Integrated sensory evaluation and feature-based molecular networking elucidate flavor improvement and associated metabolites in dark tea fermented by Eurotium cristatum and Bacillus coagulans

Secondary microbial fermentation plays a crucial role in improving dark tea flavor, yet how microorganisms regulate flavor formation and transformation remains unclear. This study proposes integrating human/electronic tongue sensory evaluation with feature-based molecular networking (FBMN) to address this issue. Sensory evaluation showed that Eurotium cristatum fermentation improved the flavor of Liubao tea by increasing sweetness and reducing bitterness, whereas Bacillus coagulans enhanced its aged flavor. FBMN identified 358 compounds, including ten novel ethyl-pyrrolidinone-substituted flavan-3-ol B-ring fission analogs (eps-FBRFAs). E. cristatum fermentation significantly increased FBRFAs, eps-FBRFAs, and non-ester-type catechins, while decreasing ester-type catechins. Correlation analysis revealed that these upregulated metabolites were positively associated with sweetness, with TAS1R2/TAS1R3 as a potential target of FBRFAs and eps-FBRFAs. B. coagulans fermentation increased linear peptides, which were positively associated with the aged flavor. These findings improve understanding of microbial contributions to dark tea flavor and key metabolites, providing insights for product quality improvement.