大豆β-伴球蛋白酶解过程中的抗原性变化及其寡糖链结构在抗原活性中的作用

BACKGROUND: Soybean protein has the immunogenicity, which is an important safety problem in food processing. The traditional heat treatment and enzymatic hydrolysis can not completely eliminate the antigenicity, but greatly reduce the processing performance of soybean protein in food. Soybean antigenic protein is glycoprotein, but the relationship between the sugar chain and the antigenicity of soybean protein or its enzymatic hydrolysates is an unsolved scientific problem. . OBJECTIVE: This project takes soybean β-conglycinin (the highest activity of soybean protein) as the object, In this project, the changes of glycoprotein composition and allergenicity of soybean β-conglycinin and its deglycosylated form during the enzymatic hydrolysis, the oligosaccharide chains compositions and glycopeptide combination mode of the hydrolysates will be researched. From the results, the role of the oligosaccharide on antigen activities of β-conglycinnin and its hydrolysate will be revealed.. METHOD: The allergenicity of soybean protein and its hydrolysates will be detected with ELISA kit (Enzyme-Linked Immuno-Sorbent Assay) specified for different allergenic protein or peptide. The allergenic composition in the hydrolysats will be identified with Western blotting and specific IgE binding in SDS-PAGE (Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis). The GFC (Gel Filtration Chromatography) and RP-HPLC(Reverse Phase –High Performance Liquid Chromatography) will be used to isolate the allergenic component, and MALDI-TOF-MS (Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry) will be used to analysis the peptide mass fingerprinting (PMF) and sugar chain sequences sequece of allergenic glycopeptides. Glycoside hydrolase will be used to degrade oligosaccharide chains in the allergenic glycopeptides, and GC(Gas Chromatography) method will be used to determine the type of sugar. The β-elimination reaction will be used to determine sugar- peptide binding mode. Based on the information above and the bioinformatics principle, we should describe the sequence epitope structure and its position in soy allergenic protein.. SIGNIFICANCE: This work may be useful to select an effective enzymatic hydrolysis method of eliminating soy protein antigenicity and also maintaining its good processing properties. These researches are innovatively different from other enzymatic hydrolysis of soybean protein reported in content and method.

背景：大豆蛋白抗原性是食品加工中的重要安全问题。传统加热和酶解方法不能完全消除其抗原性，却大大降低其食品加工性能。大豆抗原蛋白是糖蛋白，但糖链在该蛋白及其酶解后抗原活性变化中的作用是未解决的科学问题。目的：以抗原活性最高的β-伴球蛋白为对象，首次通过对天然的和脱糖链的β-伴球蛋白在酶解过程中分子组成和抗原性变化规律，以及糖链成分、糖肽结合方式的研究，揭示糖链在β-伴球蛋白及其酶解产物抗原活性中的作用。方法：以SDS-PAGE和PAS法分析蛋白分子组成，ELISA法分析糖蛋白/肽抗原活性，免疫印迹法确定抗原成分，GFC、RP-HPLC和2D-SDS-PAGE分离抗原成分；糖苷酶降解寡糖链、GC法确定糖种类，β-消去反应确定糖-肽结合方式，MALDI-TOF-MS鉴定抗原活性肽质量指纹图谱和糖肽链中糖的序列等。意义：为探讨有效消除大豆蛋白抗原性、且保留其良好加工性能的酶解方法提供理论基础。

本项目首先研究并确定多级分离等电点沉淀法分离纯化大豆β-伴球蛋白。 通过β-消去反应和PNGase F酶脱糖链后GC-MS分析，β-伴球蛋白中糖与肽链的连接方式有N-型和O-型连接，其中糖链成分有岩藻糖，甘露糖等。ELISA试剂盒分析表明，β-伴球蛋白脱糖链后仍保留约60%抗原活性，表明糖链的存在对该蛋白具有抗原活性有贡献。用多种商品蛋白酶制剂分别水解原β-伴球蛋白、脱糖链β-伴球蛋白以及大豆分离蛋白（其中主要成分是β-伴球蛋白），发现在蛋白质分子成逐渐降解的同时，抗原活性在酶解的前期间内快速减少，但其后酶解过程中，蛋白质分子成分和抗原活性变化较小，即使酶解3小时以上，蛋白质的某些亚基仍然完整存在，而且新生成的一些肽链分子量不再发生变化，表明这些肽链表现出抗酶解特性，且抗原活性也不会完全消失。不同种类酶制剂水解时变化均略有差异，详见研究报告正文。首次证明脱糖链后的该大豆蛋白更容易被蛋白酶水解，且抗原活性更低，也表明糖链的存在对β-伴球蛋白抗原性有贡献。首次通过MALDI-TOF-MS分析和大豆白质数据库、抗原数据检索，分别确认了三种酶制剂水解产物中抗酶解肽链的来源，发现β-伴大豆球蛋白的α-、α′-和β-亚基均可产生抗酶解片段；首次鉴定出这些抗酶解肽链中存在完整的序列型抗原表位。详见报告正文。通过LC-MS/MS分析，发现在α-亚基的480位、β-亚基的37位和371位的天冬酰胺上有糖基化位点。用LC-MS/MS分析鉴定出来12个N-糖基化位点，其中5个在α-亚基上，3个在α′-亚基上，4个位于β-亚基上。本项目还首次研究了大豆发芽过程中,在大豆内源性蛋白酶作用下，大豆蛋白成分降解和抗原性变化规律，发芽7天，β-亚基也难以完全消失，与商品酶制剂酶解结果相似。动物营养应用效果表明，大豆蛋白经多种蛋白酶联合水解产物对动物的生长发育和健康有利。上述结果从蛋白质分子水平上揭示了酶解不能完全消除β-伴大豆球蛋白抗原性的根本原因。推测大豆蛋白的抗原性是大豆自我保护、抵御外来生物侵蚀的一种机制。