvinylpolypyrrolidine. Conventional clari cation with activated charcoal treatment was found to be the most ef - cient process, resulting in a patulin reduction of 40.9%. Reductions achieved with the ultra ltration process were always,11% regardless of whether the process was combined with other treatments. The adsorption kinetics of patulin on activated carbon have been characterized as a physical, endothermic process (72). The temperature dependence of the adsorption kinetics was described with the use of the Langmuir model. Additives used in raw juice also in uence the decrease in patulin levels. After depectinization, clari cation, ning, and ltration, patulin reductions of 25% for apple juice, 50% for apple juice with 500 mg of ascorbic acid per kg, and 42% for apple juice with 100 mg of sulfur dioxide per kg were observed by Aytac and Acar (17). In the same study, no patulin reduction occurred during concentration and pasteurization. However, more severe heat treatment leads to a degradation of patulin. The heating of apple juice

7 1520 DRUSCH AND RAGAB J. Food Prot., Vol. 66, No. 8 TABLE 4. Mycotoxins in fruit juices Reference Commodity Toxin(s) No. of samples positive/no. of samples analyzed Maximum concn 4 Apple juice Guava juice A atoxins B1, G1 5/5 2/5 30 mg/liter 12 mg/liter 67 Grape juice, white Grape juice, red Apple juice Orange juice Black currant juice Tomato juice Ochratoxin A 21/27 56/64 0/33 0/30 3/19 3/ mg/liter 5.3 mg/liter 0.06 mg/liter mg/liter 132 Grape juice, red Grape juice, white Apple juice Orange juice Ochratoxin A 7/8 0/3 0/2 0/ mg/liter 97 Grape must Ochratoxin A 8/ mg/liter 38 Grapefruit juice Ochratoxin A 1/ mg/liter 8 Grape must Patulin 30/ mg/liter 21 Apple juice, conventional Apple juice, organic Apple juice with pulp Patulin 3.03 mg/kg 28.2 mg/kg 1,150 mg/kg 28 Apple and mixed fruit juice Patulin 101/241 1,130 mg/liter 54 Baby juice Juice drink Pure apple juice Mixed juice 95 Apple juice Apple-acerola juice Grape juice Sour cherry juice Blackcurrant juice Orange juice Patulin 0/9 0/11 59/71 0/14 Patulin 10/10 1/1 2/2 1/1 1/1 1/ mg/liter 26.0 mg/liter 0.7 mg/liter 5.2 mg/liter 0.2 mg/liter 0.1 mg/liter 0.1 mg/liter 109 Patulin Apple juice Pear juice 9/9 0/ mg/liter 58 Patulin Apple juice 8/31 45 mg/liter 202 Patulin Apple juice Black currant juice Cherry must 42/106 3/10 1/5 200 mg/liter 200 mg/liter 200 mg/liter 6 Patulin Fruit juices (positive samples of apple, 8/44 60 mg/liter pineapple, passion fruit juice) 41 Patulin Apple juice concentrate, diluted 215/ mg/liter 8 Patulin Grape must 21/ mg/liter 59 Patulin Quince juice Apple juice Pear juice 0/8 0/10 0/2 36 Patulin Apple juice and products Pear juice 69 Patulin Orange juice Grapefruit juice Pineapple juice Apple juice Pear juice Grape juice, red Grape juice, white 73/ /27 0/19 0/21 0/17 0/16 0/12 0/8 402 mg/kg 1 20 mg/kg 70 Patulin Apple juice, from concentrate Apple juice, directly produced 39/ / mg/liter 171 mg/liter 131 Patulin Apple juice 27/ mg/liter 33 Alternariol Alternariol methyl ether Apple juice concentrate 17/32 17/ mg/liter 1.71 mg/liter 127 Alternariol, alternariol methyl ether, altenuen, altertoxin I Apple products Tomato products Other fruit products (pears, grapes, red currants, strawberries, cherries, plums, peaches, oranges, grapefruits) 0/18 0/10 0/12 Mixed fruit products 0/7

8 J. Food Prot., Vol. 66, No. 8 MYCOTOXINS IN FRUIT PRODUCTS 1521 for nearly 3 h at 1008C resulted in a patulin reduction of 33% (52). As can be seen from Table 4, the occurrence of ochratoxin A was limited to grape juice, especially red grape juice. Zimmerli and Dick (132) suggested that ochratoxin A in wines from southern European regions results from the growth of mold after the grapes have been harvested. The climatic conditions in these regions are not favorable for the growth of ochratoxin A producing fungi. Majerus et al. (67) pointed out that long-time enzymatic treatment of crude juice and berries at increased temperatures to improve the color of red grape juices and red wine favors fungal growth and ochratoxin production. In contrast to patulin, ochratoxin A remained stable during alcoholic fermentation (132). Stability in juice. In 1968, Scott and Somers (102) observed that within 3 weeks of storage, initial concentrations of patulin decreased to 25, 75, and 65% in canned orange, apple, and grape juices, respectively. These authors concluded that these decreases during storage could be attributed to the levels of sulfhydryl groups present in the juices. Ascorbic acid present in the samples probably contributed to patulin degradation. Different studies in which ascorbic acid has been added to apple juice or buffer solutions clearly show increased patulin reductions in the presence of ascorbic acid (17, 24, 110). This reaction is believed to involve a metal-catalyzed oxidation of ascorbate or ascorbic acid (24). Free radicals are generated from this reaction and may attack the conjugated double bond of the patulin. Experimental evidence for this reaction is still lacking. The addition of sulfur dioxide also leads to a reduction in patulin levels in juices, although this effect is not as pronounced as it is with ascorbic acid. For sulfur dioxide, a reversible binding and an irreversible binding of patulin, depending on ph, have been described (110). In general, the detected levels of patulin contamination of fruit juices presented in Table 4 are low, re ecting highquality products and the use of good quality assurance and management systems in the production chain. A high incidence of patulin contamination has recently been reported by a Turkish research group (131). The incidence of apple juice contamination at levels of.50 mg/liter was 44%, and the maximum concentration detected was 733 mg/liter. These results indicate that poor-quality fruits may have been used for these products, and this conclusion is reinforced by the patulin results presented above for fresh fruits. Another point that needs further investigation is Beretta et al. s (21) nding that patulin levels are signi cantly higher in apple juice made from organic apples than in apple juice made from conventionally farmed apples. These authors conclude that the use of fungicides in conventional farming may be responsible for the lower levels of mycotoxin contamination. The natural occurrence of a atoxins in orange juice has been described (4). Ragab (88) investigated the fate of aflatoxins in oranges inoculated with A. parasiticus during processing. This author showed that up to 69% of the single a atoxins remain in the peel, up to 13% remain in the pulp, and only up to 35% appear in the clear juice. Pasteurization did reduce a atoxin levels in juice by 9 to 20% for the single toxins, and sterilization at 1008C for 30 min decreased a atoxin levels by up to 73%. The strongest heat resistance was observed for a atoxin B1, and the next strongest heat resistance was observed for a atoxin G1. Storage of the product at 25 to 358C for 20 weeks resulted in an 87% reduction in a atoxin B1 and a 100% reduction in a atoxin G1. In an early investigation of Alternaria toxins in fruit juices, no contamination was detected in any of the samples tested (127). It seems likely that the detection limit of the method used (which ranged, e.g., from 10 to 100 mg/kg, depending on the fruit matrix, for alternariol) was not suf- cient. Scott (101) reported that alternariol has been detected in apple juice, raspberry drink, grape juice, cranberry juice, raspberry juice, and prune nectar. Alternariol methyl ether has been detected in apple juice and prune nectar. Alternariol and alternariol methyl ether were shown to be stable in apple juice with and without vitamin C over 20 days and at 808C after 20 min. Altertoxin I was found to be moderately stable (101). MYCOTOXINS IN DRIED FRUITS Most investigations involving dried fruits deal with fruits grown in warm climates, such as gs, dates, and grapes. The available data are very contradictory, indicating that many different factors affect fungal growth on and mycotoxin contamination of such fruits. Zohri and Abdel-Gawad (133) analyzed samples of dried gs, apricots, plums, and raisins for the natural occurrence of a atoxins, citrinin, ochratoxins, patulin, sterigmatosyctin, diacetoxyscirpenol, T-2 toxin, and zearalenone. Only ochratoxin