Abstract: This study integrated alpha/beta diversity analysis, principal coordinate analysis (PCoA), and SourceTracker to systematically investigate the contribution of the environmental microbiota to the microbial community of fermented grains (Jiupei) and their functional regulatory mechanisms during the fourth round of fermentation of jiang-flavor baijiu. Results revealed that the microbial community structure of Jiupei was significantly driven by environmental selection pressures. Production tools (33.14%) were the core microbial source, with Kroppenstedtia guangzhouensis and Scopulibacillus daqui carried by them dominating community assembly through contact transfer (ADONIS R2 = 0.453, P < 0.001). Daqu (starter culture, 29.96%) specifically enriched functional fungi such as Monascus purpureus, while the drying floor (26.21%) introduced osmotolerant microbes (e.g., Oceanobacillus caeni) into Jiupei via ground diffusion. The airborne microbiota contributed minimally to the microbial community in Jiupei (0.70%), reflecting their colonization limitation under high-temperature and osmotic stress. Functional enrichment analysis demonstrated that the tool-associated microbiota directly drove ethanol synthesis via pyruvate metabolism (ko00620) and the tricarboxylic acid (TCA) cycle (ko00020), whereas the airborne microbiota optimized yeast stress responses through the mitogen-activated protein kinase (MAPK) signaling pathway (ko04010). Additionally, 9.99% of unassigned microorganisms (e.g., Desmospora sp. 8437) need metagenomic analysis to elucidate their ecological roles. This study highlights the tripartite interaction mechanism of environment, process, and microbes, providing a theoretical foundation for optimizing the traditional brewing process.

Key words: jiang-flavor baijiu; fourth round of fermentation; environmental microbiota; microbial community function; synergistic regulatory mechanisms