CN108576502A - 利用鳀鱼皮中活性多肽制备的复配物的方法 - Google Patents
利用鳀鱼皮中活性多肽制备的复配物的方法 Download PDFInfo
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Abstract
本发明公开一种利用鳀鱼皮中活性多肽制备的复配物的方法，包括：鳀鱼活性多肽制备，复配物制备和检验保存，鳀鱼皮中活性多肽制备步骤为：取新鲜鳀鱼，取鳀鱼皮洗净，匀浆，于‑20℃至‑30℃冷藏备用；解冻并将匀浆后的鳀鱼皮按固液比4‑6:1加入去离子水，于42‑50℃条件下恒温搅拌，调pH值至6.8‑7.2，加入酶制剂，酶解4‑6h，酶解过程中在酶解液中通入氮气；灭酶处理，调pH至中性，离心，取上清液，得鳀鱼活性多肽，通过本发明的方法制备的复配物能消除胀、健脾益胃，成分中芳香性氨基酸含量高，复配物还具有对自由基清除的效果。
Description
技术领域
本发明属于饮料制备技术领域，具体涉及一种利用鳀鱼皮中活性多肽制备的复配物的方法。
背景技术
随着社会发展的加速，生活节奏的加快，人们经常因生活作息或包含不正常引起上火，短时间内的工作压力大、睡眠不足时，也会出现火气大的现象，随着全球变暖，夏季的温度越来越高，许多人不同出现不同程度的消化不良、食积气滞、脘腹胀满等症状，而目前市场上的饮料虽然种类繁多，但是具有消食除胀、健脾益胃补肾并能预防肿瘤的的饮料却并不多，无法满足人们的需要。
发明内容
本发明的目的在于提供一种利用鳀鱼皮中活性多肽制备的复配物的方法，制备的复配物能消除胀、健脾益胃，成分中芳香性氨基酸含量高，复配物还具有对自由基清除的效果。
本发明为实现上述目的所采取的技术方案为：利用鳀鱼皮中活性多肽制备的复配物的方法，包括：鳀鱼活性多肽制备，复配物制备和检验保存，鳀鱼皮中活性多肽制备步骤为：取新鲜鳀鱼，取鳀鱼皮洗净，匀浆，于-20℃至-30℃冷藏备用；解冻并将匀浆后的鳀鱼皮按固液比4-6:1加入去离子水，于42-50℃条件下恒温搅拌，调pH值至6.8-7.2，加入酶制剂，酶解4-6h，酶解过程中在酶解液中通入氮气；灭酶处理，调pH至中性，离心，取上清液，得鳀鱼活性多肽，上述方法通过酶解法提取鳀鱼活性多肽，反应条件温和、反应效率高，不存在逆反应，提取的鳀鱼活性多肽分子量在2000-3000Da之间，由天门冬氨酸、苏氨酸、谷氨酸、甘氨酸、丙氨酸、异亮氨酸、酪氨酸、苯丙氨酸、组氨酸、赖氨酸和精氨酸等十一种氨基酸组成，具有很好的体外抗氧化能力，经试验发现，提取的鳀鱼活性多肽能有效提高血清及组织的 SOD 活性、GSH-Px活性和T-AOC 值。
优选的，去离子水中含有0.1-0.2wt%的L-苯基丁氨酸和D-苯基丁氨酸的混合体，L-苯基丁氨酸和D-苯基丁氨酸重量比为53~58:0.3~0.5，通过在去离子水中加入的混合体可使鳀鱼细胞膜下形成小孔，致使细胞内的物质泄漏，提高活性多肽的得率，并且避免了破碎处理过程中需因升温及机械力引起的蛋白质变性。
优选的，通入氮气操作为：每隔30-40min在酶解液中通入氮气，通入量为20-30g/h，通入的氮气可使鳀鱼活性多肽结构中的芳香族氨基酸的酚腔基基团能够作为有效电子供体，提高制备的鳀鱼活性多肽中的芳香性氨基酸含量，增强鳀鱼活性多肽的抗氧化效果。
优选的，酶制剂由以下成分及重量份组成：中性蛋白酶4-8份、木瓜蛋白酶5-7份和胰蛋白酶1-2份，利用生物酶催化的酶促反应所具有的专一性特点，选择中性蛋白酶、木瓜蛋白酶和胰蛋白酶，将鳀鱼蛋白分解为多肽，提高多肽得率和多肽中的氨基酸数量。
