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Journal ofFood Protection, Vol. 44, No.9, Pages 702-710 !September 1981) Copyright©, International Association of Milk, Food, and Environmental Sanitarians

Toxins of Penicillium Species Used in Manufacture

P.M. SCOTT

Food Directorate, Health Protection Branch, Health and Welfare Canada, Ottawa, Ontario, Canada KIA OL2

(Received for publication October 24, 1980) Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/9/702/1654438/0362-028x-44_9_702.pdf by guest on 01 October 2021

ABSTRACT have aflatoxins B1, B2, G 1 or G 2 been detected in blue The known metabolites of strains of cheese or -type cheese (13,47,84). Paren­ associated with blue-veined are penicillic acid, thetically, any cheese may be contaminated with roquefortine, isofumigaclavines A and B, PR toxin and related aflatoxin M 1, originating from metabolism of aflatoxin metabolites, and siderophores. Of these, B1 present in dairy cattle feed (32,74). Engel and von penicillic acid and PR toxin, one of the most acutely toxic Milczewski (1 7) extended the screening of fungal cultures metabolites of P. roqueforti, are unstable in cheese. to include sterigmatocystin, diacetoxyscirpenol, ochra­ Roquefortine, isofumigaclavine A, mycophenolic acid and the toxin A, patulin, penicillic acid, , and citreo­ siderophore ferrichrome have been detected in at viridin, in addition to aflatoxins B1, B2, G 1, G 2 and M 1, low ppm levels. Cyclopiazonic acid is a metabolite of but none of these were produced by 17 Penicillium caseicolum (P. camembertz), the used in the strains of P. roqueforti, 36 of P. caseicolum, and five of manufacture of Camembert-type cheeses. Low concentrations P. camemberti. Mintzlaff and Machnik (50) similarly of this have been detected in the cheese crusts. Although no acute health hazard can be identified from the detected no aflatoxins B1 and G 1, ochratoxin A, patulin, presence of these known fungal metabolites in -ripened penicillic acid, citrinin, rubratoxin B, tremortin A or cheeses, there are conflicting reports on the carcinogenicity of zearalenone in extracts from 16 strains of P. caseicolum P. camemberti cultures and one report on sub-acute toxicity of and seven strains of P. roqueforti used for cheese lipids from an Egyptian blue cheese. manufacture; six of the latter strains were toxigenic to chick embryos. Leistner and Eckardt (43) screened 80 strains of P. roqueforti and 69 strains of P. camemberti There are essentially two species of Penicillium used to isolated from foods and feeds for 20 Penicillium manufacture mold-ripened specialty cheeses. Spores of mycotoxins (not including aflatoxins) but only known Penicillium roqueforti are inoculated into curd or milk in metabolites of these species (see below) were detected. the production of many varieties of blue cheese, such as Many of the strains were cheese starter cultures. , Stilton, Danish Blue, and METABOLITES AND MYCOTOXINS FROM PENICIL­ Gammelost. Penicillium caseicolum, synonymous with LIUM ROQUEFORT! and the name used by cheesemakers, Penicillium candidum (51, 76), is em­ Patulin, penicillic acid and citrinin ployed in the ripening of and Camembert. Less well Notwithstanding the negative surveys of Engel and von known are lightly bluish strains of P. camemberti sold in Milczewski (1 7) and Mintzlaff and Machnik (50) referred France under the name of Penicillium album for making to above, patulin and penicillic acid (Fig. 1), which are certain goat cheeses (51). Also, the fungus isolated from toxic produced by several Penicillium and an Italian blue-veined cheese, Dolce Verde, was species (77), are certainly known metabolites described as being related to and of the species P. roqueforti. They have been detected not toP. roqueforti (1 4). Although there appear to be no together in cultures of an atypical strain of P. roqueforti reports of toxic effects in humans following consumption isolated from Cheddar cheese (66). Penicillic acid was of fungally-processed cheese, current scientific interest in found in two of six strains of P. roqueforti isolated from mycotoxins has focused increasing attention on the blue cheese (55), in one of 80 strains isolated from foods production of toxic metabolites by these Penicillium and feeds (43), and crystalline penicillic acid was species. obtained from Penicillium suavolens (=P. roquefortz) It is of some reassurance to note that neither P. (30. 76). Twelve of the 80 isolates of P. roqueforti from roqueforti nor P. caseicolum form aflatoxins (55,84), nor foods and feeds, including meat products, and seven

