CN111455007A - 一种从鱼籽中制备的改善记忆功能的多肽 - Google Patents
一种从鱼籽中制备的改善记忆功能的多肽 Download PDFInfo
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Abstract
本发明提供一种从鱼籽中制备的,具有改善记忆功能的多肽;是采用海洋鱼籽作为原料制备的,能够改善记忆的功能肽。本发明所提供的功能肽,是将鱼籽进行匀浆,得到匀浆液;匀浆液中加入胰蛋白酶和胶原蛋白酶进行酶解,得到酶解液,离心取上清液;上清液进行膜过滤,过滤液即为鱼籽功能肽溶液。本发明制备具有改善记忆功能的鱼籽肽,制备方法步骤简单,可操作性强,产量高,易于工业化生产;得到的产品改善记忆功能明显。
Description
技术领域
本发明属于天然产物提取制备技术领域,具体涉及一种从鱼籽中制备的改善记忆功能的多肽。
背景技术
我国是水产品加工的大国,在鱼类加工过程中,产生了大量的鱼类副产物如鱼鳞、鱼头、内脏和鱼籽。这些副产物主要用于生产鱼油、鱼粉、肥料、宠物食品和鱼饲料。据报道,海洋中的副产物是保健品和功能性食品配料最好的来源。目前,有关鱼皮和内脏等的高值化利用较多,而鱼籽的利用报道很少。
鱼籽是鱼类加工业中的一种非常重要的副产物,富含高品质的必须氨基酸和脂肪酸。据报道,鱼籽中蛋白质约占其干重的50-85%,脂肪约占干重的8-32%。鱼籽多肽是鱼籽蛋白经蛋白酶水解后得到的多肽混合物。如果仅以蛋白质或多肽作为开发鱼籽的用途,其营养价值和经济价值有效,无法达到鱼籽资源的高值利用,因此亟需开发鱼籽多肽的功能,提高其营养价值和经济价值。
发明内容
本发明的目的是提供一种从鱼籽中制备的,具有改善记忆功能的多肽;是采用海洋鱼籽作为原料制备的,能够改善记忆的功能肽。
本发明所提供的功能肽,其制备方法如下:
1)匀浆:将鱼籽进行匀浆,得到匀浆液;
所述的鱼籽为海水鱼的鱼籽;
作为实施例的具体记载,所述的鱼籽为黄鱼的鱼籽或鮟鱇鱼的鱼籽;
2)酶解:向步骤1)的匀浆液中加入胰蛋白酶和胶原蛋白酶进行酶解,得到酶解液,离心取上清液;
其中胰蛋白酶和胶原蛋白酶在匀浆液的加入量比为1:50—5:1,但不局限在这个比例范围内;
作为优选,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;
作为实施例的一种具体记载,是在50℃酶解5h;
所述的离心的转速为6000-12000rpm;
3)膜过滤:对步骤2)制备的上清液进行膜过滤,过滤液即为鱼籽功能肽溶液。
所述的步骤1)中匀浆的条件如下:高速剪切机转速为8000-12000rpm,剪切时间为30min;
上述步骤3)中膜分离是先用3500Da的膜分离,然后用360Da的膜分离。
本发明所提供的功能肽用于制备用于改善记忆的制品。
本发明制备具有改善记忆功能的鱼籽肽,制备方法步骤简单,可操作性强,产量高,易于工业化生产;得到的产品改善记忆功能明显。
附图说明
图1:分子量的标准曲线图;
图2:本发明制备的鱼籽功能肽分子量分布图。
具体实施方式
本发明中所涉及的名词,解释如下:
多肽:是指分子量在50-2000Da的含有酰胺键的化合物和蛋白质。
胰蛋白酶:为蛋白酶的一种,EC 3.4.4.4,是从牛、羊、猪的胰脏提取的一种丝氨酸蛋白水解酶。本发明中使用的是GB2760-2014表中规定的胰蛋白酶。
胶原蛋白酶:是一类专门酶解胶原蛋白的水解酶。本发明中使用的是GB2760-2014表中规定的胶原蛋白酶。
东莨菪碱致记忆障碍大鼠:是利用东莨菪碱抑制神经递质传导,诱导大鼠神经细胞受损,造成记忆障碍大鼠,用于鱼籽功能肽的功效筛选。
通过以下具体实施例对本发明做进一步的具体说明。
实施例1:复合酶制备的鱼籽多肽
1)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,向上述匀浆液中分别加入表1中所记载的组合酶,50℃酶解5h,每个30min搅拌一次,酶解结束后加热至90℃灭酶,酶解液8000rpm离心后10min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,干燥得到鱼籽多肽。
2)筛选具有改善记忆力的鱼籽多肽
上述结果中得到的不同鱼籽多肽,利用东莨菪碱致记忆障碍大鼠逃避潜伏期实验进行筛选,如表1所示,实验结果显示,由胰蛋白酶和胶原蛋白酶酶解得到的鱼籽多肽,具有明显改善东莨菪碱致记忆障碍,其功能明显优于其他酶解方法得到的鱼籽多肽。
表1:鱼籽多肽对AD大鼠遇避潜伏期的影响数据表
注:与模型组(东莨菪碱)比较,**P<0.01,*P<0.05。
最终确定使用胰蛋白酶和胶原蛋白酶和酶解鱼籽来制备具有改善记忆功能的多肽。
实施例2
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,向上述匀浆液中加入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。
实施例3