优选的，灭酶处理步骤为：将酶解液置于98-100℃条件下加热3-6min，终止酶解反应，防止酶解反应过度。
优选的，复配物的制备过程为：按重量份计取：苦荞、陈皮、莱菔子、红枣、火龙果皮、蛇皮、黄芪，置于容器中加入蒸馏水，文火煎煮2-3h，冷却，过滤，在滤液中加入蜂蜜、苹果汁和鳀鱼活性多肽常温搅拌3-5h，搅拌速度为70-80r/min，巴氏灭菌，装瓶，通过煎煮的方式将选用的原材料中的有效成分溶于蒸馏水中，并将蜂蜜、苹果汁和鳀鱼活性多肽通过常温搅拌的方式加入药液中配置成复配物，便于人们饮用，改善制备的复配物口感和香味同时避免蜂蜜、苹果汁和鳀鱼活性多肽受高温效果被破坏。
优选的，复配物由以下成分及重量份组成：苦荞12-15份、陈皮3-6份、莱菔子2-4份、红枣20-35份、火龙果皮10-15份、蛇皮1-3份、黄芪3-5份、蒸馏水80-160份、蜂蜜5-14份、苹果汁3-7份、鳀鱼活性多肽5-10份，上述复配物具有润肠通便、降火解毒、消食除胀、健脾益胃、明目的功效，同时通过添加鳀鱼活性多肽使复配物具有很好的抗氧化性能，鳀鱼活性多肽中的芳香性氨基酸含量很高，有效提高复配物对DPPH自由基的清除能力，使复配物具有一定的预防肿瘤的作用。
优选的，复配物保存温度为2-5℃，通过低温保存有效保证鳀鱼活性多肽的活性，利于保持复配物的口感及活性多肽及复配物其余成分在人体肠胃中的吸收效果。
与现有技术相比，本发明的有益效果为：选用以红枣为主原料制备复配物并通过添加中草药成分丰富复配物功效再配合蜂蜜、苹果汁、鳀鱼活性多肽等成分进一步丰富复配物的口感，同时避免使用白糖，制备所得的复配物具有润肠通便、降火解毒、消食除胀、健脾益胃、明目的功效，同时通过添加鳀鱼活性多肽使复配物具有很好的抗氧化性能，鳀鱼活性多肽中的芳香性氨基酸含量很高，有效提高复配物对DPPH自由基的清除能力，人们在长期饮用复配物的过程中，复配物可促进人体未活化的淋巴细胞增殖，起到预防肿瘤的效果，很适合老人、年轻人、儿童饮用。
附图说明
图1：实施例4中不同鳀鱼肽剂量对DPPH自由基的清除率折线图；
图2：实施例4中各组小鼠组织SOD活性统计图。
具体实施方式
以下结合实施例对本发明作进一步详细描述：
实施例1：
利用鳀鱼皮中活性多肽制备的复配物的方法，包括：鳀鱼活性多肽制备，复配物制备和检验保存，具体步骤如下：
1）鳀鱼皮中活性多肽制备：取新鲜鳀鱼，取鳀鱼皮洗净，匀浆，于-24℃冷藏备用；解冻并将匀浆后的鳀鱼皮按固液比4.5:1加入去离子水，去离子水中含有0.14wt%的L-苯基丁氨酸和D-苯基丁氨酸的混合体，L-苯基丁氨酸和D-苯基丁氨酸重量比为55:0.4，于47℃条件下恒温搅拌，调pH值至6.9，加入酶制剂，酶制剂由以下成分及重量份组成：中性蛋白酶7份、木瓜蛋白酶5份和胰蛋白酶1份，酶解4h，酶解过程中在酶解液中通入氮气，每隔34min在酶解液中通入氮气，通入量为25g/h，将酶解液置于98℃条件下加热4min，调pH至中性，于4℃条件下9000r/min离心12min，取上清液，得鳀鱼活性多肽；
2）复配物的制备：按重量份计取：苦荞13份、陈皮4份、莱菔子3份、红枣27份、火龙果皮14份、蛇皮2份、黄芪4份，置于容器中加入蒸馏水100份，文火煎煮3h，冷却，过滤，在滤液中加入蜂蜜10份、苹果汁5份和鳀鱼活性多肽6份常温搅拌4h，搅拌速度为76r/min，巴氏灭菌，装瓶；
3）检验及保存：检查瓶体有无破裂等异常现象，贴上标签后将复配物于4℃低温保存。
实施例2：
