Substitution site dependent effects of carboxymethyl chitosan on heat-induced structural evolution and aggregation of gluten

To elucidate the mechanism by which carboxymethyl chitosan (CMCh) regulates heat-induced aggregation of gluten proteins, CMCh derivatives with distinct substitution sites (amino, hydroxyl, and dual amino/hydroxyl substitution) were prepared and systematically compared. The results demonstrated that CMCh effectively modulated gluten aggregation during heating, and the regulatory effect strongly dependent on the substitution site. The degree of substitution (DS) of the derivatives ranged from 0.58 for O-CMCh to 1.26 for N-CMCh, while N,O-CMCh exhibited a DS of 1.18 and the highest water solubility (67.98 g L −1 ). The dual substituted CMCh (N,O-CMCh) exhibited the most pronounced effect, significantly enhancing gluten network elasticity and thermal stability. The onset and peak denaturation temperatures increased to 62.56 °C and 67.88 °C, respectively, with a denaturation enthalpy of 0.70 J/g at 95 °C, markedly higher than that of the control. Furthermore, N,O-CMCh promoted more ordered disulfide cross-linking during heating, increasing the SDS-insoluble protein content (SDS-I) to 80.40% at 95 °C. This was accompanied by a controlled decrease in free sulfhydryl groups and a corresponding increase in disulfide bond formation. In contrast, the single-site substituted derivatives exhibited weaker regulatory effects: N-CMCh increased SDS-I to 75.20%, whereas O-CMCh displayed thermal behavior most similar to the control. The superior functionality of N,O-CMCh is attributed to its unique molecular structure, which enables synergistic electrostatic interactions, hydrogen bonding, and synergistic disulfide cross-linking.This study clarifies the substitution site–dependent structure–function relationship of CMCh and provides a theoretical foundation for the rational design of high-performance CMCh improvers for cereal food.