Hanseniaspora species can create a privileged niche for Oenococcus oeni during triple-strain simultaneous alcoholic-malolactic fermentation based on metabolomics analysis.

Previous studies have demonstrated that non-Saccharomyces yeasts can enhance malolactic fermentation (MLF) efficiency during co-fermentation. However, the underlying mechanisms remain unclear. Here, we demonstrate how specific strains create favorable metabolic niches through tripartite fermentation assays involving 16 strains from 8 species co-cultured with Saccharomyces cerevisiae F5 and Oenococcus oeni SD-2a, Hanseniaspora spp. emerged as optimal partners, reducing MLF duration by 50 % (≤48 h vs. 96 h in controls) while increasing O. oeni biomass by > 10-fold. Metabolomic analysis revealed that O. oeni utilizes malate as a carbon source within an ecological niche shaped by yeast-driven sugar competition, while upregulated nicotinamide metabolism enhanced NAD+ regeneration, boosting malolactic enzyme activity. Furthermore, strain-specific peptide secretion provided targeted bacterial support: H. osmophila NX39 produced quorum-sensing peptides and fumarate to activate bacterial pathways, whereas H. uvarum HN2 synthesized nutritional peptides and arabitol to alleviate auxotrophy. Exogenous amino acids (Glu/Trp/Cys) further enhanced MLF efficiency, with glutamate specifically accelerating early O. oeni growth while maintaining S. cerevisiae viability (stable at 108 CFU/mL). These findings transform co-inoculation from a high-risk practice into a robust enological strategy by establishing triple-strain simultaneous alcoholic-malolactic fermentation systems, providing a foundation for next-generation precision enology.