CHEMICAL AND BIOCHEMICAL STUDIES OF TOXIC FLUORESCENT SUBSTANCE PRODUCED BY ASPERGILLUS FLAVUS (LINK) WHEN CULTURED ON PALMSAP

Abstract

Czapek medium, Czapek-Dox medium, glucose-ammonium nitrate medium and palmwine of different ages have been used in the culture of Aspergillus flavus (Link ex Fries). Of these growth media, unfermented palmwine (palmsap) seemed to be the best for the growth of the mould. This medium also gave the largest production of toxic substances. As a result of this finding, an attempt was made to define palmwine chemically. Using chromatographic and colorimetric techniques, sucrose, glucose, fructose and maltose were shown to be present in high concentrations. Growth promoting factors such as sodium, potassium, coper, iron, calcium, zinc, manganese, cobalt, iodine, phosphorus, sulphur, ascorbic acid, nicotinic acid and some other vitamins were also present. Evidence is presented to show that unfermented palmwine is nutritionally better than the fermented samples for growing the fungus and producing the toxins. Toxins were not found in naturally-occuring palmsap. Two other fluroscent substances B₀ and G₀, were extracted from the fermentation medium with chloroform and purified by thin layer chromatography and crystallination. They form plate crystals which have low melting paints. Chemical evidence proves that they are not aromatic. These findings are supported by evidence from infrared, ultraviolet, nuclear magnetic resonance and mass spectroscopies. Palmtoxins B₀ and G₀ have been suggested as long chain unsaturated fatty acids (diones) which may be spatial isomers of each other. Each of these exhibits a mass peak corresponding to N⁺ =380, an ion for which the molecular formula C₂₄ H₄₄ O₃ has been suggested. The molar extinction coefficients of palmotoxins B₀ and G₀ were found and compared with those of aflatoxins. The output of toxins was measured with a spectrophotometer. It has been found that aflatoxins B₁ in the major metabolite of A.flavus is a stationary culture of palmwine. Comparatively, palmotoxins B₀ and G₀ are produced in minute quantities. In order to compare the toxicities of the various substances, appropriate experiments were performed using 5 species of 6-day old avian embryos. Aflatoxins B₁ has been found to be as toxic as palmotoxins B₀. Palmotoxin G₀ is the least toxic of these substances.