CN109221982A - 一种基于关键氨基酸定向修饰的低致敏性螃蟹加工方法 - Google Patents
一种基于关键氨基酸定向修饰的低致敏性螃蟹加工方法 Download PDFInfo
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Abstract
一种基于关键氨基酸定向修饰的低致敏性螃蟹加工方法，涉及食品加工。螃蟹加盐前处理；关键氨基酸定向修饰；产品包装。将螃蟹经不同方式的熟制加工后，提取粗蛋白，以SDS‑PAGE电泳和Dot Blot分析不同程度热加工后的粗蛋白，以兔抗TM多克隆抗体的结合活性为指标，根据免疫结合程度判断不同加工方式下螃蟹的致敏性，筛选出获得低致敏螃蟹的加工方式。利用简单的抗原抗体结合反应，确定螃蟹过敏原经不同热加工后的致敏性变化情况，筛选出一种基于关键氨基酸定向修饰的低致敏性螃蟹加工方法，工艺简单、可操作性强、易于实现规模化生产。
Description
技术领域
本发明涉及食品加工，尤其是涉及一种基于关键氨基酸定向修饰的低致敏性螃蟹加工方法。
背景技术
甲壳类食物过敏是指外源性食物蛋白进入机体，发生抗原抗体的特定免疫应答反应，致使机体出现荨麻疹、哮喘、鼻炎、腹泻、呕吐等，严重的还伴随虚脱、休克，甚至死亡等症状。食物过敏蛋白与致敏反应相关的仅为其抗原决定簇中的数个至数十个氨基酸残基。大部分过敏原均为分子量小于90kDa、具酸性等电点的糖蛋白，研究表明，糖蛋白的结构在参与抗原表位形成的过程中扮演着不可或缺的重要作用(Tarentino,AL,et al.,Deglycosylation of asparagine-linked glycans by peptide:N-glycosidaseF.Biochem,1985,24(17):4665-4671)。因此通过还原糖定向修饰，使引起致敏的抗原表位不再为机体免疫系统所识别，是开发低致敏性食品的一个重要方向。
青蟹、梭子蟹具有生长快、个体大、适应性强等特点，其肉质鲜美，营养价值高。2015 年全国捕捞量在13.6万吨，2016年捕捞量在14.3万吨，而蟹类过敏发病率也逐年攀升(农业部渔业局.2017中国渔业统计年鉴[M].北京:中国农业出版社,2017:38-40)。早在上个世纪，就已经有相关文献证明原肌球蛋白(Tropomyosin，TM)是蟹类中主要过敏原(Leung,PSC,et al.,Identification and molecular characterization of Charybdisferiatus tropomyosin,the major crab allergen.J.Allergy Clin.Immunol,1998,102(5):847-852)，但其氨基酸序列非常保守，能引起甲壳类、软体类及尘螨类的交叉过敏，同时TM也是一种热稳定性良好的盐溶性蛋白，耐胃液消化。
已经报道的过敏原消减方式，大多针对于纯化的过敏原，集中在实验室范围内大量研究，忽略了食品基质间的相互作用，不适于工业化的扩大生产。
发明内容
本发明的目的在于提供可消减加工后螃蟹过敏原致敏性的一种基于关键氨基酸定向修饰的低致敏性螃蟹加工方法。
本发明包括以下步骤：
1)螃蟹加盐前处理；
在步骤1)中，所述螃蟹加盐前处理的具体方法可为：鲜活螃蟹为原料，清除泥沙及污物，清水漂洗干净；蒸煮器中预先放入0.5％～2.0％食盐水，螃蟹于95～100℃煮制3～9min；煮制后的螃蟹经冰水漂洗，沥干；所述螃蟹可选用青蟹、梭子蟹等。
2)关键氨基酸定向修饰；