JOURNAL OF FOOD PROTECTION. VOL. 44. SEPTEMBER l 981 PENICILLIUM-TOXINS IN CHEESE 703

o unstable in moist grains (}6); although stability studies in cheese have not been carried out, it is not likely to be a contaminant of blue cheese in view of the reports noted ~ above on its non-production by strains of P. roqueforti associated with blue cheese (1 7,50). The known a0 OH secondary metabolites of P. roqueforti are summarized in Table 1. Discussion of the remainder of these follows. PATULIN Alkaloids PENICILLIC ACID P. roquejorti was first shown to produce alkaloids in 1958 (89) and this observation was later confirmed by OH Abe et al. (3) and Bekmakhanova (9). Only recently, HOOC however, have crystalline alkaloids been isolated and structurally characterized. Roquefortine and isofumig­ H aclavine A were isolated from the of P. 0 roqueforti in France and assigned the chemical Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/9/702/1654438/0362-028x-44_9_702.pdf by guest on 01 October 2021 structures shown in Fig. 2 (6,81,82). Roquefortine is CH3 identical with isolated by Ohmomo et al. CITRININ in Japan (62,63,65) while isofumigaclavine A and its hydrolysis product, insofumigaclavine B, are the same as Figure 1. Chemical structures of patulin, penicillic acid, and citrinin. roquefortines A and B, respectively, also obtained by these workers (62,63,65). The known clavine alkaloid isolates from grape (but not six strains from blue cheese) festuclavine and one of the two stereoisomeric 12, produced patulin (}7,43). In a separate report (44), one of 13-dihydroroquefortines, named roquefortine D, have three meat isolates that produced patulin also formed the been isolated by the Japanese group and, more recently, nephrotoxic citrinin (Fig. 1) (77). There are in the Soviet Union (36,60,62,63) (Fig. 2). Roquefortine D indications of possible carcinogenicity of patulin and is probably a precursor of roquefortine (C) (61). It should penicillic acid from subcutaneous injection experiments be noted that the nomenclature roquefortines A-D in rodents (77). However, with patulin, a recently proposed by the Japanese workers covers two different completed long-term study found no carcinogenic action structural types of indole alkaloids. Marcfortine A when it was administered orally to rats (70). Further­ (Fig. 2) is a novel alkaloid very recently obtained from P. more, the infrequent formation of penicillic acid and roqueforti (73). non-formation of patulin by P. roqueforti strains from blue cheese, their instability in cheese (45,88), and the Roquefortine is prpduced by most strains of P. fact that cheese is not a good substrate for their roquiforti isolated from blue cheese or used as cheese production (45,66,88), suggest there is virtually no starters (19,80), by a small percentage of strains from potential health hazard from blue cheese directly due to meat (43), and has also been detected in cultures of patulin and penicillic acid. Citrinin, like patulin, is Penicillium notatum (19), (95) and (94). Both roquefortine and TABLE 1. Secondary metabolites ofPenicillium roqueforti. isofumigaclavine A have actually been detected in commercial samples of blue cheese. Scott and Kennedy Presence in blue cheese Metabolite (79) found roquefortine in 16 of 16 samples from seven countries at a mean concentration of 0.80 !J.glg, usually Patulin not looked for (unlikely) accompanied by isofumigaclavine A (mean 0.61 1-Lg/g), Penicillic acid not looked for (unlikely) and with traces of isofumigaclavine B in some samples. Citrinin not looked for (unlikely) The highest concentrations of alkaloids (6.8 1-Lg/g for Roquefortine (roquefortine C) up to 6.8 ).lg/g roquefortine and 4.7 !J.glg for isofumigaclavine A) were Dihydroroquefortine (roquefortine D) not looked for Isofumigaclavine A (roquefortine A) up to 4.7 ).lg/g present in selected subsamples containing as much mold Isofumigaclavine B (roquefortine B) traces as possible. Detection limits of the method were Festuclavine traces 0.03-0.05 1-Lglg for roquefortine and about half of this for Marcfortine A not looked for isofumigaclavine A. Ohmomo et al. (63) detected PR toxin not detected (unstable) roquefortine A (isofumigaclavine A) in five samples of Eremofortins A, B, C and D not looked for (unlikely) foreign and domestic blue cheese at concentrations of Mycophenolic acid up to 14.3/Ag/g 0.2-3.6 !J.glg together with traces of roquefortine B, Coprogen no festuclavine and roquefortine (C). Average levels of Ferrichrome yes; 5-10 !Aglg roquefortine found by Ware et al. (96) in 12 samples of siderophore activity blue cheese and two samples of blue cheese dressing were Toxins 1, 2 and 3 not looked for 0.42 and 0.0451-Lg/g, respectively. Ergosterol; ergothioneine and not looked for The toxic properties of these alkaloids have not been hercynine extensively investigated (Table 2), and there are

