Two monoclonal antibodies KTM-205-POD and KTM-240-POD which are highly specific to 3,4,15-triacetyl-NIV (TANIV) and TANIV + 3,15-diacetyl-DON (DADON), respectively, were used for the determination of nivalenol (NIV) and deoxynivalenol (DON) production by Fusarium spp. Sixteen strains of F. graminearum isolated from wheat and barley grains were cultured on polished rice substrate. NIV, DON and their acetylated derivatives present in the culture extract were partially acetylated into TANIV and DADON, respectively, before direct competitive enzyme-linked immunosorbent assay (ELISA). Results determined by ELISA, expressed as NIV and DON equivalents, were subsequently confirmed by gas chromatography-mass spectrometry. The correlation between the two methods for concentrations of total NIV and total DON was r2 = 0.9875 (p<0.001). The results show that ELISA, at a cut-off limit of 25 mg/kg, can be used as a rapid and simple method for the screening of NIV- and DON-producing strains of F. graminearum.
The natural occurrence of type B trichothecenes (deoxynivalenol (DON), 3-acetyldeoxynivalenol(3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV) and fusarenone-X (F-X)) was investigated in formula feeds and feedstuffs. Twenty-one formula feed samples and 44 feedstuff samples (maize, milo, barley, wheat, rye, wheat bran, soybean meal, rapeseed meal, and cottonseed) were collected from the Japanese Feed Industry. These samples were analyzed by gas chromatography after purification with a multifunctional cleanup (MFC) column and trimethyl silyl derivatization. DON was detected in formula feed, cereals and bran samples. The concentrations in positive samples were 16~190 μg/kg in formula feeds and 14~2,200 μg/kg in cereals and wheat bran. 15-ADON was detected in formula feed, maize, wheat and wheat bran samples at concentrations of 10~94 μg/kg in positive samples. NIV was detected in the samples of formula feed, milo, barley, rye, wheat and wheat bran samples at concentrations of 26~140 μg/kg in positie samples. 3-ADON and F-X were not detected in any sample. None of the samples of cottonseed, soybean meal and rapeseed meal contained these trichothecenes.
On Penicillium expansum, germination of the spore (conidia), growth using fruit components, production of various enzymes and enzyme action for apple fruit were examined. Xylan- and pectin-degrading enzymes were easily liberated in the buffer solution from the spore. The levels of these enzyme activities were largely affected by carbon sources in the sporogenesis culture medium. The spore harvested from the culture of the xylan medium showed the high xylanase activity and the spore from the pectin medium showed the high pectinase activity. The carbon source of germination medium and the enzyme level of the spore were related to the elongation of germ tube. The mold grew well in the medium containing xylan or pectin, and abundantly formed the spore. Unlike this, its growth using soluble sugars (glucose, fructose, sucrose) was poor. The mold produced gluconate from glucose, and glucose oxidase and catalase were produced in the culture broth. The fruit tissue remarkably browned, when it was incubated with glucose oxidase and catalase. This browning reaction was promoted in the presence of xylan-degrading enzyme. Two xylanases and β-xylosidase were purified from the culture filtrate as homogeneous proteins. The reducing sugar was liberated from the fruit when it was incubated with these enzymes.
In recent year, the Joint FAO/WHO Codex Alimentarius Commission (CAC), an international body formed to develop standards, has carried out active discussions, and established standards, for some mycotoxins in food. In response to a request of the CAC, the Joint FAO/WHO Expert Committee on Food Additives evaluated the safety of various mycotoxins in February 2001. An approach to standards setting is expected to be advanced by the global scientific community in the near future. Under such circumstances, Japan has established standards for deoxynivalenol and patulin a guideline level for deoxynivalenol in wheat in May 2002, and a standard for patulin in apple juice and in apple juice ingredients for other nonalcoholic beverages in November 2003. It is very important for Japan to continue to investigate various mycotoxins, and to develop standards for food mycotoxins, in order to prevent health hazards.