在步骤2)中，所述关键氨基酸定向修饰的具体方法可为：螃蟹于1.0％～5.0％还原糖中浸渍后放入压力锅，经压力处理，达到定向修饰螃蟹过敏原中抗原表位关键氨基酸的目的；所述还原糖的pH为6.5～8.5；所述浸渍的时间可为0.5～2.0h，螃蟹与糖水的配比可为1g ︰(10～20)mL，其中螃蟹以质量计算，糖水以体积计算；所述压力处理的条件可为在0.08 MPa下压力处理3～9min。
3)产品包装；
在步骤3)中，所述产品包装的方法可为：加工处理后的螃蟹，沿中轴线对开，真空包装灭菌，-20℃冻存，得冻蟹。
本发明采用复杂食品基质中过敏原关键氨基酸定向修饰加工，以消减过敏原的致敏性，具有一定的实用价值，可为进一步开发低致敏性制品提供参考。
本发明将螃蟹经不同方式的熟制加工后，提取粗蛋白，以SDS-PAGE电泳和DotBlot分析不同程度热加工后的粗蛋白，以兔抗TM多克隆抗体的结合活性为指标，根据免疫结合程度判断不同加工方式下螃蟹的致敏性，筛选出获得低致敏螃蟹的加工方式。
本发明的优点是：利用简单的抗原抗体结合反应，确定螃蟹过敏原经不同热加工后的致敏性变化情况，筛选出一种基于关键氨基酸定向修饰的低致敏性螃蟹加工方法，本发明工艺简单、可操作性强、易于实现规模化生产。
附图说明
图1为加工前后螃蟹产品实物图。在图1中，A为加工前生鲜蟹图，B为关键氨基酸定向修饰加工后螃蟹图，C为真空包装后螃蟹产品图。
图2为实施例2加工前后螃蟹过敏原的致敏性变化情况(聚丙烯酰胺凝胶电泳(SDS-PAGE))。在图2中，M：蛋白分子量标准；1：加工前的粗提蛋白；2：煮制6min的粗提蛋白；3：氨基酸定向修饰6min的粗提蛋白；4：压力处理6min的粗提蛋白；5：煮制 6min的水溶液；6：氨基酸定向修饰6min的水溶液；7：压力处理6min的水溶液。
图3为过敏原定向修饰的关键氨基酸位点图。在图3中，根据NCBI上的蟹过敏原TM的氨基酸序列，通过SWISS-MODEL软件生成其3D模拟图，再利用Pymal软件进行氨基酸的标识，其中箭头所示标识R255，R139，R101，R91，K49为还原糖修饰的氨基酸。
图4为点印迹法(Dot Blot)示意图。
具体实施方式
以下结合附图和具体实施例对本发明作进一步的说明。
实施例1
1)螃蟹加盐前处理：鲜活螃蟹(青蟹、梭子蟹)为原料，清除泥沙及污物，清水漂洗干净；蒸煮器中预先放入0.5％食盐水，螃蟹于95～100℃煮制3min；煮制后的螃蟹经冰水漂洗，沥干。
2)关键氨基酸定向修饰：螃蟹于1.0％还原糖(pH 6.5)中浸渍0.5h；螃蟹与糖水之比为 1︰20(w:v)；浸渍后的螃蟹放入压力锅，经0.08MPa压力处理3min，达到定向修饰螃蟹过敏原中抗原表位关键氨基酸的目的。
3)产品包装：加工处理后的螃蟹，沿中轴线对开，真空包装灭菌，-20℃冻存，得冻蟹。
实施例2
1)螃蟹加盐前处理：鲜活螃蟹(青蟹、梭子蟹)为原料，清除泥沙及污物，清水漂洗干净；蒸煮器中预先放入1.0％食盐水，螃蟹于95～100℃煮制6min；煮制后的螃蟹经冰水漂洗，沥干。
2)关键氨基酸定向修饰：螃蟹于3.0％还原糖(pH 7.5)中浸渍1.0h；螃蟹与糖水之比为 1︰15(w:v)；浸渍后的螃蟹放入压力锅，经0.08MPa压力处理6min，达到定向修饰螃蟹过敏原中抗原表位关键氨基酸的目的。
3)产品包装：加工处理后的螃蟹，沿中轴线对开，真空包装灭菌，-20℃冻存，得冻蟹。
实施例3
1)螃蟹加盐前处理：鲜活螃蟹(青蟹、梭子蟹)为原料，清除泥沙及污物，清水漂洗干净；蒸煮器中预先放入2.0％食盐水，螃蟹于95～100℃煮制9min；煮制后的螃蟹经冰水漂洗，沥干。
2)关键氨基酸定向修饰：螃蟹于5.0％还原糖(pH 8.5)中浸渍2.0h；螃蟹与糖水之比为 1︰10(w:v)；浸渍后的螃蟹放入压力锅，经0.08MPa压力处理9min，达到定向修饰螃蟹过敏原中抗原表位关键氨基酸的目的。
