超高压及热处理对β-伴大豆球蛋白致敏性的影响机理研究

β-conglycinin is considered as major allergenic proteins in soybean. The ultra-high pressure and heat treatment during food processing could have an influence on the allergenicity of β-conglycinin. However, the influence mechanism on allergenicity has not been illustrated now. In this study, β-conglycinin is firstly extracted and purified from soybean, and then be treated by ultra-high pressure, heat treatment and ultra-high pressure combined with temperature. Antigenicity and allergenicity of the treated protein are qualitatively and quantitatively analyzed by Enzyme Linked Immunosorbent Assay (ELISA) and Western blotting. The molecular weight distribution and composition characteristics are analyzed by means of SDS-PAGE, 2-D electrophoresis and Size-exclusion Chromatography (SEC). The change regulations of the secondary and advanced structure are determined by Fluorescence spectroscopy, Circular Dichroism Spectrum (CD), Fourier Transform Infrared spectrum (FTIR) and other analysis technologies. The microstructure and aggregation morphology are observated by scanning electron microscopy and AFM. The structure-activity relationship between the molecular structure of the treated protein and its allergenicity will be explored. Finally, the allergenicity changes of crucial epitopes in the treated protein will be analyzed with epitope antibody constructed by bioinformatics and immunology methods. The research will reveal the influence mechanism of ultra-high pressure and heat treatment on the allergenicity of β-conglycinin, and provide an important theoretical evidence for the development of hypoallergenic soybean protein products in practial production.

β-伴大豆球蛋白是大豆中的主要致敏蛋白，在食品加工中超高压及热处理对β-伴大豆球蛋白致敏性会产生一定影响，但其机理目前尚未阐明。本项目首先从大豆中提取纯化β-伴大豆球蛋白，经超高压、热处理及超高压联合热处理后，采用ELISA和Western-Blot方法对处理后蛋白的过敏特性进行定性和定量分析；利用SDS-PAGE、2-D电泳、SEC色谱分析处理后蛋白的分子组成特性，采用荧光光谱、CD光谱、傅里叶红外光谱（FTIR）等研究其二级和高级结构变化，扫描电镜及AFM观测其微观结构及聚集形态，探究超高压及热处理过程中β-伴大豆球蛋白的分子结构与其过敏特性的构效关系；最后通过生物信息学及免疫学手段构建表位抗体，深入分析处理后蛋白关键性抗原表位致敏性的变化规律。该研究将揭示超高压及热处理影响β-伴大豆球蛋白致敏性的作用机理，为实际生产中开发低敏性大豆蛋白制品提供重要理论依据。

大豆蛋白具有较高的营养价值，但同时也会导致食物过敏反应，其中β-伴大豆球蛋白是大豆中的主要致敏蛋白。本项目研究超高压（HHP）及热处理对β-伴大豆球蛋白致敏性的影响及作用机理。采用ic-ELISA检测HHP及热处理后β-伴大豆球蛋白与抗体的结合能力；利用生物信息学结合免疫学技术确定β-伴大豆球蛋白β亚基上的优势抗原表位，制备表位多克隆抗体，分析加工过程中蛋白抗原表位致敏性的变化规律；利用电泳及光谱技术表征β-伴大豆球蛋白的结构特性，分析加工后蛋白过敏性与结构变化之间的相关性；构建体外消化体系及小鼠致敏模型，探究HHP及热处理对β-伴大豆球蛋白致敏性的影响机理。结果表明，筛选并鉴定了β-伴大豆球蛋白β亚基上的十个优势抗原表位，制备了表位抗体。随着热处理温度升高，β-伴大豆球蛋白的IgG结合能力降低，但其IgE结合能力以及与9种表位抗体的结合能力增强。300 MPa（20 ℃，40 ℃和60 ℃）和400 MPa（20 ℃和60 ℃）的HHP处理抑制了β-伴大豆球蛋白与IgG及部分表位抗体的结合，但其IgE结合能力没有明显变化。HHP和热处理均改变了β-伴大豆球蛋白的空间结构，暴露出更多的疏水基团，结构变得无序，导致原有抗原表位被掩盖或暴露。体外消化结果表明，HHP和热处理促进了β-伴大豆球蛋白的消化，大量抗原表位被破坏，但部分表位能够抵抗消化而存在。经消化后，热处理β-伴大豆球蛋白与IgG、IgE及表位抗体的结合能力均显著下降；400 MPa 60 ℃的HHP处理后β-伴大豆球蛋白显示出最低的IgG和IgE结合能力，但与表位抗体结合能力增加。此外，HHP及热处理能够减弱β-伴大豆球蛋白的致敏性，使小鼠过敏症状得到有效缓解，从而阻断食物过敏反应发生。该项目研究结果将为实际加工中开发低敏性大豆制品提供重要理论依据。