A was detected in all samples of apricots (50 to 110 mg/kg), gs (60 to 120 mg/kg), and plums (210 to 280 mg/kg) tested. All raisin samples were free of mycotoxins. MacDonald et al. (66) determined levels of ochratoxin A and a atoxins in 60 samples of retail dried vine fruits purchased in the United Kingdom. Ochratoxin A concentrations exceeded 0.2 mg/kg in 19 of 20 currant samples, in 17 of 20 sultana samples, and in 17 of 20 raisin samples, with an overall incidence of 88%. The maximum level detected was 53.6 mg of ochratoxin A per kg; no contamination with a atoxins was observed. Differences in the contamination levels found in these two studies may be due to different detection limits for the methods used. Herry and Lemetayer (48) detected a atoxin B1 at low levels in 2 of 28 date samples and in 1 of 52 raisin samples. Youssef et al. (130) examined 100 dried-raisin samples of for mycotoxin contamination. A atoxin B1 was detected in two of these samples at concentrations of 300 and 220 mg/kg, and ochratoxin A was found in one sample at 250 mg/kg. Ragab et al. (89) analyzed 40 samples of dates and date products for a atoxin contamination. Two of ve samples of pitted date fruits stuffed with peanuts contained aflatoxin B1 at concentrations of 4.8 and 6.2 mg/kg. These authors attributed the presence of a atoxin to previous con-

9 1522 DRUSCH AND RAGAB J. Food Prot., Vol. 66, No. 8 TABLE 5. Occurrence of mycotoxins in dried gs Mycotoxin(s) Frequency a Concn Reference Total a atoxins 1/187 6/67 1/8 24.0% 9.0% 32/83 8/42 A atoxin B1 1/350 1/140 94/386 8/16 6.4% 2/4 6/54 8/15 37/52 8/42 A atoxin B2 6.4% 2/54 A atoxin G1 49/386 3/16 6.4% 2/54 15/52 10 mg/kg 14 mg/kg 19 mg/kg mg/kg mg/kg.100 mg/kg 325 mg/kg.100 mg/kg.100 mg/kg mg/kg mg/kg 112 mg/kg 11.8 mg/kg 124 mg/kg 1.9 mg/kg 5 76 mg/kg 260 mg/kg 51 mg/kg 27 mg/kg mg/kg mg/kg 61 mg/kg 345 mg/kg mg/kg A atoxin G2 2/54 59 mg/kg 99 Ochratoxin A 12/52 11/54 4/4 0.1% 5 12 mg/kg 185 mg/kg mg/kg Traces 6,900 mg/kg 370 7,860 mg/kg Kojic acid 52/ ,900 mg/kg 111 a Number of samples positive/number of samples analyzed or percentage of samples positive. tamination of the peanut kernels with which the date fruits had been stuffed. Head et al. (46) reported that copra contained,20 mg of a atoxin B1 per kg. Both a atoxin and free fatty acid contents increased with increasing degrees of contamination with A. avus. As can be seen from Table 5, several investigations on mycotoxin contamination in gs have been conducted. As is the case for other dried fruits, a few contaminated gs may result in the contamination of a whole batch of fruits. Özay and Alperden (81) examined 103 samples collected from various orchards and at various stages of g processing, including samples of dried gs and g paste produced from these dried gs. Overall, a atoxins B1, B2, G1, and G2 were present in 29% of the samples examined at levels of 0.5 to 63.0 mg/kg, 0.5 to 37.7 mg/kg, 0.5 to 78.3 mg/kg, and 0.5 to 12.5 mg/kg, respectively. Ochratoxin A was detected in only 3% of the samples at levels of 5.2 to 8.3 mg/ kg. In samples collected during the sun drying of gs, only a atoxin B1 and G1 were detected. In the g paste, after the dried fruits had been homogenized, all a atoxins and ochratoxin A were found at concentrations lower than those in gs at previous production stages. The occurrence of a atoxins is not necessarily associated with visible changes in fruit. From a batch of 160 kg of dried gs, 386 gs (8.6 kg) were randomly taken and analyzed individually for a atoxin contamination by Steiner et al. (112). A atoxins B1 and G1 were found in 94 and 49 gs, respectively, at concentrations of 0.2 to 30 ng/g. To determine whether the a atoxin contamination was associated with the dark-colored gs, 16 dark, discolored gs were sorted out and analyzed individually by the same investigators. Only eight of these gs contained a atoxin B1, and three of them also contained a atoxin G1. In a different study, Steiner et al. (111) analyzed dried gs showing bright greenish yellow uorescence under UV light for the presence of a atoxins, cyclopiazonic acid, kojic acid, and ochratoxin A. BGY uorescence is not emitted