Comparison of Different Preparation Methods for Selenium-Enriched Spirulina 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 spirulina 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 H 2 O 2 -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 Akkermansia and Blautia . 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.