Establishment of a workflow for high-throughput identification of anti-inflammatory peptides from sea cucumbers.

Developing an effective workflow for screening anti-inflammatory peptides is crucial for discovering novel food-derived anti-inflammatory peptides and optimizing the screening and identification process of bioactive peptides. Virtual screening identified three major yolk proteins as target precursor proteins for anti-inflammatory peptides in sea cucumbers. A portfolio of 170 peptides was identified from hydrolysates after 9 h of alcalase treatment by combining antioxidant activity determination and peptidomics analysis. Among these, 12 high-confidence anti-inflammatory peptides were identified through virtual screening. Three of these peptides were shown to effectively inhibit the production of NO and the release of pro-inflammatory cytokines in RAW264.7 cells. Molecular docking demonstrated that these three peptides exerted their anti-inflammatory effects primarily by binding to the active sites of cyclooxygenase-2 and inducible nitric oxide synthase through hydrophobic interactions. This study provided a reference workflow for screening anti-inflammatory peptides, facilitating the discovery of novel anti-inflammatory peptides and the high-value utilization of sea cucumber cooking liquid.