JOURNAL OF FOOD PROTECTION. VOL 44, SEPTEMBER 1981 704 SCOTT conflicting reports on the toxicity of roquefortine. Two observations concerning conditions for pro­ However, no potential acute human health hazard can be duction of the major P. roqueforti alkaloids that may be extrapolated from the amounts of roquefortine present in useful in preparing them for future research are (a) blue cheese. Studying the antimicrobial effect of cultivation at 30 C rather than 22 C favored production roquefortine on strains of bacteria that appear in blue of roquefortine while hindering formation of isofumiga­ cheese, Kopp and Rehm (34) found that its activity was clavine A (65) and (b) yields of isofumigaclavine A were limited to inhibiting growth of gram-positive organisms three times greater at 15 C than at 25 C (80). containing hemins. PR toxin and related metabolites TABLE 2. Toxicity ofPenicillium roqueforti alkaloids in the PR toxin (Fig. 3) is a consistently detected metabolite mouse. of P. roqueforti, including many isolates from blue Metabolite LDso (mg/kg, i.p.) Toxic effects Reference cheese (43,68, 72,80.98,100,102), although cheese starter Roquefortine 15-20 convulsions (79) strains have been found that appear not to produce the (50-100 mg/kg) toxin (68, 71). It is mainly present in the culture medium

rather than the mycelium (80). PR toxin is one of the Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/9/702/1654438/0362-028x-44_9_702.pdf by guest on 01 October 2021 169-189 no observed (5) most acutely toxic metabolites known to be formed by P. neurotoxicity roqueforti. Acute toxicological effects in rodents (Table 3) include degenerative changes in the liver and kidney of Isofumigaclavine A 340 muscle relaxant, (63) the rat (102); oral administration of 0.5 mg per day to antidepressant, 60-g weanling rats for 2 months did not, however, etc. produce any visible toxic effects (72). PR toxin inhibits in Isofumigaclavine B 1000 (63)

ROQUE FORTINE 12, 13-DIHYDROROQUEFORTINE

ISOFUMIGACLAVINE A ( R =OCOCH3) MARCFORTINE A I SOFUMIGACLAVINE B ( R = OH) FESTUCLAVI NE ( R =H)

Figure 2. Chemical st~ctures q(alkaloids from Penicillium roqueforti.

JOURNAL OF FOOD PROTECTlON. VOL 44. SEPTEMBER 1981 PENICILLIUM-TOXINS IN CHEESE 705

TABLE3. ofPR toxin. Organism Effect Reference

C57mouse LD 50 1-2 mg/kg (i.p.) (5) C57 mouse LD 50 72-100 mg/kg (oral) (5) Swiss-Webster mouse LD 503.5-4.6 mg/kg (i.p.) (5) Weanling mouse LD 50 5.8 mg/kg (i.p.) x10 (oral) Weanling rat } (101) LD 50 11 mg/kg (i.p.) Weanling rat LD 50 14.5 mg/kg (i.p.) (72) Ciliate protozoan Minimal active dose 0.25 ).lg/ml (15) (Colpidium campylum) vivo and in vitro protein synthesis (56,101) and in vitro nor in cheese processed with known producers of PR nucleic acid synthesis, including irreversible inhibition of toxin (1 8, 72, 78). Detection limits in two of these studies

