转谷氨酰胺酶交联协同热加工调控豆腐致敏性的研究

Enzymatic cross-linking and thermal processing are the effective methods to control the allergenicity of food allergies. This proposal targets to eliminate the allergenicity of tofu. Firstly, raw soymilk will be produced by soaking and grinding of soybean, and enzymatic tofu will be prepared by heat-treatment and using transglutaminase (MTG) as coagulant. During the course of tofu production, the allergenicity is regarded as an indicator for optimizing technological parameters of thermal processing and enzymatic cross-linking. Followed by controllable processing research of enzymatic tofu, the change of composition and chemical structure of the main allergenic protein and conjuates in tofu will be characterized by electrophoresis, mass spectrometry, circular dichroism, UV spectroscopy and fluorescence spectroscopy. Based on the above work, the digestion stability and allergenicity of tofu will be evaluated through in vitro immunological and animal experiments. Finally, the association between the structure of the main allergen in tofu and its allergenicity will be analyzed. In this proposal, the relationship between the structure and the allergenicity of tofu caused by thermal processing and cross-linking of MTG will be illustrated, and it will reveal the discipline about regulating allergenicity by thermal processing and MTG cross-linking. Furthermore, this proposal may provide theoretical basis for producing hypoallergenic tofu and maximally eliminate the allergenicity of soybean allergens during the production of tofu.

酶交联和热加工是调控过敏食物致敏性的有效方法。本项目以消减豆腐致敏性为研究目标，拟以大豆为原料，经浸泡、磨浆后制成生豆浆，再经热加工、并以转谷氨酰胺酶（MTG）为促凝剂制成酶交联豆腐。在制作豆腐过程中，以致敏性为评价指标，通过优化热加工和酶交联工艺参数，完成酶交联豆腐可控加工研究。其后，借助电泳、质谱、圆二色光谱、紫外光谱、荧光光谱等技术解析和表征豆腐中主要过敏蛋白及交联产物组成和化学结构变化；在此基础上，对豆腐进行体外消化稳定性评价分析，并通过体外免疫实验和动物实验对所制豆腐的致敏性进行科学评估；最后完成豆腐中主要过敏蛋白结构改变与其致敏性变化的关联分析。 通过本项目研究，可阐明热加工和MTG交联导致豆腐致敏性变化与其过敏蛋白组成、结构改变的关系，揭示MTG交联协同热加工调控豆腐致敏性的规律，为低致敏豆腐的研发提供理论依据，期望能最大程度地在豆腐生产中消减大豆过敏蛋白的致敏性。

豆腐中蛋白丰富，营养价值高。然而大豆，又是八大过敏食物之一，易引起人体过敏。加热和添加促凝剂是豆腐加工的关键环节。加热是控制食物致敏性常用的方法，但因大豆中过敏原热稳定性较好，仅凭加热难以有效降低其致敏性。微生物来源转谷氨酰胺酶(MTG)是一种酶类促凝剂，研究表明：过敏原致敏性表位可被MTG交联破坏或掩埋，导致其致敏性降低。本研究探讨加热和MTG交联对豆浆中过敏原结构和致敏性的影响，通过加热和MTG交联制备豆腐，表征豆腐的理化特性和在胃肠道的消化性；然后以KU812细胞脱颗粒评估豆腐消化产物的致敏性，再以BABL/c小鼠为动物模型评估豆腐致敏性。结果表明：两步加热后豆浆的粘度明显大于一步加热，加热后豆浆蛋白的二级、三级结构发生改变，蛋白内部的疏水性残基暴露于分子外部；加热显著降低豆浆的致敏性，且两步加热致敏性降低更显著。豆浆经75℃-5min/95℃-10min两步加热、pH6.0和50℃交联3h，产物致敏性进一步降低；交联后蛋白中α-螺旋和β-转角含量升高，无规则卷曲含量减少，紫外吸光值和疏水性增强。在优化条件下制作了单一促凝剂豆腐和复合促凝剂豆腐，理化性质表明，添加MTG的复合促凝剂豆腐弹性更好、结构更均一、品质更细腻。模拟胃肠消化显示，酶交联豆腐在胃内难消化，而在肠内消化与非酶豆腐无显著差别。不同豆腐胃消化产物IgE结合能力有显著性差异，其中内酯和MTG两种豆腐的胃消化产物IgE结合能力较低，但胃肠消化产物IgE结合能力无显著差异。KU812细胞脱颗粒评价豆腐消化产物致敏性的结果表明：复合促凝剂豆腐尤其是内酯+MTG型豆腐的胃肠消化产物促细胞脱颗粒释放活性介质含量最少，致敏性最低。动物实验部分，由于小鼠宰杀、血清和相关标本的采集结束不久，部分送检指标和数据处理仍在进行中。研究表明MTG在改善传统豆腐品质和降低致敏性方面具有一定的实用价值。