695

J. Milk Food Techno[, Vol. 38, No. 11, Pages 695-705 (November, 1975) Copyright © 1975, International Association of Milk, Food, and Environmental Sanitarians

Patulin: A of Potential Concern in Foods1

W. T. STOTT and L. B. BULLERMAN Department ofFood Science and Technology University ofNebraska, Lincoln, Nebraska 68503

(Received for publication April21. 1975)

ABSTRACT discovery of (82). Human mycotoxicoses have Downloaded from http://meridian.allenpress.com/jfp/article-pdf/38/11/695/2396903/0022-2747-38_11_695.pdf by guest on 29 September 2021 Patulin is a metabolite of numerous Aspergillus and Penicillium also been recorded. "St. Anthony's Fire" of the middle species. It is toxic to a wide range of biological systems including ages was caused by ingestion of sclerotia of Claviceps microorganisms. plants and animals. Patulin has also been shown to be purpurea in rye grain infested by Ergot. In Russia in the carcinogenic to mice. The susceptibility of n1an to this toxin is unknown mid 1940's, as much as 10% of the population was at this time. However. when the numerous biological systems affected affected by a disease known as Alimentary Toxic Aleukia by patulin are considered, it is likely that man would also be susceptible to the toxic etlects of this compound in some manner. Thus, patulin (AT A), with an accompanying high mortality rate (61). must be considered a potential health hazard. This disease was caused by the consumption of The fact that patulin can be produced by a number of penicillia at overwintered grains that were molded by Fusarium and refrigeration temperatures is a source of possible concern. Many of Cladosporium species. More recently, dietary intake of these are found on refrigerated food products and the possibility aflatoxins has been linked to the high incidence of liver that patulin production could occur during refrigerated storage is very real. However, the reactivity of patulin with sulfhydryl group> and other disease in Uganda and Thailand (3, 116). Another food components may cause its apparent disappearance from a food metabolite, patulin, is causing increasing concern as a product. Whether the compound breaks down and disappears or is potential toxic fungal contaminant of food and feed simply chemically altered to a non-detectable form is not known. products. Apparently toxicity is lessened, though it is not known if low pH or reactions with digestive might reverse these reactions or HISTORY liberate free patulin in the gut. Certain foods such as cheeses and cured meats which are low in carbohydrates and high in protein do not Discovery appear to support patulin production to any great extent. This Patulin was first described as an during combined with the reactivity of patulin with sulfhydryl groups seems to lessen the danger of patulin occurrence in these foods. However. apple scientific endeavors in the 1940's to find additional products and grain based products may pose more of a potential following the discovery of penicillin by problem. Apple products are of particular concern since common apple Fleming in 1929 (43). Patulin was isolated by several rotting molds are frequent producers of patulin and because of the workers from a variety of molds which gave rise to several stability of patulin in these products. synonyms for the compound. Table llists the discoveries Further work is necessary to determine more completely the foods most likely to be contaminated with patulin. Presently, foods high in of various molds that produce patulin and the different carbohydrates would appear to be of greater concern than foods low in names given the compound. carbohydrates and high in protein. Refrigerated storage of foods will By 1944 comparisons were being made of these not necessarily guarantee that they will remain free of patulin since variously reported antibiotics and it was found that they many molds capable of producing patuiin can do so at low all appeared to be the same compound. Berge! et al. (15) temperatures. Futiher work is also needed to determine the fate of patulin as it undergoes reactions with sulfhydryl groups and other food claimed clavatin, clavifonnin, and patulin were identical. components to determine if there are conditions under which patulin Chain et al. (32) supported this claim, and found can be regenerated or recovered in a toxic form. daviformin and patulin to be identical compounds. Anslow et al. (6) believed expansin to be patulin; Mold grm-vth on foods and feeds is a common Oosterhuis and Luyken (89) confirmed this finding and occurrence which may result in organoleptic showed that expansin and claviformin were identical. deterioration of products, economic losses, and potential Clavacin and patulin were reported by Hooper et a!. (56), health hazards through production of toxic mold and Katzman et al. (64) to be alike, and the School of metabolites known as "." Diseases caused by Pathology in Oxford, England, reported expansin to be poisoning of animals or humans with preformed toxic identical with patulin (2). Lastly, gigantic acid was metabolites produced by molds growing in food products determined to be claviformin by Florey ct al. (44). are referred to as mycotoxicoses. Animal mycotoxicoses Therapeutic potential have occurred throughout history; the most recent major Possible therapeutic uses of patulin for combating outbreak occurred in England in 1960, and Jed to the human diseases were investigated soon after its 'Published as Paper No. 3983. Journal Series. Nebraska Agriculturnl discovery. One worker observed personal relief from a Experiment Station, Lincoln. Research was conducted under Project "common cold'' by nasal douching of a 0.01% aqueous No. 16·22 and was supported by Public Health Research Grant No. CA14260 from the National Cancer Institute. solution of patulin (98). He, and serveral of his 696 STOTT AND BULLERMAN

TABLE l. Summary of patulin producing molds and synonyms for patulin CHEMISTRY, TOXICITY AND BIOCHEMICAL ACTIVITY

Species offu:tgus Reference Chemical nature Penicillium expansum Luijk (130) The chemical nature of patulin has been thoroughly Anslow et a!. (6) clavacin investigated. It can be isolated as colorless to white Kent and Heatley (67) clavacin crystals from etheral extracts which have no optical Penicillium sp. Atkinson (J 0) penicidin activity. Patulin has a melting point of 110.5 C, and P. clavif'orme Chain et al. (3 1) claviformin Berge! et al. (15) clavatin absorption maxima in the ultraviolet region have been P. patulum Raistrick et al. (98) patulin reported at 275 (36), 277 (94) and 276 nm (64, 139). An Berge! et al. (15) clava tin infrared scan of patulin has absorption bands in the P. melinii Karow and Foster (63) clavacin double bond region of 5.6, 5.9 and 6.1 microns (36, 139). P. w1icae Kent and Heatley (67) clavacin Patulin is unstable in alkali and loses its biological Downloaded from http://meridian.allenpress.com/jfp/article-pdf/38/11/695/2396903/0022-2747-38_11_695.pdf by guest on 29 September 2021 P. equinum and P. novae-zeelanlial Burton and Pausacker activity (10, 31, 56, 63). It is also unstable in contact with (26) 502 (94), but is stable in acid (31. 59). It is soluble in ether, chloroform, ethyl acetate, and ethanol (133). P. leucopus Umezawa et al. (129) patulin Woodward and Singh (I 39, 140) are credited with the P. cyclopium, Singh (1 17) patulin P. griseofulvum, elucidation of the structure of patulin (Fig. 1). Patulin is P. equinum, P. divergens and P. lapidosum Aspergillus clavatus Wiesner (137) clavacin Waksman et al. (I 34) clavacin Umczawa et al. (129) Patulin Berge! et a!. (I 5) clava tin A. giganteus Philpot (93) gigantic acid 0 Florey eta!. (44) claviformin A. terreus Kent and Heatley (67) clavacin Byssochlamys nivae Karow and Foster (63) clavacin Mold sp. c colleagues, reported an immediate clearing of the nasal passages and cessation of all symptoms within 24 h. In a study involving application of a patulin solution to a person with early cold symptoms, no cure was evident but nasal congestion was relieved. In a much larger study involving 300 naval men with common colds, successful results were reported using a nasal spray containin~ patulin (98). Patulin concentrations ranging from 0.02 to 0.005% gave up to a 58% cure rate compared to 9.4% for controls. However, these results were not supported by 0 OH findings of an extensive Army trial when compared to the natural progression of the disease (120). In addition, a clinical trial on the therapeutic value of patulin held in 1944 found it to be of little or no value as a cold cure (77). Patulin Figure l. Structure of patulin. Several studies involving the use of patulin as a treatment for dermal infections have also been reported. Jennings (60) utilized vaseline preparations of patulin in an unsaturated lactone and has the empirical formula of skin tests, and found that a 0.1 o/o preparation caused no C7H 60 4 with a molecular weight of 154. Patulin has been reaction, but a 1.0% preparation produced edema, given the name 4-hydroxy-4H-furo [3, 2c] pyran-2(6H)­ redness, and a roughness of the skin which lasted up to one. two weeks. de Wit (38) used etheral and ointment patulin Toxicity preparations to combat fungal skin infections. All Patulin is toxic to many biological systems. All etheral solutions of from 0.025 to 0.005% produced bacterial species tested have been found to be sensitive to severe irritation while less than 0.005% ointments were this antibiotic to some degree, irrespective of Gram type. tolerated and produced a regression of the infections. The inhibitory effect of patulin on several human However, use of patulin as a fungicidal agent was pathogens was quantitated by Chain et aL (31). questioned by Hopkins et al. (57) who found it no better Bacterium coli (Jischerichia colt) and Staphylococcus than other agents in use at the time. aureus were completely inhibited by a 0.1 o/o PATULIN 697 concentration of patulin in 10 min. Chain et al. and urticae, which produced patulin, causing reduced wheat W aksman et al. (133, 134) reported that patulin was a seed germination rate and plant size (85). Indeed, wheat bactericidal compound. Indeed, over 75 species of seeds and seedlings have been shown to be sensitive to as bacteria have been demonstrated to be sensitive to little as 20 ppm patulin (40, 84, 127). patulin by several workers (33). Patulin has also been shown to be toxic to animal Patulin has also been shown to be an active yeast and systems. Intravenous injection of patulin into mice and mold antagonist, and can inhibit germination of mold rats gave LD 50 values varying from 0.3-0.7 mg patulin/20 spores (102). Several Pythium sp. have been oberved to be g body weight, and 1 mg per mouse was always lethal (17, quite sensitive to patulin, even at concentrations as low 31, 98, 120). Lethal dose for mice by intraperitoneal as 0.00025% (6, 130). Katzman et al. (64) noted a strong injection was reported by Lochhead et al. (71) to be fungistatic activity by patulin against Rhizopus 0.1-0.2 mg patulin, and by Stansfeld et al. (120) to be nigricans, Monilia albicans, Saccharomyces cerevisiae, 0.25 mg patulin per mouse. Similar amounts as used for

