Targeted Microbial Co-Inoculation Enhances Flavor and Community Structure in Nongxiangxing Baijiu Fermentation

Distilled Nongxiangxing Baijiu (NXB), produced from lower-layer fermented grains (FG), consistently exhibits superior quality, whereas upper-layer FG often yield variable and inferior sensory attributes. Biofortification of fermentation by inoculation with complementary microorganisms is a promising approach for stabilizing and enhancing NXB quality. This study compared single-strain inoculations with Bacillus subtilis (B group), Pichia kudriavzevii (P group), and mix-strain inoculations with Bacillus subtilis and Pichia kudriavzevii (BP group) in terms of physicochemical properties, volatile flavor metabolites, and microbial community structure of the upper-layer FG during NXB fermentation. Mantel and correlation analyses were used to assess the correlations among these parameters. Results showed that both inoculants significantly improved ethanol yield, with the greatest enhancement observed in the BP60 treatment (inoculated with Bacillus subtilis and Pichia kudriavzevii). The semi-quantified relative concentrations of key volatile organic compounds, including ethyl butyrate and ethyl hexanoate, were significantly elevated in the BP60 FG. Mantel and correlation analyses identified moisture content and reducing sugar concentration as the dominant physicochemical drivers shaping microbial community composition and collaborative interactions, which were mainly related to acid-, alcohol-, and ester-associated metabolism, whereas fungi were more closely linked to ester, aromatic, and phenolic compound formation. Collectively, these findings demonstrate that co-inoculation with Bacillus subtilis and Pichia kudriavzevii promotes the accumulation of signature flavor metabolites during NXB fermentation. This mechanistic insight provides a foundation for rational process optimization and targeted quality control for strategies in industrial-scale NXB production. PRACTICAL APPLICATIONS: The co-inoculation with Bacillus subtilis and Pichia kudriavzevii promotes the accumulation of signature flavor metabolites during NXB fermentation.