An integrated Peptidomics approach to analysing the formation mechanism of bitter taste in walnut protein hydrolysates.

Enzymatic hydrolysis enhances the functionality of walnut protein; however, the presence of bitter peptides limits its application. This study compared the hydrolytic efficiency, peptide patterns and bitterness of eight food-grade proteases, and revealed the mechanism behind the bitter taste of walnuts. Neutral protease performed best (60% degree of hydrolysis, 58% yield), producing mainly <3 kDa peptides. Trypsin yielded the most <0.5 kDa fragments, while acidic protease/trypsin released the most free amino acids. Papain, bromelain, and pepsin showed low activity, producing >3 kDa products. Electronic tongue analysis combined with orthogonal projections to latent structures discriminant analysis (OPLS-DA) identified chymotrypsin hydrolysate as the most bitter. Bitterness showed a positive correlation with M2(0.5-1 KDa)/M3(1-3KDa) peptides. Terminal sequencing revealed that hydrophobic residues predominated at the N-termini, while the C-termini contained both hydrophobic and basic residues. The motifs Phe-Phe and Arg-Pro were significantly linked to bitterness, with C-terminal hydrophobicity and Phe-Phe motifs contributing most significantly. Chymotrypsin preferentially cleaved the hydrophobic region of A0A2I4FU77, which was identified as a potential hotspot for bitter peptides. Peptide profiling revealed an 'enzyme-protein-site' coupling mechanism, providing molecular-level insights into the formation of bitterness in walnut peptide hydrolysates.