and Sporotrichium schenkii but no effect upon intravenous injection have produced death in mice and Downloaded from http://meridian.allenpress.com/jfp/article-pdf/38/11/695/2396903/0022-2747-38_11_695.pdf by guest on 29 September 2021 Aspergillus clavatus, a patulin producer, even at rats by subcutaneous injection (71). The LD 50 by oral concentrations up to 1 mg patulin/ml. These results were administration to mice is 0.7 mg patulin, and 2.5 mg also found by Sanders (107) who observed several human patulin was always fatal (1 7). Rats have given pathogenic fungi to be sensitive to concentrations of 0.01 comparable oral responses; death took up to 4 days in to 0.0006o/o of patulin. Patulin has also been evaluated these studies, and if not immediate resulted in severe for control of several plant pathogens, including downy pathological changes in animals that did not die. mildew of cucumbers (7), damping off of safflower (47), Pathological changes reported were lungs edematous crown gall (68), and loose smut of wheat (127). with hemorrhaging; capillary damage in the liver, spleen, Acute toxicity of patulin to cells in vitro has been and kidney; and edema of the brain (2, 64). observed by many workers. Toxicity to leucocytes at Several other animals have also been observed to be concentrations ranging from 0.1 to 0.00012% has been sensitive to patulin. Studies have found 10-15 mg reported (31, 98, 132). Rabbit corneal cells have also been patulin/kg fatal for cats, rabbits, and mice (17. 41). reported to be inhibited by patulin in concentrations of Chicken embryos (55, 79, 80, 81), chickens and quail (74, 0.02% (132). Some stimulatory activity has also been 79, 80), rabbit skin (55), guppies (64), the crustacean noted. Mouse leucocytes and rabbit epithelium cultures Cyclops fuscus (100), brine shrimp (101), and zebra fish were stimulated at 20-40 f..l.g of patulin/ml but inhibited larvae (l) have also been found to be sensitive to patulin at 100-200 f..l.g patulin/ml, and a 50% inhibition of the in various degrees. An insecticidal activity of patulin has multiplication of rat and mouse fibroblasts in culture has also been noted (34). been noted by as little as 154 ng of patulin/ml (90, 97). Besides the overt toxicity demonstrated by patulin to Chick fibroblasts and heart cultures were inhibited by animals, carcinogenic activity has been observed with 100 and 10 p.g patulin/ml, respectively, in another study sublethal dosages. Mice, when repeatedly injected with (2). No effect on any particular phase of replication of sublethal doses of patulin, were observed to form HeLa cells treated with patulin was noted by Kawasaki et localized tumors after 15 months (39). Thus it was al. (66), but rather a slowing of the entire cell cycle. concluded that patulin is also a potential to Cancer cells may also be affected by patulin. Ehrlich animals. carcinoma cells and mouse ascites tumor cells were reportedly inhibited by 20-40 and 60 p.g patulin/ml, Biochemical activity respectively (70). Tissues described as malignant have Much work has been done to determine the precise also been observed to be inhibited by a 0.004% mechanism of the toxic activity of patulin. Inhibition of concentration of patulin (132). aerobic respiration by patulin in several systems has been Patulin is also toxic to higher plants. Iyengar and observed and reviewed by Singh (118). Bacteria, fungi, Starky (58) reported that cucumber seeds were sensitive guinea pig kidney slices, brain homogenates, and to patulin solutions and were unable to germinate or phagocytic cells have all been found susceptible to were stunted in root and stem length after exposure to respiration inhibition by patulin solutions. Cell free patulin. Berestets'kyi and Synyts'kyi {14) have found 90% extracts of Claviceps purpurea were found to be inhibited of culture filtrates of 85 strains of Penicillium urticae much more rapidly (40 min) than complete mycelia (3-6 Banier to be toxic to seedlings of maize, peas, and flax, h) suggesting that a membrane barrier to patulin was and patulin at 1 ppm to be toxic to sugar beets. present in whole cultures. Duckweed plant (Lemna minor) was inhibited by less The effect of patulin on semipermiability of cell than 1 p.g patulin/g (83). Patulin at SOp.g/g wilted membranes has been studied. Potassium ion absorption safflower seedlings {47), and at 15 J.tglg inhibited pea by erj1hrocytes (62) and glucose uptake by fungal (;lscochyta pisi) seed germination (135), tomato seedlings mycelia (117) have been shown to be interrupted by (78), and germinated corn (84). But perhaps the most patulin. However, no leakage of metabolites such as significant observation of phytotoxicity was that inorganic phosphorous, carbohydrates, amino acids, etc., exhibited upon wheat. It was shown that stubble from C. purpurea mycelium or of hemoglobin from mulching of fields supported extensive growth of P. bovine erythrocytes was noted in preparations treated 698 STOTT AND BULLERMAN with patulin (49). It has been pointed out by Singh (118) patulin with sulfhydryl groups (13, 28, 29, 30, 48, 105). that inhibition of respiration would halt the natural Patulin has been observed to react with cysteine, uptake of several necessary substrates. The lack of glutathionine, thioglycolate, and dimercaptopropanol metabolite leakage in treated preparations was suggested (12, 13, 27, 37, 48, 55, 78, 105). It has been theorized that by Singh (118) to indicate that no altering of membrane by reacting with critical sulfhydryl groups in the active transport systems occurs. sites of enzymes a toxic activity would be exhibited. Inhibition of protein and RNA syntheses were not Conversely, an excess of sulfhydryl groups would detoxify observed in the multiplication of influenza virus in mice patulin, supposedly by binding all of the patulin treated with patulin (106). In another study, total lipids, molecules before they could react with a vital group. carbohydrates, RNA, DNA, proteins, and chitin analyses Andraud and Andraud (4) attributed the loss of ATPase of C. purpurea cultures treated with patulin showed no activity to this reaction and mixtures of cysteine and differences from controls, even though generation times patulin were not inhibitory to lactic dehydrogenase (9).

