Feature-Based Molecular Networking Analysis of the Metabolites Produced by in vitro Solid-State Fermentation Reveals Pathways for the Bioconversion of Epigallocatechin Gallate

Flavan-3-ol B-ring fission derivatives (FBRFDs) are secondary metabolites that contribute to the unique properties of fermented dark teas. However, the FBRFD precursors and biochemistry are unclear. Fungal strains cultured from Fuzhuan brick tea (FBT) were incubated in an in vitro solid-state fermentation system containing β-cyclodextrin-embedded epigallocatechin gallate (EGCG), a potential precursor of FBRFDs. The produced metabolites were analyzed through a combination of targeted chromatographic isolation, non-targeted spectroscopic identification, and Feature-based Molecular Networking (FBMN) in the Global Natural Products Social Molecular Networking (GNPS) platform. Dihydromyricetin was identified for the first time, indicating that fungi possess a flavan-3-ol C-ring oxidation pathway. EGCG was verified as the precursor of dihydromyricetin and FBRFDs such as teadenol A and fuzhuanin A. The conversion was driven by the fungi rather than a hygrothermal effect. Isolates from Pezizomycotina showed much stronger abilities to convert EGCG to the B-/C-ring oxidation products than those from Saccharomycotina or Basidiomycota.