Enzymatic formation of ether linkage producing shoyuflavones from genistein and (+/-)-trans-epoxysuccinic acid.

The production mechanism of shoyuflavones, conjugated ethers of isoflavones with tartaric acid and isolated from fermented soy sauce, was studied. In the high molecular weight fraction of the culture extract of Aspergillus oryzae, genistein was transformed into shoyuflavone B in the presence of (+/-)-trans-epoxysuccinic acid but not in the low molecular one. Asp. sojae and Asp. tamarii showed high activity similar to Asp. oryzae but none of Asp. niger, Rhizopus oligosporus, and Mucor praini did. The contents of epoxysuccinic acids in the starting materials of soy sauce and the cultures of various Asp. fungi were determined as dimethyl 2-chloro-3-hydroxysuccinate derivatives by GC-MS. Although epoxysuccinic acids were contained in Asp. oryzae, Asp. sojae, and Asp. tamarii cultures, they were not found in soybeans and wheat. A possible producing mechanism for shoyuflavones by enzymatically conjugating isoflavones to (+/-)-trans-epoxysuccinic acid with ether linkage was suggested.

Differentiation of soy sauce types by HPLC profile pattern recognition. Isolation of novel isoflavones.

Nonvolatile minor components in various brands of Japanese fermented soy sauce were analyzed by gradient RP-HPLC and monitored at 280 nm. Chemometric pattern recognition techniques, such as cluster analysis, linear discriminant analysis (LDA), LDA using genetic algorithm (GA-LDA) and soft independent modelling of class analogy (SIMCA), succeeded in differentiating the resulting HPLC profiles according to soy sauce brands. Three components playing key roles in the differentiation were isolated by preparative HPLC and purified by gel-filtration chromatography, or simply repeated preparative HPLC. FAB-MS, 1H-, 13C-NMR and IR spectra suggested that these three components having molecular weights of 386, 402 and 418 were isoflavone derivatives. By applying HMBC spectral analysis, these isoflavones were identified as conjugated ethers of tartaric acid with daidzein, genistein and 8-hydroxygenistein. These new isoflavone derivatives are produced by some strains of Aspergillus fungi.

[Specific odor component produced by Mycobacterium lepraemurium on Ogawa yolk medium].

When Mycobacterium lepraemurium is grown on the 1% Ogawa yolk medium, it produces a specific odor. This odor was not observed in other easily cultivable acid-fast bacilli. Therefore, identification of the components responsible for the specific odor produced by M. lepraemurium was attempted. The odor components were extracted for overnight with sterilized and distilled water from the Ogawa yolk medium on which M. lepraemurium had been cultivated for two months. The odor components in the extract was adsorbed on refined charcoal. After washing with distilled water for three times, the charcoal was dried. Then the odor components were eluted from the charcoal with ethanol and the eluate was condensed under nitrogen gas flow at 40 degrees C. The condensate was analyzed by Gas-Chromatography-Mass-Spectrum (GC-MS). Phenylethanol and phenylacetic acid were identified as major odor components. A mixture of authentic phenylacetic acid, its methyl and ethyl esters, smelled similar to the odor of cultivated medium of M. lepraemurium. Thus, phenylacetic acid was identified as the key odor component produced by M. lepraemurium. When initial isolation culture of M. lepraemurium from murine leproma was cultivated on the Ogawa yolk medium by adding phenylacetic acid, growth inhibition was brought by the compound.