利用鳀鱼皮中活性多肽制备的复配物的方法，包括：鳀鱼活性多肽制备，复配物制备和检验保存，具体优选步骤如下：
1）鳀鱼皮中活性多肽制备：取新鲜鳀鱼，取鳀鱼皮洗净，匀浆，于-28℃冷藏备用；解冻并将匀浆后的鳀鱼皮按固液比5:1加入去离子水，去离子水中含有0.1wt%的L-苯基丁氨酸和D-苯基丁氨酸的混合体，L-苯基丁氨酸和D-苯基丁氨酸重量比为58:0.5，于48℃条件下恒温搅拌，调pH值至7.0，加入酶制剂，酶制剂由以下成分及重量份组成：中性蛋白酶8份、木瓜蛋白酶6份和胰蛋白酶1份，酶解5h，酶解过程中在酶解液中通入氮气，每隔38min在酶解液中通入氮气，通入量为27g/h，将酶解液置于98℃条件下加热4min，调pH至中性，于4℃条件下8000r/min离心12min，取上清液，得鳀鱼活性多肽；
2）复配物的制备：按重量份计取：苦荞13份、陈皮4份、莱菔子3份、红枣30份、火龙果皮12份、蛇皮2份、黄芪4份，置于容器中加入蒸馏水120份，文火煎煮2.5h，冷却，过滤，在滤液中加入蜂蜜5份、苹果汁7份和鳀鱼活性多肽10份常温搅拌5h，搅拌速度为80r/min，巴氏灭菌，装瓶；
3）检验及保存：检查瓶体有无破裂等异常现象，贴上标签后将复配物于5℃低温保存。
实施例3：
利用鳀鱼皮中活性多肽制备的复配物的方法，包括：鳀鱼活性多肽制备，复配物制备和检验保存，具体步骤如下：
1）鳀鱼皮中活性多肽制备：取新鲜鳀鱼，取鳀鱼皮洗净，匀浆，于-20℃冷藏备用；解冻并将匀浆后的鳀鱼皮按固液比4:1加入去离子水，去离子水中含有0.2wt%的L-苯基丁氨酸和D-苯基丁氨酸的混合体，L-苯基丁氨酸和D-苯基丁氨酸重量比为55:0.3，于47℃条件下恒温搅拌，调pH值至7.2，加入酶制剂，酶制剂由以下成分及重量份组成：中性蛋白酶6份、木瓜蛋白酶5份和胰蛋白酶1份，酶解6h，酶解过程中在酶解液中通入氮气，每隔34min在酶解液中通入氮气，通入量为28g/h，将酶解液置于100℃条件下加热4min，调pH至中性，于4℃条件下10000r/min离心14min，取上清液，得鳀鱼活性多肽；
2）复配物的制备：按重量份计取：苦荞14份、陈皮4份、莱菔子3份、红枣28份、火龙果皮14份、蛇皮2份、黄芪4份，置于容器中加入蒸馏水120，文火煎煮3h，冷却，过滤，在滤液中加入蜂蜜14份、苹果汁5份和鳀鱼活性多肽8份常温搅拌4h，搅拌速度为70r/min，巴氏灭菌，装瓶；
3）检验及保存：检查瓶体有无破裂等异常现象，贴上标签后将复配物于3℃低温保存。
实施例4：
对本发明实施例2提取的鳀鱼活性多肽进行试验：试验所用试剂均购买于北京协和生物工程研究所。
试验共选取普通级昆明种小鼠60只，体质量 18~22g，小鼠在试验开始之前适应性喂养一周，自由饮水和进食，一周后，60只小鼠被随机分成3组：1组空白组、1组鳀鱼肽高剂量组（实验组1）、1组鳀鱼肽低剂量组（实验组2），每组20只小鼠，在4周的试验过程中，每天下午4点至5点，实验组1 组的小鼠被灌胃5 mg/ g·d）的鳀鱼多肽，实验组2 组的小鼠被灌胃 0.5 mg/（g·d）的鳀鱼多肽，而空白组的小鼠则被灌胃蒸馏水，在灌胃后，小鼠被允许休息30 min，然后进行游泳训练，第 1 周游泳训练时间为20 min，以后每周增加5 min；游泳池的尺寸为50 cm×50 cm×50 cm，水深为40 cm，水温保持在（25±1）℃，用玻璃棒不停地搅动池中的水以使小鼠不停地游动. 当小鼠头部沉入水中后7s仍不能正常地把头伸出水面，则认为此时小鼠已进入力竭状态，记录从小鼠开始游泳到力竭状态的时间，该时间为力竭游泳时间。
