Sea cucumber viscera-derived antifreeze peptides: identification, ice recrystallization inhibition activity, and...

Sea cucumber viscera were investigated as a source of natural antifreeze peptides. Activity-guided hydrolysis identified Alcalase as the most suitable protease for producing sea cucumber viscera antifreeze peptides (SCVAFPs) with enhanced antifreeze-related activity and enriched low-molecular-weight components. Subsequent ultrafiltration and Sephadex G-25 purification further identified LMW-II as the key active fraction with the strongest cryoprotective effect. Its superior activity, initially reflected by improved catalase retention during repeated freeze-thaw cycles, was further verified by direct ice recrystallization inhibition (IRI) analysis, which revealed reduced crystal coarsening, smaller crystal size and area, and more uniform ice morphology. HPLC-MS/MS combined with bioinformatic screening further identified 81 previously unreported candidate antifreeze peptides from LMW-II. Molecular docking and molecular dynamics simulations highlighted GRESNWQAGVGDWR, QDGKLIVWDSYTTN, and SLVDGFDNSDFTLH as representative peptides, and further indicated a putative dual antifreeze mechanism involving multipoint interfacial association with the ice surface through hydrogen-bond-dominated interactions, with acidic residues, particularly Asp, contributing prominently to interfacial binding, together with interference with the water-water hydrogen-bond network and weakening of the ordering tendency of surrounding water molecules, thereby delaying ice-front propagation. These findings provide mechanistic support for the development of sea cucumber viscera-derived antifreeze peptides as natural cryoprotectants in frozen food systems and highlight a value-added route for sea cucumber viscera utilization.