Covalent and non‐covalent interaction of myofibrillar protein and cyanidin‐3‐O‐glucoside: focus on structure, binding sites and in vitro digestion properties

Abstract BACKGROUND The aim of this study was to investigate the effects of covalent and non‐covalent interactions between myofibrillar protein (MP) and cyanidin‐3‐ O ‐glucoside (C3G) on protein structure, binding sites, and digestion properties. Four methods of inducing covalent cross‐linking were used in the preparation of MP‐C3G conjugates, including tyrosinase‐catalyzed oxidation, alkaline pH shift treatment, free radical grafting, and ultrasonic treatment. A comparison was made between MP‐C3G conjugates and complexes, and the analysis included sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), C3G binding ratio, liquid chromatography–tandem mass spectrometry (LC–MS/MS), protein side‐chain amino acids, circular dichroism spectroscopy, three‐dimensional fluorescence, particle size, and in vitro simulated digestion. RESULTS Covalent bonding between C3G and amino acid side chains in MP was confirmed by LC–MS/MS. In covalent bonding, tryptophan residues, free amino groups and sulfhydryl groups were all implicated. Among the 22 peptides covalently modified by C3G, 30 modification sites were identified, located in lysine, histidine, tryptophan, arginine and cysteine. In vitro simulated digestion experiments showed that the addition of C3G significantly reduced the digestibility of MP, with the covalent conjugate showing lower digestibility than the non‐covalent conjugate. Moreover, the digestibility of protein decreased more during intestinal digestion, possibly because covalent cross‐linking of C3G and MP further inhibited trypsin targeting sites (lysine and arginine). CONCLUSION Covalent cross‐linking of C3G with myofibrillar proteins significantly affected protein structure and reduced protein digestibility by occupying more trypsin binding sites. © 2023 Society of Chemical Industry.