Soybean Protein Amyloid Fibrils as Natural Cryoprotectants: Structural Characterization and Water Interaction Mechanisms

Self-assembled proteins can significantly inhibit ice recrystallization, offering potential for cryoprotection. Here, soybean protein amyloid fibrils (SAFs) were fabricated via combined germination and acid-heat-induced fibrillation. Germination enhanced the fibrillation efficiency of soybean protein isolate (SPI). SAFs with the strongest ice recrystallization inhibition (IRI) activity were prepared from SPI of two-day germinated soybeans after 20 h of acidic-heat treatment (SAF-20). SAF-20 exhibited concentration-dependent IRI activity, with stronger inhibition of ice crystal growth at higher concentrations. It showed high ice-affinity adsorption and ice nucleation activity without altering ice crystal morphology. Structural analyses revealed that self-assembly promoted protein aggregation and increased surface hydrophobicity and β-sheet content. These changes strengthened hydrogen bonding at the ice-water interface, forming ordered interfacial water layers that disrupted long-range water ordering and inhibited ice crystal growth. Furthermore, SAF-20 significantly improved post-thaw recovery of cryopreserved Caco-2 cells, demonstrating its cryoprotective efficacy.