Comparative metabolomics-based analysis and functional mechanisms of the natto fermentation process as regulated by Bacillus subtilis strains CGMCC 34222 and A9

Natto is a soybean product fermented by Bacillus subtilis , and strain-specific differences can markedly affect its functional properties and sensory quality. In this study, a high-performance strain CGMCC 34222 and an industrial strain A9 were subjected to a multidimensional comparative analysis integrating genomics, metabolomics, and functional performance evaluation. Compared with A9, CGMCC 34222 possesses distinctive genetic evolutionary features and encodes a specific serine protease gene. 347 and 375 differential metabolites were identified during natto fermentation by CGMCC 34222 and A9, mainly involved in amino acid metabolism, lipid and lipid-like molecule metabolism, and carbohydrate metabolism. Among these metabolites, palmitic acid, stearic acid, and leucine were significantly enriched, accompanied by enhanced glutamate and phenylalanine metabolism. Volatile compound analysis further indicated that 2,5-dimethylpyrazine is a major contributor to the characteristic aroma of natto, and the relative proportion of soybean-derived amine off-flavor compounds was markedly reduced during fermentation. In addition, natto fermented by CGMCC 34222 exhibited higher nattokinase activity and improved odor and sensory. Overall, the molecular and metabolic characteristics of CGMCC 34222 and A9 reveal the strain-dependent mechanisms underlying functional activity and flavor quality in natto fermentation. This study establishes a genomics–metabolomics-based evaluation framework for the rational screening of high-quality natto-fermenting strains.