Rice, supplemented with Dendrobium officinale, was subjected to cofermentation using Saccharomyces cerevisiae FBKL2.8022 (Sc) and Wickerhamomyces anomalus FBKL2.8023 (Wa). The alcohol content was determined with a biosensor, total sugars with the phenol-sulfuric acid method, reducing sugars with the DNS method, total acids and total phenols with the colorimetric method, and metabolites were analyzed using LC-MS/MS combined with multivariate statistics, while metabolic pathways were constructed using metaboAnalyst 5.0. It was found that the quality of rice wine was higher with the addition of D. officinale. A total of 127 major active substances, mainly phenols, flavonoids, terpenoids, alkaloids, and phenylpropanoids, were identified. Among them, 26 substances might have been mainly metabolized by the mixed-yeasts fermentation itself, and 10 substances might have originated either from D. officinale itself or from microbial metabolism on the newly supplemented substrate. In addition, significant differences in metabolite could be attributed to amino acid metabolic pathways, such as phenylalanine metabolism and alanine, aspartate, and glutamate metabolism. The characteristic microbial metabolism of D. officinale produces metabolites, which are α-dihydroartemisinin, alantolactone, neohesperidin dihydrochalcone, and occidentoside. This study showed that mixed-yeasts cofermentation and fermentation with D. officinale both could increase the content of active substances in rice wine and significantly improve the quality of rice wine. The results of this study provide a reference for the mixed fermentation of brewer's yeast and non-yeast yeasts in rice wine brewing.
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