饮料中提取酪蛋白的方法选择

Selection of extraction method for casein in beverages

目的探索4种沉淀蛋白质的方法在提取饮料酪蛋白过程中对其复溶率的影响,为建立超高效液相色谱-三重四极杆串联质谱法鉴别饮料中酪蛋白亚型及相应含量的测定寻找最佳前处理方法。方法选取乳粉、以乳粉或牛乳为原料的饮料、添加酪蛋白或酪蛋白酸钠的饮料作为测试样品,用pH 8.5的Tris-HCl溶液溶解,采用等电点沉淀法、乙腈沉淀法、丙酮沉淀法和乙醇沉淀法提取样品中的蛋白质,低温离心后对沉淀的蛋白质进行复溶,用考马斯亮蓝法分别测定沉淀前和复溶后样品溶液中可溶性蛋白质的含量,计算类酪蛋白的复溶率。结果乳粉和3类饮料中的酪蛋白经等电点沉淀法提取后,乳粉的类酪蛋白复溶率在64.95%~66.20%之间,3种饮料中的类酪蛋白复溶率在69.73%~84.95%之间,等电点沉淀法优于其他3种沉淀方法。αs-酪蛋白标准物质和β-酪蛋白标准物质的复溶率分别为70.16%和87.17%,且经高分辨质谱确证其复溶后的主成分为酪蛋白。另外对45种含酪蛋白饮料中的类酪蛋白含量进行了测定,其含量在0.10~42.36 mg/g(mg/mL)之间。结论等电点沉淀法提取类酪蛋白的复溶率较其他3种沉淀方法相对稳定,为饮料中酪蛋白的亚型鉴别及相应含量测定提供了回收率较高的前处理方法,为今后测定酪蛋白含量的液相质谱法的研究奠定了较好基础。

Objective To explore the effect of 4 kinds of proteins precipitation methods on the solubility of casein in the extraction process, and to determine the best pretreatment method for identifying casein isoforms and their relative content in beverages by ultra performance liquid chromatography with three quadrupole mass spectrometry. Methods The testing samples were milk powder, beverage with milk or milk powder as raw materials and beverage with casein or sodium caseinate, which were firstly dissolved in Tris-HCl buffer solution（pH 8.5）. Then the proteins of samples were precipitated using isoelectric precipitation method, acetonitrile precipitation method, acetone precipitation method and ethanol precipitation method, respectively, and followed by the redissolution after low temperature centrifugation. Finally, The content of soluble protein were measured before precipitation and after rehydration in the sample solution by Bradford method and the corresponding redissolution percentages of casein were calculated. Results The casein in the milk powder and 3 kinds of beverages were extracted by isoelectric precipitation method. The redissolution percentages of casein from milk powder ranged from 64.95% to 66.20%, and the redissolution percentages of casein from three kinds of beverages ranged from 69.73% to 84.95%. Therefore, isoelectric precipitation was superior to other three pretreatment methods. The redissolution percentages of αs-casein and β-casein were 70.16% and 87.17%, respectively. It was confirmed by high resolution mass spectrometry that the primary component of redissolved compound was casein. Besides, the content of casein in 45 kinds of casein beverages ranged from 0.10 to 42.36 mg/g（mg/mL）. Conclusions The redissolution percentages of casein using isoelectric precipitation method is relatively more stable than the other three precipitation methods. It provides a better pretreatment method for identification and determination of casein in beverages, and lays a good foundation for the determination of casein content by liquid chromatography-mass spectrometry in the future.