Combining molecular simulations and bitter receptor modelling reveals the bitter-taste mechanism of walnut protease hydrolysates.

Walnut protein hydrolysates (WPHs) have attracted considerable attention owing to their functional properties. However, the enzymatic hydrolysis process frequently leads to the formation of bitter peptides, which impart an undesirable taste to the hydrolysates, consequently limiting their applications. To elucidate the mechanisms underlying bitterness perception, this study used a combined computational and experimental approach to systematically explore bitter peptides derived from walnut proteins and their interaction mechanisms with bitter-taste receptors (type 2 taste receptors, T2Rs). Initially, a computer-based prediction process was adopted to screen 596 potential bitter peptides from chymotrypsin hydrolysates. The screening led to the identification of T2R1, T2R14 and T2R41 as the primary candidate receptors. The combination of molecular docking and molecular dynamics simulations showed that hydrogen bonds and hydrophobic interactions between the peptides and receptors are crucial for stabilising the complex. Notably, three peptides-LHFR, KIVNKDGKPY (KIV10), and LIGEAAKNQ (LIG9)-exhibited stable binding with low energies. Further experiments confirmed that these three peptides markedly increased calcium flux in HEK293T cells overexpressing T2Rs (T2R1, T2R14 and T2R41). Transcriptome analysis revealed that KIV10 significantly activates the bitter-signal transduction cascade in HEK293T cells overexpressing T2R14. This study provides a theoretical model for understanding the generation of hydrolysed bitter taste in food proteins by systematically elucidating the bitter-taste mechanisms of walnut protein-derived bitter peptides.