DNA synthesis in rat liver cells (8,101); it was found to be were 0.2-0.25 t-tglg. PR toxin can be converted into the Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/9/702/1654438/0362-028x-44_9_702.pdf by guest on 01 October 2021 cytotoxic in rat liver, porcine and human cell lines (8,46), markedly less toxic PR imine in the presence of blue mutagenic to Salmonella typhimurium (S8,91), showed cheese (S,57, 78), presumably by reacting with ammonia DNA-attacking ability in the rec assay (90), and was a or ammonium salts naturally present in the cheese. PR direct-acting mutagen to three strains of Saccharomyces imine is also unstable in the cheese (78). Compounds cerevisiae and Neurospora crassa (99). It is not yet known other than ammonia that might be responsible for the whether PR toxin is carcinogenic, although Polonelli et instability of PR toxin in blue cheese include amino al. (72) indicated that tests to determine this would be acids, amines and casein and its breakdown products. carried out. Metabolites related to PR toxin, named PR toxin does react readily with neutral and basic amino eremofortins A, B, C and D (Fig. 3), have been obtained acids, glutathione and a mixture of casein and peptone from a strain of P. roqueforti used to make Auvergne (5,18,59,78) and limited studies so far indicate much blue cheese (7,52,53). Eremofortins A, B and C were not lower acute toxicity in the mouse of model reaction acutely toxic to mice at respective doses of 15, 15 and 50 products with L-leucine or L-a-alanine (S). PR toxin also mg/kg (intraperitoneal injection) and did not inhibit in reacts with butterfat to some extent (18). Taking into vitro protein and RNA syntheses (11,57). Also, in account all the foregoing considerations, any potential contrast to PR toxin, eremofortins A, B, C and D were health hazard from blue cheese due to PR toxin appears not toxic to the ciliate protozoan Colpidium campylum to be negligible. However, other substrates in which PR (15). toxin were more stable might be subject to contamina­ There has been understandable concern in view of the tion by PR toxin - Still (86) found approximately 8 t-tg/g above properties that PR toxin might be found in blue in one of seven silage samples examined. cheese. However, conditions under which blue cheese is Mycophenolic acid made- feeble aeration, low carbohydrate concentration First isolated in 1896 from Penicillium brevi­ and presence of sodium chloride - do not favor PR toxin compactum and subsequently from a number of other production (25, 71, 72); it should be noted that low Penicillium species (.15,106), the antibiotic mycophenolic temperature (8 or 15 C) would not in itself lower PR toxin acid (Fig. 4) showed promise as a drug in the treatment production, merely delay it (18,80). Furthermore, even if of psoriasis (49), and is not generally regarded as a potent PR toxin were formed at some stage during maturation mycotoxin because of oral LD values found in the of blue cheese, it is not stable in blue cheese (18, 78) and it 50 mouse and rat of 2500 and 700 mg/kg, respectively has not been detected by TLC in commercial blue cheese (12,105). There were no apparent signs of toxicity in rabbits dosed orally 5 days/week with 320 mg of ~~ mycophenolic acid/kg of body weight for one year (4). CH3COO~R I CH3 CH3 CH3

PR TOXIN (R;CHOl EREMOFORTIN B EREMOFORTIN A (RoCH3l EREMOFORTIN C (R=CH20Hl HOOC

0 OH

'"'coo f>yf~\ ~3CH3

EREMOFQRTIN D Figure 3. Chemical structures of PR toxin and related MYCOPHENOLIC ACID metabolites ofPenicillium roqueforti. Figure 4. Chemical structure ofmycophenolic acid.

