高压脉冲电场结合糖基化对β-乳球蛋白抗原性与结构的影响

Effect of the Pretreatment Combining Glycation and Pulsed Electric Fields on the Antigenicity and Structure of β-Lactoglobulin

摘要:

β-乳球蛋白（β-lactoglobulin，β-Lg）是一种营养丰富的牛乳清蛋白，但也是导致牛乳过敏的主要过敏原；本文以β-Lg为研究对象，通过酶联免疫吸附法、电泳、圆二色谱及荧光光谱等方法，在蛋白与糖的质量比为1:2、修饰温度60 ℃、反应时间3 h、反应环境pH 7.4和不同高压脉冲电场（pulsed electric fields，PEF）强度反应条件下，对β-Lg-半乳糖复合物中β-Lg的抗原性变化及反应产物的结构特性进行研究。结果表明：经过PEF结合糖基化处理后，β-Lg抗原性显著降低，且PEF预处理会促进β-Lg抗原性降低，在电场强度25 kV/cm下预处理90 μs后进行糖基化，β-Lg抗原性降低最多，降低了约72.9%；其分子量增大；α-螺旋和β-转角结构减少，而β-折叠和无规则卷曲结构逐渐增多；表面疏水性和内源性荧光强度均降低；自由巯基含量先升高后下降，这为制备低致敏性β-Lg提供了一种新的方法。

Abstract:

β-Lactoglobulin (β-Lg) is a nutrient-rich bovine whey protein, but it is a major allergen in milk. In this study, we investigated β-Lg, and the changes in the antigenicity of the β-Lg in β-Lg-galactose conjugates and the structural properties of reaction products as determined using indirect competitive enzyme-linked immunosorbent assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism spectroscopy, and fluorescence spectroscopy. The reaction conditions were as follows: mass ratio of β-Lg and galactose, 1:2, modification temperature, 60 ℃, reaction time, 3 h and reaction pH 7.4. The pulsed electric field (PEF) intensity was varied. The results showed that after the pretreatment with glycation and PEF, the antigenicity of β-Lg significantly reduced, and PEF pretreatment promoted the decrease in antigenicity. When the sample was treated with PEF at 25 kV/cm for 90 μs, followed by glycation, the largest drop in the antigenicity of β-Lg was achieved (about 72.9%). After pretreatment with glycation coupled with PEF, the molecular weight of β-Lg increased. The contents of α-helix and β-turns decreased while the content of β-sheet and random coil increased. The intrinsic fluorescence intensity and surface hydrophobicity decreased, and the free sulfhydryl content first increased and then decreased. In this study, we describe a novel method for producing low-allergic β-Lg.