were slowed (117). It has been noted, however, that Cysteine but not glutathione or thioglycolate was Downloaded from http://meridian.allenpress.com/jfp/article-pdf/38/11/695/2396903/0022-2747-38_11_695.pdf by guest on 29 September 2021 patulin plus penicillic acid may induce DNA strand observed to inhibit patulin by Cavallito and Bailey (28). In breakage in HeLa cells after 1 h of incubation (128). another study the reaction with cysteine was reportedly Since the effects of patulin on respiration appear to be slow although reducing the UV absorption of patulin critical to its toxic activity, systems associated (117). The reaction of patulin with glutathione was with respiration have been investigated. These data were observed to be pH dependent to some extent, with the reviewed by Singh (118). Dehydrogenase activities in reaction being slower at pH 5.0 than at 7.0 (55). Products mouse ascites tumor cells were found to be inhibited at 20 of this reaction were less toxic to mice, rabbit skin, and J-ig patulin/ml in one study and unaffected by 30-120 1-'g chicken embryos. This theory has been challenged, patulin/ml in another. C. purpurea succinate oxidase however, by several findings. Ashoor and Chu (8) showed and dehydrogenase were inhibited up to 90o/o by 1,155 J-ig that patulin, bonded to cysteine, could still bond and patulin/mg protein. NADH oxidase, succinate cyto­ inhibit muscle aldolase, and Singh (117) observed that chrome C reductase, and cytochrome oxidase were patulin did not react with glyceraldehyde-3-phosphate inhibited by approximately 30% at a much higher dehydrogenase, an enzyme with sulfhydryl groups in its concentration, 7,000 1-'g patulin/mg protein. Rabbit active site. Singh (118) has reported that the muscle aldolase was inhibited by patulin with an cysteine-patulin readion is a very slow one, even at high inhibition constant (Kj) of 1.3 x w-s M (8), while lactic cysteine concentrations and has suggested that a dehydrogenase was inhibited with a Kj of 6.2 X w-6 M modified form of patulin may be the toxic form of the (9). Adenosine triphosphatase (A TPase) from human compound. erythrocytes has also been shown to be inhibited by patulin (4). Conversely, glucose oxidase and glyceralde­ PRODUCTION AND BIOSYNTHESIS hyde-3-phosphate dehydrogenase were not inhibited by Production conditions patulin (1 17). Singh (118) has reasoned that because of Production of patulin by mold cultures in laboratory the relative insensitivity of the terminal electron media has been investigated by numerous workers. transport enzymes (NADH oxidase, cytochrome C Czapeks-Dox broth, at pH 6.9, has been utilized to reductase, and oxidase), the sensitivity of anaerobic produce patulin in still cultures of A. clavatus, bacteria to patulin (37), and the sensitive nature of Aspergillus terreus and Penicillium patulum incubated oxygen consumption to patulin, the site of action of in the dark at 25 C for 2 weeks (3!. 72, 137). Variations of patulin in biological systems is before the terminal stages this medium have included addition of 3% corn steep of respiration. liquor (63) and use of maltose or glucose as the One suggested mode of action of patulin, once at the carbohydrate source (142). site of activity. has been the reported reactability of Successful production of patulin by several Aspergillus

TABLE 2. Summary q(sub:>trates

Incubation temperature TimP Patulin "1cdium Species (days! (mg;ml) References ------Raulin-Thom ?. patulum 12-14 0.7 -1.26 (98) P. patulum + P. expansum 24 1"' 0.02-(J.37 (6) P. urticae 25 14 O.l (88) P. patulum 25 14 1.37 (123) potato extract Penicillium 27-28 8-10 !.33 (127} Yt:ast extract-sucrose P, expansurn 0.48 (8]) P_ expansun1 22-24 2l (119) P. urticae 25 14 2.7 (811) P. patulunl 2S 14 2.8 (123) a Prepared from raw potatoes according to the method of Norstadt and McCalla (86) PATULIN 699

0 NAOPH+H• NAoP• Cc-c~ ();CH3 OCHzOHNAOP4'NAOPH CHO CHO [ Do OH 900Hl c2<> Acetyi-CoA ~ I I ~ +HillAI HOA HOI ~ 3Malon~I-CoA ~ ~ ~ ~ --=---"" v--Vo-+ = I L --+ 3~~ 6-Me!t!y!sa!t;.: ACid ~ ~-HO·Be: m·HO-Be~d!l Gent~ehv;t CH~ggH - CH20HCHO J 0 QH 4CoA-SH alcohol Pre-patulin Patulin Figure 2. The major pathway for patulin biosynthesis in Penicillium urticae NRRL 2159A (45). sp. has been reported using glucose-nitrate-trace three malonyl-CoA molecules combine, undergo mineral broth. Aspergillus fumigatus and A. clavatus reduction and decarboxylation, to produce 6-MS (Fig. 2). produced maximum amounts of patulin in still cultures Upon further decarboxylation and oxidation, the 6-MS at 28 C after 8 and 6 days, respectively, as measured by molecule is converted to m-OH-benzaldehyde which then antibiotic activities of the filtrates. Variations have undergoes a rearrangement resulting in one molecule of included use of brown sugar instead of glucose and patulin. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/38/11/695/2396903/0022-2747-38_11_695.pdf by guest on 29 September 2021 incubation for 7 to 10 days at 22 to 28 C in shallow Bu'Lock et al. (23) further characterized the cultures (35) and addition of 0.2% corn steep liquor or biosynthesis of patulin as the result of secondary 1 %yeast extract, at pH 4.5, with 5 to 11 days incubation metabolism. Growth of P. urticae was explained in terms at 24 C (64). of two phases: (a) tropophase, in which mycelial nitrogen Several substrates have also been utilized to produce and phsophorous uptake occurs, and RNA and patulin by Penicillium spp. (Table 2). Studies of sulfhydryl containing compounds appear with the rapid optimum production of patulin by penicillia have shown utilization of glucose, primarily by the hexose that with inorganic salts media. glucose and iron salts monophosphate pathway; and (b) idiophase, in which a stimulated patulin production while most organic reduced assimilation of nitrogen and phosphorous materials such as yeast extract, corn steep liquor, and occurs, RNA and sulfhydryl levels are lower, glucose peptone depressed toxin production (71, 127). However, oxidation slows and occurs primarily by glycolysis, fatty potato extract prepared from raw potatoes resulted in acids begin to accumulate, and special phenolic very high yields of patulin from P. urticae (86, 88). In metabolites derived from 6-MS appear. The transition general, glucose has been found to be the most suitable between phases is sharp, and occurs between 24 and 36 h carbohydrate source for patulin production by of growth, at which time 6-MS appears. Bu'Lock et al. Penicillium spp., with sucrose, lactose, dextrin, and (25) have shown by selective protein synthesis inhibition starch about half as effective. Greater yields of patulin that the formation of 6-MS is due to a 6-MS synthetase have been obtained with stationary than with submerged enzyme system which is metabolically stable. This cultures, and the optimum temperature for patulin enzyme is formed during replicatory growth and production has been reported to be 20 to 25 C, though activated later. Feedback control ofthis enzyme by 6-MS lesser amounts can be produced at 5 C. At 25 C highest has also been noted (24). yields of patulin have been obtained in 12 to 14 days of The conversion of 6-MS to gentisaldehyde and to incubation. patulin was observed to involve metabolically labile More recently Stott and Bullerman (123) found that enzymes formed later in culture development and which patulin production by P. patulum followed a similar were regulatable by both induction in the presence of pattern. Glucose supported higher yields of patulin than substrate and repression by high nutrient levels (25). A lactose in a salts medium. and more patulin was key enzyme in this conversion has been identified as produced at 25 than at 5 C. An inorganic nitrogen source m-OH-benzyl alcohol dehydrogenase which requires supported more patulin production at 25 C than organic NADP+ cofactor and catalyzes the oxidation of nitrogren from milk, but the organic nitrogen supported m-OH-benzyl alcohol to m-OH-benzaldehyde (46). A more toxin production at 5 C. Growth of the on possible feedback inhibitor of this enzyme was thought to broth containing organic nitrogen was always very be gentisaldehyde. Scott and Beadling (110) also reported extensive when compared to growth on broth containing the activity of m-OH-benzyl alcohol dehydrogenase and inorganic nitrogen or potato dextrose broth, but toxin observed patulin production from gentisaldehyde in cell production was less. Patulin was produced on a broth free systems from P. patulum. It was suggested that a medium containing 1% casein, but was not detectable in dehydrogenase system is involved in the synthesis of a 25% slurry of casein. patulin from gentisaldehyde.

