Integrative metagenomic, metabolomic, and transcriptomic analyses unravel flavor development driven by a defined starter cocktail in cocoa fermentation.

This study investigated the impact of a defined starter cocktail (Hanseniaspora uvarum XY23.1, Saccharomyces cerevisiae XY23.2, Lactiplantibacillus plantarum XY23.1, and Gluconobacter oxydans XY23.2) on Trinitario cocoa fermentation in Hainan, China. A multi-omic approach integrating shotgun metagenomics, metabolomics (LC-MS and GC-MS), and transcriptomics was employed to comprehensively assess microbial succession, metabolite dynamics, and functional gene expression. Starter inoculation accelerated cocoa fermentation and stabilized microbial succession, and the four introduced strains remained dominance during the process. Metabolomic analysis revealed that the inoculated group had a remarkable increase in the content of the aromatic volatiles phenylethyl alcohol, 3-methylbutyl acetate, and delta-decalactone, which contributes to fruity and floral aroma characteristics. Shotgun metagenomics revealed enrichment of genes involved in carbohydrate utilization, amino acid catabolism, and acetate ester biosynthesis. Transcriptomics also indicated that S. cerevisiae upregulated Ehrlich pathway and glyoxylate cycle under co-culture conditions, demonstrating its central role in aroma formation and redox balance, while H. uvarum upregulated sugar transporters and stress-response genes, demonstrating a putative regulatory role in nutrient competition and interspecies interaction. Overall, our findings elucidate that defined starter cultures can shape cocoa fermentation toward improved consistency and aroma outcomes, providing a foundation for designing function-driven microbial consortia for controlled fermentation applications.