The Effects of Iterative Freeze–Thaw Cycles on the Structure, Functionality, and Digestibility of Grifola frondosa Protein

To improve the processing performance and application potential of <i>Grifola frondosa</i> protein (GFP), this study employed multiple freeze-thaw (F-T) cycles to modify GFP and systematically evaluated the changes in its structure, functional properties, and digestive behavior. The results indicated that F-T treatment induced significant oxidation and structural unfolding in GFP, as evidenced by an increase in carbonyl content from 0.75 ± 0.05 nmol/mg to 1.77 ± 0.04 nmol/mg, a decrease in α-helix content from 40.23% to 36.78%, disruption of the microstructure, and degradation of some low-molecular-weight proteins. In terms of functional properties, F-T treatment significantly enhanced the emulsifying performance of GFP, with the best effect observed after 3 F-T cycles. Emulsifying ability increased from 21.83 ± 1.14 m²/g to 26.11 ± 1.61 m²/g, and emulsifying stability improved from 18.36 ± 1.78% to 25.37 ± 0.84%. This was accompanied by favorable changes in the emulsion's interfacial properties, including a reduction in average particle size (480.5 ± 30.5 nm) and an increase in absolute ζ-potential (-55.5 ± 0.2 mV). These changes were closely related to the dynamic evolution of free sulfhydryl groups and surface hydrophobicity. In vitro digestion experiments revealed that the protein digestibility and soluble peptide content of GFP peaked after two F-T treatments, reaching 64.88 ± 0.86% and 1.99 ± 0.09118 mg/mL, respectively, and then gradually declined; its antioxidant activity also showed an initial increase followed by a decrease with increasing F-T cycles. In summary, an appropriate number of F-T treatments can effectively enhance the emulsifying properties and digestive characteristics of GFP; this research provides a theoretical basis for the physical modification of GFP and broadens its potential applications in food emulsion products.