Effect of Prolonged Frozen Storage on the In Vitro Digestion of Minced Pork: Insights from Protein Structural Changes

Long-term frozen storage is widely used for pork preservation, yet its impact on protein digestibility remains inadequately explored. This study investigated the effect of frozen storage duration (0, 3, and 12 months) on the changes in digestive properties and protein structure of minced pork during in vitro digestion. With extended freezing, the hardness, chewiness, and shear force of pork significantly increased, while protein digestibility decreased. A confocal laser scanning microscope showed an increase in the particle size of digesta. After 12 months of frozen storage, the digestibility of the pork samples decreased. The extent of reduction reached 11.31% under pepsin digestion and 11.33% under pepsin-trypsin digestion, compared to the fresh samples. Structural analysis indicated that prolonged freezing led to protein denaturation and aggregation, as confirmed by a decrease in α-helix contents, an increase in the β-sheet and random coil structures, and the formation of high-molecular-weight aggregates. These structural alterations hindered protease accessibility, resulting in reduced digestibility. The detrimental effects on protein structure and digestibility became more pronounced with longer frozen storage.