Milk protein-derived immunomodulatory peptides: Enzymatic preparation, sequence-function mapping, and antioxidant...

This study generated multifunctional peptides by hydrolyzing three milk proteins (α-lactalbumin, β-lactoglobulin, and casein) using either alkaline protease or a pepsin-trypsin mixture. Highly active hydrolysate fractions were characterized by LC-MS/MS, and molecular docking was used to provide mechanistic insights. A strong correlation was observed between immunomodulatory activity and antioxidant capacity in vitro. Among all fractions, the F2 fraction from the α-lactalbumin alkaline protease hydrolysate exhibited the highest in vitro antioxidant capacity (2.0547 ± 0.0321 μmol TE/mg). Three peptides identified from F2 (GRFF, WDSF, and SVPGPMGPSGPR) significantly enhanced RAW264.7 phagocytosis and promoted the secretion of pro-inflammatory mediators, and they also showed notable in vitro antioxidant capacity (1.4320 ± 0.0140 to 1.8450 ± 0.0070 μmol TE/mg). Docking analyses suggested that these peptides may interact with TLR2/TLR4 and the antioxidant regulator Keap1, supporting a potential link between immune activation and redox modulation. Overall, this work provides a basis for the discovery and utilization of high-value, candidate multifunctional bioactive peptides derived from milk proteins.