Functional division of labor within defined yeast consortia drives flavor formation during early solid-state fermentation of sichuan shai vinegar

Sichuan Shai vinegar (SSV) is a traditional fermented product with complex microbial ecology. This study elucidated the functional division of labor within the yeast microbiota during the early stage (days 1-5) of solid-state fermentation in SSV. Investigating of four key yeast strains (Saccharomyces cerevisiae, Pichia kudriavzevii, Kazachstania humilis, and Brettanomyces bruxellensis) via co-culturing, metabolomics, and simulated fermentation revealed distinct roles: Pichia kudriavzevii dominated ester synthesis, Brettanomyces bruxellensis primarily produced characteristic flavor compounds (e.g., acetaldehyde, 4-ethylguaiacol), Kazachstania humilis efficiently produced acids accelerating acidification, and Saccharomyces cerevisiae produced ethanol, which served as a precursor for ester synthesis by other yeasts. The triple-strain combination of Pichia kudriavzevii, Kazachstania humilis, and Brettanomyces bruxellensis exhibited optimal synergy, achieving peak total acid (10.96 g/100 g DW) and acetic acid (3.54 g/100 g DW) content while significantly enhancing characteristic flavor profiles. Untargeted metabolomics indicated that this combination efficiently regulated multiple flavor biosynthesis pathways through pyruvate-mediated metabolic hubs. This systematic clarification of functional roles within the yeast community provides an experimental foundation for designing synthetic microbial starters to modulate flavor profiles and advance the standardization of fermented food production.