Microbial interactions and sequential inoculation of Metschnikowia koreensis and Saccharomyces cerevisiae shape cider flavor development

Mixed-culture fermentation can enhance cider aroma, but direct co-culture often limits the persistence of non- Saccharomyces yeasts. Among these species, Metschnikowia koreensis is noteworthy for its strong protease and lipase activities that release aroma precursors, yet its interaction with Saccharomyces cerevisiae has not been well characterized. This study evaluated sequential inoculation of M. koreensis and S. cerevisiae with 0 h, 24 h, and 48 h delays. A 48-h delay (SM 48 ) extended M. koreensis viability to 9 days, increased ethanol production, and produced sensory attributes characterized by lower sourness and astringency and higher umami and richness. The SM 48 group produced the most abundant and diverse volatile compounds, with key fruity and floral esters elevated. OPLS-DA identified these esters as major discriminant contributors, whereas compounds commonly associated with off-flavors such as acetic acid and hexanal were reduced. Metabolic pathway analysis indicated enhanced Ehrlich and esterification pathways with reduced acetate production. Multi-method validation supported that SM 48 cider exhibits more complex volatile profiles, stronger fruity and floral notes, and improved color. These results demonstrate that controlled sequential inoculation is an effective approach for improving cider aroma complexity and overall product quality.