[关键词]
[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。
[Key word]
[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.
[中图分类号]
[基金项目]
国家863科技计划项目(2013AA102205)