高静压诱导鱿鱼原肌球蛋白Tod p 1和血蓝蛋白结构变化机理及致敏性研究

Squid is a high-value but high allergenicity food. Tropomyosin Tod p 1(TTp1) and haemocyanin (HC) have been identified as the major allergens in squid. The secondary and tertiary structure characteristics of allergenic proteins are crucial to their allergenic potential. So that appropriate food process techniques could be performed on food to modify its protein structure and, hence, change the allergenicity. High hydrostatic pressure (HPP) is an emerging nonthermal food processing and preservation technique which provides potential benefits for processing aquatic product. The project is proposed to 1) identify changes of the secondary and tertiary structure of TTp1 and HC in squids treated by 100-800 MPa HPP through circular dichroism spectra, laser Raman spectroscopy, fluorescence spectra, etc.;2）develop predicted model on three dimensional structure changes of allergenic proteins by small angle X-ray scattering, etc.; 3) characterize the IgE-specific binding activity, simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) digestion stability of allergenic protein and the specific composition by sulfate-polyacrylamidegel electrophoresis (SDS-PAGE), western blotting, IgE-immunoblotting, inhibition enzyme-linked immunosorbent assay (ELISA), and MALDI-TOF-MS spectra. On the basis of all the investigations above, the HHP-induced action mechanism on conformation modifications of allergenic proteins as well as the structure change-allergenicity of squid will be clarified for the first time. This study is critical for establishing the scientific basis of controlling squid allergies, providing the new technologic support for the application of HPP in aquatic products.

鱿鱼营养丰富，但其高致敏性亦导致严重的食品安全问题。原肌球蛋白 Tod p1和血蓝蛋白是鱿鱼过敏原，其二、三级结构特征对致敏性起决定作用。因此可通过适宜的加工手段来改变过敏原蛋白二、三级结构，从而影响致敏性。高静压是当前最有前景实现工业化的非热加工新技术，适用水产品加工。本项目以鱿鱼为原料，进行100-800MPa高静压处理。用圆二色谱、激光拉曼光谱、荧光光谱等技术研究高静压引起的原肌球蛋白 Tod p1和血蓝蛋白二、三级结构变化，用小角X-射线散射测试等手段建立表征过敏蛋白三维构象的模型及其变化，采用电泳、免疫印迹、ELISA、肽质量指纹谱等表征过敏原蛋白免疫活性、胃肠液消化稳定性的变化及鉴定特征组分。在此基础上，本研究将首次揭示高静压诱导蛋白质二、三级结构和三维构象变化的作用机理及其与致敏性的关系。研究成果将为水产品致敏性控制提供理论依据，并为高静压在水产品加工中的应用提供科学指导。

鱿鱼营养丰富，但其高致敏性亦导致严重的食品安全问题。原肌球蛋白和血蓝蛋白是鱿鱼过敏原，其二、三级结构对致敏性起决定作用。食品加工手段能够改变过敏原蛋白结构，影响食品过敏性。高静压是当前最有前景实现工业化的非热加工新技术，适合水产品加工。本项目以鱿鱼为原料，进行 100-600 MPa 的高静压处理，用圆二色光谱、小角 X-射线散射、荧光分光光度等分析高静压引起的原肌球蛋白/血蓝蛋白二、三级结构变化，采用电泳、免疫印迹、 ELISA、肽质量指纹谱等手段，检测过敏原蛋白免疫活性、胃肠液消化稳定性的变化及其降解特征组分，以揭示高压诱导过敏蛋白质降解、结构变化机理及其与过敏性的关系。高静压处理可以改变鱿鱼原肌球蛋白和血蓝蛋白结构，破坏其α 螺旋结构，使其表面疏水性增加，自由巯基含量降低；高静压处理通过改变蛋白结构使其致敏性降低。本研究表明高静压破坏了原肌球蛋白/血蓝蛋白的结构，导致了该蛋白致敏性的下降，为高静压在生产低致敏性鱿鱼上的应用提供了科学依据。