Microbial succession and functional partitioning during eight rounds of stacking fermentation in sauce-flavor Baijiu

Stacking fermentation is a crucial stage in the production of sauce-flavor baijiu, a traditional Chinese distilled liquor known for its complex aroma. However, continuous microbial succession and functional dynamics throughout the complete eight-round stacking process remain insufficiently resolved. This study systematically investigated physicochemical properties, volatile compounds, microbial community structure, and functional gene profiles across eight consecutive rounds of stacking fermentation. Moisture, acidity, and temperature increased during fermentation, whereas starch content declined markedly, indicating progressive substrate transformation. A total of 82 volatile compounds were identified, with esters and acids dominating and peaking in rounds 4-6, which are associated with high-quality baijiu. Metagenomic sequencing enabled species-level characterization of microbial succession. Bacterial communities shifted from lactic acid bacteria (LAB) to Kroppenstedtia eburnea , Thermoactinomyces vulgaris , and Bacillus spp., while fungal succession progressed from initial yeast dominance to mold co-occurrence and then back to yeast dominance. Functional gene annotations revealed glycolysis and pyruvate metabolism as central pathways. Co-occurrence networks and correlation analyses suggested potential functional partitioning among microbial groups: molds were associated with starch metabolism and ethyl phenylacetate formation, yeasts with ethanol and ester production, and LAB with organic acid metabolism. This study provides a comprehensive, species-resolved analysis of microbial succession and functional partitioning across all eight rounds of stacking fermentation in sauce-flavor baijiu. The findings highlight the roles of specific microbial taxa in shaping fermentation outcomes and offer mechanistic insights for optimizing flavor formation and product quality in traditional baijiu production.