Comparison of Different Preparation Methods for Selenium-Enriched <i>Spirulina</i> Peptides: Structure and in Vitro and in Vivo Bioactivity Assessments

This study systematically compared the impact of direct (D_SeSPs) and indirect (I_SeSPs) enzymatic hydrolysis on the preparation and bioactivity of selenium-enriched <i>spirulina</i> peptides. Comprehensive evaluation revealed that direct hydrolysis yielded peptides with a higher degree of hydrolysis, selenium content (238.42 μg/g), distinct structural features, including enriched hydrophobic residues and retained selenocysteine motifs, and superior in vitro free radical scavenging capacity. In a H₂O₂-induced Caco-2 cell model, D_SeSPs demonstrated superior antioxidant activity by activating the Keap1-Nrf2 pathway and enhancing intracellular antioxidant defenses. Furthermore, in a murine model of ulcerative colitis, D_SeSPs alleviated colonic oxidative damage, preserved intestinal barrier integrity, and increased the abundance of beneficial bacteria, including <i>Akkermansia</i> and <i>Blautia</i>. These findings highlight the critical influence of preparation methods on peptide efficacy and identify direct enzymatic hydrolysis as an effective strategy for producing high-performance spirulina-derived selenium supplements for functional food applications.