Identification of antioxidant oligopeptides targeting Nrf2 from gastrointestinal digestion of casein glycomacropeptide

The gastrointestinal tract is constantly exposed to various harmful factors, and Nrf2 plays a crucial role in mediating cellular defense against these factors, particularly oxidative stress. Glycomacropeptide (GMP), which naturally occurs in milk and is also produced from κ-casein during cheese production, is a bioactive peptide known for its health-promoting effects. This study aimed to identify antioxidant oligopeptides (composed of 2–10 amino acids) targeting Nrf2 from GMP gastrointestinal digestion. LC-MS/MS analysis identified a total of 85 peptides, including 27 oligopeptides, from GMP gastrointestinal digestion in vitro. Three oligopeptides, namely EINTVQVTS, SPPEINTVQ, and KNQDKTEIPT, were selected based on their high interaction scores with Keap1 protein, which is critical for regulating Nrf2 activation. Cell-based studies validated the ability of these GMP-derived oligopeptides to activate Nrf2 and downstream antioxidant gene expression. Furthermore, the animal study demonstrated in vivo antioxidant capacity enhancement by GMP via Nrf2 signaling and intestinal release of homologous peptides with these three oligopeptides. Our findings indicate that GMP could potentially increase intestinal antioxidant capacity and treat oxidative stress-related disorders by releasing oligopeptides targeting Nrf2 from gastrointestinal digestion.