A method has been developed for the production of aflatoxin by growing Aspergillusflavus strain NRRL 2999 on the solid substrate rice. Optimal yields, more than 1 mg of aflatoxin B1 per g of starting material, were obtained in 5 days at 28 C. A crude product containing aflatoxins was isolated by chloroform extraction and precipitation with hexane from concentrated solutions. The crude product consisted of 50% aflatoxin in the following ratio: B1-B2-G-G2, 100:0.15:0.22:0.02. Aflatoxin B1 was separated from almost all the impurities and from the other aflatoxins by chromatography on silica gel with 1% ethyl alcohol in chloroform. Analytically pure aflatoxin B1 was recrystallized from chloroform-hexane mixtures.

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Corn from an Arkansas farm, where three horses died and others became sick, was investigated for causative principles. Necropsy of the three horses revealed what appeared to be severe hepatic necrosis. Histopathological examination indicated a pattern of hepatic lesions that was suggestive of aflatoxin contamination of the feed. Mycological examination of the corn by dilution plating revealed 95% of the colonies as Aspergillus flavus. Chemical analysis of the corn for mycotoxins was positive for aflatoxin B1, B2, and M1 at concentrations of 114, 10, and 6 micrograms/Kg, respectively. Cyclopiazonic acid, sterigmatocystin, and the Fusarium toxins, vomitoxin (deoxynivalenol), T-2 toxin, and diacetoxyscirpenol, were not detected. The presence of aflatoxin metabolites in the moldy corn and the presence of appropriate lesions were compatible with the diagnosis, equine aflatoxicosis.

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A method has been developed for the production of aflatoxin by growing Aspergillus flavus strain NRRL 2999 on the solid substrate rice. Optimal yields, more than 1 mg of aflatoxin B 1 per g of starting material, were obtained in 5 days at 28 C. A crude product containing aflatoxins was isolated by chloroform extraction and precipitation with hexane from concentrated solutions. The crude product consisted of 50% aflatoxin in the following ratio: B 1 -B 2 -G 1 -G 2 , 100:0.15:0.22:0.02. Aflatoxin B 1 was separated from almost all the impurities and from the other aflatoxins by chromatography on silica gel with 1% ethyl alcohol in chloroform. Analytically pure aflatoxin B 1 was recrystallized from chloroform-hexane mixtures.

A method has been developed for the production of aflatoxin by growing Aspergillus flavus NRRL 3145 on solid substrate wheat. Optimal yields of 900 jug of aflatoxin G1 and 900 jug of aflatoxin B1 per g of substrate were obtained in 4 to 5 days at 28 C. A study of aflatoxin production on hulls and groats of oats and on whole oats by A. flavus strains NRRL 2999, NRRL 3000, and NRRL 3145 revealed that aflatoxin was produced on all three substrates, although production was very slight on hulls. Strain NRRL 3145 grown on solid substrate groats produced the largest amounts of aflatoxin: 580 Ag of B1 and 450 ug of G1 per g of substrate. A densitometric method for reading thin-layer chromatographic plates is described; this is more objective and more accurate than the visual methods previously used for the determination of all four aflatoxins.

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A method for aflatoxin M1 in dairy products is presented. It decreases the analysis time compared to currently accepted methods. Samples are extracted in a blender or separatory funnel for 1 min with chloroform and saturated sodium chloride solution. The chloroform extracts are partially purified on a small silica gel column (2 g), and M1 is determined by thin layer chromatography (TLC) and densitometry. Recoveries of M1 added to milk are about 80%. Recovery of M1 from cheeses is variable depending on the type of cheese. The method gave results for a naturally contaminated powdered milk comparable to analyses by accepted methods. The determination limit of the method is about 0.1 μg/kg.