Untargeted metabolomic profiling of Aspergillus sojae 3.495 and Aspergillus oryzae 3.042 fermented soy sauce koji and effect on moromi fermentation flavor

Aspergillus sojae 3.495 and Aspergillus oryzae 3.042 are essential microbial starters in fermented foods due to their high hydrolytic enzyme activities. However, their metabolic characteristics and differences during soy sauce fermentation are still unclear. This study identified 42 non-volatile differential metabolites based on UHPLC-MS/MS, among which 10 amino acids and derivatives and 4 non-volatile acids were high in A. sojae 3.495 koji, while 3 sugar alcohols, 5 flavonoids, and 4 fatty acids were prominent in A. oryzae 3.042. Further analysis revealed that higher acidic protease (1887.43 U/g) in A. sojae 3.495 and higher glucoamylase (2413.36 U/g) and cellulase (890.78 U/g) in A. oryzae 3.042 dominated the formation of differential metabolites. Metabolic pathway analysis showed that shikimate and phenylalanine metabolic pathways in A. sojae 3.495 were more active and enhanced 3-methyl-butanal and 2-phenylethanal in moromi fermentation. The more prominent pathways of sugar alcohol metabolism and flavonoid biosynthesis in A. oryzae 3.042 koji enhanced 2-methyl-butanoic acid and ethyl 3-methylbutyrate in moromi and significantly elevated reducing sugars (1.56 g/100 mL). Overall, comparative metabolomics elucidated the metabolic framework and flavor formation mechanisms of different inocula in soy sauce koji, providing ideas for scientific control of soy sauce fermentation and multi-strain koji-making for flavor enhancement.