Bovine β-lactoglobulin (β-Lg) is one of major allergens in cow's milk. Previous study showed that ultrasound treatment induced the conformational changes of β-Lg and promoted the glycation in aqueous solutions, which is, however, less efficient compared with dry-state. In this work, the effect of ultrasound pretreatment combined with dry-state glycation on the IgG and IgE binding of β-Lg was studied. Dry-state glycation with mannose after ultrasound pretreatment at 0-600 W significantly reduced the IgG and IgE binding of β-Lg, with the lowest values observed at 400 W. The decrease in the IgG and IgE binding of β-Lg was attributed to the increase in glycation extent and the changes of secondary and tertiary structure, which reflected in the increase of UV absorbance, α-helix and β-sheet contents, as well as the decrease of intrinsic fluorescence intensity, surface hydrophobicity, β-turn, and random coil contents. Moreover, ultrasound pretreatment promoted the reduction of IgG and IgE binding abilities by improving glycation, reflecting in the increase of the glycation sites and the degree of substitution per peptide (DSP) value determined by Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Ultrasound pretreatment at 400 W showed the most significantly enhanced glycation extent. Besides, the results suggested FTICR-MS could provide insights into the glycation at molecular level, which was conducive to the understanding of the mechanism of the reduction in the IgG and IgE binding of β-Lg. Therefore, ultrasound pretreatment combined with dry-state glycation may be a promising method for β-Lg hyposensitization.

Bovine α-lactalbumin (α-LA) is one of major food allergens in cow's milk. The present work sought to research the effects of ultrasonic pretreatment combined with dry heating-induced glycation between α-LA and galactose on the immunoglobulin E (IgE)/immunoglobulin G (IgG)-binding ability and glycation extent of α-LA, determined by inhibition enzyme-linked immunosorbent assay and high-resolution mass spectrometry, respectively. The IgE/IgG-binding ability of glycated α-LA was significantly decreased as a result of ultrasonic pretreatment, while the average molecular weight, incorporation ratio (IR) value, location and number of glycation sites, and degree of substitution per peptide (DSP) value were elevated. When the mixtures of α-LA and galactose were pretreated by ultrasonication at 150 W/cm, glycated α-LA possesses seven glycation sites, the highest IR and DSP values, and the lowest IgE/IgG-binding ability. Therefore, the decrease in the IgE/IgG-binding ability of α-LA depends upon not only the shielding effect of the linear epitope found to be caused by the glycation of K13, K16, K58, K93, and K98 sites but also the intensified glycation extent, which reflected in the increase of the IR value, the number of glycation sites, and the DSP value. Moreover, allergenic proteins and monosaccharides pretreated by ultrasonication and then followed by dry-state glycation were revealed as a promising way of achieving lower allergenicity of proteins in food processing.

Bovine β-lactoglobulin (β-Lg) is a major allergen existing in milk and causes about 90% of IgE-mediated cow's milk allergies. Previous studies showed that pulsed electric field (PEF) treatment could partially unfold the protein, which may contribute to the improvement of protein glycation. In this study, the effect of PEF pretreatment combined with glycation on the IgG/IgE-binding ability and the structure of β-Lg was investigated. The result showed that PEF pretreatment combined with glycation significantly reduced the IgG and IgE binding abilities, which was attributed to the changes of secondary and tertiary structure and the increase in glycation sites and degree of substitution per peptide (DSP) value determined by electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD/FTICR-MS). Unexpectedly, glycation sites (K47, K91 and K135) added by two mannose molecules were identified in glycated β-Lg with PEF pretreatment. Moreover, the results indicated that PEF pretreatment at 25 kV cm for 60 μs promoted the reduction of IgG/IgE-binding capacity by increasing the glycation degree of β-Lg, whereas single PEF treatment under the same conditions markedly enhanced the IgG/IgE-binding ability by partially unfolding the structure of β-Lg. The results suggested that ECD/FTICR-MS could help us to understand the mechanism of reduction in the IgG/IgE-binding of β-Lg by structural characterization at the molecular level. Therefore, PEF pretreatment combined with glycation may provide an alternative method for β-Lg desensitization.