Enzymatic and Nonenzymatic Conjugates of Lactoferrin and (−)-Epigallocatechin Gallate: Formation, Structure, Functionality, and Allergenicity

The impact of covalent attachment of (−)-epigallocatechin gallate (EGCG) to lactoferrin (LF) on the structure, morphology, functionality, and allergenicity of the protein was studied. These polyphenol–protein conjugates were formed using various enzymatic (laccase- and tyrosinase-catalyzed oxidation) and nonenzymatic (free radical grafting and alkali treatment) methods. The preparation conditions for forming the enzymatic conjugates were optimized by exploring the influence of order-of-addition effects: protein, polyphenols, and enzymes. The total phenol content of the LF–EGCG conjugates was quantified using the Folin–Ciocalteu method. The nature of the conjugates formed was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared (FTIR) spectroscopy analyses. These studies showed that enzymatic cross-linking was a highly effective means of forming LF–EGCG conjugates. Analysis of these conjugates using various spectroscopic methods showed that conjugation to EGCG changed the molecular structure of LF. Atomic force microscopy showed that the four covalent cross-linking methods affected the size and morphology of these LF–EGCG conjugates formed. The antioxidant activity and emulsifying stability of LF were significantly improved by conjugation to EGCG. Finally, an enzyme-linked immunosorbent assay (ELISA) and a western blot assay indicated that conjugation of EGCG reduced the binding capacity of LF to immunoglobulin E (IgE) and immunoglobulin G (IgG), which is consistent with a decrease in allergenicity. Overall, this study suggests that LF–EGCG conjugates formed using enzymatic or nonenzymatic methods have potential applications as functional ingredients in foods.