Properties of TCA-insoluble peptides in kimoto (traditional seed mash for sake brewing) and conditions for liberation of the peptides from rice protein.

It was found that a large amount of TCA (trichloroacetic acid)-insoluble peptides were liberated into the supernatant of kimoto on the 7th-10th day of mashing. These TCA-insoluble peptides had five polypeptide groups (12, 21, 31, 38, and 55 kDa) on SDS-PAGE (SDS-polyacrylamide gel electrophoresis), and a large amount of high molecular weight peptides, higher than 10,000, were observed upon gel filtration chromatography using TSKgel G2000swxl (Tosoh Co.). Four of these peptides (12, 21, 31, and 38 kDa on SDS-PAGE) appeared specifically in kimoto, and were not detected at all either in sokujo-moto or in the main mash for sake brewing. These TCA-insoluble peptides were fractionated from the supernatant of kimoto on the 9th day, and it was revealed that free amino acids were produced abundantly from them in the presence of the enzyme of rice-koji. Therefore, it was assumed that the peptides are related to the abundant production of free amino acids in kimoto. For the liberation of these TCA-insoluble peptides from rice protein, the enzyme of rice-koji was indispensable. The enzyme liberating the TCA-insoluble peptides from rice protein was purified from rice-koji, and was presumed to be identical with acid protease (AP) of rice-koji. The presence of a high concentration of glucose (higher than 20%) was also indispensable for the liberation of the TCA-insoluble peptides. Furthermore, it was revealed that the peptides were liberated from rice protein under a limited pH of around 4.5.