## 3 Results  
### Dynamic Changes in Physicochemical Factors  
Throughout the stacked fermentation process, physicochemical factors--including moisture content, total acidity, starch content, glucose content, lactic acid concentration, ethanol concentration, and temperature--exhibited dynamic alterations. These changes influence microbial metabolic activities, which in turn shape the production of flavor compounds [25; 26]. Moisture content is a critical parameter in Baijiu production, significantly influencing microbial succession [27]. As illustrated in Figure 1A, the moisture content of the stacked fermented grains in workshop H ranged from 45% to 48%. No significant changes were observed during the first three days, followed by an increase on the fourth day. Conversely, the moisture content in workshop L ranged from 40% to 44%, and exhibited a decreasing trend during the first three days, with a subsequent increase on the fourth day. This difference may be attributed to the high evaporation rate in workshop L due to elevated fermentation temperatures during the early stage, followed by increased microbial activity and starch degradation in later stages, which contributed to higher moisture retention. Starch, the primary energy source for microbial growth during Baijiu brewing, directly correlates with Baijiu yield. Starch content exhibited an increasing trend initially, followed by a decrease in both workshops (Figure 1B). Workshop L showed higher starch levels compared to workshop H, suggesting a lower abundance of amylase-secreting microorganisms in workshop L. This indicates that the starch consumption and utilization rates were less efficient in workshop L, highlighting the limited influence of stacking fermentation on starch content. Glucose content reflects the balance between saccharification and fermentation rates. In both workshops, glucose content increased significantly during the fourth round of stacked fermentation, rising from 0.71% to 1.26% in workshop H and from 0.55% to 1.2% in workshop L, respectively (Figure 1C). This upward trend indicates the vigorous microbial decomposition activity and accelerated conversion of raw materials into glucose, outpacing their utilization rates. Acidity modulates fungal structure and diversity and influences the production of volatile metabolites during Baijiu fermentation [28]. As depicted in Figure 1D, total acid remained relatively stable throughout the fermentation process, reflecting a dynamic equilibrium. This stability likely results from ongoing microbial metabolism, which produces organic acids while contributing to ester formation [29]. Nonetheless, the total acid in workshop H was consistently lower than in workshop L.  
Lactic acid, a predominant acidic substances in sauce-flavor Baijiu, plays a crucial role in fermentation [30]. As shown in Figure 1E, lactic acid concentration declined in both workshops, with higher levels observed in workshop H. The declining trend is attributed to elevated stacked fermentation temperatures, which attenuated the activity of acid producing strains. Additionally, lactic acid was consumed for ester synthesis, contributing to its depletion. Ethanol was identified as a key factor governing microbial succession during fermentation, shaping Baijiu's flavor profile [31]. Ethanol levels increased significantly in both workshops throughout the stacked fermentation process, with the most prominent rise observed in workshop H (Figure 1F). Temperature is a paramount indicator of stacked fermentation efficiency [32]. During the initial stages, the temperature in workshop H was lower than in workshop L. However, as fermentation progressed, the temperature in workshop H increased more rapidly (Figure 1G). It was discovered that during the stacking fermentation process, the temperature exhibited a significant positive correlation with the yeast count. As the stacking time extended, the number of yeasts increased substantially. The metabolic heat generated by  
Figure 1: Dynamics of physicochemical characteristics.