JOURNAL OF FOOD PROTEC110N. VOL 44, SEPTEMBER 1981 706 SCOTT

However, rats given daily oral doses of 30 mg/kg were ferrichrome (Fig. 5), which was identified in the cheese dead within 9 weeks and rhesus monkeys receiving 150 together with an unknown negatively charged sidero­ mg/kg daily developed abdominal colic, bloody diarrhea, phore. Ferrichrome was obtained in crystalline form weight loss and anemia after 2 weeks (12). Some adverse from commercial blue mold powder and coprogen clinical reactions, including diarrhea, cramps and (Fig. 5) was isolated from P. roqueforti culture medium nausea, were noted in 35 human patients with psoriasis after addition of ferric sulfate. Coprogen is also a known treated with high oral doses of mycophenolic acid (2.4g to metabolite of P. camemberti (107). Ong and Neilands 7.2 g daily) for 52-104 weeks (49). Mycophenolic acid (67) speculated on the possible nutritional and toxicolo­ induced mutations and chromosome aberrations in a gical relevance of siderophores in food and suggested mouse mammary carcinoma cell line (92), but was not they might complex iron, making it unavailable to the mutagenic in Salmonella systems (58,97). Only recently body, or a hydroxamate siderophore such as ferrichrome has mycophenolic acid been reported as a metabolite of might be hydrolysed to a mutagenic alkyl hydroxylamine. P. roqueforti, produced by all of 16 strains isolated from In common with otlber fungi, P. roquejorti produces blue cheese by Lafont et al. (38). An unidentified the water-soluble betaines, ergothioneine and hercynine Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/9/702/1654438/0362-028x-44_9_702.pdf by guest on 01 October 2021 material toxic to the chicken embryo was present in (23) (Fig. 6). addition to the mycophenolic acid in older cultures at + 15 C, but not at 25 C. One hundred samples of blue N(CH3l3 cheese of various types were then analysed for H I mycophenolic acid, which was found in 38 of them in HS~~N~H2CH-COO- concentrations of up to 14.3 !lglg (40). The method was N-- sensitive to 0.02 llg of mycophenolic acid/g (85). ERGOTHIONEI NE HERCYNINE Roquefort cheese showed the highest incidence of Figure 6. Chemical structures of the betaines, ergothioneine contamination (84o/o). There was a correlation between and hercynine. levels of mycophenolic acid in a cheese sample and in a culture of the corresponding isolated strain of P. Miscellaneous toxicological studies on Penicillium roqueforti, indicating that use of tested strains for cheese manufacture would eliminate any potential problem roqueforti posed by mycophenolic acid contamination. Kanota (29) purified three substances from the culture filtrate of a toxic strain of P. roqueforti isolated from Siderophores and betaines rice. "Toxin 1", a low molecular weight, spectroscopi­ Siderophores are low molecular weight ferric ion cally characterized crystalline compound, "Toxin 2", a transport agents formed by a variety of microorganisms brown oil, and "Toxin 3", these last two both growing in media containing low concentrations of uncharacterized, had weak acute toxicity to mice. available iron. Such a medium is cheese. Ong and Umeda et al. (93) isolated two non-toxic crystalline Neilands (67) detected siderophore activity in Oregon materials and an uncharacterized cytotoxic fraction from blue cheese at levels of S-10 llg/g, estimated as P. roqueforti. In addition, there are reports of toxigenic NHCOCH3 I strains of P. roqueforti isolated from chestnuts (103), HO(CHz)z C=CHCON(CH 2l3CHCOO(CH 2l2 C=CHCO pecans (104), meat products (43) and blue cheese (39), I I I I CH3 OH CH3 N{OH) using day-old cockerels to indicate toxicity in the first two studies. 0 I Estimates of the frequency of general acute toxino­ OH HN~(CHz)3 genesis of P. roquejorti strains associated with blue HO(CH 2 l 2 ~=CHCO~(CHz)3 --l .NH cheese vary with the study and with the bioassay. Lafont CH3 y et al. (39) observed that extracts from 10 of 27 strains 0 showed marked toxic effects in at least one of three DEFERRICOPROGEN biological tests: the chick embryo (three strains), mouse (two strains) and rat hepatoma cell cultures (nine strains); certain other unidentified Penicillium strains isolated from cheese were also toxigenic. As indicated previously, Mintzlaff and Machnik (50) found that mycelium extracts from 6 of 7 cheese starter strains were toxic to the chick embryo, Moubasher et al. (55) observed that 2 of 6 P. roqueforti strains from blue cheese produced unidentified factors toxic to the chick embryo, while Krusch et al. (37) did not detect any morphological changes when extracts of 17 blue cheese strains were FERRI CHROME tested in cultures of two human cell lines. Figure 5. Chemical structures of siderophores from Pencill­ Frank et al. (20.21) concluded that long term feeding ium roqueforti. of P. roqueforti cultures to Rainbow trout for 100 days

