Microbiologia

APPLIED

AND

ENVIRONMENTAL MICROBIOLOGY, Dec. 1979, p. 1052-1055

Vol. 38, No. 6

0099-2240/79/12-1052/04$02.00/0

Cyclopiazonic Acid Production by Penicillium camemberti Thom and Natural Occurrence of This Mycotoxin in Cheese
JOSEPH LE BARS

Station de Pharmacologie-Toxicologie, INRA, 31300 Toulouse, France
Received for publication 24 September 1979

Every Penicillium camembertistrain freshly isolated from 20 commercial cheese brands produced cyclopiazonic acid in two culture media at 25, 13, and 4°C; the toxin yield was greatly dependent on the strain and environmental parameters (medium, temperature, and incubation time). The toxigenic ability appeared as a log-normal distribution. This mycotoxin was found in the crust (0.05 to 0.1 ,ug/g in three samples, 0.1 to 0.2,ug/g in five samples, and 0.4, 1, and 1.5 ,Lg/g in three other samples) but not in the inner part. When its acute toxicity is considered, doses eventually ingested by consumers are very low (lower than 4 ,ug). Means for prevention are discussed. A highly toxigenic strength and rate appear to be necessary features leading to natural contamination in cheeses. The distribution of toxigenic abilitymakes possible without delay a choice of weakly toxic strains. Cyclopiazonic acid, isolated from Penicillium cyclopium Westling (4), is toxic to rats per os (12); we found this toxin in laboratory cultures of 25% of P. cyclopium strains from feedstuffs. Other fungal species are also known to produce cyclopiazonic acid: Aspergillus versicolor (10), Aspergillus oryzae (11), and Aspergillus flavus (7).Recently, Still et al. (14) described cyclopiazonic acid in Penicillium camemberti cultures and in cheeses stored at 25°C, an unusual condition. Several types of cheeses (Brie, Camembert, Carre de l'est, etc.) are fermented by Penicillium caseicolum Bainier (9), which is now regarded as a synonym of P. camemberti Thom
In relation to the hygienic hazard and the economic incidence of cyclopiazonicacid contamination, the purpose of this study was to examine toxin bioproduction by freshly isolated strains of P. camemberti from conmmercial cheeses and to investigate the possible occurrence of this toxin in these foods.
glucose, 10; succinic acid, 10; KH2PO4, 1; MgSO4. 7H20, 0.3; NH40H to pH 5.6. Fungi were grown in 30 ml of medium in 100-ml Erlenmeyer flasks at 25°C (optimal temperature forcyclopiazonic acid bioproduction) (14) for 25 days and at 13°C (ripening temperature for cheeses) and 4°C (refrigerator temperature) for 33 days. Cyclopiazonic acid analysis. The development of cyclopiazonic acid concentration was examined in culture filtrates, and the yield in mycelia was measured at the end of each incubation period. Thin-layer chromatographic (TLC) adsorbents were as follows:Silica Gel 60, 0.25-mm thickness, on glass plates (P), and Silica Gel 60, 0.25-mm thickness, on glass plates with fluorescent indicator (F), both impregnated with oxalic acid (0) or tartaric acid (T). Developer solvents were as follows: (solvent I) ethyl acetate-2-propanol-NH40H (20:15:10); (solvent II) CHCl3-isobutylmethylketone (4:1); (solvent III) toluene-ethyl acetate-formic acid (5:4:1);(solvent IV) CHCl3-acetone (95:5). Cyclopiazonic acid was visualized as a violet spot, in ordinary light, after spraying with Ehrlich reagent (10 g of 4-dimethylaminobenzaldehyde in 100 ml of HCI, extemporaneously diluted with 4 volumes of acetone). Extraction procedures were as follows. A 1-ml portion of culture filtrate, adjusted to pH 3 with aqueous HCI (50:50), was extracted twice with 4 ml ofCHC13; after drying and evaporation, 10 ul of the extract, dissolved in 0.1, 0.25, or 1 ml of CHC13, was spotted on TLC plates (P) and developed with solvent I. At the end of the incubation period, mycelial mats were extracted by soaking and agitation in an azeotropic CHCl3-MeOH mixture; after drying and evaporation, extracts were dissolved in CHC13 for TLC; defatting was accomplished on TLC plates...