by the a atoxin itself but by kojic acid, a compound produced by A. avus at the same time a atoxin is produced. This uorescence is commonly used in the food industry as a screening device for the detection of a atoxins. The removal of BGY- uorescent gs from a 56-kg batch effectively lowered the contamination level from 22.6 to 0.3 mg of a atoxin B1 per kg. All 52 BGY-positive gs contained kojic acid at concentrations of 8 to 6,900 mg/kg. Thirtyseven (71%) fruits were contaminated with a atoxin B1 at concentrations of 5 mg/kg and 76 mg/kg, and 15 (29%) fruits contained a atoxin G1 at levels of 5 mg/kg to 180 mg/kg. Ochratoxin A concentrations ranging from 5 mg/kg to 12 mg/kg were detected in 12 (23%) fruits at cone. These results demonstrate that dried gs exhibiting BGY uorescence are likely to be contaminated with mycotoxins. Factors involved in the high contamination levels for dried fruits. It is evident from Table 5 that dried gs are a high-risk commodity among dried fruits. Incidence of contamination for a fruit is presumably in uenced by factors such as the suitability of the fruit as a substrate and the frequency of infection of the fruit with toxigenic molds (as discussed above), as well as harvesting and drying conditions and moisture content or water activity (a w ) value. Harvesting and drying conditions. The traditional method applied for the harvesting and drying of gs in Turkey was described by Özay and Alperden (81) as follows. The ripe gs are left on the trees until they shrivel. The gs fall to the ground and are gathered up and dried further in the sunlight on a drying device. This sun-drying process takes almost 5 days. After drying, the sound fruits are sorted out from the damaged ones and stored in farm storehouses. Obviously, the environmental conditions associated with these procedures seem favorable for mycotoxin production in infected fruits. Anton (13) reported that the temperature in the g cultivation regions during the ripening, harvesting, and drying of the fruits ranged from 27 to 308C, which is close to the optimum temperature for mold growth and mycotoxin formation. Generally, if the moisture content of the stored crop exceeds 13 to 18%, mycotoxin formation can occur (27). As is the case for mycotoxigenic fungi and their toxins in grains and seeds, critical points in the formation of mycotoxins in dried gs during storage are moisture content, storage conditions,

10 J. Food Prot., Vol. 66, No. 8 MYCOTOXINS IN FRUIT PRODUCTS 1523 TABLE 6. Minimum a w values for the growth and mycotoxin formation of some potentially toxic molds (16, 19, 27, 73 78, 81, 83, 86) Mold Aspergillus avus A. parasiticus A. ochraceus A. nomius A. versicolor Penicillium P. viridicatum P. expansum P. patulinum P. clavatus P. citrinum Minimum a w value for: Fungal growth Mycotoxin formation and, with respect to the analysis, heterogeneous distribution within a lot of fruits. Dried gs are comparable large fruits, and therefore the moisture content within a fruit and within a lot may vary. Moistened parts offer the opportunity for fungal growth with subsequent accumulation of mycotoxins and water resulting from the respiratory activity of the molds. So-called hot spots with very high a atoxin concentrations can develop in stored fruits (81). Steiner et al. (113) investigated the distribution of a atoxins in an a a- toxin-contaminated lot of gs and found concentrations of 0.4 to 37.0 mg/kg in different sublots. These results show that very few highly contaminated gs may lead to a signi cant concentration of a atoxins in an entire lot. Özay et al. (79) studied the in uence of harvesting methods, pretreatments, sun drying, and solar drying on the mold and mycotoxin contamination of gs. These authors concluded that harvesting the fruits by hand and drying them as soon as possible with a solar drier minimizes environmental contamination, results in homogenous drying, and is helpful in reducing a atoxin contamination in dried gs as well as in improving other quality characteristics in comparison with those of samples treated otherwise. Moisture content and a w. A common feature of dried fruits is that a low a w