JOURNAL OF FOOD PROTECTION. VOL. 44, SEPTEMBER 1981 PENICILLIUM-TOXINS IN CHEESE 707

and to rats over their life span did not show any harmful specially prepared Camembert cheeses with low fat effects c;l0,21). The latter investigation was a cancer content produced no significant increase in tumors study and included an experiment with weekly either. Cultures of the three strains were also fed to subcutaneous injection of a suspension of P. roqueforti Rainbow trout for 100 days without any ill effects (21). mycelium (2.9 g/animal/week) for 52 weeks. No chemical No toxins were formed by 41 strains of P. caseicolum (or analysis of the mycelium for any of the metabolites camembertt), using two human cell lines for detection described above was made. (37), nor by a further 16 strains of P. caseicolum tested in Lipids and flavors of blue cheese the chick embryo (,50); Lafont et al. (39) found 1 of 2 strains of P. camemberti yielded extracts that were of The action of P. roqueforti lipase and, if not moderate acute toxicity to mice by intraperitoneal inactivated by pasteurization, milk lipase, on milk fat injection. Apart from one report that strains of P. triglycerides produces free fatty acids. These are camemberti can produce an antibiotic that may be precursors of methyl ketones and secondary alcohols, citrinin (10), the only mycotoxin known to be produced which are important components of blue cheese and blue by this species is cyclopiazonic acid, which is discussed

cheese flavor, used as a food additive (33,54). Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/9/702/1654438/0362-028x-44_9_702.pdf by guest on 01 October 2021 below. Biological activity of 43 of 69 isolates of P. Concentrations of methyl ketones in fermented blue camemberti from various foods and feeds, using the cheese flavor can reach 7-12 times those in blue cheese chicken embryo and brine shrimp larvae as test (33,54). Various substrates have been used for this organisms, was probably due to this mycotoxin (43). fermentation, including lypolized milk fat (16). There appear to be no toxicological studies or search for Cyclopiazonic acid mycotoxins in this type of product. Abdel Kader et al. Cyclopiazonic acid (Fig. 7) is acutely toxic to rats with

