Rubratoxin Formation in Soybean Substrates

Sterile soybeans, low and full fat soy meal, soy residue, soy curd, and soy whey fortified with different nutritional supplements were inoculated with spore suspensions of Penicillium rubrum P-3290. Yeast extract-sucrose (YES); glucose-malt extract; maltose-soytone; soy whey containing 1.75, 3.5, and 5.5% dissolved solids; and a maltose-salts medium were also inoculated. Cultures were incubated quiescently at 28 C for 7, 10, or 14 days. Rubratoxins were extracted with ethyl acetate and diethyl ether and resolved by thin-layer chromatography. Greatest yields of toxin were obtained from natural rather than chemically defined media. Semisynthetic media supported formation of moderate amounts of rubratoxins. Malt extract and glucose supported greatest production of both rubratoxins A and B. Unsupplemented soy whey or soy milk did not support rubratoxin production. Although soy meal supported production of both rubratoxins A and B (0.12–0.24 mg/g and 0.55–0.71 mg/g), low fat soy meal was more suitable for toxin formation than was full fat meal. Both rubratoxins A and B were produced by P. rubrum grown in soy whey fortified with soytone, malt extract, or glucose; only rubratoxin B was produced when yeast extract or sucrose served as supplements. YES broth supported formation of only rubratoxin B. Production of toxin in soybeans was influenced by time of incubation and temperature. Maximum yields of toxin on soybeans were obtained after 14 days of incubation at 25 C, after 28 days at 28 C, after 7 days at 32 C, and after 3 days at 37 C. Rubratoxin was not produced at 40 C. Yields of rubratoxin A in soy whey media ranged from 35–40 and from 15–28 mg/100 ml when malt extract or glucose were added; values for rubratoxin B ranged from 55–100 mg/100 ml and 65–125 mg/100 ml.