Study On The Glycation Of Chicken Egg Yolk Immunoglobulin And The Mechanism Of Its Function

Chicken egg yolk of immunoglobulin, short for Ig Y. It is easily to get and produce.Also, it does not interact with rheumatoid factors or activate mammalian complement. It has become a considerable potential alternatives of antibiotics. However, the application of Ig Y is limited due to its susceptibility to temperature, p H and enzymes. The glycan structure of Ig Y plays an important role in maintaining the physicochemical properties and antigenic properties. Glycation in Ig Y can change its conformation, thus improve its physical and chemical properties, and other functions. Therefore, the glycation of Ig Y is of great importance. The objective of this study was to develop a simple, safe and large-scale separation method for Ig Y from egg yolk. Then, Ig Y was glycated with various kinds of sugars and sugar chain length and study the different effects of the stability of Ig Y and modified production. The results as follows:1. The objective of this chapter was to develop a simple, safe and large-scale separation method for extraction Ig Y from egg yolk. Egg yolk was diluted with delipidation solutions, which was 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 monosacchrides, respectively. SDS-PAGE, Western blotting and high-performance liquid chromatography(HPLC) was used for Ig Y quantitative analysis. The optimal condition of all monosaccharide concentration for pectin,λ-carrageenan, dextran sulfate, and carboxymethylcellulosewas 0.1%. The yield could reach 8.36 mg/m L, 9.31 mg/m L, 7.64 mg/m L and 8.1 mg/m L, respectively. And purity could reach 83.3%, 80.1%, 80.94% and 79.94%. For methylcellulose, optimal isolation was at 0.15%, the yield and purity was 7.65 mg/m L and 78.17%, respectively. The immunoreactivity of Ig Y was measured by ELISA, and ELISA indicated that the isolated Ig Y maintained a relative high immunoreactivity. The results showed that yield, purity,and immunoreactivity varied with types and concentrations of polysaccharides. The best results were obtained in the presence of 0.1% pectin. Under this condition, yield andpurity can reach 8.36 mg/m L egg yolk and 83.3%, respectively, and the negative effect of Ig Y on immunoreactivity can be minimized. The procedure of isolation was simple with a higher yield of Ig Y, avoiding energy and time-consuming methods. Therefore, the isolation condition under study has a great potential for food industry production of Ig Y on a large scale.2. Four different monosaccharides(glucose, mannose, fucose, and fructose) and four different chain length of dextran(5000K、20000K、40000K、70000K) were used to prepare protein-sugar conjugates with Ig Y via the Maillard reaction. UV-vis and SDS-PAGE demonstrated that the advanced glycation end products(AGEs) formation increased with the extension of incubation time, confirming that the formation of glycated products was a time-dependent process.The result showed that the extent of cross-linking of Ig Y by fructose was significantly greater than by the other monosaccharides, and the extent of cross-linking of Ig Y strengthed with the increase of the length of chain of dextran. Fourier transform infrared spectroscopy(FT-IR), circular dichroism(CD) and fluorescence spectroscopy were used to monitor the glycation-induced structural changes in the whole reaction process. The results of fluorescence spectroscopy showed that fluorescence emission spectra of the Ig Y-sugar conjugates reduced as the incubation time increased, which indicated the proteins underwent conformational change. CD showed the content of α-helixes and random coils increased, and β-sheets and β-turns decreased after glycation. Amino acid analysis showed the contents of lysine and arginine declined after glycation.3. This chapter studied the thermal stability of Ig Y-sugar conjugates after glycation.Differential scanningcalorimetry(DSC) showed that the thermal stability of Ig Y was improved after glycation. The Td reached the highest when the Ig Y-Glc conjugate was at18d(79.8 °C), Ig Y-Man was at 24d(77 °C), and Ig Y-Fuc(78.5 °C) and Ig Y-Fru(79 °C)were at 12 d. Meanwhile, the value of the enthalpy changes(ΔH) presented a slight decrease with the time extension, which indicated less energy was required for the thermal denaturation. The Td reached the highest when the Ig Y-5000 K conjugate was at24 d(77.3 °C), Ig Y-20000 K was at 18 d(75.9 °C), and Ig Y-40000 Kat 18 d(74.5 °C) and Ig Y-70000K(74 °C) were at 12 d.4. This chapter studied the immunoreactivity of Ig Y-sugar conjugates after glycation.The enzyme-linked immunosorbentassay(ELISA) indicated theimmunoreactivity improved at first, then suffered a loss after glycation. Ig Y glycated with Fru, Man, Fuc and Fru reached the highest at 6 d, increased 30.3%, 13.7%, 12%, 22.4%. However, Ig Y glyacted with dextran suffered a loss after glycation. Ig Y-5000 K conjugate reached the highest when it was at 24 d, reduced 14.3%. Ig Y-20000 K, 40000 K and 70000 K was at 6 d,reduced 37.4%, 40.4%, 44.3%, respectively. The immunoreactivity of Ig Y-sugar conjugates: G5000K>G20000K>G40000K>G70000.5. This chapter studied the stability of Ig Y and Ig Y-sugat conjugates in the digestive systerm of artificial simulated gastrointestinal. ELISA indicated the rate ratio of activity reservation of Ig Y was 20.1% after digested. Ig Y glycated with Glc and Fuc reached the highest at 18 d, increased 201% and 121%, respectively. Ig Y glycated with Man reached the highest at 12 d, increased 200%. Ig Y glycated with Fru was at 24 d, increased 180%.However, Ig Y glycated with dextran suffered a loss after glycation to an extent.