Integration of correlation analysis and untargeted metabolomics reveals microbial interactions driving flavor formation in bacteria-yeast Co-fermented Lithocarpus litseifolius [hance] chun (L. litseifolius) kombucha

L. litseifolius , a sweet-leafed primarily distributed in southern China, exhibits potential as a novel substrate for kombucha fermentation. This study developed a two-stage co-fermentation to enhance the flavor of L. litseifolius kombucha, by using Lactobacillus plantarum , Acetobacter pasteurianus , and Kluyveromyces marxianus as co-starters. Co-fermentation significantly altered free amino acids (FAAs), volatile flavor compounds (VFCs), and organic acids (OAs), attenuating the pungent acetic acid odor and imparting a distinctive floral, fruity, and fatty aroma. High-throughput sequencing analysis revealed that Acetobacter pasteurianus maintained dominance throughout the fermentation. Correlation analysis and redundancy analysis (RDA) definitively confirmed the synergistic effects of bacteria and fungi on shaping the final flavor profile, particularly contributing to the formation of floral-fruity aroma, umami, and sourness. Furthermore, untargeted metabolomics identified 40 differential metabolites in triple-strain co-fermentation, including cis-4-coumaric acid, 2-isopropylmalic acid, caffeic acid et al. KEGG pathway enrichment analysis mapped probable biosynthetic pathways for these compounds, particularly those involving FAAs (e.g., alanine, aspartate) and OAs (e.g., coumarate, succinate). This study provides insights into flavor formation mechanisms in L. litseifolius co-fermentation, offering a scientific basis for developing novel plant-based fermented beverages.