Proteins and polyphenols can form complexes through two main types of interactions: non-covalent and covalent interactions. Non-covalent interactions include hydrogen bonding, hydrophobic interactions, electrostatic interactions, and van der Waals forces (Figure 1). These interactions are generally reversible and depend on protein structure, the chemical characteristics of polyphenols, and environmental conditions. Although they do not form stable chemical bonds like covalent bonds, they play an important role in protein–polyphenol complex formation and can alter protein properties in food systems.
Hydrogen bonds form between the hydroxyl (–OH) groups of polyphenols and polar groups on amino acid side chains or the polypeptide backbone of proteins (Figure 1). In addition, the aromatic rings of polyphenols can interact hydrophobically with nonpolar regions of proteins, particularly those containing hydrophobic amino acids such as leucine, alanine, isoleucine, methionine, valine, tryptophan, and phenylalanine. Electrostatic interactions also contribute to complex formation. At appropriate pH values, negatively charged polyphenol groups can interact with positively charged amino acid residues, such as lysine, arginine, and histidine. Van der Waals forces further contribute to stabilizing these molecular interactions.
These types of interactions may occur simultaneously, with their relative contributions depending on the structure and chemical characteristics of the polyphenols, amino acid composition, and protein conformation. Consequently, the formation of protein–polyphenol complexes can alter protein conformation and physicochemical properties without the formation of covalent bonds.
Covalent bonds between proteins and polyphenols are generally stable and irreversible, and can form through three pathways: enzymatic oxidation, oxidation under alkaline conditions, and free-radical-induced coupling. Figures 2A and 2B illustrate the mechanisms of enzymatic and alkaline oxidation of polyphenols, respectively. In enzymatic oxidation (Figure 2A), polyphenols are converted into highly reactive intermediates, such as semiquinones or quinones, through the action of phenol-oxidizing enzymes, particularly laccase and tyrosinase. Similarly, under alkaline conditions (typically at pH ≥ 9; Figure 2B), polyphenols can undergo oxidation to form these intermediates. The resulting quinones then react with nucleophilic groups on proteins, leading to the formation of covalent bonds.
The functional groups involved in these reactions may include the amino groups of lysine and sulfur-containing groups of cysteine; methionine and tryptophan have also been implicated in this mechanism. Conjugation can form C–N or C–S bonds, resulting in mono-, dimeric, or polymeric adducts. When intermolecular cross-linking occurs, more complex protein–polyphenol networks may be formed. Covalent conjugation can alter the functional properties of proteins. For example, laccase-mediated conjugation of soy protein with gallic acid increases radical-scavenging activity and reducing power, whereas conjugation of peanut albumin with EGCG improves emulsifying properties. Other protein–polyphenol systems have also shown improvements in antioxidant activity, thermal stability, storage stability, and foam stability. However, these effects depend on the nature of the protein and polyphenol, as well as the reaction conditions.
Although covalent conjugation produces stable complexes, excessive cross-linking may lead to the formation of large protein networks or undesirable by-products. Therefore, oxidation conditions, pH, and the extent of conjugation should be carefully controlled when this approach is used to modify the functional properties of proteins in food applications.
In addition to enzymatic and alkaline oxidation, covalent bonds between proteins and polyphenols can also be formed through free-radical-induced coupling (Figure 3). This enzyme-free method uses H₂O₂ and ascorbic acid together as a redox system to generate hydroxyl radicals (HO•). These radicals oxidize amino acid residues on protein side chains, generating protein radicals that can react with polyphenols to form covalent bonds. As illustrated in the proposed mechanism, ascorbic acid participates in redox reactions, generating intermediates such as ascorbate radicals and dehydroascorbic acid. In the presence of H₂O₂, these reactions contribute to the formation of HO•, thereby initiating protein oxidation and the formation of protein–polyphenol complexes.
Unlike enzymatic or alkaline oxidation (Figures 2A and 2B), in which polyphenols are first oxidized to quinones or semiquinones before reacting with nucleophilic groups on proteins, the free-radical pathway begins with protein oxidation. The resulting protein radicals subsequently react with polyphenols to form covalent bonds. Some studies have reported that conjugation of wheat gluten hydrolysate with chlorogenic acid improves thermal stability, emulsifying properties, and foaming properties. In addition, conjugation of walnut proteins with EGCG, quercetin, ferulic acid, or resveratrol reduces their binding to immunoglobulin E (IgE) under in vitro conditions. These findings suggest that free-radical-induced coupling may be used to modify the functional and biological properties of proteins, depending on the protein–polyphenol system and processing conditions.
Non-covalent and covalent interactions between proteins and polyphenols can affect not only protein structure and physicochemical properties but also protein digestion. The formation of protein–polyphenol complexes may mask enzyme-accessible sites on protein molecules, while free or protein-bound polyphenols may interact with digestive enzymes and alter their catalytic activity. These effects may influence the extent of protein hydrolysis and the peptides generated during digestion.
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