Self-decreasing pH hydrolysis produces absorbed soybean peptides with enhanced hepatoprotection: The indispensable role of a tunable aromatic-pro-Arg/Lys motif.

This study aimed to investigate the anti-alcoholic liver injury efficacy of soybean peptides prepared by different enzymatic processes after simulated gastrointestinal digestion and intestinal absorption. Three distinct hydrolysis strategies were evaluated: self-decreasing pH hydrolysis, simultaneous multi-enzyme hydrolysis, and sequential multi-step hydrolysis using alcalase, papain, and flavourzyme. A Caco-2/HepG2 co-culture model revealed that although the sequential hydrolysis P-A-F group exhibited the greatest apparent permeability coefficient at 44.47 × 10-7 cm/s, the self-decreasing pH hydrolysis A↘P↘F absorbed peptides demonstrated the most potent hepatoprotective activity in ethanol-injured HepG2 cells. These peptides significantly reduced AST and ALT levels to 5.01 U/L and 0.91 U/L, respectively. Mass spectrometry and functional validation identified PFPRPQP as a key peptide mitigating hepatocellular damage, while PIPFPR showed enhanced free radical scavenging capacity. Molecular docking and amino acid substitution experiments demonstrated that hepatoprotective activity depended on the conformational integrity of the aromatic-Pro-Arg/Lys (Φ-P-R/K) core motif and adjacent C-terminal sequences. Specifically, PFPRPQP activated the Nrf2 pathway through Keap1 binding, whereas PIPFPR shifted its target to CYP2E1, potentially exerting enzyme inhibition. This study established an integrated research paradigm from process screening to mechanistic elucidation, providing a strategic foundation for developing targeted anti-alcoholic liver injury peptides with high oral bioavailability.