Abstract: This study focused on the sixth round of stacking fermentation of outer-layer fermented grains (Jiupei) during jiangxiangxing baijiu production. Based on temperature, the process was divided into two stages: medium-temperature fermentation (MT; ≤ 30 ℃) and high-temperature fermentation (HT; > 30 ℃). Using integrated metagenomics and untargeted metabolomics, Jiupei was analyzed for microbial succession and changes in and differential characteristics of metabolite profile at the different temperature stages. The results revealed that during the transition from MT to HT, the fungal community, dominated by Monascus, Aspergillus, Rasamsonia, Pichia, and Zygosaccharomyces, was substantially influenced by temperature, whereas the bacterial community, dominated by Kroppenstedtia, Desmospora, and Lentibacillus, exhibited greater stability compared with the fungal community. Linear discriminant analysis effect size (LEfSe) analysis identified 8 differential bacterial genera (including Saccharopolyspora, Streptomyces, and Brevibacterium) and 16 differential fungal genera (including Kazachstania, Aspergillus, and Monascus) as key taxa driving the distinction between the MT and HT stages. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis based on metagenomic data demonstrated that high-temperature stacking fermentation drove niche differentiation of the microbial communities, with yeasts gradually becoming the dominant group, shifting from “bacterial broad-spectrum metabolism” in the MT stage to “yeast core metabolism” in the HT stage. Differential accumulated metabolites (DAMs) analysis showed that under the combined effects of high temperature and microbial activity, glycerolipids including diacylglycerol (18:1/18:4) and diacylglycerol (16:0/20:5), amino acids and their derivatives including L-pyroglutamate and glycyl-valyl-asparagine, riboflavin, and nicotinamide-N-oxide accumulated significantly during the HT stage. Correlation analysis showed that the bacterial and fungal genera associated with the greatest number of DAMs were Brevibacterium (15) and Penicillium (24), respectively. Differential dominant genera including Brevibacterium, Penicillium, and Aspergillus showed highly significant positive correlations (P < 0.01) with glycyl-valyl-asparagine, glycyl-leucine, diacylglycerol (18:1/18:4), and diacylglycerol (16:0/20:5). Furthermore, the fungal community, represented by molds (Penicillium, Aspergillus) and yeasts (Pichia, Kazachstania), displayed higher connectivity complexity with the metabolic network compared with the bacterial community. In conclusion, this study found that at different temperature stages, the microbial succession, functional characteristics, and metabolite expression in outer-layer Jiupei varied across the different temperature stages of stacking fermentation, providing a reference for further research on the stacking fermentation mechanism of jiangxiangxing baijiu.

Key words: jiangxiangxing baijiu; stacking fermentation; temperature; microbial community; metabolite; correlation