Tartary buckwheat protein-phenol conjugate prepared by alkaline-based environment: Identification of covalent binding sites of phenols and alterations in protein structural and functional characteristics

Tartary buckwheat protein-rutin/quercetin covalent complex was synthesized in alkaline oxygen-containing environment, and its binding sites, conformational changes and functional properties were evaluated by multispectral technique and proteomics. The determination of total sulfhydryl and free amino groups showed that rutin/quercetin can form a covalent complex with BPI and could significantly reduce the group content. Ultraviolet-visible spectrum analysis showed that protein could form new characteristic peaks after binding with rutin/quercetin. Circular dichroism spectrum analysis showed that rutin and quercetin caused similar changes in the secondary structure of proteins, both promoting β-sheet to α-helix, β-ture and random coil transformation. The fluorescence spectrometry results showed that the combination of phenols can cause the fluorescence quenching, and the combination of rutin was stronger than the quercetin. Proteomics showed that there were multiple covalent binding sites between phenols and protein. Rutin had a high affinity for arginine, and quercetin and cysteine had high affinity. Meanwhile, the combination of rutin/quercetin and protein had reduced the surface hydrophobic ability of the protein, and improved the foaming, stability and antioxidant properties of the protein. This study expounded the mechanism of the combination of BPI and rutin/quercetin, and analysed the differences of the combination of protein and phenols in different structures. The findings can provide a theoretical basis for the development of complexes in the area of food.