Mechanism Insights in Freeze-Thaw Process Impacting Cold Denaturation of Gluten Proteins During Frozen Storage.

Cold denaturation of gluten proteins during prolonged frozen storage or repeated freeze-thaw cycles can severely affect the quality of frozen cereal products. While both processes have been studied individually, their combined effects and underlying mechanisms remain unclear. This study systematically evaluated the hydration properties and conformational changes in gluten proteins stored at -73 °C and -23 °C, with or without freeze-thaw cycling. Compared to continuous storage, freeze-thaw cycles reduced water-holding capacity by 9.1-12.2% and increased oil-holding capacity by 5.3-10.3%, indicating aggravated structural damage. Ultra-low temperature storage (-73 °C) suppressed ice crystal growth, preserved hydration, and limited hydrophobic residue exposure. Spectroscopic analyses revealed a temperature-dependent shift from α-helices to β-sheets and β-turns, which was accelerated by freeze-thaw cycles. Enhanced hydrophobic interactions and tryptophan exposure further indicated destabilization. Molecular dynamics simulations showed that increased hydrogen bonding between proteins and water contributed to unfolding at low temperatures, while temperature fluctuations intensified denaturation through repeated hydrogen bond breakage and reformation. These results underscore the critical role of thermal instability in cold denaturation and offer mechanistic insights for improving cryoprotection strategies in frozen food systems.