A New Insight Into the Structure‐Activity Relationship of Antifreeze Activity in Bovine Hide Collagen Peptides: Based on Molecular Weight Differences

This study investigates the structure-activity relationship between molecular weights and antifreeze activity in collagen peptides derived from bovine hide collagen. The peptides were prepared using ultrasound-assisted enzymatic hydrolysis technology, followed by ultrafiltration fractionation (unsized (US), <1 kDa, 1-3 kDa, and >3 kDa). The antifreeze activity and structural characteristics of collagen peptides with different molecular weights were determined. The results revealed that the <1 kDa fraction demonstrated optimal hypothermic protective activity on catalase activity and exhibited the smallest contact angle, while the 1-3 kDa fraction showed the highest thermal hysteresis activity. Comparative analysis demonstrated that low molecular weight peptides (<3 kDa) contained significantly higher proportions of both hydrophilic and hydrophobic amino acids (P < 0.05), along with enhanced solubility and surface hydrophobicity compared to their high molecular weight counterparts (>3 kDa). Notably, molecular weight reduction correlated with progressive collagen crystal structure fragmentation, accompanied by increased β-turn and random coil content. These structural modifications facilitated the exposure of internal chromophores, elevated fluorescence intensity, and generated uniformly dispersed peptide particles. Through isolation and purification of low molecular weight fractions, six novel antifreeze peptides were identified. Molecular docking simulations revealed their binding mechanism to ice crystals via hydrogen bonds, with peptide AAGPPGTP exhibiting superior antifreeze potential through the formation of six hydrogen bonds with ice crystal molecules. These findings provide novel insights for the strategic development of cryoprotective agents using collagen-derived peptides. PRACTICAL APPLICATION: This study systematically investigated the effect of the molecular weight of collagen peptides on antifreeze performance and elucidated the structural basis underlying their functional variations. Finally, the molecular weight range of peptide segments that is more conducive to exerting the antifreeze effect was determined. Furthermore, the research provides a concise production process flow. Remarkably, six antifreeze peptide sequences with safety, reliability, and non-cytotoxicity were characterized after screening, and their physical and chemical properties were determined. These breakthroughs significantly facilitate the industrial-scale production of antifreeze peptides.