CN109221973A - 一种低致敏性鱼肉干的制备方法 - Google Patents
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Abstract
一种低致敏性鱼肉干的制备方法，涉及鱼肉干。鱼肉的前处理；过敏原抗原表位定点改性；鱼肉烘干包装。采用预煮结合盐糖水浸渍对鱼肉进行处理，通过高温使得还原糖对过敏原抗原表位进行定向修饰降低其致敏性，并将产品进行隔水压力处理和烘干包装。在鱼肉干的生产加工过程中采用煮制处理，还原糖修饰结合隔水压力处理，显著降低鱼肉干产品的致敏性，且具有操作简便、易实现规模化生产等特点。
Description
技术领域
本发明涉及鱼肉干，尤其是涉及一种低致敏性鱼肉干的制备方法。
背景技术
中国是亚洲最大的水产品捕捞及生产国，近年来鱼类养殖和捕捞量持续上升。近海中上层鱼，包括金色小沙丁鱼(鰛仔)、蓝圆鲹(巴浪鱼)、脂眼鲱、鲐鱼(花威)、颌圆鲹(竹叶巴浪)、竹荚鱼(大目鲭)、羽鳃鲐(白柄仔)、大甲鲹(铁甲)等，它们的鱼肉中富含蛋白质、多肽、多糖等营养物质，且其年产量大、加工生产成本低、市场需求量大，在食品产业开发中具广阔市场前景。
同时，相关调查显示食物过敏发病率呈现逐年增长的趋势，食物过敏已经上升为全球性公共卫生问题，成为食品安全问题中亟需防控的环节。早在上个世纪，WHO就已将鱼类为作为八大过敏性食物之一，研究表明，92.5％的鱼类过敏是由小清蛋白(Parvalbumin，PV)引起的，其症状包括过敏性休克等急性疾病及过敏性皮炎和嗜酸性肠胃病等慢性疾病(Sharp,MF,et al.Immunological cross-reactivity between four distantparvalbumins-impact on allergen detection and diagnostics.Molecul.Immunol,2015,63(2):437-448)。PV是一种钙结合蛋白，存在于脊椎动物肌肉骨骼细胞中，是肌浆蛋白的主要成分，在肌肉的舒张运动中起着重要作用，其分子量常见于10～14kDa。通常来讲，过敏原中决定抗原特异性的特殊氨基酸基团称为抗原表位，一般由20个以下的氨基酸残基组成。在过敏反应过程中，抗原表位与IgE特异性结合，促使机体产生过敏反应。美拉德反应又称非酶褐变，是蛋白质或氨基酸与还原糖在加热或长时间存放时发生的反应，可以使食品中的蛋白质变性，产生特殊的香气和色泽，是食品加工中一种重要反应，常见的还原糖有葡萄糖、果糖、半乳糖、麦芽糖等。由于其生产工艺成熟、价格低廉，且对人体无毒无害，已被广泛应用于食品的生产加工过程中。同时有研究表明，其可以改变食物的过敏性(Ma,XJ,etal.,Conformation affects the potential allergenicity of ovalbumin afterheating and glycation.Food Addit.Contam.A,2013,30(10):1684-1692)。
发明内容
本发明的目的是提供工艺简单、可操作性强、易实现规模化生产的一种低致敏性鱼肉干的制备方法。
本发明包括以下步骤：
1)鱼肉的前处理；
在步骤1)中，所述鱼肉的前处理的具体方法可为：选用新鲜或冷冻的鱼类为原料，去鱼鳞、鱼内脏、鱼头、鱼尾和鱼骨，洗净后的鱼肉蒸煮，经冰水漂洗，在盐糖水浸渍；所述鱼类可采用近海中上层鱼类；所述蒸煮可将洗净后的鱼肉放入蒸煮器中，95～100℃下煮制3～9min；所述盐糖水的温度可为0～4℃，所述盐糖水的组成可为1.0％～5.0％还原糖、0.5％～2.0％食盐，余量为水，pH 6.5～8.5；所述浸渍的时间可为0.5～2.0h；所述鱼肉与盐糖水的配比可为1g︰(5～10)mL，其中鱼肉以质量计算，盐糖水以体积计算。
2)过敏原抗原表位定点改性；
在步骤2)中，所述过敏原抗原表位定点改性的具体方法可为：经过浸渍的鱼肉放入压力锅内，还原糖修饰结合0.08MPa隔水压力处理3～9min，冰水漂洗。
3)鱼肉烘干包装；
在步骤3)中，所述鱼肉烘干包装可将鱼肉按规格分别放在容器内烘干，并进行真空包装。
本发明中采用预煮结合盐糖水浸渍对鱼肉进行处理，通过高温使得还原糖对过敏原抗原表位进行定向修饰降低其致敏性，并将产品进行隔水压力处理和烘干包装。
本发明的优点是：在鱼肉干的生产加工过程中采用煮制处理，还原糖修饰结合隔水压力处理，显著降低鱼肉干产品的致敏性，且具有操作简便、易实现规模化生产等特点。
附图说明
图1为低致敏性鱼肉干的实物图。在图1中，A为清洗后的鱼肉样品，B为真空包装的鱼肉干成品。
图2为实施例1～3加工前后鱼肉过敏原致敏性的变化。在图2中，A为聚丙烯酰胺凝胶电泳(SDS-PAGE)，B为点印迹法(Dot Blot)，一抗为兔抗鱼PV，二抗为羊抗兔；M：蛋白分子量标准；1：加工前的鱼肉粗提蛋白；2：煮制3min后的鱼肉上清；3：煮制6min后的鱼肉上清；4：煮制9min后的鱼肉上清；5：按实施例1方法加工后的鱼肉粗提蛋白；6：按实施例2方法加工后的鱼肉粗提蛋白；7：按实施例3方法加工后的鱼肉粗提蛋白；8：直接0.08MPa隔水压力处理6min后的鱼肉粗提蛋白。
