Unraveling the Cryoprotective Molecular Mechanisms of Antifreeze Peptides: New Insights from Simultaneous Interactions with Ice Crystals and Lactalbumin Proteins

Traditional cryoprotectants such as sucrose and phosphates are limited by safety and flavor concerns, creating a demand for food-grade alternatives. Here, <i>Bos taurus</i> domesticus gelatin antifreeze peptides (BtAFP) were investigated for their dual cryoprotective mechanisms. Rheological analysis showed that BtAFP enhanced gel elasticity and delayed freezing, exhibiting superior cryoprotection compared with bovine serum albumin. LC-MS/MS identified 452 peptides, from which five nontoxic collagen-derived candidates with amphiphilic α-helix/β-sheet motifs were selected. Ice recrystallization assays showed that the majority of ice crystals in purified peptide solutions were concentrated below 1000 μm². Differential scanning calorimetry indicated that GKEAFP, GLPAFP, and GLRAFP most effectively inhibited ice growth. Molecular docking demonstrated stable peptide-ice and peptide-α-lactalbumin interactions, with GLPAFP notably stabilizing protein structures through extensive hydrogen bonding and surface interactions. These findings suggest that gelatin-derived antifreeze peptides suppress ice recrystallization and stabilize proteins via amphiphilic ice-binding motifs, providing promising natural cryoprotectants for frozen food applications.