CN113481272A - 一种使用橄榄油和核桃油制备的改善记忆的制品 - Google Patents
一种使用橄榄油和核桃油制备的改善记忆的制品 Download PDFInfo
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Abstract
本发明提供一种使用橄榄油和核桃油制备的，具有改善记忆功能的制品，所提供的功能性食品制品，是将鱼籽肽添加到溶液中制备的；所述的鱼籽肽，是将大黄鱼的鱼籽先使用胰蛋白酶和胶原蛋白酶的复合酶进行酶解；酶解产物离心后，离心液用3500Da的膜分离，然后用360Da的膜分离制备的。本发明在制备的具有改善记忆功能的鱼籽肽的基础上，使用橄榄油和核桃油制成溶液，将鱼籽肽添加到溶液中制成可进行长期保存的制品。所提供的制品具有预防和改善记忆衰退的潜在功能。
Description
技术领域
本发明属于功能食品制备技术领域，具体涉及一种使用橄榄油和核桃油制备的改善记忆的制品。
背景技术
随着我国人口老龄化的发展，阿尔茨海默病已的发病率越来越高。截止2019年，我国有超过1000万名阿尔茨海默患者，是全球AD患者数量最多的国家。而且，目前人们的工作和生活节奏加快，压力增加导致长期处于应激状态中，导致大脑认知学习记忆能力下降。因此，提供具有改善提高学习记忆的生物制品就具有良好的市场前景。
再之前的研究中，对来自于海水鱼的鱼籽进行酶解，制备获得了具有改善记忆功能的鱼籽肽。但所制备的鱼籽肽还存在着成分不清楚，以及如何开发成能够上市销售的产品的问题。
发明内容
本发明提供一种使用橄榄油和核桃油制备的，具有改善记忆功能的制品，从而弥补现有技术的不足。
本发明提供的功能性食品制品，是将鱼籽肽添加到溶液中制备的；
所述的鱼籽肽，是将大黄鱼的鱼籽先使用胰蛋白酶和胶原蛋白酶的复合酶进行酶解；酶解产物离心后，离心液用3500Da的膜分离，然后用360Da的膜分离制备的。
所述的鱼籽肽在溶液中的浓度为10-50μg/ml。
所述的胰蛋白酶和胶原蛋白酶的酶活比为1:10；
所述的酶解条件，其一种是在35-55℃酶解3-5h；
所述的离心的转速为6000-12000rpm；
本发明所使用的鱼籽肽，其一种肽段的氨基酸序列为LYEFLLYMDNYF；
所述的溶液，其中包含有橄榄油和核桃油；
作为优选，所述的橄榄油在溶液中的体积百分比为1-5％；核桃油在油性溶液中的体积百分比为1-10％；
更进一步的，所述的溶液中还添加有用于提高口感或气味的、食品领域中常用的佐剂；
所述的佐剂，包含有甜味剂；例如蜂蜜。
本发明在制备的具有改善记忆功能的鱼籽肽的基础上，使用橄榄油和核桃油制成溶液，将鱼籽肽添加到溶液中制成可进行长期保存的制品。所提供的制品具有预防和改善记忆衰退的潜在功能。
附图说明
图1：本发明制备的鱼籽功能肽分子量分布图；
图2：反相高效液相色谱C18纯化肽段图。
具体实施方式
下面通过具体实施例对本发明进行详细的描述。
实施例1：制备具有改善记忆功能的鱼籽肽及肽段纯化测序
1、制备鱼籽肽
称取黄鱼干鱼籽100g，加入1000ml蒸馏水，利用高速剪切机剪切匀浆30min，高速剪切机转速为10000rpm。向制备的匀浆液中加入3.0g胰蛋白酶和胶原蛋白酶(酶活比1:10)，50℃酶解5h，边酶解边搅拌。酶解结束后加热至90℃灭酶，酶解液8000rpm离心后，上清液用3500Da和360Da膜过滤仪分离，得到的样品经浓缩至原体积的1/10，喷雾干燥得到49.8g鱼籽功能肽。
根据国标(GB/T 22729-2008)方法测定制备鱼籽功能肽的分子量，结果显示分子量小于2000Da的鱼籽功能肽占87.52％(图1)。
采用东莨菪碱致记忆障碍大鼠作为实验动物，水迷宫实验结果显示，使用制备的鱼籽肽的治疗组均能够提高东莨菪碱致记忆障碍大鼠的记忆能力。
2、肽段的纯化和测序
考虑到酶解制备的鱼籽肽是多肽混合物，同样浓度下，其改善记忆的功能相比于纯化的多肽明显偏低。为了提高活性，对制备的鱼籽肽进行纯化。
1)将鱼籽肽用Sephadex G-25凝胶柱进行分离纯化，洗脱液选用去离子水，洗脱速度为0.4ml/min，在220nm下收集洗脱峰。共分离到六个分离峰A-F，分别测定活性，收集活性最高的组分备用。
测定结果表明在相同蛋白浓度下峰D的活性最大，将峰D进行浓缩，进一步经过Sephadex C-25阳离子交换柱进行纯化。
