Impacts of glycation and transglutaminase-catalyzed glycosylation with glucosamine on the conformational structure and allergenicity of bovine β-lactoglobulin

beta-Lactoglobulin (-LG) is recognized as the major milk allergen. In this study, the effects of transglutaminase (TGase) and glucosamine (GlcN)-catalyzed glycosylation and glycation on the conformational structure and allergenicity of -LG were investigated. The formations of cross-linked peptides were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and GlcN-conjugated modification was identified using matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Structural analysis revealed that glycosylation and glycation of -LG induced unfolding of the primary protein structure followed by a loss of the secondary structure. As revealed by circular dichroism (CD) spectroscopy, glycosylated -LG exhibited the highest increase in the -sheets from 32.6% to 40.4% (25 degrees C) and 44.2% (37 degrees C), and the percentage of -helices decreased from 17.7% to 14.4% (25 degrees C) and 12.3% (37 degrees C), respectively. The tertiary and quaternary structures of -LG also changed significantly during glycosylation and glycation, along with reduced free amino groups and variation in surface hydrophobicity. Immunoblotting and indirect enzyme-linked immuno sorbent assay (ELISA) analyses demonstrated that the lowest IgG- and IgE-binding capacities of -LG were obtained following glycosylation at 37 degrees C, which were 52.7% and 56.3% lower than that of the native protein, respectively. The reduction in the antigenicity and potential allergenicity of glycosylated -LG was more pronounced compared to TGase treated- and glycated -LG, which correlated well with the structural changes. These results suggest that TGase-catalyzed glycosylation has more potential compared to glycation for mitigating the allergenic potential of milk products.