We screened sake yeasts Kyokai no. 6 and 7 for novel spontaneous alcohol-tolerant mutant strains. Stress-tolerance-related tests were performed to evaluate properties of the strains K6AT1–K6AT8 and K7AT1–K7AT5. All the K6AT and K7AT strains, except K7AT5, possessed higher alcohol and heat stress tolerance than each parental strain. We analyzed the general properties and the content of organic acids and volatile compounds using sake samples obtained from a small-scale sake brewing test. Their high acidity was consistent with their characteristic organic acid contents, showing a higher concentration of malic, succinic, and lactic acids than the sake sample obtained using each parental strain. A lower amino acidity would reflect a higher ratio of surviving yeast cells at the end of mash. The contents of volatile compounds in the sake samples produced by the K6AT strains were similar to those in the sake sample produced by K6. That of the sake samples produced by the K7AT strains varied for each strain. A taste sensor analysis reflected the chemical content characteristics for each sake sample. Throughout the tests, K7AT5 showed substantially different characters from the other mutant strains. Although it showed higher alcohol tolerance than K7 at the screening stage, the results of several subsequent tests indicated that its alcohol tolerance had been lost. Additionally, K7AT5 gained a unique brewing characteristic compared with the other K7AT strains. These findings implied that a suppressor mutation of alcohol tolerance should occur and spread within the K7AT5 strain.
As a result of quantitating the amount of D-amino acid in tane koji and ingredients, D-Glu of 0.088 mmol/kg was contained in the defatted soybean. However, D-Asp, D-Glu, D-Ser, D-Ala and D-Leu were included in a total of 4.053 to 4.591 mM in raw soy sauce. The amount of D-amino acid was increased in the koji making process, moromi fermentation, and maturation process. Among microorganisms isolated from the manufacturing process, Tetragenococcus halophilus showed a high ability to convert L- to D-amino acid. In addition, Staphylococcus gallinarum and S. saprophyticus, which were present in the koji also showed this high ability.