Molecular Basis of Bitterness Modulation during Black Tea Fermentation

Abstract Black tea fermentation involves enzymatic oxidation of catechins into characteristic phenolics such as theaflavins and thearubigins, which profoundly influence bitterness perception. However, the molecular mechanisms by which progressive phenolic oxidation modulates bitterness remain incompletely understood. In this study, controlled oxidation model systems were investigated using an integrated strategy combining cell-based calcium imaging, molecular simulations, and AFM-based single-molecule force measurements. Cell-based assays showed that tyrosinase-mediated oxidation enhanced TAS2R14 activation, whereas endogenous enzyme and laccase mediated oxidation markedly reduced intracellular calcium responses. Molecular simulations revealed that theaflavins exhibit higher binding stability, lower binding free energy, and stronger activation-related conformational effects on TAS2R14 than catechins. Consistently, AFM measurements confirmed stronger adhesion forces for theaflavin–TAS2R14 interactions. In contrast, further oxidative dimerization imposed steric constraints that weakened receptor binding and activation. Collectively, this study clarifies how progressive phenolic oxidation during tea fermentation differentially modulates bitterness at the molecular and receptor levels.