图3为抗原表位上的氨基酸修饰示意图。在图3中，根据NCBI上的过敏原PV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，标识K55，K84，K88，K97，K108为还原糖修饰的在PV抗原表位上的关键氨基酸。
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)鱼肉的前处理：选用新鲜或冷冻的近海中上层鱼类为原料，去鳞、内脏、头、尾和骨，洗净后的鱼肉放入蒸煮器中，95～100℃煮制3min；煮制的鱼肉经冰水漂洗，于0～4℃下的盐糖水(1.0％还原糖、0.5％食盐，98.5％水，pH 6.5)浸渍2.0h，鱼肉与盐糖水的配比为1g︰10mL。
(2)过敏原抗原表位定点改性：经过浸渍的鱼肉放入压力锅，还原糖修饰结合0.08MPa隔水压力处理3min，冰水漂洗。
(3)鱼肉烘干包装：鱼肉按规格分别放在容器内烘干，并进行真空包装。
图1给出低致敏性鱼肉干的实物图。
实施例2：
(1)鱼肉的前处理：选用新鲜的或冷冻的近海中上层鱼类为原料，去鳞、内脏、头、尾和骨，洗净后的鱼肉放入蒸煮器中，95～100℃煮制6min；煮制的鱼肉经冰水漂洗，于0～4℃下的盐糖水(3.0％还原糖、1.2％食盐，95.8％水，pH 8.5)浸渍0.5h，鱼肉与盐糖水之比为1g︰7.5mL。
(2)过敏原抗原表位定点改性：经过浸渍的鱼肉放入压力锅，还原糖修饰结合0.08MPa隔水压力处理6min，冰水漂洗。
(3)鱼肉烘干包装：鱼肉按规格分别放在容器内烘干，并进行真空包装。
实施例3
(1)鱼肉的前处理：选用新鲜的或冷冻的近海中上层鱼类为原料，去鳞、内脏、头、尾和骨，洗净后的鱼肉放入蒸煮器中，95～100℃煮制9min；煮制的鱼肉经冰水漂洗，于0～4℃下的盐糖水(5.0％还原糖、2.0％食盐，93％水，pH 7.5)浸渍1.5h，鱼肉与盐糖水之比为1g︰5mL。
(2)过敏原抗原表位定点改性：经过浸渍的鱼肉放入压力锅，还原糖修饰结合0.08MPa隔水压力处理9min，冰水漂洗。
(3)鱼肉烘干包装：鱼肉按规格分别放在容器内烘干，并进行真空包装。
实施例4加工前后鱼肉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.粗蛋白的提取：
取鱼肉于磷酸盐缓冲液(pH 7.0)中，进行组织捣碎，离心后取上清留样，SDS化后用凝胶电泳检测蛋白变化，免疫印迹法测定致敏性变化。
2.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。
3.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(0.7×0.7cm)，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×5min，加入兔抗PV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)于室温下孵育1.0h。
(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，显色后用凝胶成像仪进行拍照。
4.结果分析
结果如图2所示，经关键表位定向修饰后的鱼肉，其粗蛋白中耐热过敏原PV的IgG免疫结合活性显著低于未加工的鱼肉中的PV。图3表明本发明中所用的还原糖进行定向修饰的氨基酸在其抗原表位上，解释了本加工方法可消减鱼肉中过敏原的致敏性。
Claims (10)
1.一种低致敏性鱼肉干的制备方法，其特征在于包括以下步骤：
1)鱼肉的前处理；
2)过敏原抗原表位定点改性；
3)鱼肉烘干包装。
2.如权利要求1所述一种低致敏性鱼肉干的制备方法，其特征在于在步骤1)中，所述鱼肉的前处理的具体方法为：选用新鲜或冷冻的鱼类为原料，去鱼鳞、鱼内脏、鱼头、鱼尾和鱼骨，洗净后的鱼肉蒸煮，经冰水漂洗，在盐糖水浸渍。
3.如权利要求2所述一种低致敏性鱼肉干的制备方法，其特征在于所述鱼类采用近海中上层鱼类。
4.如权利要求2所述一种低致敏性鱼肉干的制备方法，其特征在于所述蒸煮是将洗净后的鱼肉放入蒸煮器中，95～100℃下煮制3～9min。
5.如权利要求2所述一种低致敏性鱼肉干的制备方法，其特征在于所述盐糖水的温度为0～4℃。
6.如权利要求2所述一种低致敏性鱼肉干的制备方法，其特征在于所述盐糖水的组成为1.0％～5.0％还原糖、0.5％～2.0％食盐，余量为水，pH 6.5～8.5。
7.如权利要求2所述一种低致敏性鱼肉干的制备方法，其特征在于所述浸渍的时间为0.5～2.0h。
8.如权利要求2所述一种低致敏性鱼肉干的制备方法，其特征在于所述鱼肉与盐糖水的配比为1g︰(5～10)mL，其中鱼肉以质量计算，盐糖水以体积计算。
9.如权利要求1所述一种低致敏性鱼肉干的制备方法，其特征在于在步骤2)中，所述过敏原抗原表位定点改性的具体方法为：经过浸渍的鱼肉放入压力锅内，还原糖修饰结合0.08MPa隔水压力处理3～9min，冰水漂洗。
10.如权利要求1所述一种低致敏性鱼肉干的制备方法，其特征在于在步骤3)中，所述鱼肉烘干包装是将鱼肉按规格分别放在容器内烘干，并进行真空包装。
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