Monitoring glycation-induced structural and biofunctional changes in chicken immunoglobulin Y by different monosaccharides.

Chicken IgY has been applied in the food industry as an important supplement. Glycation of IgY is of special interest due to its possible influence on the structure and functionality of IgY. IgY was subjected to in vitro glycation with 4 different monosaccharides, including glucose (Glu), mannose (Man), fucose (Fuc), and fructose (Fru). The objective of this paper was to characterize the formation of IgY-sugar conjugates using ultraviolet spectra, fluorescence spectra, circular dichroism spectra, Fourier transform infrared spectra, and SDS-PAGE and differential scanning calorimetry analysis. The antigen epitopes of native and glycated IgY was determined by enzyme-linked immunosorbent assay (ELISA). The existence of broad bands in stacking gel and sample well demonstrated that reducing monosaccharides covalently bound to IgY. The secondary structure of IgY was altered from a well-defined ß-sheet structure to a random coil structure. Fluorescence spectra showed that IgY was more hydrophilic after glycation. Thermal stability of glycated IgY was remarkably increased over that of native IgY. However, ELISA results would suggest that the antigen epitopes recognized by the polyclonal antibody and overall conformation changed in the IgY molecule due to a decrease in polyclonal antibody binding to glycated IgY. Data suggested that glycation induced by reducing sugars significantly affects the structure and antigen-binding ability of IgY, which could reduce the utility of IgY in certain applications.

A simple method for isolating chicken egg yolk immunoglobulin using effective delipidation solution and ammonium sulfate.

Chicken egg yolk immunoglobulin (IgY) is a superior alternative to mammalian immunoglobulin. However, the practical application of IgY in research, diagnostics, and functional food is limited due to complex or time-consuming purification procedures. The objective of this study was to develop a simple, safe, large-scale separation method for IgY from egg yolk. Egg yolk was diluted with 6-fold delipidation solutions made of different types (pectin, λ-carrageenan, carboxymethylcellulose, methylcellulose, and dextran sulfate) and concentrations (0.01, 0.05, 0.1, 0.15, and 0.2%) of polysaccharides, respectively. The yolk solution was adjusted to pH 5.0, and then kept overnight at 4°C before being centrifuged at 4°C. The resulting supernatant was added to 35% (w/v) (NH4)2SO4 and then centrifuged. The precipitant, which contained IgY, was dissolved in distilled water and then dialyzed. SDS-PAGE and Western blotting were utilized to conduct qualitative analysis of IgY; high-performance liquid chromatography (HPLC) was used for quantitative analysis. The immunoreactivity of IgY was measured by ELISA. The results showed that yield, purity, and immunoreactivity varied with types and concentrations of polysaccharides. The optimal isolation of IgY for pectin, λ-carrageenan, dextran sulfate, and carboxymethylcellulose was at the concentration of 0.1%; for methylcellulose, optimal isolation was at 0.15%. The best results were obtained in the presence of 0.1% pectin. In this condition, yield and purity can reach 8.36 mg/mL egg yolk and 83.3%, respectively, and the negative effect of IgY on immunoreactivity can be minimized. The procedure of isolation was simplified to 2 steps with a higher yield of IgY, avoiding energy- and time-consuming methods. Therefore, the isolation condition under study has a great potential for food industry production of IgY on a large scale.