Anti-Inflammatory Protein Hydrolysate Screened from Crayfish Shells Attenuates Colitis by Oxidative Stress Suppression, Inflammation Reduction and Barrier Reconstruction

Inflammatory bowel disease (IBD) is associated with intestinal inflammation and oxidative stress. Protein-rich crayfish shell (CS) by-products may provide a sustainable source of bioactive hydrolysates. This study screened five protease-derived hydrolysates and evaluated the alkaline protease-derived hydrolysate (ACSP) in lipopolysaccharide-stimulated IEC-6 cells and mice with DSS-induced colitis. ACSP scavenged DPPH and ABTS•+ radicals, with IC50 values of 0.888 ± 0.004 and 0.853 ± 0.003 mg/mL. It also enhanced superoxide dismutase and catalase activities, and inhibited NO release and albumin denaturation in vitro. In IEC-6 cells, ACSP increased viability, reduced iNOS, IL-1β, IL-6, and TNF-α expression, and increased IL-10 expression. In mice, ACSP attenuated weight loss, disease activity, colon shortening, histopathological injury, and goblet cell depletion. The high-dose group had disease activity index and histological scores of 3.60 ± 0.55 and 4.80 ± 0.40 compared with 9.40 ± 1.14 and 10.40 ± 0.80 in the model group. ACSP reduced pro-inflammatory cytokines, myeloperoxidase activity, and NO levels while restoring antioxidant enzyme activities and tight junction protein expression. Peptidomic analysis identified 516 peptide sequences, including peptides containing hydrophobic, acidic, and C-terminal basic residues. These findings support further investigation of ACSP for intestinal health applications and the value-added utilization of CS by-products.