2)峰D经过Sephadex C-25阳离子交换柱进行纯化，得到四个峰，其中第二个峰的水迷宫实验结果表明其具有最大的活性。
3)将第二个峰的样品进行脱盐，通过反相高效液相色谱C18半制备柱继续分离纯化，得到了活性肽P1(图2)；检测其IC50值为0.0512mg/ml。
纯化后的P1多肽使用电喷雾四级杆正交加速-飞行时间串联质谱仪进行测序，Q_TOF2配备钠升喷雾源。测定在离子方式下进行，进样流速为1μl/min，质谱仪Glu_fib串联质谱碎片校正。使用氮气作为雾化气体，氩气作为碰撞气体，检测温度为80℃，锥孔电压50V，毛细管电压3000v，TOF加速电压为9.1kv，MCP检测器电压为2200V。
采用Micromass的转用软件MaxEnt3，Biolynx及MasSeq对MS/MS谱进行分析；确定P1肽段的序列为LYEFLLYMDNYF，其分子量为1600Da。
3、检测P1肽段改善记忆的功能
将P1肽段通过上海生工进行合成后，合成的多肽用Ph7.0的PBS缓冲溶液配置成浓度为40μg/ml的P1肽段溶液，同时将制备的鱼籽肽也制备成浓度为40μg/μl的鱼籽肽溶液。
使用P1肽段溶液和鱼籽肽溶液对东莨菪碱致记忆障碍大鼠进行水迷宫行为学测试。在进行水迷宫训练前10h分别注射P1肽段溶液(0.5μl、1μl)和鱼籽肽溶液(1μl、1.5μl)；每组注射10只实验大鼠。同时选用10只东莨菪碱致记忆障碍大鼠，注射PBS缓冲液作为对照组。
连续注射训练5天，在训练结束后进行水迷宫行为测试实验，统计不同注射组的逃避潜伏期和跨原平台次数。
结果显示，注射了P1肽段溶液(0.5μl、1μl)大鼠，相比于鱼籽肽溶液实验组，表现为逃避潜伏期明显缩短；跨越平台的次数增加(P<0.01)，表明纯化的单一多肽的P1肽段的治疗效果明显好于多肽段混合的鱼籽肽溶液组(表2)。
表1：P1肽段溶液和鱼籽肽溶液对AD大鼠逃避潜伏期和跨越平台数统计表
实施例2:制备功能性液体制品
将实施例1中制备的P1肽和/或鱼籽肽添加到溶液中，制成具有改善记忆功能的制品。所述的溶液，其中包含有橄榄油和核桃油；
其中一种具体的制品的制备过程如下：
1)取橄榄油2份、核桃油3份、食品用乳化剂10份、水86份进行混合后搅拌制成溶液；
2)将P1肽和/或鱼籽肽添加到溶液中，添加浓度为10-50μg/ml。
更进一步的，所述的溶液中还添加有用于提高口感或气味的食品领域中常用的佐剂；如甜味剂。
将制备的溶液(其中添加有10μg/ml的P1肽)每天饲喂东莨菪碱致记忆障碍大鼠，进行跳台实验。学习成绩实验中，投喂溶液组与对照组比较，大鼠的反应时间均缩短，错误次数均减少，差异具有统计学意义(P<0.01)。实验结果使用本发明的P1肽段和鱼籽肽制备的溶液具有提高早老性痴呆大鼠跳台记忆成绩的效果，具有潜在开发成用于改善记忆功效的功能制品的潜力。
Claims (10)
1.一种功能性制品，其特征在于，所述的制品是将鱼籽肽添加到溶液中制备的；所述的鱼籽肽，是将大黄鱼的鱼籽先使用胰蛋白酶和胶原蛋白酶的复合酶进行酶解；酶解产物离心后，离心液用3500Da的膜分离，然后用360Da的膜分离制备的。
2.如权利要求1所述的功能性制品，其特征在于，所述的鱼籽肽在溶液中的浓度为10-50μg/ml。
3.如权利要求1所述的功能性制品，其特征在于，所述的胰蛋白酶和胶原蛋白酶的酶活比为1:10。
4.如权利要求1所述的功能性制品，其特征在于，所述的酶解，是在35-55℃酶解3-5h。
5.如权利要求1所述的功能性制品，其特征在于，所述的离心的转速为6000-12000rpm。
6.如权利要求1所述的功能性制品，其特征在于，所述的鱼籽肽中一个肽段的氨基酸序列为LYEFLLYMDNYF。
7.如权利要求1所述的功能性制品，其特征在于，所述的溶液中包含有橄榄油和核桃油。
8.如权利要求7所述的功能性制品，其特征在于，所述的橄榄油在溶液中的体积百分比为1-5％；核桃油在油性溶液中的体积百分比为1-10％；。
9.如权利要求1所述的功能性制品，其特征在于，所述的溶液中还添加有食品领域中常用的佐剂。
10.如权利要求9所述的功能性制品，其特征在于，所述的佐剂包含有甜味剂。
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