Effect of pyrogallic acid (1,2,3-benzenetriol) polyphenol-protein covalent conjugation reaction degree on structure and antioxidant properties of pumpkin (Cucurbita sp.) seed protein isolate

Pumpkin (Cucurbita sp.) seed protein isolate (PSPI) and pyrogallic acid (1,2,3-benzenetriol) covalent conjugation with various ratios was prepared at pH 9. The degree of covalent conjugates in the reaction system was determined by polyphenol bound equivalents and SDS-PAGE. When increasing the polyphenols concentration, the number of bound equivalents was increased from 14.30 +/- 3.35 to 131.56 +/- 4.47 nmol/mg, while SDS-PAGE showed all the bands of the protein-polyphenol slightly shifted to the higher molecular weight direction, indicating that the reaction could lead to cross-linking of PSPI and PA to increase its molecular weight. The results of FTIR spectra indicated that the amino group participated in the covalent conjugates, and the structure became closer by the formation of hydrogen bonds during the reaction. According to the established mechanism model, it could be speculated that the covalent interaction between PSPI and pyrogallic acid (PA) relied on free amino groups of protein. In addition, the thermal stability and antioxidant properties got significant improvement. These protein-polyphenol covalent conjugates had potential to be used as new antioxidants in the food industry. At the same time, the conclusion that the polyphenol loading could be controlled was proved.