We previously developed a peptide-enriched soy sauce-like seasoning called Fermented Soybean Seasoning (FSS) with high-temperature fermentation, and we have reported the antihypertensive effects of FSS. Seryl-tyrosine (Ser-Tyr) and glycyl-tyrosine (Gly-Tyr) were identified from FSS as active constituents in the antihypertensive effects. They were found to be particularly enriched in FSS; more so than in regular soy sauce. In the present study, we clarified one of the mechanisms underlying the accumulation of these bioactive peptides during high temperature soy sauce brewing. Crude enzyme extracts were prepared from model soy sauce mash (moromi) fermented at various temperatures. Leucine aminopeptidase-I, II, and seryl-tyrosine hydrolytic activity were found to decrease in the moromi incubated at the fermentation temperature of FSS whereas almost no decrease was observed in that of regular soy sauce. The concentrations of ACE inhibitory peptides, Ser-Tyr and Gly-Tyr, in the moromi incubated at high temperature were revealed to be higher than those at low temperature through quantitative LC-MS/MS analysis. These results suggested that the peptidases responsible for degrading low molecular weight bioactive peptides were inactivated during the high temperature fermentation, thus, these peptides would be likely to remain in the high temperature fermentation. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.