Biosynthesis DETECTION AND ANALYSES The biosynthetic pathway of patulin from 6-methyl­ Physicochemical methods salicylic acid (6-MS) was elucidated by Bu'Lock and Many methods have been reported for extraction, Ryan (22) using [14('] glucose. Forrester and Gaucher (45) identification, quantitation, and bioassay of patulin from and Scott et al. (109) confirmed these results and also laboratory media as well as some food products. Early reported a number of side reactions which could occur. methods of extracting patulin from liquid culture media In the synthesis of patulin one acetyl-CoA molecule plus involved the use of a norite absorption (1 %) with acetone 700 STOTT A~D BULLERMA~

elution giving crystalline patulin (63). Present methods p-anisaldehyde, o.l J-lg (113), n-methylbenzthiazolone-(2)­ simply use ethyl acetate extraction and concentration hydrazone, 0.06 J-lg (103), and fluorescence quenching (73, 84, 119), or diethyl ether extraction and alumina on fluorescent silica gel, 0.04 J-lg (95). Patulin recovered (80-200 mesh) column clean up (86). Extraction methods from flour and fruit juice has been quantitated by from grains have involved the use of acetonitrile-water derivatization with phenylhydrazine (1 14), and 3-methyl- (9 + l) combined with defatting using isooctane (121), or 2-benzothiazolinone hydrazone hydrachloride (115), acetonitrile-hexane (4 + 1) extraction with preparative from meat with 1 o/o diphenylborinic acid-B amino-ethyl TLC clean up (95). Tauchmann et al. (126) reported a ester (126), and from grain by 3 o/o ammonium hydroxide method for the extraction of patulin from sausages using then 4 o/o phenylhydrazine (95). Detection limits reported acetonitrile-hexane (20 -+- 9) as the extracting . with these methods were 0.02-0.05, 0.01, 0.1, and 0.12 J-lg Extracts were put on a celite column, defatted with patulin, respectively. hexane, and eluted with ethyl acetate. Extraction from Bioassay methods several flours and fruit juices using ethyl acetate Downloaded from http://meridian.allenpress.com/jfp/article-pdf/38/11/695/2396903/0022-2747-38_11_695.pdf by guest on 29 September 2021 In addition to chemical means of analysis, several extraction with silica gel (G-60) column clean up with biological methods have been reported for the benzene-ethyl acetate (3 + 1) (115) or ethyl acetate observance ofthe toxicity of patulin, but few quantitative elution (112) has been reported. Direct liquid-liquid methods exist. Chicken embryos have been widely extraction from apple cider with ethyl acetate has also utilized to test a variety of compounds, including been reported (96). mycotoxins, for toxicity. Potentially toxic compounds in Identification and quantitation of patulin in food and aqueous or certain organic test solutions can be injected culture extracts has been reported using several into the volk or air sac (131). Chicken embryos have been techniques. Patulin can be identified using gas-liquid found s~nsitive to at least 8J-lg of patulin and have been chromatography of the patulin silyl ether, acetate, and used to detect patulin and measure its biological activity chloroacetate derivatives which have lower detection (20. 21, 72, 79, 80, 81, 141). Feeding of patulin to mice limits of 100, 60, and 40 ng, respectively (96). This and ducklings was used in another study to measure method can be used to quantitate patulin by preparing a toxicity (111). Rabbit skin, quail, mice, white leghorn standard curve. With this method, recovery of patulin cockrels, and brine shrimp have also been used by from apple juice was found to have a lower detection numerous workers to demonstrate toxicity due to their limit of 0. 7 11g/ml of juice. Gas chromatography (GC) of sensitivities to patulin as previously discussed. patulin without derivatization has also been reported (I 14). Although no detection limits were given, 1 ppm of A quantitative bioassay using germination of wheat patulin recovered from apple juice was detectable by this seeds to measure the toxicity of patulin was reported by method. Pero et a!. (91) and Pero and Harvan (92) Norstadt and McCalla (88). The Cheyenne strain of described GC methods for patulin along with penicillic wheat seed was found to be sensitive to about 20 J-lg

acid from corn and patulin alone as a trimethylsilyl ether patulin/mi. Patulin has an LD 50 of 18J-lglml to zebra fish for qualitative data in corn. Gas chromatography of larvae, and this system has also been proposed as an patulin and its derivatives has been applied to the assay system for patulin (1). Brine shrimp were found quantitation of patulin extracted from rice (125). Use of sensitive to as little as 10 11g patulin and might therefore high pressure liquid chromatography (HPLC) for also have potential application as a bioassay system (51). isolation of patulin, combined with detection using Several bacterial systems have been utilized to assay ultraviolet adsorption of 254 nm has also been reported patulin. These have been reported using the streak plate (136). Using this method, levels of patulin as low as 11 method with Bacillus subtilis, Escherichia coli, and S. 11g/liter of apple juice could be detected with a lower aureus (48, 64), and the cylinder plate method with S. detection limit of 4 ng. aureus and E. coli (54, 127). With these methods Mass spectrometry was used to confirm the recovery of the potency of the sample was recorded as the highest patulin from apple juice by Scott et al. (114). The dilution of the substance which prevented gro\\

OCCURRENCE IN FOODS patulin to be stable at 100 C for 15 min. The effect of pH on stability of patulin was investigated by Lovett and Reactions of patulin with food components Peeler (75). Patulin was found to be resistant to thermal Several compounds common to certain foods, have been destruction at pH 3.5, 4.5, and 5.5. Both D and Z values observed to affect patulin, though not all contain increased as the pH decreased indicating increased heat sulfhydryl groups. Peptone, casein hydrolysate, serum, stability at acidic pH values. liver infusion, glycine, methionine, asparagine, p-amino Stott and Bullerman (124) found that when pure benzoic acid (5, 12, 32, 37, 64, 134), sodium sulfate, patulin was added to Cheddar cheese, there was an sodium thiosulfate, and sodium pyrosulfate (78) display immediate decrease in the amount of patulin that could an inhibitory effect on patulin. Opposed to these are be recovered by solvent extraction. The decrease was the several compounds which are reported to enhance greatest during the first 3 h. From 12 to 48 h there was no toxicity. These are tryptophan, urea, and thiourea (37). further substantial decrease in the percent of patulin

Stability of patulin in food products recovered. Neither storage temperature nor heat Downloaded from http://meridian.allenpress.com/jfp/article-pdf/38/11/695/2396903/0022-2747-38_11_695.pdf by guest on 29 September 2021 The stability of patulin in several food systems has sterilization of the cheese had any effect on recovery of been observed, relative to time and temperature. the patulin. Disappearance of patulin was attributed to Pohland and Allen (94) have observed the stability of chemical reaction with the cheese. patulin in apple juice and in several grain systems (Fig. Contamination offoods with patulin producing molds 3). At 4-8 1-1-g patulin per gram, patulin was stable in Toxigenic molds capable of producing patulin have

I been isolated from a variety of commercial food products IOOJf-X-X~ X-X and from poultry feed. Flour has yielded A. terre us, A. JT---x x x clavatus, and P. urticae contaminants (50). P. expansum, A. clavatus, A. terreus, and P. urticae have \~ ~~APPLE JUICE (400fLg150mll been isolated from cereal grains and legumes (III). P. ~ \ ~ ~DRY CORN (3001'-g/50g l expansum has also been isolated from inshell pecans (108); from apricots, crab apples, and persimmons (JI9); ~ 60~~ \ i WET CORN (3001'-g/SOg ) from pears and grapes (6. 119); and from apples (6, 52, ~ I \~ ~DURHAM WHEAT(2t:l

