Process optimization for regulating ethyl acetate synthesis via co-fermentation of Wickerhamomyces anomalus and...

To enhance the synthesis efficiency of ethyl acetate, a key flavor compound in Baijiu (Chinese liquor), a co-fermentation system was developed by integrating the high ethyl acetate-producing yeast Wickerhamomyces anomalus YF1503 with the high ethanol-producing yeast Saccharomyces cerevisiae YF1914. Metabolic interactions and process parameters influencing ethyl acetate biosynthesis were systematically investigated. In synchronously inoculated 1:1 mixed culture, S. cerevisiae YF1914 growth was unaffected, whereas W. anomalus YF1503 biomass decreased significantly. Sequential inoculation strategies markedly inhibited growth of delayed-inoculated strains. Cell-free filtrate assays confirmed that inhibition arose not only from carbon source competition but also from antagonism over other essential nutrients. Optimal conditions were identified as sorghum hydrolysate medium (12 °Bx), initial pH 3.0, 1:1 inoculation ratio (2 × 10⁶ CFU/mL total), and a two-stage fermentation: static incubation of W. anomalus YF1503 (28 °C, 24 h) followed by S. cerevisiae YF1914 inoculation and shaking incubation (28 °C, 180 rpm, 36 h). Under these conditions, ethyl acetate yield reached 4.30 g/L, representing a 3.8-fold increase over single-culture fermentation. This study elucidates the metabolic interaction patterns of functional yeasts in Baijiu fermentation and introduces a targeted co-fermentation strategy for directional enhancement of ethyl acetate production, providing a framework for aroma optimization in traditional liquor manufacturing.