(1,2) have shown that lipids extracted from an Egyptian an oral LD 50 of 36 mg/kg in males and 63 mg/kg in blue cheese were toxic when fed to rats, causing liver females. Intraperitoneal injection produced hyperesthe­ injury and 100o/o mortality after 6 weeks. The toxic sia and convulsions. Purchase (75) concluded that lesions factors were located mainly in the unsaponifiable matter produced in the liver did not resemble those expected but remain unidentified. Clearly, further work is needed from a hepatocarcinogen such as aflatoxin or sterigmato­ in this area. The only study in which animals have been cystin. The grounds for a statement by Orth (69) that fed blue cheese itself was the cancer study of Frank et al. cyclopiazonic acid is carcinogenic are therefore obscure. (20) where a total lifetime dose of 8 kg of cheese/rat Cyclopiazonic acid was first isolated from Penicillium produced no evidence of carcinogenic effects. cyclopium and later from Aspergillus versicolor, Major lipids found in P. roqueforti mycelium by Aspergalus jlavus and Aspf;rgillus oryzae c;l2,28,48,64, Shimp and Kinsella (83) were phospholipids (35.3o/o) and 69). Flavutoxin, from A. jlavus, is a metal chelate of triglycerides (33.8 o/o) while non-saponifiable material was cyclopiazonic acid (22). All of a total of 63 isolates of P. 8.1 o/o. Kaufmann et al. (31), using different culture camemberti from cheese that have been tested produced conditions, reported that phospholipids were only cyclopiazonic acid (41,87). Sixty-nine isolates from foods 12-18 o/o of total mycelium lipids for both P. roqueforti and feeds, including numerous cheese starter strains, and P. caseicolum and that free sterols, including also produced cyclopiazonic acid (43), confirming that P. ergosterol, comprised 16-17 o/o ofthe lipids. camemberti is a consistent producer of this mycotoxin. Still et al. (87) detected cyclopiazonic acid in the crust of PENICILLIUM CASEICOLUM [CAMEMBERTI] Camembert cheese itself (up to 4 p.g/g) if it were stored 5 days at 25 C after normal ripening, but not during the Gibel et al. (24) reported that a strain of P. camemberti ripening and storage at 14-18 C and 8 C, respectively. var. candidum dosed three times a week by stomach tube None was detected in 12 different brands of commercial or subcutaneous injection to Wistar rats showed cheeses, using a method capable of detecting 0.1 p.g of carcinogenic and leukemogenic effects with tumor cyclopiazonic acid/g. Le Bars (41), on the other hand, induction times of 445-754 days. Further investigations found cyclopiazonic acid in 11 out of 20 Camembert by Gibel (cited in ref. 84) on two other strains used for cheese crusts in concentrations of 0.05 to 1.5 p.g/g; it was production of Camembert cheese indicated that one of not detected (<0.02 p.g/g) in the inner parts of four these also showed carcinogenic activity. These observa­ cheeses containing the highest levels of cyclopiazonic tions were not confirmed by Frank et al. (20), who acid in the crust. LUck et al. (47) also did not find it in the retested Gibel's original strain, grown on the same wort inner part of four Camembert cheeses and one Brie. agar, by administering it to Sprague-Dawley rats by Toxigenic character of the P. camemberti strain isolated gavage once weekly for life or by weekly subcutaneous from a given cheese was a necessary but not sufficient injection for 52 weeks. Increased incidences of tumors condition for contamination of the cheese by cyclopia· compared to the controls were attributed to longer life zonic acid (41). LeBars (41,42) recommended selection of span of the test animals. Similar tests on two other strains that are weakly toxigenic at 13 C, have a low rate commercial starter strains for Camembert cheese as well of toxinogenesis, and which are less responsive to a as lifetime feeding tests with five types of commercial temperature increase, to limit or prevent occurrence of Brie or Camembert cheese in weekly alternation and two cyclopiazonic acid in Camembert cheese. In any event,

JOURNAL OF FOOD PROTECTION, VOL 44, SEPTEMBER 1981 708 SCOTT consumers would only eat 3 to 4 JJ,g of cyclopiazonic acid 8. Aujard, C., E. Morel-Chany, C. Icard, and G. Trincal. 1979. in a portion (one-eighth) of a highly contaminated Effects of PR toxin on liver cells in culture. Toxicology 12: Camembert cheese sample (41). 313-323. 9. Bekmakhanova, N.E.l974. Testing different substrates used for selecting microscopic fungi, potential producers of alkaloids. Mikol. Fitopatol. 8: 152-155. 10. Binder, W. 1954. The formation of an antibiotic substance in CH3--~---N----~ of the Camembert, Gorgonzola and Roquefort types of cheese. Milchwiss. Ber. 4: 88-129. Chern. Abstr. 49, 3431b (1955) 11. Cacan, M., S. Moreau, and R. Tailliez. 1978. Etude in vitro de Ia glucuronoconjugaison de Ia toxine de Penicillium roqueforti (P .R.T.) et de ses metabolites associes. Biochimie 60: 685-689. 12. Carter, S. B., T. J. F~anklin, D. F. Jones, B. 1. Leonard, S.D. Mills, R. W. Turner, and W. B. Turner. 1969. Mycophenolic acid: an anti-cancer compound with unusual properties. Nature (London) 223: 848-850.

13. Corbion, B., and J. M. Fremy. 1978. Recherche des aflatoxines Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/9/702/1654438/0362-028x-44_9_702.pdf by guest on 01 October 2021

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Bullerman, can·r.fromp. 701

other toxic substances produced by Aspergillus species. The contributors to the symposium, all experts in their A paper on the trichothecenes and other mycotoxins specific areas, have attempted to review the literature in produced by Fusarium and Stachybotrys species was these areas and provide an update on what is known included in the symposium but does not appear here. about a number of mycotoxins other than the aflatoxins.

JOURNAL OF FOOD PROTECTION. VOL. 44. SEPTEMBER 1981