Glycine as a promising phosphate alternative: Molecular simulation reveals superior cryoprotection via ice inhibition and myosin stabilization in frozen crayfish

Frozen storage is widely used to preserve crayfish ( Procambarus clarkii ), but repeated freeze-thaw (F-T) cycles cause quality deterioration. Replacing phosphate-based cryoprotectants with safer natural alternatives has become a major research focus. This study explored the cryoprotective role of glycine (10 g/L) in frozen crayfish and its associated mechanisms. Glycine lowered nucleation temperature, crystallization enthalpy, and freezing point more effectively than sodium tripolyphosphate (STPP), while showing strong ice recrystallization inhibition (IRI) activity. Such regulation of ice crystals is crucial for reducing mechanical damage to muscle fibers during repeated F-T cycles. Besides, it also inhibited lipid and protein oxidation, improved water retention and texture, and better-preserved muscle microstructure compared with STPP. Molecular dynamics simulations indicated that glycine can stabilize myosin via electrostatic interactions and act through the water replacement hypothesis to reduce protein-water contact and ice-induced damage. Moreover, glycine may adsorb onto ice crystal surfaces through electrostatic forces, disrupting crystal structure and modulating ice growth. Overall, glycine has potential as a natural, safe cryoprotectant alternative to phosphate antifreeze agents in frozen crayfish processing. • Glycine lowered nucleation temperature, crystallization enthalpy, and freezing point. • Glycine showed stronger IRI activity, yielding smaller ice crystals. • Glycine preserved muscle microstructure integrity better than STPP. • Glycine inhibited lipid and protein oxidation, improving water retention and texture. • Glycine stabilizes myosin via electrostatic interactions.