Characterization of cold plasma-induced covalent conjugation of diverse polyphenols to β-lactoglobulin: Impacts on functionality and antigenicity

The promotion of covalent grafting of seven structurally distinct polyphenols to β-Lactoglobulin (β-LG) via cold plasma (CP) treatment, and their influence on functionalities and antigenicity of β-LG were systematically investigated. Results indicated that covalent β-LG-polyphenol conjugates were generated after undergoing 45 s of CP treatment, with the highest (135.49 ± 1.87 μmol/g) and lowest (2.56 ± 1.43 μmol/g) polyphenol content in β-LG-(−)-epigallocatechin gallate (β-LG-EGCG) and β-LG-syringic acid (β-LG-SA) conjugates, respectively. This result clearly demonstrated that the binding capacity is directly related to the number of phenolic hydroxyl (-OH) groups in polyphenols. Multispectral analysis revealed that the β-LG's spherical conformation unfolded upon covalent grafting with polyphenols. Further analysis of functionalities and antigenicity revealed that improvement in functionalities and reduction in antigenicity of β-LG was positively correlated with the grafted polyphenol content. The β-LG conjugates modified with EGCG displayed a maximum decrease in antigenicity (58.40 ± 1.03 %), a nearly seven-fold increase in DPPH, and a five-fold increase in ABTS free radical-scavenging capabilities when compared to native β-LG. According to LC-MS/MS and immunoblot analysis, the primary cause of the decrease in antigenicity was the disruption of the linear IgE epitope in β-LG as a result of conjugated EGCG's masking and steric hindrance impact. These findings demonstrate that CP is a reliable method for promoting covalent conjugation between polyphenols and proteins. Moreover, the binding affinity was found to be positively correlated with the number of -OH groups present in the polyphenol, which is a key structural determinant for enhancing functional properties and for reducing the antigenicity of the resulting protein-polyphenol complexes.