Effect of mixed starter cultures of Bacillus on sensory characteristics of bacterial-douchi: insights from free amino acids, electronic sensory, flavor profiling and quorum sensing

This study aimed to investigate the effect of mixed starter cultures of cellulase-producing Bacillus licheniformis ND38 and protease-producing Bacillus subtilis NB13 on flavor characteristics of bacterial-douchi. Mixed starter cultures increased total titratable acidity, amino-nitrogen, and protease activity while decreasing total sugar and crude protein in douchi, compared with single starter. Higher viable counts and activity of quorum sensing molecule autoinducer-2 (AI-2) were observed in douchi with combined Bacillus starters. Furthermore, the total free amino acid (FAA) content in douchi with mixed fermentation was 9.02 % higher than that with ND38 single-strain and 21.34 % higher than that of the NB13 single-strain fermentation. Electronic sensory analysis showed that mixed Bacillus fermentation conferred the complementary flavor formation of douchi. Headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS)analysis revealed that mixed starter cultures combined the complementary volatile profiling of each single strain and conferred characteristic nutty, roasted, creamy and floral aromatic notes. Especially, volatile compounds such as pyrazines, alcohols, aldehydes, and esters were increased. The findings indicate that mixed Bacillus starter cultures can increase free amino acids generation and improve flavor formation, probably mediated with microbial interaction, showing their potential application in douchi production. • Mixed fermentation boosts free amino acids, indicating enhanced microbial metabolism. • AI-2 quorum sensing activity suggests microbial interaction between the two Bacillus strains. • E-tongue analysis confirms improved and complementary taste profiles in mixed fermentation douchi. • E-nose and HS-SPME-GC-MS reveal enriched volatile components compared to single-strain fermentation.