X been identified as the Gymnoascus species reported by J \I Karow and Foster (63) and Kuehn (69) to produce ; patulin. Atkinson (11) reported that patulin producing ' X-- 0I I --- ~ penicillia came mainly from fruits and vegetables. But 8 12 ELAPSED TIME :DAYS) more recently, dried sausages were found to be contaminated with P. expansum, P. urticae, and P. Figure 3. Recovery of patulin from various commodities (94). melinii (79, 80, 8I, 126). P. expansum has been isolated apple juice and dry corn up to 14 days. Spiked Durham from aged, cured hams (14I) and other meats (42). Lovett wheat, wet corn, and sorghum gave an 80o/o decrease in (72) has reported the isolation of A. terreus and P. detectable toxin after 14 days. Timonin (I27) has shown patulum from poultry feed. that patulin is also unstable in wheat flour. Scott and Patulin producing strains of molds isolated from foods Somers (112) investigated the stability of patulin in generally make up a low percentage of the total isolates. several fruit juices at 22 and 80 C. At 4 ppm, patulin was Only 1% of the penicillia isolated from flour and bread observed to be stable in apple and grape juices up to 3 by Bullerman and Hartung (19) were able to produce weeks at 22 C. Greater than SO% of the patulin in apple chemically detectable patulin in vitro. Six of 422 (1.4%) and grape juices was detectable after 10 min at 80 C, and of the total molds isolated from European style dry 45% after 20 min. Harwig et a!. (53) reported that patulin sausages were observed by Mintzlaff et al. (8I) to be disappeared more rapidly in apple juice undergoing capable of producing 0.02 to 0.48 mg patulin/ml of by Saccharomyces cerevisiae and Sac­ laboratory media. Of the 116 molds isolated from charomyces ellipsoides than in non-fermented juice. cornmeal only 0.9% were reported to produce patulin by However, the disappearance could not specifically be Bullerman eta!. (21). Thirteen of 349 (4 %) molds isolated related to cell growth or ractions with yeast metabolites, from Cheddar cheeses were found to produce patulin, though these were given as possible explanations. The but no patulin producing isolates were found in a survey authors concluded that it would be unlikely for patulin to of Swiss cheese (18, 20). However, 42 of 61, or 66%, of be detectable in the fermented type of apple cider they naturally rotted apples yielded P. expansum isolates had studied. which were observed to produce patulin (52). Thus, apple The thermal stability of patulin has been studied in products appear to be the foods most likely to be pure systems. Wiesner (137) noted the resistance of contaminated with patulin with a lesser chance of patulin to boiling and Heatley and Philpot (54) reported contamination existing for grain products and 702 STOTT AND BULLERMAN

refrigerated food products such as cheese and cured meat samples molded by P. expansum gave no detectable meats. patulin (42). Finally, whole meal wheat-germ and linseed Patulin production at refrigerated temperatures breads contaminated with P. expansum and analyzed for Several molds isolated from foods have been reported patulin after growth showed that the toxin could be to produce patulin at refrigerated temperature in detected after 10 days of growth but not after 20 days laboratory media (76). Three strains of A. clavatus, and (I 04). The lack of or decrease in patulin evidenced in two each of P. expansum and P. patulum produced in these studies was attributed to the reaction of the toxin excess of 400 JJg patulin/ml media after 100 days of with sulfhydryl groups present in the foods making it incubation at 1. 7 C. Patulin production in excess of 70 chemically undetectable and of lessened toxicity. Stott JJglml was observed from P. claviforme at 7.2 C and P. and Bullerman (124) found that when P. patulum was griseofulvin at 12.8 C after 55 days incubation. P. grown on Cheddar cheese, patulin was detected in the claviforme and P. expansum produced more patulin at mold mycelia and the first 2-3 mm of cheese in samples 7.2 C than at 12.8 C, and detectable toxin was observed stored at 25 C for 2 weeks. The amounts of patulin found Downloaded from http://meridian.allenpress.com/jfp/article-pdf/38/11/695/2396903/0022-2747-38_11_695.pdf by guest on 29 September 2021 by 10 days at 1.7 C, 7 days at 7.2 C, and 5 days at 12.8 C. ranged from 1.3 to 0.2 JJg patulin/g of mycelia and P. urticae has been reported capable of producing up to cheese. However, no patulin could be detected in mycelia 630 J-Ig patulin/mlliquid medium in 3 weeks at 10 C and or cheese in samples stored at 5 C for 6 weeks. up to 250 JJg/ml at 5 C. Levels of 60 J-Ig and 43 J-Ig of the toxin was observed per milliliter of medium within 4 davs REFERENCES at these temperatures, respectively (88). In a simil~r 1. Abedi, Z. H., and P. M. Scott. 1969. Detection of toxicity of afla­ study using P. expansum in liquid media, maximum toxin, , and other fungal toxins by lethal action patulin production of 0.6 mg/ml media was observed on zebra fish larvae. J. Ass. Offic. Anal. Chern. 52:963. after 5-7 weeks incubation at 10 C with 0.15 mg/ml 2. Abraham, E. P., and H. W. Florey. 1949. Substances produced by Fungi Imperfecti and Ascomycetes. In H. W. Florey, E. Chain, produced within 2 weeks. At 0 C approximately 0.02 mg N. G. Heatley, M.A. Jennings, A. G. Sanders, E. P. Abraham, patulin/ml of media was noted after 2 weeks, 0.6 mg/ml and M. E. Florey (eds.). Antibiotics. Vol. 1, p. 273. Oxford Uni­ after 12 weeks, and a maximum amountof 0.7 mg/ml versity Press, London, New York and Toronto. media after 18 weeks incubation (119). Stott and 3. Alpert, M. E., M.S. R. Hutt, G. N. Wogan, and C. S. Davidson. Bullerman (123) found that P. patulum produced up to 1971. Association between content of food and hepa­ toma frequency in Uganda. Cancer 28:253. 674 JJg patulin/ml of potato dextrose broth at 5 C when 4. Andraud, J., and G. Andraud. 1971. Effect of some lactones on incubated for 6 weeks. the activity of (Na• - K•)-adenosine-triphosphatase of human erythrocytes. C. R. Seances Soc. Bioi. Fil. 165:301. Natural occurrence of patulin in foods 5. Andraud, C., R. Cuvelier, P. Tranche, and J. Couquelet. 1965. Though patulin producing molds have been found on Inhibition of liver sulfhydryl groups in the rat by patuline, iso­ a variety of foods and wide temperature ranges, only patuline and some homologous substances. C. R. Seances Soc. Bioi. 158:2341. apple sap, apple juice, and apple cider have been found 6. Anslow, W. K., H. Raistrick, and G. Smith. 1943. Antifungal to be naturally contaminated by this toxin. Brian et al. substances from moulds. Part I. Patulin. a metabolic product of (16) discovered patulin in excess of 1000 ppm and Harwig Penicillium patulum Banier and Penicillium expansum (Link). et al. (52) found 0.02 to 17.7 mg per apple from the sap of Thorn. Trans. Soc. Chern. Ind. 62:236. 7. Ark, P. A., and J. B. Thompson. 1957. Control of downy mildew apples rotted by P. expansum. Apple tissue was found to of cucumber and antibiotics. Plant Dis. Rept. 41:452. contain up to 125 J-Ig patulin/g of tissue molded by P. 8. Ashoor, S. H., and F. S. Chu. 1973. Inhibition of muscle aldo­ expansum, but this was substantially lowered when lase by patulin and penicillic acid in vitro. Food Cosmet. Toxicol. 11:995. incubated in atmospheres modified to 2%02 or 7.5% C02 (119). Scott et al. (I 14) isolated 1 ppm patulin from one of 9. Ashoor, S. H., and F. S. Chu. 1973. Inhibition of alcohol and lactic dehydrogenases by patulin and penicillic acid in vitro. Food 12 commercially available "Sweet Apple Cider" samples Cosmet. Toxicol. 11:617. analyzed. Wilson and Nuovo (138) reported finding up to 10. Atkinson, N. 1942. Antibacterial substances produced by 45 ppm patulin from "Organic Apple Cider." In the moulds. I. Penicidin, a product of the growth of a Penicillium. Washington, D.C. area levels of 49 to 309 ppb patulin Aust. J. Exp. Bioi. and Med. Sci. 20:287. were found in eight of 13 commercial apple cider samples 11. Atkinson, N. 1943. Antibacterial substances produced by some analyzed in another study (136). common Penicillia. Aust. J. Exp. Bioi. and Med. Sci. 21:15. 12. Atkinson, N., and N. Stanley. 1943. Antibacterial substances Despite these findings, there are not reports of patulin produced by moulds. IV. The detection and occurrence of sup­ being present in foodstuffs other than apple juice. This is pressors of penicidin activity. Aust. J. Exp. Bioi. and Med. Sci. true even though extensive growth of patulin producing 21:249. fungi has taken place upon the product. Mintzlaff et al. 13. Atkinson, N., and N. Stanley. 1943. Antibacterial substances (81) could find no chemically detectable patulin in produced by moulds. The mechanism of the action of some peni­ cidin suppressors. Aust. J. Exp. Bioi. Med. Sci. 21:255. fermented dried sausages even after extensive growth of 14. Berestets'kyi, 0. 0., and Synyts'kyi. 1973. Phytotoxic activity in P. expansum had occurred. P. expansum is a known different strains of Penicillium urticae Banier. Mikrobiol. Zh. 35: patulin producing mold often isolated from this type of 349. sausage and has even been recommended as a starter for 15. Berge!, F., A. L. Morrison, R. Klein, A. R. Moss, H. Rinder­ mold fermented sausages in Europe. In another study, knecht, and J. L. Ward. 1943. An antibiotic substance from PATULIN 703

