Effects of pile size on the fermentation characteristics of zaopei during sauce-flavor baijiu production: An integrated analysis of physicochemical parameters, microbial communities, and volatile flavor compounds.

Stacking fermentation is a distinctive production process during the brewing of sauce-flavor baijiu. Fermentation pile structure can shape physicochemical heterogeneity and microbial succession in zaopei during stacking fermentation, but the effects of different pile sizes on zaopei fermentation characteristics and their underlying mechanisms remain unclear. In this study, two types of fermentation pile size were established (D-group: single-pile group; S-group: double-pile group), and their effects on the physicochemical properties, volatile flavor substances, and microbial community composition of zaopei during the 4th-6th fermentation rounds of sauce-flavor baijiu production were systematically investigated. The results showed that titratable acidity, moisture, starch, reducing sugar, and organic acids exhibited generally similar variation trends in the D-group and S-group. However, at the end of pit fermentation (CJ stage), higher levels of titratable acidity, lactic acid, and total alcohols were observed in the S-group, whereas relatively higher contents of total esters and pyrazines were detected in the D-group. Similar succession patterns were observed for the dominant microbial communities under the two fermentation pile size conditions, mainly including Kroppenstedtia, Acetilactobacillus, Lentibacillus, Oceanobacillus, Pichia, Monascus, Aspergillus, and Kazachstania. During the stacking fermentation stage, relatively higher abundances of Acetilactobacillus and Pichia were observed in the D-group, whereas relatively higher abundances of Lentibacillus, Kroppenstedtia, and Kazachstania were observed in the S-group. Mantel test analysis showed that acidity and moisture were among the important physicochemical factors potentially driving changes in important microorganisms under the two fermentation pile size conditions. Random forest analysis further showed that genera such as Oceanobacillus, Acetilactobacillus, Bacillus, Schizosaccharomyces, and Aspergillus were closely associated with changes in acidity. PICRUSt2 prediction suggested that higher potential was present in the D-group for starch degradation related functions, whereas higher predicted levels were observed in the S-group for organic acid metabolic functions. In summary, different fermentation pile size may influence the formation of flavor and quality in sauce-flavor baijiu by regulating the microecological structure of zaopei and its potential metabolic functions. Results presented in this study provided a theoretical basis for process regulation of stacking fermentation in sauce-flavor production.