高静压结合酶法降低凡纳滨对虾虾仁致敏性机理研究

Under the financial aids of "Eleventh Five-Year" National Science and Technology Supporting Program, our previous research gained much achievement, including having effectively removed over 90% anaphylactogen from whole Penaeus vannamei peeled prawn by high static pressure combined with enzymatic treatment, and also determined the related technical conditions of the preparing low-allergen Penaeus vannamei peeled prawn. But there still exist some uncertainty which needs further research, such as the relationship between structural variation of allergen protein and its subsequent allergenicity, the effects of other ingredients in shrimp on allergen protein abatement, and how inner allergens of peeled prawn have been decreased, during the reduction of anaphylactogen caused by high static pressure.. In this schedule, it is to reveal the molecular mechanism how the application of high static pressure combined with enzymatic treatment can reduce the allergen of Penaeus vannamei. That is, it is to evaluate the relationship between the high-static-pressure-caused structural change of tropomyosin, the major allergenic protein in peeled prawn, and its allergenicity, and also the relationship between the linear epitopes changes of tropomyosin caused by high static pressure together with emzymetic treatment and its allergenicity. In addition, it also to reveal the mechanism by which high static pressure combined with enzymatic treatment can lower allergization of peeled prawn, such as the effects of peeled prawns allergen protein dissolution, protease penetration into peeled prawns, the variation of protease activity and other ingredients of peeled prawns on the results of anaphylactogen alleviation. This study could be a guide in the development and production of low-allergen or non-allergen shrimp products.

在国家"十一五"科技支撑项目的支持下，采用高静压结合酶法处理整体虾仁，有效地消减了过敏原，其致敏性降低90%以上，并确定了制备低致敏性虾仁的相关技术参数。但对于虾仁过敏原消减过程中，高静压引发过敏原蛋白结构变化与变态反应原性关系；虾仁中其他成分对过敏原蛋白消减的影响；以及虾仁内部过敏原如何被消减等机理问题还需要深入探讨。.本项目采用高静压与蛋白酶结合的方法处理凡纳滨对虾主要过敏原-原肌球蛋白，通过研究高静压处理原肌球蛋白引发结构变化与其变态反应原性的关系，以及高静压结合酶法处理原肌球蛋白引起线性抗原表位的变化与变态反应原性关系，从分子水平揭示高压结合酶法对凡纳滨对虾过敏原消减的机理。研究高静压条件下虾仁过敏原蛋白的溶出、蛋白酶向虾仁内部渗透、蛋白酶活力变化、及虾仁成分对过敏原消减效果的影响等，揭示高压结合酶法降低虾仁致敏性的机理。该研究对开发低过敏原或无过敏原的虾类制品具有指导作用。

在国家自然科学基金面上项目的支持下，对高静压结合酶法降低凡纳滨对虾虾仁致敏作用机理研究。采用DE-52阴离子与D152阳离子交换层析柱串联纯化凡纳滨对虾主要过敏原-原肌球蛋白（TM），获得样品纯度大于95%。经鉴定，纯化样品氨基酸序列与虾的TM同源性为98%。在此基础上，用不同压力条件（压力0.1-800MPa，温度10-37℃，时间10-40min）处理TM，研究TM结构变化与其致敏性的关系。结果显示，高压处理能够引发TM的致敏性和三级结构的变化，而其二级结构相对含量无显著变化；且TM致敏性变化与其三级结构有显著的相关性。. 用不同蛋白酶在高压下处理TM，研究高压结合酶法对TM致敏性消减效果。结果显示，在一定高压下处理蛋白酶，对酶活力有提高作用。在37℃下保压20min，胰蛋白酶在300MPa处理 TM，致敏性消减98％；菠萝蛋白酶在200MPa处理TM，致敏性消减85％；木瓜蛋白酶在200MPa处理TM，致敏性消减97％。高压下酶处理TM，其线性表位消减90%左右。. 以整体虾仁为原料，研究高静压条件下虾仁蛋白的溶出与虾仁致敏性消减关系。结果表明，高静压能影响虾仁蛋白的溶出，在200MPa、室温下保压30min，溶出蛋白量最高为3.79g/100g虾，是0.1MPa处理的1.53倍。采用高压（常温下0.1-900MPa）处理虾仁，当压力在0.1-300MPa范围内，虾仁致敏性的消减与蛋白溶出正相关；当压力在300-900MPa范围内，虾仁致敏性的消减与虾蛋白变性有关。. 高压下用胰蛋白酶处理虾仁，能够使蛋白酶向虾仁内部渗透；在200MPa、 40℃下，加酶量3000U/（g虾仁）、处理30min，虾仁致敏性消减95.27%。另外，处理条件的不同，其作用机理不同，在200MPa下消减虾仁致敏性主要以虾蛋白溶出和蛋白酶的水解作用为主；而600MPa下消减虾仁过敏原是高压变性（改变构象）和蛋白水解共同作用的结果。. 因此，通过研究，揭示了高压结合酶法降低虾仁致敏性的机理。