β-Lactoglobulin (BLG) is a main allergen in cow’s milk, which can cause severe allergic reactions. This study aimed to evaluate the effect of co-modification with ferulic acid (FA) and docosahexaenoic acid (DHA) on the antigenicity and structural properties of BLG. A BLG-DHA-FA complex was obtained by non-covalent binding and its structural characteristics and antigenicity were investigated by various spectroscopic techniques, molecular docking and indirect competitive enzyme-linked immunosorbent assay. The results showed that the combination of FA and BLG-DHA changed the microenvironment surrounding aromatic amino acids, enhancing the polarity. FA and DHA bound to the hydrophobic cavity of BLG through hydrophobic interaction and hydrogen bonding, thereby changing the secondary structure of BLG. Specifically, the contents of β-sheets and random coils were reduced, and the β-turn content was increased. Compared with BLG and BLG-DHA, the addition of FA significantly reduced the antigenicity, and the highest inhibition rate against the immunoglobulin G (IgG) binding ability of 40% was obtained when the FA concentration was 0.08 mg/mL.

The structural characteristics of proteins change when they are complexed with polyphenols and polysaccharides. This study investigated the synergistic effects of ferulic acid (FA), a dietary polyphenol, and xanthan gum (XG) at different mass concentrations on the structural characteristics and antigenicity of β-lactoglobulin (β-LG). The structural changes of β-LG after binding with FA and XG were analyzed using ultraviolet-visible (UV-Vis) absorption spectroscopy, intrinsic fluorescence spectroscopy, synchronous fluorescence spectroscopy, three-dimensional fluorescence spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and molecular docking simulation. The impact of β-LG-FA-XG binding on protein antigenicity was evaluated using an indirect competitive enzyme-linked immunosorbent assay. The results demonstrated significant changes in the microenvironments around tryptophan and tyrosine residues after the complexation, along with an increase in the polarity. Furthermore, the secondary structure of the protein changed; as the mass concentration of XG increased, the relative content of β-sheets first increased and then decreased, while the relative contents of α-helices and β-turns first decreased and then increased. The relative content of random coils showed no clear trend. In addition, compared with β-LG, the β-LG-FA-XG ternary complex exhibited a significantly reduced binding capacity to immunoglobulin G (IgG), indicating decreased antigenicity. The highest IgG binding capacity inhibition rate of 17.97% was observed at an XG mass concentration of 0.10 mg/mL.

β-Lactoglobulin (β-LG) can cause severe allergic reactions. In this study, the effects of combined modification of β-LG with xanthan gum (XG) and docosahexaenoic acid (DHA) on its antigenicity and structural characteristics were investigated. DHA-β-LG and DHA-β-LG-XG complexes were obtained through non-covalent binding. Their antigenicity and structural characterization were carried out using indirect competitive enzyme linked immunosorbent assay, particle size analysis, three-dimensional fluorescence spectroscopy, Fourier transform infrared (FTIR) spectroscopy, simultaneous fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy and molecular docking. The results showed that the antigenicity of the ternary complex was significantly reduced compared with those of β-LG and the binary complex (P < 0.05). The highest inhibition rate, (24.81 ± 0.27)%, of immunoglobulin G (IgG) binding capacity was observed at 2.5 mg/mL XG concentration. The ternary complex was more stable than the binary complex. As the concentration of XG increased, the particle size of the ternary complex increased and then decreased. Under the coexistence of XG and DHA, the relative contents of β-sheet and β-turn increased, and the relative contents of α-helix and random coil decreased. Furthermore, the exposure levels of both fluorophores and peptide bonds in β-LG showed a linear positive correlation with XG concentration. XG was inserted into the only hydrophobic cavity of β-LG, and DHA bound to the hydrophobic pocket on the surface of β-LG. Both XG and DHA formed the ternary complex with β-LG through hydrogen bonding, electrostatic interactions, and hydrophobic interactions. These structural changes affect the antigen binding epitopes of β-LG, thereby reducing its antigenicity.

β-Lactoglobulin (β-LG) is a major allergen in cow’s milk. In this study, synchronous fluorescence spectroscopy, three-dimensional fluorescence spectroscopy, circular dichroism (CD) spectroscopy, molecular docking, and indirect competitive enzyme-linked immunosorbent assay were used to explore the interaction of docosahexaenoic acid (DHA) with β-LG and its influence on the allergenicity of β-LG. The results showed that the binding of DHA to β-LG significantly changed the microenvironment of the aromatic residues of β-LG and increased its polarity. DHA bound to β-LG to form a stable complex through hydrophobic interactions, changing the secondary structure of β-LG and resulting in folding of its backbone. The relative content of β-sheet increased and then decreased with increasing concentration of DHA, while the relative content of α-helix showed an opposite trend. However, the relative contents of β-turn and random coil showed irregular changes. In addition, the antigenicity of β-LG decreased after combination with DHA, and the immunoglobulin E (IgE) binding ability inhibition rate was about 29% by 3 mmol/L DHA.