Synergistic co-fermentation of non-Saccharomyces yeasts enhanced fermentation performance and aroma characteristics of citrus wine

Non- Saccharomyces yeast are gaining attention in winemaking for enhancing aromatic complexity, but their industrial use is limited by inefficient metabolism. This study used metabolomics and transcriptomics to analyze fermentation kinetics and aroma profiles in citrus wines fermented with non- Saccharomyces yeast included Candida ethanolica, Hanseniaspora guilliermondii , and Hanseniaspora thailandica . Results showed that using two non- Saccharomyces yeast strains in mixed cultures improved flavor, ethanol, organic acids, and volatile aromas more than single-strain or three-strain fermentations. Metabolomic analysis showed that co-fermenting with H. guilliermondii and H. thailandica boosted fermentation, increasing ethanol production by 33.3% and volatile aromatic compounds by 28.2%, along with a significant rise in total organic acids (p<0.05), the vitamin C content significantly increased (149.36 mg/L). Transcriptomic analysis indicated that the mixed fermentation of H. guilliermondii and H. thailandica improved fermentation performance by upregulating fatty acid and glycolytic metabolic pathways, while also modifying glycine and porphyrin metabolism to produce a greater quantity of flavor compounds. The present study represents the inaugural evaluation of the fermentation capacity and aroma characteristics of citrus fruit wine utilizing exclusively non- Saccharomyces yeast. The findings provide a foundational basis for optimizing mixed culture strategies in fruit wine fermentation and for broadening the industrial applications of non-conventional yeasts. • Citrus wine's flavor quality from sole non- Saccharomyces fermentation was assessed. • Co-fermenting H. guilliermondii and H. thailandica improved fermentation efficiency. • Upregulation of fatty acid and glycolytic pathways to boost fermentation efficiency. • Co-fermenting two non- Saccharomyces boosted aromatic compounds and polyphenols. • Glycine and porphyrin metabolism enhanced aromatic compounds and polyphenols.