Gastrointestinal digestion modulates antioxidant activity of edible insect peptides: Molecular weight-dependent effects and in vivo validation in Caenorhabditis elegans

Edible insects are sustainable protein sources for functional foods; however, comparative evaluation of digestive stability and antioxidant mechanisms across species remains critically needed. This study provides: (1) systematic molecular weight fractionation with simulated gastrointestinal digestion (SGD), (2) species-specific analysis across three insects, (3) in vivo validation using Caenorhabditis elegans, and (4) LC-MS/MS characterization. Objectives were to: (1) evaluate SGD effects on antioxidant activities of fractionated peptides from adult house cricket (AHCK), black soldier fly larvae (BSFL), and silkworm pupae (SWP), (2) identify bioactive sequences, and (3) validate efficacy under oxidative stress. Proteins underwent enzymatic hydrolysis (2SD protease, 3% w/w, 50 °C, 2 h) and tangential flow filtration (>10, 5-10, 3-5, <3 kDa). Fractions underwent SGD (gastric: pepsin, pH 2.0, 2 h; intestinal: pancreatin/bile salts, pH 7.0, 2 h) and evaluation via DPPH, ABTS, and metal chelating assays. The <3 kDa fractions exhibited highest activities: DPPH IC₅₀ 1.45-2.18 mg/mL, ABTS IC₅₀ 0.82-1.15 mg/mL, metal chelation 85.3-97.6% (P < 0.05). SGD induced species-specific responses: AHCK showed 97.58% metal chelation; SWP maintained 56.6% DPPH retention. LC-MS/MS identified 14 peptides (412-689 Da) including conserved YPLDL and PSLPA sequences. SGD-treated <3 kDa peptides (1 mg/mL) extended C. elegans median survival to 750-780 min (acute stress) and lifespan by 2-3 days (chronic stress) versus controls. These findings support applications in protein beverages, functional bars, and dietary supplements as natural antioxidants. Key challenges include species-specific digestive responses and processing stability, requiring optimization of molecular weight fractionation parameters.