当训练的小鼠在小游泳池中游泳 30 min，然后捞出，眼眶取血，测量血液中血糖（GLU）含量、血乳酸（LA）含量和血清尿素氮（BUN）的含量，以及肌酸激酶（CK）和乳酸脱氢酶（LDH）的活性，并且在试验第28天禁食空腹过夜，称质量后眼眶采血，低温离心分离血清（4℃，6 000r/min，10 min），-80 ℃保存待测，取血后的小鼠颈椎脱臼处死，用冰生理盐水洗去心、肝、肾上面的浮血，用滤纸吸干，称质量后用液氮迅速冷冻，以0.2 mol/L、pH 7.4 的PBS 缓冲液做匀浆介质在低温下将各器官组织按照 1∶9（m/V）匀浆，于4 ℃，6 000 r/min离心 10 min，取上清， -80 ℃保存，测定不同鳀鱼肽剂量对DPPH自由基的清除率，如图1所示，测定各组小鼠组织SOD活性，如图2所示。
上述实施例中的常规操作为本领域技术人员所熟知，在此不进行赘述。
以上所述的实施例对本发明的技术方案进行了详细说明，应理解的是以上所述仅为本发明的具体实施例，并不用于限制本发明，凡在本发明的原则范围内所做的任何修改、补充或类似方式替代等，均应包含在本发明的保护范围之内。
Claims (8)
1.利用鳀鱼皮中活性多肽制备的复配物的方法，包括：鳀鱼活性多肽制备，复配物制备和检验保存，其特征在于：鳀鱼皮中活性多肽制备步骤为：取新鲜鳀鱼，取鳀鱼皮洗净，匀浆，于-20℃至-30℃冷藏备用；解冻并将匀浆后的鳀鱼皮按固液比4-6:1加入去离子水，于42-50℃条件下恒温搅拌，调pH值至6.8-7.2，加入酶制剂，酶解4-6h，酶解过程中在酶解液中通入氮气；灭酶处理，调pH至中性，离心，取上清液，得鳀鱼活性多肽。
2.根据权利要求1所述的利用鳀鱼皮中活性多肽制备的复配物的方法，其特征在于：所述去离子水中含有0.1-0.2wt%的L-苯基丁氨酸和D-苯基丁氨酸的混合体，L-苯基丁氨酸和D-苯基丁氨酸重量比为53~58:0.3~0.5。
3.根据权利要求1所述的利用鳀鱼皮中活性多肽制备的复配物的方法，其特征在于：所述通入氮气操作为：每隔30-40min在酶解液中通入氮气，通入量为20-30g/h。
4.根据权利要求1所述的利用鳀鱼皮中活性多肽制备的复配物的方法，其特征在于：所述酶制剂由以下成分及重量份组成：中性蛋白酶4-8份、木瓜蛋白酶5-7份和胰蛋白酶1-2份。
5.根据权利要求1所述的利用鳀鱼皮中活性多肽制备的复配物的方法，其特征在于：所述灭酶处理步骤为：将酶解液置于98-100℃条件下加热3-6min。
6.根据权利要求1所述的利用鳀鱼皮中活性多肽制备的复配物的方法，其特征在于：所述复配物的制备过程为：按重量份计取：苦荞、陈皮、莱菔子、红枣、火龙果皮、蛇皮、黄芪，置于容器中加入蒸馏水，文火煎煮2-3h，冷却，过滤，在滤液中加入蜂蜜、苹果汁和鳀鱼活性多肽常温搅拌3-5h，搅拌速度为70-80r/min，巴氏灭菌，装瓶。
7.根据权利要求6所述的利用鳀鱼皮中活性多肽制备的复配物的方法，其特征在于：所述复配物由以下成分及重量份组成：苦荞12-15份、陈皮3-6份、莱菔子2-4份、红枣20-35份、火龙果皮10-15份、蛇皮1-3份、黄芪3-5份、蒸馏水80-160份、蜂蜜5-14份、苹果汁3-7份、鳀鱼活性多肽5-10份。
8.根据权利要求1所述的利用鳀鱼皮中活性多肽制备的复配物的方法，其特征在于：所述复配物保存温度为2-5℃。
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