3)产品包装：加工处理后的螃蟹，沿中轴线对开，真空包装灭菌，-20℃冻存，得冻蟹。
实施例4加工前后螃蟹的免疫结合活性检测(以实施例1～3的成品作为检测对象)，它包括以下步骤：
1.SDS-PAGE检测加工前后的蛋白变化情况
(1)样品的SDS化：测定粗蛋白及水溶液中蛋白含量，调整样品至相同浓度(2mg/mL)，与适量的4×SDS Loading buffer混匀。
(2)电泳过程：考虑到样品为粗蛋白，分子量大小不等，本发明中所用到的聚丙烯酰胺凝胶浓度为15％，电流10mA，电泳时间1.5h。
(3)成像过程：电泳结束后，切去多余梳子部分，用考马斯亮蓝R-250对凝胶染色，染至深蓝色，后换用适量脱色液，脱色至显示出清晰的蓝色蛋白条带，凝胶成像仪拍照保存。
2.采用Dot Blot检测产品的免疫结合活性
(1)蛋白点样：将硝酸纤维素膜(NC膜)分为长宽相等的正方形小格(0.7cm×0.7cm)，调整蛋白至相同浓度(2mg/mL)，每个样品2μL直接点样于NC膜，室温晾干。
(2)一抗孵育：晾干后经5％脱脂奶于摇床上封闭1.5h后的NC膜，经TBS-T漂洗3次×5 min，加入兔抗青蟹TM一抗(1︰100 000稀释)于室温孵育1.0h。
(3)二抗孵育：一抗孵育结束后的NC膜，用羊抗兔特异性IgG(1︰20 000)室温孵育1.0 h。
(4)ECL显色：二抗孵育后，将漂洗后的NC膜与ECL显色液反应2min后，显色拍照。
(5)结果分析：结果如图1～3所示，经关键氨基酸定向修饰后的螃蟹，其粗蛋白中耐热过敏原TM的免疫结合活性显著低于未加工螃蟹和仅经过压力处理后的螃蟹。表明本发明中所用的关键氨基酸定向修饰的加工方法可使螃蟹肌肉致敏性明显消减。
表1
点印迹法(Dot Blot)如图4所示，在图4中，一抗为兔抗蟹TM，二抗为羊抗兔。存留条带的灰度分析如表1所示。
Claims (9)
1.一种基于关键氨基酸定向修饰的低致敏性螃蟹加工方法，其特征在于包括以下步骤：
1)螃蟹加盐前处理；
2)关键氨基酸定向修饰；
3)产品包装。
2.如权利要求1所述一种基于关键氨基酸定向修饰的低致敏性螃蟹加工方法，其特征在于在步骤1)中，所述螃蟹加盐前处理的具体方法为：鲜活螃蟹为原料，清除泥沙及污物，清水漂洗干净；蒸煮器中预先放入0.5％～2.0％食盐水，螃蟹于95～100℃煮制3～9min；煮制后的螃蟹经冰水漂洗，沥干。
3.如权利要求2所述一种基于关键氨基酸定向修饰的低致敏性螃蟹加工方法，其特征在于所述螃蟹选用青蟹、梭子蟹。
4.如权利要求1所述一种基于关键氨基酸定向修饰的低致敏性螃蟹加工方法，其特征在于在步骤2)中，所述关键氨基酸定向修饰的具体方法为：螃蟹于1.0％～5.0％还原糖中浸渍后放入压力锅，经压力处理，达到定向修饰螃蟹过敏原中抗原表位关键氨基酸的目的。
5.如权利要求4所述一种基于关键氨基酸定向修饰的低致敏性螃蟹加工方法，其特征在于所述还原糖的pH为6.5～8.5。
6.如权利要求4所述一种基于关键氨基酸定向修饰的低致敏性螃蟹加工方法，其特征在于所述浸渍的时间为0.5～2.0h。
7.如权利要求4所述一种基于关键氨基酸定向修饰的低致敏性螃蟹加工方法，其特征在于所述螃蟹与糖水的配比为1g︰(10～20)mL，其中螃蟹以质量计算，糖水以体积计算。
8.如权利要求4所述一种基于关键氨基酸定向修饰的低致敏性螃蟹加工方法，其特征在于所述压力处理的条件为在0.08MPa下压力处理3～9min。
9.如权利要求1所述一种基于关键氨基酸定向修饰的低致敏性螃蟹加工方法，其特征在于在步骤3)中，所述产品包装的方法为：加工处理后的螃蟹，沿中轴线对开，真空包装灭菌，-20℃冻存，得冻蟹。
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