Abstract: A total of 56 strains with high protease activity were isolated from high-salt liquid-state moromi (soy sauce mash) by the casein plate method. Among these, the strain CS1.11 with the highest amylase activity, CS1.13 with the highest protease activity and CS1.17 with the highest cellulase activity were selected by the Oxford-cup method. According to the 16S rRNA gene sequence analysis and morphology, CS1.11, CS1.13, and CS1.17 were identified as B. velezensis, B. methylotrophicus and B. subtilis, respectively. The three strains and the control strain B. subtilis CS1.03 were separately subjected to salt brine fermentation after koji-making. It was found that the fermented soy sauce inoculated with CS1.13 had the highest ammonia nitrogen content (5.12 g/L), and the fermented soy sauce inoculated with CS1.11 had the highest reducing sugar content (27.20 g/L). The results were consistent with the strains’ enzymatic activities. In addition, the four strains had little effect on the total acid content of soy sauce. All the fermented soy sauce samples were qualitatively and quantitatively analyzed by solid-phase microextraction-coupled to gas chromatography-mass spectrometry (SPME-GC-MS) using the internal standard method. It was found that all four strains were advantageous for producing pyrazines and their precursors (acetoin and 2,3-butanediol), the three test strains being superior to the control strain. Pyrazines could impart a good flavor and health factors to soy sauce, and were the characteristic flavor substances of fermented soy sauce inoculated with Bacillus spp. Bacillus strains CS1.11, CS1.13, and CS1.17, thanks to their potential to promote moromi fermentation, enrich the flavor of soy sauce and increase health factors, will hold great promise for application in soy sauce production.

Key words: Bacillus spp.; promoting moromi fermentation; screening by Oxford-cup method; protease activity; pyrazines