Comprehensive analysis of spatiotemporal heterogeneity reveals the effects of physicochemical and biological factors on temperature rise during the Moutai-flavor Baijiu stacking fermentation process

In the brewing process of Maotai-flavor Baijiu (MFB), high-temperature stacking fermentation, which spontaneously captures and enriches environmental microorganisms, plays a crucial role in subsequent fermentation and the quality of the final product. This study focused on a analysis of comprehensive spatiotemporal heterogeneity in fermented grains under different temperature-rise patterns using absolute quantitative amplicon sequencing. For the first time, the temperature-rise process of MFB fermentation was visualized, and the spatiotemporal heterogeneity in high (HT, > 50 degrees C) and low (LT, < 40 degrees C) peak temperature groups was revealed. Acidity, lactic acid, and acetic acid were negatively correlated with fermentation temperature (P < 0.001, rho < 0), and these factors were identified as the primary physicochemical contributors to the differential temperature-rise patterns between the HT and LT groups (Variable Importance in Projection (VIP) > 1). Furthermore, the spatial differences of acid distribution mediated the spatiotemporal heterogeneity in dominant microbial communities, driving microbial succession. In the pile center with higher acidity, Lactobacillales became the absolutely dominant species, altering microbial assembly patterns, species richness (Chao1) and evenness (Pielou). A random forest regression model was established to identify the characteristic microorganisms influencing the temperature rise during fermentation. Through isothermal microcalorimetry, Saccharomycetales was identified as a positive biological factor driving the temperature rise during stacking fermentation in MFB, whereas latic acid and Lactobacillales acted as negative factors. This study provides constructive suggestions for process optimization and quality control strategies in the production of MFB.