Elucidating the astringency perception mechanism of protocatechuic acid, a key astringent component in black rice, based on polyphenol-salivary protein interaction

Black rice is rich in bran layer polyphenols, which enhance bioactivity but may reduce its consumer acceptability due to astringency. Polyphenol-salivary protein aggregation is the molecular basis of astringency. In this study, a saliva-induced precipitation approach with liquid chromatography-tandem mass spectrometry was used to identify protocatechuic acid (PA) as the key astringent component in black rice, accounting for 68% of total precipitated polyphenols. With α-amylase as a representative, the PA-salivary protein interaction was analyzed using fluorescence spectroscopy, isothermal titration calorimetry (ITC), and molecular docking. Fluorescence spectroscopy showed a quenching constant (K _q) of 7.8451 × 10¹¹ L⋅mol^-1⋅s^-1, and ITC revealed a binding constant (K) of 2.65 × 10³ L/mol with 1.32 binding sites, indicating the interaction was characterized by strong, spontaneous, noncovalent binding, static quenching, and multiple binding sites. Atomic force microscopy, contact angle, and small amplitude oscillatory shear rheology revealed that PA increased salivary film surface roughness (contact angle from 36.2° to 43.3°) and weakened viscoelasticity, thereby facilitating film rupture and loss of lubrication underlying astringency perception through tactile stimulus pathway. Molecular dynamics simulations further revealed stable PA binding to human astringency receptor human taste 2 receptor member 16 (hTAS2R16), indicating an additional astringency perception gustatory pathway.