Insights into Physicochemical Characteristics, Flavor Development, and Microbial Succession During the Natural Fermentation of Sichuan-Style Black Soybean Soy Sauce

Sichuan-style black soybean soy sauce is a traditional fermented condiment renowned for its complex and regionally distinctive flavor profile. This study systematically investigated the physicochemical properties, flavor compounds, and microbial succession during six months of natural fermentation to elucidate the mechanisms underlying its unique flavor formation. Results showed that the amino acid nitrogen level increased to a peak of 1.37 g/100 mL before stabilizing at 1.01 g/100 mL, accompanied by a continuous rise in total acidity (0.69-2.75 g/100 mL). A total of 132 volatile compounds were identified, with esters (e.g., hexanoic acid, methyl ester, hexadecanoic acid, and methyl ester), alcohols (e.g., (E)-2-hepten-1-ol and trans-2-undecen-1-ol), and aldehydes (e.g., benzaldehyde and benzeneacetaldehyde) serving as key differentiating components. Nine taste-active (TAV ≥ 1) and 22 odor-active (ROAV ≥ 1) compounds were recognized as major flavor determinants, among which methional (ROAV = 4.77-119.05), 1-octen-3-ol (ROAV = 40.68-149.35), and 4-ethyl-2-methoxyphenol (ROAV = 4.70-36.26) were dominant contributors imparting sauce-like, mushroom-like, and smoky-clove notes, respectively. Microbial succession revealed a transition from <i>Weissella</i> and <i>Aspergillus</i> dominance in the early stage to salt-tolerant <i>Tetragenococcus</i> and aroma-producing yeasts (<i>Kodamaea</i> and <i>Zygosaccharomyces</i>) in later phases. Beyond organic acids and fermentation parameters (e.g., pH and salinity), microbial interactions were identified as critical drivers shaping community assembly and succession. Metabolic pathway analysis revealed a stage-dependent mechanism of flavor formation. During the initial stage (0-2 months), <i>Aspergillus</i>-mediated proteolysis released free amino acids as key taste precursors. In the later stages (3-6 months), <i>Tetragenococcus</i> and aroma-producing yeasts dominated, synthesizing characteristic esters (e.g., benzoic acid and methyl ester, correlated with <i>Tetragenococcus</i>; r = 0.71, <i>p</i> < 0.05), phenolics (e.g., 4-ethyl-2-methoxyphenol, correlated with <i>Wickerhamomyces</i>; r = 0.89, <i>p</i> < 0.05), and sulfur-containing compounds (e.g., methional, correlated with <i>Wickerhamomyces</i>; r = 0.83, <i>p</i> < 0.05).