Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea.
Poult Sci. 2014 Apr;93(4):1010-7. doi: 10.3382/ps.2013-03649.
Ovotransferrin and ovomucoid were separated using 2 methods after extracting the ovotransferrin- and ovomucoid-containing fraction from egg white. Diluted egg white (2×) was added to Fe(3+) and treated with 43% ethanol (final concentration). After centrifugation, the supernatant was collected and treated with either a high-level ethanol (61% final concentration) or an acidic salt combination (2.5% ammonium sulfate and 2.5% citric acid) to separate ovotransferrin and ovomucoid. For the high-level of ethanol method, ovotransferrin was precipitated using 61% ethanol. After centrifugation, the precipitant was dissolved in 9 vol. of distilled water and the residual ethanol in the solution was removed using ultrafiltration. The supernatant, mainly containing ovomucoid, was diluted with 4 vol. of water, had ethanol removed, and was then concentrated and used as the ovomucoid fraction. For the acidic salt precipitation method, the ethanol in the supernatant was removed first. The ethanol-free solution was then concentrated and treated with a 2.5% ammonium sulfate and 2.5% citric acid combination. After centrifugation, the precipitant was used as the ovotransferrin and the supernatant as the ovomucoid fraction. The ovomucoid fraction from both of the protocols was further purified by heating at 65°C for 20 min and the impurities were removed by centrifugation. The yields of ovomucoid and ovotransferrin were >96 and >92%, respectively. The purity of ovomucoid was >89% and that of the ovotransferrin was >88%. The ELISA results confirmed that the activity of the separated ovotransferrin was >95%. Both of the protocols separated ovotransferrin and ovomucoid effectively and the methods were simple, fast, and easy to scale up.
从蛋清中提取含有卵转铁蛋白和卵类黏蛋白的馏分后，使用 2 种方法分离卵转铁蛋白和卵类黏蛋白。将稀释的蛋清（2 倍）添加到 Fe(3+)中，并使用 43%乙醇（最终浓度）处理。离心后，收集上清液，并用高浓度乙醇（最终浓度 61%）或酸性盐组合（2.5%硫酸铵和 2.5%柠檬酸）处理以分离卵转铁蛋白和卵类黏蛋白。对于高浓度乙醇法，使用 61%乙醇沉淀卵转铁蛋白。离心后，将沉淀物溶解在 9 体积的蒸馏水中，并使用超滤去除溶液中的残留乙醇。上清液（主要含有卵类黏蛋白）用 4 体积的水稀释，去除乙醇，然后浓缩并用作卵类黏蛋白馏分。对于酸性盐沉淀法，首先去除上清液中的乙醇。然后去除乙醇的溶液浓缩并用 2.5%硫酸铵和 2.5%柠檬酸组合处理。离心后，沉淀物用作卵转铁蛋白，上清液用作卵类黏蛋白馏分。两种方案得到的卵类黏蛋白馏分均通过在 65°C 加热 20 分钟进一步纯化，并通过离心去除杂质。卵类黏蛋白和卵转铁蛋白的产率分别大于 96%和大于 92%。卵类黏蛋白的纯度大于 89%，卵转铁蛋白的纯度大于 88%。ELISA 结果证实分离的卵转铁蛋白的活性大于 95%。两种方案均能有效分离卵转铁蛋白和卵类黏蛋白，方法简单、快速，易于放大。