Aspergillus clavatus and Penicillium claviforme and its probable series of reactive lactones and related substances. Brit. J. Cancer identity with patulin. Nature 152:750. 15:85. 16. Brian, P. W., G. W. Elson, and D. Lowe. 1956. Production of 40. Ellis, J. R., and T. M. McCalla. 1970. Patulin effects on wheat patulin in apple fruits by Penicillium expansum. Nature 178:263. plants in field treatments. Bacteriol. Proc. 1970, A9. 17. Broom, W. A., E. Biilbring, C. J. Chapman, J. W. F. Hampton, 41. Ernst, A. M. 1946. Unpublished. Quoted in deWit. 1946. In A.M. Thomson, J. Ungar, R. Wien, and G. Woolfe. 1944. The Modern development of chemotherapy. p. 158. Monographs on pharmacology of patulin. Brit. J. Exp. Pathol. 25:195. the progress of research in Holland, No. 4. Elsevier Pub!. Co., 18. Bullerman, L. B. 1975. Examination of Swiss cheese for incid­ Inc., New York and Amsterdam. ence of mycotoxin producing mold. J. Food Sci. (submitted for 42. Fiedler, H. 1974. Mould and mycotoxin detection in meat prod­ publication). ucts. Archiv fur Lebensmittelhygiene 24:180. 19. Bullerman, L. B., and T. E. Hartung.1973. Mycotoxin producing 43. Fleming, A. 1929. On the antibacterial action of cultures of a potential of molds from t1our and bread. Cereal Sci. Today 18: Penicillium, with special reference to their use in the isolation of 346. B. injluenzae. Brit. J. Exp. Pathol. 10:226. 20. Bullerman, L. B., and F. J. Olivigni. 1974. Mycotoxin producing 44. Florey, H. W., M.A. Jennings, and F. J. Philpot. 1944. Clavi­ potential of molds isolated from Cheddar cheeses. J. Food Sci. formin from Aspergillus giganteus Wehm. Nature 153:139. 39:1166. 45. Forrester, P. I., and G. M. Gaucher. 1972. Conversion of 6- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/38/11/695/2396903/0022-2747-38_11_695.pdf by guest on 29 September 2021 21. Bullerman, L. B., J. Baca, and W. T. Stott. 1975. An evaluation of methylsalicylic acid into patulin by Penicillium urticae. Biochem. potential mycotoxin producing molds in corn meal. Cereal Food 11:1102. World 20:248. 46. Forrester, P. I., and G. M. Gaucher. 1972. m-Hydroxybenzyl 22. Bu'Lock, J. D., and A. J. Ryan, 1958. The biogenesis of patulin. alcohol dehydrogenase from Penicillium urticae. Biochem. 11: Proc. Chern. Soc. 158:222. 1108. 23. Bu'Lock, J. D., D. Hamilton, M. A. Hulme, A. J. Powell, H. N. 47. Gattani, M. L. 1957. Studies on the control of damping-off of Smalley, D. Shepherd, and G. N. Smith. 1965. Metabolic devel­ safflower with antibiotics. Plant Dis. Rept. 41:160. opment and secondary biosynthesis in Penicillium urticae. Can. 48. Geiger, W. B., and J. E. Conn. 1945. The mechanism of the anti­ J. Microbiol. 11:765. biotic activity of clavacin and penicillic acid. J. Amer. Chern. 24. Bu"Lock, J.D., M.A. Hulme, and D. Shepherd. 1966. Inhibition Soc. 67:112. and possible feedback by 6-methylsalicylic acid. Nature 211:1090. 49. Gottlieb, D., and J. Singh. 1964. The mechanism of patulin inhi­ 25. Bu'Lock, J.D., D. Shepherd, and D. J. Winstanley. 1969. Regula­ bition of fungi. Riv. Pathol. Vegetale, Ser. III 4:455. tion of 6-methyl salicylate and patulin synthesis in Penicillium 50. Graves, R. R., and C. W. Hesseltine. 1966. Fungi in flour and urticae. Can. J. Microbiol. 15:279. refrigerated dough products. Mycopathol. Mycol. Appl. 29:277. 26. Burton, H. S., and B. F. Pausacker. 1947. Unpublished. Quoted 51. Harwig, J.. and P.M. Scott. 1971. Brine shrimp (Artemia salina by E. P. Abraham and H. W. Florey. 1949. Substances produced L.) larvae as a screening system for fungal toxins. Appl. Mi­ by Fungi Imperfecti and Ascomycetes. In H. W. Florey, E. Chain, crobiol. 21:1011. N. G. Heatley, M. A. Jennings, A. G. Sanders, E. P. Abraham 52. Harwig, J., Y-K Chen, B. P. C. Kennedy, and P.M. Scott. 1973. and M. E. Florey (eds.). Antibiotics. Vol. 1, p. 273. Oxford Uni­ Occurrence of patulin and patulin-producing strains of Penicil· versity Press, London, New York and Toronto. lium expansum in natural rots of apple in Canada. Can. Inst. 27. Bustinza, L. F., and A. C. Lopez. 1947. Preliminary tests in the Food Sci. Techno!. J. 6:22. study of the int1uence of antibiotics on the germination of seeds. 53. Harwig, J., P.M. Scott, B. P. C. Kennedy, and Y-K Chen. 1973. Proc. Fourth Intern. Congr. Microbiol. p. 160. July 20-26. Copen­ Disappearance of patulin from apple juice fermented by Sac· hagen. charomyces spp. Can. Inst. Food Sci. Techno!. J. 6:45. 28. Cavallito, C. J., and J. H. Bailey. 1944. Preliminary note on the in­ 54. Heatley, N. G., and F. J. Philpot. 1947. The routine examination activation of antibiotics. Science 100:390. for antibiotics produced by moulds. J. Gen. Microbiol. 1:232. 29. Cavallito, C. J., J. H. Bailey, T. H. Haskell, J. R. McCormick, and 55. Hofmann, K., H. J. Mintzlaff, I. Alperden and L. Leistner. 1971. W. F. Warner. 1945. The inactivation of antibacterial agents and Untersuchung iiber die Inactivierung des Mykotoxins Patulin their mechanism of action. J. Bacteriol. 50:61. durch Sulfhydrylgruppen. Die Fleischwirtschaft. 51:1534. 30. Cavallito, C. J., and T. H. Haskell. 1945. The mechanism of 56. Hooper, I. R., H. W. Anderson, P. Skell, and H. E. Carter. 1944. action of antibiotics. The reaction of unsaturated lactones with The identity of clavacin with patulin. Science 99:16. cysteine and related compounds. J. Amer. Chern. Soc. 67:1995. 57. Hopkins, J. G., J. K. Fisher, A. B. Hillegas, R. B. Ledin, G. C. 31. Chain, E., H. W. Florey, and M.A. Jennings. 1942. An antibac­ Rebell, and E. Camp. 1946. Fungistatic agents for the treatment terial substance produced by Penicillium claviforme. Brit. J. Exp. of dermatophytosis. J. Invest. Dermatol. 7:239. Pathol. 23:202. 58. Iyengar, M. R. S., and R. L. Starky. 1953. Synergism and antago­ 32. Chain, E .. H. W. Florey, and M.A. Jennings. 1944. Identity of nism of auxin by antibiotics. Science 118:357. patulin and claviformin. Lancet i. 112. 59. Jefferys, E. G. 1952. The stability of antibiotics in soils. J. Gen. 33. Ciegler, A., R. W. Detroy, and E. B. Lillehoj. 1971. Patulin, peni­ Microbiol. 1:295. cillic acid and other carcinogenic lactones. In A. Ciegler, S. Kadis 60. Jennings, M. A. 1944. Unpublished. Quoted by E. P. Abraham and S. J. Ajl (eds.). Microbial toxins. Vol. 6, p. 409. Academic and H. W. Florey. 1949. Substances produced by Fungi Imper­ Press, New York and London. fecti and Ascomycetes. In H. W. Florey, E. Chain. N. G. Heatley, M.A. Jennings, A. G. Sanders, E. P. Abraham and M. E. Florey 34. Cole, M., and G. N. Rolinson. 1972. Microbial metabolites with (eds.). Antibiotics. Vol. 1, p. 273. Oxford University Press, Lon­ insecticidal properties. Appl. Microbiol. 24:660. don, New York and Toronto. 35. Conn, J. E., and W. B. Geiger. 1944. Problems in the production 61. Joffee, A. Z. 1961. Biological properties of some toxic fungi and purification of clavacin. J. Bacteriol. 47:422. isolated from overwintered cereals. Mycopath. Mycol. Appl. 16: 36. Dauben, H. J.. and F. L. Weisenborn.1949. The structure of pa­ 201. tulin. J. Amer. Chern. Soc. 71:3853. 62. Kahn, J. B., Jr. 1957. Effects of various lactones and related com­ 37. DeRosnay, C. D., Martin-Dupont and R. Jensen. 1952. An anti­ pounds on cation transfer in incubated cold stored human ery­ biotic, mycoin c. J. Med. Bordeaux et Sud-Ouest 129:189. throcytes. J. Pharmacol. Exp. Therap. 121:234. 38. de Wit, J. J. 1946. In Modern development of chemotherapy. p. 63. Karow, E. 0., and J. W. Foster. 1944. An antibiotic substance 158. Monographs on the progress of research in Holland, No.4. from species of Gymnoascus and Penicillium. Science 99:265. Elsevier Pub!. Co., Inc., New York and Amsterdam. 64. Katzman, P. A., E. E. Hays, C. K. Cain, J. J. Van Wyk, F. J. 39. Dickens, F., and H. E. H. Jones. 1961. Carcinogenic activity of a Reithel, S. A. Thayer, E. A. Doisy, N.J. Wade, W. L. Gaby, C. J. 704 STOTT AND BULLERMAN

