Mixed fermentation with Saccharomyces cerevisiae and Pichia kudriavzevii has been shown to enhance wine aroma, yet the underlying mechanisms remain unclear. Monoculture of S. cerevisiae, monoculture of P. kudriavzevii, and mixed culture of S. cerevisiae and P. kudriavzevii were conducted, and the study analyzed and compared the biomass, flavor profile, and transcriptome responses of the three groups. Both yeast species exhibited growth inhibition in mixed culture, especially P. kudriavzevii. Significant differences were observed in three organic acids and the foremost 20 volatile compounds. Mixed fermentation enhanced esters (e.g., ethyl butyrate, isoamyl acetate) and volatile acids (e.g., hexanoic acid), but decreased isobutanol, phenylethyl alcohol, and quinic acid. Transcriptomic analysis revealed 294 and 332 differentially expressed genes (DEGs) in S. cerevisiae and P. kudriavzevii, respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation results indicated that DEGs in mixed fermentation were concentrated in carbohydrate metabolism and amino acid metabolism. Our integrated analysis suggested that genes such as TDH2, TDH3, and ENO2 were pivotal for ester biosynthesis. Moreover, ADH1, ADH2, HPA3, ALD6, and ARO8 were associated with quinic acid synthesis. Furthermore, ILV2, ILV5, ALD6, and others were central to the production of isobutanol and phenylethyl alcohol.

Double-round bottom fermentation (DRBF) represents an important technological innovation in strong-flavor Baijiu production, yet the microbial succession and metabolic mechanisms underlying this process remain insufficiently understood. In this study, physicochemical analyses combined with multi-omics approaches were employed to elucidate the dynamic variations in physicochemical parameters, volatile compounds, and microbial community structure and function during DRBF, as well as to reconstruct key metabolic pathways involved in fermentation. A total of 153 volatile compounds were identified, with esters, alcohols, and acids as the major components showing distinct accumulation patterns across fermentation stages. High-throughput sequencing detected 505 bacterial and 175 fungal genera, dominated by Lactobacillus, Aspergillus, and Saccharomyces. Functional annotation revealed that metabolic pathways predominated, shifting from energy- and growth-related processes in the early stage to amino acid, fatty acid, and secondary metabolite biosynthesis in the later stage. Reconstruction of metabolic pathways identified 57 key enzymes linking starch degradation, pyruvate metabolism, the tricarboxylic acid (TCA) cycle, and ester biosynthesis, indicating cooperative metabolism among bacteria, yeasts, and molds. These findings elucidate the synergistic metabolic mechanisms of flavor formation during DRBF and provide a scientific basis for optimizing fermentation control and improving Baijiu quality.