称取海洋来源的青鱼干鱼籽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,向上述匀浆液中加入5.0g胰蛋白酶和胶原蛋白酶(酶活比1:10),50℃酶解3h,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1/10,干燥得到43.46g鱼籽功能肽。
实施例4
称取鮟鱇鱼干鱼籽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8000-12000rpm,向上述匀浆液中加入5.0g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,干燥得到45.56g鱼籽功能肽。
实施例5:鱼籽功能肽的研究
一、鱼籽功能肽的分子量分析
根据国标(GB/T 22729-2008)方法测定实施例2中制备鱼籽功能肽的分子量,如图1所示,利用细胞色素C(12384Da),抑肽酶(6512Da),杆菌酶(1423Da),乙-乙-酪-精氨酸(451Da),乙-乙-乙氨酸(189Da)为标准品,建立标准曲线,得到保留时间和分子量对数值的标准曲线,方程式为y=-0.2678x+7.4913(R2=0.974)。按照标准曲线的方法,分析鱼籽功能肽的分子量分布,如图2所示,分子量小于2000Da的鱼籽功能肽占87.52%。
二、鱼籽功能肽改善记忆功能的检测
2.1鱼籽功能肽对东莨菪碱致记忆障碍大鼠逃避潜伏期和跨原平台次数的影响
水迷宫实验结果显示,模型组大鼠逃避潜伏期较对照组均明显延长,鱼籽功能肽高剂量组与模型组比较,逃避潜伏期显著小于模型组(P<0.01),中剂量组逃避潜伏期小于模型组(P<0.05)。实验第5天去掉平台,观察各组大鼠的空间探索能力,结果显示,模型组大鼠与对照组比较,跨越平台的次数显著降低(P<01),表明学习记忆能力衰退;与模型组比较,鱼籽功能肽高剂量组跨越平台的次数增加(P<0.01),中、低剂量跨越平台的次数均高于模型组,但无统计学意义,表明治疗组均能够提高东莨菪碱致记忆障碍大鼠的记忆能力(表2)。
表2:鱼籽功能肽对AD大鼠遇避潜伏期和跨原平台次数的影响(x土s)
注:与模型组(东莨菪碱)比较,*P<005。
2.2鱼籽功能肽对东莨菪碱致早老型痴呆大鼠学习记忆能力的影响
如表3所示,学习成绩实验中,模型组与对照组比较,大鼠的反应时间显著延长,错误次数明显增加,表明小鼠的学习成绩下降,差异具有统计学意义(P<0.01)。各治疗组与模型组比较,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)。高剂量组与阳性药物组比较,反应时间缩短错误次数增加,差异有统计学意义(P<0.05),高剂量组与低剂量组比较,反应时间明显缩短,错误次数减少,有统计学意义(P<0.05),表明各治疗组均能提高早老性痴呆大鼠跳台实验学习能力,且在改善潜伏时间方面鱼籽功能肽呈显著量效关系。记忆能力实验中,模型组与对照组比较,大鼠的潜伏时间显著缩短,错误次数明显增加,表明记忆成绩下降,差异具有统计学意义(P<0.01);各治疗组与模型组比较,潜伏时间均延长,错误次数均减少,差异有统计学意义(P<0.01),高剂量组与阳性药物组比较,潜伏时间延长,错误次数明显减少,差异有统计学意义(P<005),高剂量组与低剂量组比较,错误次数明显减少,但差异无统计学意义。
表3:各组大鼠学习记忆能力的比较表(x±s)
注:与模型组(东莨菪碱)比较*P<0.05。
实验结果表明各治疗组均可提高早老性痴呆大鼠跳台记忆成绩,且在增加潜伏时间有量效关系的趋势。
对本发明说明书中其它实施例中制备的鱼籽功能肽的功能进行检测,表明本发明制备的鱼籽功能肽都具有类似的改善记忆功能的效果。
Claims (10)
1.一种鱼籽功能肽,其特征在于,所述的鱼籽功能肽的制备方法如下:
1)匀浆:将鱼籽进行匀浆,得到匀浆液;
2)酶解:向步骤1)的匀浆液中加入胰蛋白酶和胶原蛋白酶进行酶解,得到酶解液,离心取上清液;
3)膜过滤:对步骤2)制备的上清液进行膜过滤,过滤液即为鱼籽功能肽溶液。
2.如权利要求1所述的鱼籽功能肽,其特征在于,所述的1)中的鱼籽为海水鱼的鱼籽。
3.如权利要求1或2所述的鱼籽功能肽,其特征在于,所述的1)中的鱼籽为黄鱼的鱼籽或鮟鱇鱼的鱼籽。
4.如权利要求1所述的鱼籽功能肽,其特征在于,所述的1)中匀浆的条件如下:高速剪切机转速为8000-12000rpm,剪切时间为30min。
5.如权利要求1所述的鱼籽功能肽,其特征在于,所述的2)中胰蛋白酶和胶原蛋白酶在匀浆液的加入量酶活比为1:50—5:1。
6.如权利要求1所述的鱼籽功能肽,其特征在于,所述的2)中胰蛋白酶和胶原蛋白酶在匀浆液的加入量酶活比为1:10。
7.如权利要求1所述的鱼籽功能肽,其特征在于,所述的2)中酶解,是在35-55℃酶解3-5h。
8.如权利要求1所述的鱼籽功能肽,其特征在于,所述的2)中酶解,是在50℃酶解5h。
9.如权利要求1所述的鱼籽功能肽,其特征在于,所述的3)中膜分离是先用3500Da的膜分离,然后用360Da的膜分离。
10.权利要求1所述的鱼籽功能肽在制备用于改善记忆的制品中的应用。
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