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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 four protein precipitation methods on the recovery rate of casein in beverage extraction and to establish a method for the determination of casein subtypes and their corresponding contents in beverage by ultra performance liquid chromatography with triple quadrupole mass spectrometry Find the best pre-treatment method. Methods Milk powder, milk powder or milk as raw material beverage, casein or sodium caseinate beverage were added as test samples and dissolved in Tris-HCl solution of pH 8.5. Isoelectric point precipitation, acetonitrile precipitation, Acetone precipitation and ethanol precipitation were used to extract the protein from the sample. After centrifugation at low temperature, the precipitated protein was reconstituted. Coomassie brilliant blue method was used to determine the content of soluble protein in the sample solution before and after sedimentation. Solubility rate. Results After the milk protein and casein in three kinds of beverages were extracted by isoelectric point precipitation, the casein re-dissolution rate of milk powder was between 64.95% -66.20%. The casein re-dissolution rates of the three kinds of drinks were between 69.73% ~ 84.95%, isoelectric point precipitation method is superior to the other three precipitation methods. The re-dissolution rates of αs-casein standard substance and β-casein standard substance were 70.16% and 87.17%, respectively, and the main component after the re-solution confirmed by high-resolution mass spectrometry was casein. In addition, the casein content of 45 kinds of casein-containing beverages was determined, which ranged from 0.10 to 42.36 mg / g (mg / mL). Conclusions The recovery rate of casein extracted by isoelectric point precipitation is relatively stable compared with the other three precipitation methods. It provides a pretreatment method with high recoveries for the identification of subtypes of casein in beverage and the determination of relative contents. The casein content of liquid chromatography mass spectrometry laid a good foundation.