Function-driven high-throughput screening and isolation of ester-producing strains for glutinous rice wine fermentation

Ester-producing strains are of great importance for enhancing the quality and flavor profiles of alcoholic beverages. However, traditional methods for screening ester-producing strains are labor-intensive and time-consuming, significantly impeding the development of alcoholic beverages industry. In this study, we selected five brands of Jiuqu to cultivate within different media, results showed that XB ( Jiuqu ) incubated with malt extract medium possessed the highest ester-producing capability, with the identification of 27 esters at the concentration of 31.44 ± 2.17 mM. Subsequently, fluorescence-activated cell sorting (FACS) was used to screen and isolate the living fungal cells which accounted for 42.80 % of total cells, followed by cultivation utilizing a culturomics approach. High-throughput screening (HTS) assays using 4-Methylumbelliferyl acetate (4-MA) were utilized to evaluate the ester-production potentials of 960 selected strains. The top10 highest ester-producing strains were sequenced, and all were identified as Saccharomyces cerevisiae , Cyberlindnera fabianii , and Wickerhamomyces anomalus. Eventually, three microbial strains were co-incubated with rice wine starter to improve the nutritional and flavor properties of glutinous rice wine. Compared to the control group, Cyberlindnera fabianii could increase reducing sugar content, up to 0.33 ± 0.01 g/mL, and significantly decrease the concentration of bitter amino acids by 55.83 %, resulting in a final concentration of 32.29 ± 1.51 mg/L. Furthermore, the glutinous rice wine with Cyberlindnera fabianii showed 38 distinct ester compounds at the content of 16.22 ± 0.51 mM, which was superior to the control group (30 ester compounds at the concentration of 11.89 ± 1.39 mM). The diversity and concentrations of flavor components, such as alcohols, aldehydes, and ketones, were also enhanced. Our findings would contribute to advancing the rapid screening of ester-producing strains, as well as providing a theoretical basis for improving the quality of glutinous rice wine.