Carroll, R. D. Muir. and L. R. Jones. 1944. Clavacin, an antibio­ gas chromatographic method for the mycotoxin penicillic acid. J. tic substance from Aspergillus clavatus. J. Bioi. Chern. 154:475. Chromatog. 65:501. 65. Kavanagh, F. 1947. Activities of twenty-two antibacterial sub­ 92. Pero, R. W .. and D. Harvan. 1973. Simultaneous detection of stances against nine species of bacteria. J. Bacterial. 54:761. metabolites from several toxigenic fungi. J. Chromatog. 80:255. 66. Kawasaki, I.. 0. Toshikazo, M. Umeda, and M. Saito. 1972. 93. Philpot, F. J. 1943. A penicillin-like substance from Aspergillus Cytotoxic efl'ect of penicillic acid and patulin on He Ia cells. Jap. J. giganteus Wehm. Nature 152:725. Exp. Med. 42:327. 94. Pohland. A. E .. and R. Allen.1970. Stability studies with patulin. 67. Kent. J.. and N. G. Heatley. 1945. Antibiotics from moulds. Na­ J. Ass. Offic. Anal. Chern. 53:688. ture 156:295. 95. Pohland, A. E., and R. Allen. 1970. Analysis and chemical con­ 68. Klemmer. H. W., A. J. Riker, and 0. N. Allen. 1955. Inhibition of firmation of patulin in grains. J. Ass. Offic. Anal. Chern. 53:686. crown gall by selected antibiotic. Phytopathol. 45:618. 96. Pohland. A. E .. K. Sanders, and C. W. Thorpe. 1970. Determina­ 69. Kuehn. H. H. 1958. A preliminary survey of the Gymnoascaceae. tion of patulin in apple juice. J. Ass. Offtc. Anal. Chern. 53:692. Mycologia 50:417. 97. Powell, A. K. 1966. Effects of propiolactone on rat ftbrocytes. 70. Lettre. H., H. Wrba, and E. Seidler. 1954. Charakterisierung von Nature 209:77. Tumorhemmstoffen mit Hilfe markierter Tumorzellen. Naturw­ 98. Raistrick, H., J. H. Birkinshaw. A Bracken, S. E. Michael, W. A. wiss. 41:122. Hopkins, and W. E. Gye. 1943. Patulin in the common cold. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/38/11/695/2396903/0022-2747-38_11_695.pdf by guest on 29 September 2021 71. Lochhead. A. G., F. E. Chase, and G. B. Landerkin. 1946. Pro­ Collaborative research on a derivative of Penicillium patulum duction of claviformin by soil Penicillia. Can. J. Res. Sect. E. Banier. The Lancet 245:625. Med. Sci. 24:1. 99. Reiss, J. 1971. Detection of patulin by thin-layer chromatog­ 72. Lovett, J. 1968. Toxigenic fungi from poultry feed and litter. Bac­ raphy. Chromatographia 4:576. terial. Proc. 1963, A76. (Abstract). 100. Reiss, J. 1972. Toxic effects of the mycotoxins aflatoxin B" rubra­ 73. Lovett, J. 1970. Biosynthesis of 1'C-patulin using Penicz1lium toxin B. patulin and diacetoxyscirpenol upon the crustacean patulum. Bacteriol. Proc. 1970, A92. (Abstract). Cyclops .fuscus. J. Ass. Offic. Anal. Chern. 55:895. 74. Lovett. J. 1972. Patulin toxicosis in poultry. Poult. Sci. 51:2097. 101. Reiss, J. 1972. Comparing investigations on the toxicity of some 75. Lovett. J .. and J. T. Peeler. 1973. Effect of pH on the thermal mycotoxins to the larvae of the brine shrimp (Artemis salina L.). destruction kinetics of patulin in aqueous solution. J. Food Sci. Zentralbl. Bakteriol. Parasitnekd. Infektionskr. Hyg. Erste. Abt. 38:1094. Orig. Reihe. B. Hyg. Praev. Med. 155:531. 76. Lovett. J .• and R. G. Thompson. 1973. Low temperature patulin 102. Reiss, J. 1973. Influence of the mycotoxins patulin and diace­ production by Aspergillus and Penicillium species. Bacterial. toxyscirpenol on fungi. J. Gen. Appl. Microbial. 19:415. Proc. 1973. E71. (Abstract). 103. Reiss. J. 1973. N -methylbenzthiazo1one-(2)-hydrazone (Besthorns 7~ '!. Medical Research Council. 1944. Clinical trial of patulin in the hydrazone) as a sensitive spray reagent for patulin. J. Chromatog. common cold. Lancett ii:373. 86:190. 78. Miescher, G. 1950. The mode of action of patulin on higher 104. Reiss. J. 1973. Mycotoxins in foodstuffs. III. The production plants, especially on tomato. Phytopath. Z. 16:369. of patulin on different kinds of sliced bread by Penicillium ex­ 79. Mintzlaff. H. J ., and W. Christ. 1972. Biological determination of pansum. Chemie, Mikrobiol. Techno!. der Leben. 2:171, mycotoxins with the chick embryo test. Die Fleischwirtschaft 105. Rinderknecht. H., J. L. Ward. F. Berge!. and A. L. Morrison. 52:1174. 1947. Bacteriological activity and possible mode of action of cer­ 80. Mintzlaff. H. J.. and L. Leistner. 1971. Untersuchung uber die tain non-nitrogenous natural and synthetic antibiotics. Biochem. Eignung von Penicillium expansum (Link) Thorn als Starterkul­ J. 41:463. tur fur die Rohwurst-Herstellung. Die Fleischwirtschaft. 51:1077. 106. Rubin, B. A., and N.J. Giarman. 1947. The therapy of experi­ 81. Mintz1aff, H. J., A. Ciegler, and L Leistner. 1972. Potential mental influenza in man with antibiotic lactones and related mycotoxin problems in mould-fern1ented sausage. Zeit. fUr compounds. Yale J. Bioi. and Med. 19:1017. Leben. Unter. und-Forsch. 150:133. 107. Sanders, A. G. 1946. Effect of some antibiotics on pathogenic 82. Nesbitt, B .. J. O'Kelly. K. Sargeant, and A. Sheridan. 1962. Toxic fungi. Lancet i:44. metabolites of Aspergil/us.flavus. Nature 195:1062. 108. Schindler, A. F., A. N. Abadie, J. S. Gecan, P. B. Mislivec, and 83. Nickell. L. G., and A. C. Finlay. 1954. Antibiotics and their P.M. Brickey.l974. Mycotoxins produced by fungi isolated from effects on plant growth. J. Agr. Food Chern. 2:178. inshell pecans. J. Food Sci. 39:213. 84. Norstadt, F. A., and T. M. McCalla. 1963. Phytotoxic substance 109. Scott, A. I., L. Zamir, G. T. Phillips, and M. Yalpani.1973. The from a species of Penicillium. Science 140:410. biosynthesis of patulin. Bioorg. Chern. 2:124. 85. Norstadt, F. A., and T. M. McCalla. 1968. Microbiological popu­ 110. Scott. A. I.. and L. Beadling. 1974. Biosynthesis of patulin. De­ lation in stubbletield mulched soil. Soil Sci. 107:188. hydrogenase and dioxygenase enzymes of Penicillium patulum. 86. Norstadt, F. A., and T. M. McCalla. 1969. Patulin production by Bioorg. Chern. 3:281. Penicillium urticae (Banier) in batch culture. Appl. Microbial. 111. Scott, De. B. 1964. Toxigenic fungi isolated from cereal and 17:193. legume products. Mycopathol. Mycol. Appl. 25:213. 87. Norstadt. F. A .. and T. M. McCalla. 1969. Penicillium urticae 112. Scott, P.M., and E. Somers. 1968. Stability of patulin and Peni­ Banier enumeration in soils. Plant and Soil 30:129. cillic acid in fruit juice and flour. J. Agr. Food Chern. 16:483. 113. Scott, P. M .. J. W. Lawrence, and W. van Walbeek. 1970. Detec­ 88. Norstadt. F. A .. and T. M. McCalla. 1971. Grov.1h and Patulin tion of mycotoxins by thin-layer chromatography: Application to formation by Penicillium urticae Banier in pure and mixed cul­ screening of fungal extracts. Appl. Microbial. 20:839. tures. Plant and Soil 34:97. 114. Scott. P.M., W. F. Miles, P. Toft. and J. G. Dube. 1972. Occur­ 89. Oosterhuis, H. K., and R. Luyken.1946. Unpublished. Quoted in rence of patulin in apple juice. J. Agr. Food Chern. 20:450. E. P. Abraham and H. W. Florey. 1949. Substances produced by 115. Scott, P.M., and B. P. C. Kennedy. 1973. Improved method for Fungi Imperfecti and Ascomycetes. In H. W. Florey, E. Chain, TLC determination of patulin in apple juice. J. Ass. Otlic. Anal. N. G. Heatley, M. A. Jennings, A. G. Sanders, E. P. Abraham Chern. 56:813. and M. E. Florey (eds.). Antibiotics. Vol. 1, p. 273. Oxford Uni­ 116. Shank, R. G. 1973. Dietary aflatoxin loads and the incidence of versity Press, London, New York and Toronto. human hepatocellular carcinoma in Thailand. Proc. of the 90. Perlman. D., N. A. Guiffre, P. W. Jackson, and F. E. Giardinello. Symp. on Mycotoxins and Mycotoxicosis, May 9, 1972. p. 112. 1959. Effects of antibiotics on multiplication of L cells in suspen­ Columbia, Missouri. sion culture. Proc. Soc. Exp. Bioi. Med. 102:290. 117. Singh, J. 1966. Mechanism of antifungal action of patulin. Ph.D. 91. Pero. R. W .. D. Harvan, R. G. Owens, and J.P. Snow. 1972. A Thesis, lJniversity of Illinois, 1966. PATULIN 705

