高效液相色谱法测定乳清蛋白中的α-乳白蛋白和β-乳球蛋白及其改性降敏

Detection ofα-lactalbumin and β-lactoglobulin using HPLC and optimization of enzymatic degradation

牛乳过敏是由牛乳中的致敏蛋白引起的一种不良反应,而乳清蛋白是从牛奶中分离的高营养价值的蛋白。本研究中,笔者建立了一种高效液相色谱技术定量检测乳清蛋白中主要致敏蛋白α-乳白蛋白和β-乳球蛋白的方法;与现行标准(T/TDSTIA 007—2019)相比,本方法采用应用更广泛的Sepax C8(4.6 mm×150 mm,5μm)色谱柱,以0.1%的三氟乙酸水溶液(A)和0.1%的三氟乙酸乙腈溶液(B)作为流动相进行梯度洗脱,色谱柱在温度28℃、波长280 nm检测条件下对目标蛋白进行分离检测。定量分析结果表明,致敏蛋白在0.125~2.000 mg/mL范围内与蛋白峰面积线性关系良好(R^2>0.99),蛋白平均回收率为92%~96%,α-乳白蛋白和β-乳球蛋白检出限分别为0.078和0.090 mg/mL。该检测方法重复性好,可用于α-乳白蛋白和β-乳球蛋白的定量分析测定。此外,考察了胰蛋白酶对2种致敏蛋白改性的影响,采用智能可视化优化方法预测出酶解最优条件,结果表明在45℃下加入2000 U/g的胰蛋白酶水解30 min后,水解率高达78.9%,2种致敏蛋白含量占牛乳总蛋白的22.7%,大大降低了乳制品中的致敏蛋白含量。

Cow milk allergy is an adverse reaction caused by allergenic proteins in milk,and whey protein is a protein with high nutritional value isolated from milk.In this study,high performance liquid chromatography was used to quantitatively determine the main allergenic proteins includeα-lactalbumin andβ-lactoglobulin in whey protein.Compared with the group standard(T/TDSTIA 007—2019),this method uses a more widely column:Sepax-C8(4.6 mm×150 mm,5μm),with 0.1%trifluoroacetic acid aqueous solution(A)and 0.1%Trifluoroacetic acid acetonitrile solution(B)as the mobile phase for gradient elution.The chromatographic column was used to separate and detect the target protein at 28℃and wavelength 280 nm.The results of quantitative analysis showed that there was a good linear relationship between allergic protein and protein peak area in the range of 0.125-2.000 mg/mL(R^2>0.99),and the average recovery of allergic protein was 92%.The detection limits ofα-lactalbumin andβ-lactoglobulin were 0.078 mg/mL and 0.09 mg/mL,respectively.The detection method has good repeatability,which can be used for the analysis and determination of the content ofα-lactalbumin andβ-lactoglobulin.Then the effect of trypsin on the modification of allergic protein was investigated,and the intelligent visual optimization method was used to predict the optimal conditions of enzymatic hydrolysis.The results showed that the hydrolysis rate was as high as 78.9%,and the content of the two allergic proteins accounted for 22.7%of the total protein,after the enzymatic hydrolysis of trypsin(2000 U/g)at 45℃for 30 minutes.The content of allergic protein in dairy products was greatly reduced.