Abstract
Caproic acid and its ester ethyl caproate are the characteristic flavor compounds of Chinese strong-flavor Baijiu (CSFB), yet their synthesis and esterification in upper-layer fermenting grains (UFG), and how these processes differ between new and aged pits, remain unclear. Compared with aged-pit UFG, the physicochemical parameters, microbial communities, and metabolite dynamics of new-pit UFG likewise followed a largely similar three-stage pattern: early acidification and starch consumption (2–8 d), mid-stage stabilization (10–20 d), and late secondary acid/ester synthesis (30–60 d). The critical divergence lay in butyric/caproic acid synthesis and the eukaryotic community: new-pit UFG produced lower amounts of butyric and caproic acids, and was dominated by taxa of unclear fermentative relevance, lacking the non-Saccharomyces yeasts (Kazachstania, Candida) enriched in aged-pit UFG, whose abundances correlated with ester accumulation. Specifically, new-pit UFG yielded lower concentrations of three of the four major ethyl esters, whereas ethyl lactate was consistently higher than in aged-pit UFG; yet, notably, ethyl butyrate accumulated despite nearly undetectable butyric acid. Correlation analyses identified candidate caproic acid producers (Lactobacillus, Pediococcus, Weissella) and esterification contributors (Candida, Mucor). These findings highlight the importance of the predicted maturity-driven metabolic–ecological network integrity in UFG, suggesting potential for synthetic-microbiome strategies in new-pit fermentation.