118. Singh, J. 1967. Patulin. In D. Gottlieb and P. D. Shaw (eds.). An­ 130. Van Luijk, A. 1938. Antagonism of Penicillium spec. versus tibiotics. I. Mechanisms of action. p. 621. Springer-Verlag, Inc., Pythuim debaryanum. Chron. Bot. 4:210. New York. 131. Verret, J. M., J. P. Maliac, and J. McLaughlin. 1964. Use of 119. Sommer, N. F., J. R. Buchanan, and R. J. Fortlage. 1974. Produc­ chicken embryos in the assay of aflatoxin toxicity. J. Ass. Offic. tion of patulin by Penicillium expansum. Appl. Microbiol. 28: Anal. Chern. 47:1003. 589. 132. Vollmar, H. 1947. Versuche iiber die Beeinflussung des Wach­ 120. Stansfeld, J. M., A. E. Francis, and C. H. Stuart-Harris. 1944. stums von Gewebe in der Gewebekultur durch Patulin. Zeitschr. Laboratory and clinical trials of patulin. Lancet ii:370. Hyg. u. Infektionskrankh. 127:316. 121. Stoloff. L., S. Nesheim, L. Yin, J. V. Rodricks, M. Stack. and A. 133. Waksman. S. A., E. S. Horning, and E. L. Spencer. 1942. The D. Campbell. 1971. A multi-mycotoxin detection method for afla­ production of two antibacterial substances, fumigacin and clava­ toxin, ochratoxin, , sterigmatocystin and patulin. J. cin. Science 96:202. Ass. Oftic. Anal. Chem. 54:91. 134. Waksman. S. A., E. S. Horning, and E. L. Spencer. 1943. Two 122. Stott, W. T., and L. B. Bullerman. 1975. Microbiological assay of antagonistic fungi, Aspergillus fumigatus and Aspergillus clava­ patulin using Bacillus megaterium. J. Ass. Offic. Anal. Chem. tus, and their antibiotic substances. J. Bacterial. 45:233. 58:497. 135. Wallen, V. R .. and A. J. Skolko. 1951. Action of antibiotics Downloaded from http://meridian.allenpress.com/jfp/article-pdf/38/11/695/2396903/0022-2747-38_11_695.pdf by guest on 29 September 2021 123. Stott. W. T., and L. B. Bullerman. 1975. Influence of carbohy­ against Ascochyta pisi. Can. J. Bot. 29:316. drate and nitrogen source on patulin production by Penicillium 136. Ware, G. M .. C. W. Thorpe. and A. E. Pohland. 1974. A liquid patulum. Appl. Microbiol. (submitted for publication). chromatographic method for the determination of patulin in 124. Stott, W. T .. and LB. Bullerman.1975. Instability of patulin in apple juice. J. Ass. Offic. Anal. Chern. 57:1111. Cheddar cheese. J. Food Sci. (submitted for publication). 137. Wiesner, B. P.1942. Bactericidal effects of Aspergillus clavatus. 125. Suzuki, T., M. Takeda, and H. Tanabe. 1971. Studies on chemi­ Nature 149:356. cal analysis of mycotoxins (!): Gas chromatography of patulin 138. Wilson, D. M .. and G. J. Nuevo. 1973. Patulin production in and its derivatives and gas chromatographic analysis of patulin in apples decayed by Penicillium expansum. Appl. Microhiol. 26: rice. J. Food Hyg. Soc. Jap. 12:489. 124. 126. Tauchmann, F., L. Toth. and L. Leistner. 1971. Eine halbquanti­ 139. Woodward, R. B., and G. Singh. 1949. The structure of patulin. tative Methode f'tir die Bestimmung von Patulin in Fleisch and J. Amer. Chern. Soc. 71:758. Fleischwaren. Die Fleischwirtschaft 51:1079. 140. Woodward, R. B., and G. Singh. 1950. The synthesis of patulin. 127. Timonin, M. I. 1946. Activity of patulin against Ustilago tritici J. Amer. Chern. Soc. 72:1428. (Pers.) Jen. Can. J. Agr. Sci. 26:358. 141. Wu. M. T., J. C. Ayres. and P. E. Koehler. 1974. Toxigenic asper­ 128. Umeda, M., T. Yamamoto, and M. Saito. 1972. DNA-strand gilli and penicillia isolated from aged, cured meats. Appl. Micro­ breakage of HeLa cells induced by several mycotoxins. Jap. J. bial. 28:1094. Exp. Med. 42:527. 142. Yamamoto. T. 1954. Studies on the poison-producing molds iso­ 129. Umezawa. H., Y. Mizuhara, K. Uekane, and M. Hagihara. 1947. lated from dry malt. I. Distribution. isolation, cultivation and A crystalline antibacterial substance from Penicillium leucopus formation of the toxic substances. J. Pharm. Soc. Japan 74:797. and four other strains of Penicillium sp. and Aspergillus clavatus 143. Yates, A. R. 1974. The occurrence of Byssochlamys sp. molds in and its probable identity with patulin. J. Penicillin (Tokyo) 1:12. Ontario. Can. Inst. Food Sci. Tech. J. 7:148.