value is the basis for their preservation. Minimum water requirements for fungal growth differ depending on the temperature and on the substrate. The minimum a w value for growth is lowest at the optimum temperature and highest near the minimum and maximum growth temperatures (27). In general, the optimum a w value for fungal growth is different from the a w value at which the maximum level of mycotoxin formation is observed. Table 6 shows the minimum a w values for the growth and mycotoxin formation of some potentially toxic molds. Several studies concerning the in uence of a w on mycotoxin formation and fungal growth were conducted by Northolt and coworkers (73 78). The minimum a w value for mycotoxin production by Penicillium species is higher than the minimum value for the production of a atoxin by Aspergillus species. The production of penicillic acid was also restricted to high a w values (0.97 to 0.99), whereas fungal growth occurred at a w values of 0.85 to 0.88 (76, 77). Pitt and Miscamble (83) studied the effects of water activity in combination with temperature on the growth of some molds. These authors found that the minimum a w values for the germination and growth of A. avus, Aspergillus oryzae, and A. parasiticus are very close to 0.82, 0.81, and 0.80, respectively, at 52, 30, and 378C, respectively. Meanwhile, for Aspergillus nomius, the minimum a w value for germination and growth is a little higher, i.e., 0.83 at both 25 and 308C but 0.81 at 378C. Dried g fruits stored in farm storehouses are quite safe in terms of their moisture contents and a w values. It can be concluded that the contamination of dried gs with mycotoxins can start on the tree and can continue in the bulk during storage as a result of poor drying and of storage conditions such as high temperatures and relative humidities, as well as the rewetting of dried fruits. Prevention and control of contamination of dried fruits. Because of the variety of factors in uencing mycotoxin contamination, such contamination is usually not associated with one production stage, but one stage may be more important than the others depending on the crop, the fungus, the mycotoxin, and the environmental conditions. Mycotoxin contamination of dried fruits may start with fungal contamination on the trees, increase during harvesting and sun drying, and continue to accumulate during storage because of rewetting and improper conditions. Swanson (116) demonstrated that environmental stress conditions such as insect infestation, drought, cultivar susceptibility, mechanical damage, nutritional de ciencies, and unseasonable temperature, rainfall, or humidity can promote mycotoxin production in growing crops. In fact, changes in farming practices in the past few decades may result in increasing stress on plants and therefore enhance fungal invasion and mycotoxin contamination. For example, the push for larger crop yields has resulted in the planting of crops more densely, with a resultant increase in competition for water and the risk of water stress. Reduced genetic diversity within cultivars and large continuous acreages of the same cultivar may also result in plants being more susceptible to stress from insects and disease pests (61). In conclusion, control management should begin in the orchard and continue until the nal product reaches the consumer. Preharvest management. From a practical point of view, the best approach to eliminating mycotoxins from foods is to prevent mold growth at all levels of production, harvesting, transport, and storage. However, such an approach is not always as simple and straightforward as it may seem (26). Swanson (116) reported that preventive management on the farm includes the use of methods of cultivating to improve plant vigor, the judicious use of insecticides and fungicides, irrigation, and cultivar selection. These methods ensure that plants will be vigorous and less vulnerable to stress, and they also help to limit the spread of mold. With regard to the preventive measures to be taken during the cultivation of fruits, Splittstoesser (106) reported that pruning, certain cultural and sanitary practices, and the