Reverse-tracing unravels microbial succession and flavor stratification driven by vertical physicochemical gradients in Zaopei fermentation.

Chinese Baijiu brewing relies on the vertical layering of Zaopei, yet its effect on microbial-flavor interactions remain unclear. This study employs result-oriented backward analysis of distinct flavor patterns to decipher how spatial differences govern metabolic networks and flavor distribution. GC-IMS/GC-MS revealed spatial divergence: the surface layer concentrated 62.89 % of total volatile flavors, significantly higher than the bottom layer (18.88 %) and the middle/upper layers (8.67-9.55 %). Fungal communities played a key role in layer differentiation (p = 0.002) through temperature and chemical changes. Deterministic selection dominated in hot and acidic (3.39 g/mol) surface layer (40.74 % |βNTI| > 2), while random drift prevailed in cool and high-ethanol (3.49 % v/v) bottom layer (NST = 78.14 %), with limited dispersal linking the layers. Five key flavor-related microbes were identified, highlighting the role of microbial metabolism in flavor development. Overall, the vertical structure of Zaopei guides the evolution of Baijiu's complex, multi-layered flavor profile.