Tong Chenyao, Geng Fang, He Zhenjiao, Cai Zhaoxia, Ma Meihu
National Research and Development Center for Egg Processing, College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China.
National Research and Development Center for Egg Processing, College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China
Poult Sci. 2015 Jan;94(1):104-10. doi: 10.3382/ps/peu005. Epub 2014 Dec 25.
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.
鸡卵黄免疫球蛋白（IgY）是哺乳动物免疫球蛋白的一种优质替代品。然而，由于纯化程序复杂或耗时，IgY在研究、诊断和功能性食品中的实际应用受到限制。本研究的目的是开发一种从蛋黄中分离IgY的简单、安全、大规模的方法。分别用由不同类型（果胶、λ-卡拉胶、羧甲基纤维素、甲基纤维素和硫酸葡聚糖）和浓度（0.01%、0.05%、0.1%、0.15%和0.2%）的多糖制成的6倍脱脂溶液稀释蛋黄。将蛋黄溶液调节至pH 5.0，然后在4°C下保存过夜，再于4°C下离心。将所得上清液加入35%（w/v）硫酸铵，然后离心。含有IgY的沉淀物溶解于蒸馏水中，然后进行透析。利用SDS-PAGE和蛋白质免疫印迹法对IgY进行定性分析；使用高效液相色谱法（HPLC）进行定量分析。通过酶联免疫吸附测定法（ELISA）测量IgY的免疫反应性。结果表明，IgY的产量、纯度和免疫反应性随多糖的类型和浓度而变化。果胶、λ-卡拉胶、硫酸葡聚糖和羧甲基纤维素分离IgY的最佳浓度为0.1%；甲基纤维素的最佳分离浓度为0.15%。在0.1%果胶存在的情况下获得了最佳结果。在此条件下，产量和纯度分别可达8.36 mg/mL蛋黄和83.3%，并且IgY对免疫反应性的负面影响可降至最低。分离程序简化为两步，IgY产量更高，避免了耗能和耗时的方法。因此，所研究的分离条件在食品工业大规模生产IgY方面具有巨大潜力。
