Some oligopeptides and amino acids have a strong influence on the sensory qualities of sake, but the formation process of such compounds in sake mash has not been well elucidated. In this study, we investigated the formation process of bitter-tasting peptides derived from rice proteins in sake mash, because knowledge about their formation may contribute to the quality control of sake. We analyzed rice protein hydrolysates in sake mash, as well as in the enzymatic digest of steamed rice grains digested by either sake-koji or by crude enzyme extracted from sake-koji. SDS-PAGE showed that a smaller amount of polypeptides (> M.W. 10,000) accumulated in the supernatant of sake mash than in either enzymatic digest. The concentration of peptides in the supernatant of sake mash increased gradually from the early stages of fermentation. Five bitter-tasting peptides (No. 9, < QLFNPS; No.13, < QLFNPSTNP; No. 17, < QLFNPSTNPVVH; No. 18, < QLFNPSTNPVVHSP; No. 20, < QLFGPNVNPWFINP), which were previously found in sake mash, were not found in significant amounts in sake-koji. On the other hand, these peptides accumulated at the early stages of both sake mash fermentation and the enzymatic digests, although the levels in sake mash were higher than those in the digests. The present study demonstrated that the 5 bitter-tasting peptides formed in high concentrations when steamed rice grains were digested under conditions of sake mash fermentation with yeast. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.