CN114507702B - 一种海洋南极磷虾肽及其应用 - Google Patents
一种海洋南极磷虾肽及其应用 Download PDFInfo
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Abstract
本发明涉及功能性多肽制备技术领域,具体涉及一种海洋南极磷虾肽及其应用;是以新鲜南极磷虾或冷冻南极磷虾为原料,经过粉碎后,分别用胰蛋白酶、胃蛋白酶和木瓜蛋白酶进行酶解得到的南极磷虾蛋白水解肽。本发明所制备的多肽具有降胆固醇的功效,从而提高南极磷虾的应用开发价值。
Description
技术领域
本发明属于功能多肽制备技术领域,具体涉及一种海洋南极磷虾肽及其应用。
背景技术
南极磷虾(Euphausia superba)是磷虾科、磷虾属虾类。南极磷虾具有典型的高蛋白、低脂肪的特点,其蛋白和脂肪含量分别为16.31%和1.3%。并且磷虾中矿物质含量丰富,矿物质含量为2.76%,高于日本对虾(1.6%)、蛤蜊(2.2%)等多种海产品。南极磷虾的蛋白水解产物中氨基酸种类丰富,含有18种氨基酸,其中包含人体所需的8种必需氨基酸。其中谷氨酸含量最高,赖氨酸次之。南极磷虾的脂类中以极性脂含量最高,甘油三酯次之,分别为总脂肪含量的56-81%、12-38%。南极磷虾中还含有多种活性物质,如蛋白消化酶、类胞菌素氨基酸等。因此南极磷虾具有重要的经济和食用价值。
对于海洋活性多肽的获取方法,包含有直接提取、体外降解、工程菌进行发酵、酶水解或化学合成等。蛋白质的酶法水解不会导致营养方面的损失,也不会产生毒理上的问题;同时酶作用具有特异性、高效性和专一性,可以在温和的条件下进行,能耗也低,所以酶法水解在食品加工中应用非常广泛。
利用酶法水解南极磷虾蛋白质制备南极磷虾活性肽的技术已有报道。例如申请号为CN201410075726.1的中国发明专利中公开了冰鲜南极磷虾的蛋白水解产物的制备及其用途,上述发明中公开了对南极磷虾的原料进行破碎、采用碱性蛋白酶获得酶解液,而后进行固液分离和干燥得到南极磷虾肽粉,从而进一步加工制备其他功能制品。但上述发明中对南极磷虾进行酶解是采用的单酶,并且在未进一步提纯的情况下直接固液分离和干燥得到南极磷虾肽粉。而且,在活性肽的制备过程中,酶的选择以及酶解液提纯是核心技术,不同的工艺所得的肽的产品活性是不一样的。
发明内容
本发明提供了一种海洋南极磷虾肽及其应用,所制备的多肽具有降胆固醇的功效,从而提高南极磷虾的应用开发价值。
本发明所提供的一种降胆固醇南极磷虾肽,是以新鲜南极磷虾或冷冻南极磷虾为原料,经过粉碎后,分别用胰蛋白酶、胃蛋白酶和木瓜蛋白酶分步进行酶解得到南极磷虾蛋白水解肽液,经喷雾干燥或真空冷冻干燥制备的;
更进一步的,所述的南极磷虾肽是将南极磷虾蛋白水解肽液通过分子量为1000Da的超滤膜过滤后,获得的过滤液经喷雾干燥或真空冷冻干燥制备的;
进一步的,所述的胰蛋白酶的水解条件如下:温度为50℃,pH值为7.0,酶解时间为7h;
进一步的,所述的胃蛋白酶的水解条件如下:温度为50℃,pH值为7.0,酶解时间为7h;
进一步的,所述的木瓜蛋白酶的水解条件如下:温度为55℃,pH值为7.0,酶解时间为6h;
进一步的,所述的南极磷虾肽,其氨基酸序列分别为EIEVDENDEH、NTGDPVIREDE、SYVGQKDAQGED。
本发明所提供的南极磷虾肽用于制备降胆固醇的制品;
本发明还提供一种具有降胆固醇效果的制品,所述的制品中包含有药理有效浓度的上述的南极磷虾肽。
本发明制备的降胆固醇南极磷虾肽经C57BL/6高血脂模型雄性SD大鼠灌胃实验证明具有较好的辅助降胆固醇功效。该降胆固醇南极磷虾肽的生产工艺,可操作性强,产品质量可控,蛋白质酶解程度完全,所得的降胆固醇南极磷虾肽活性高,可用于食品和医疗保健等领域。
附图说明
图1:为本发明实施例3中南极磷虾活性肽P液组分分析鉴定图;
图2:为本发明实施例3中大鼠血清中甘油三酯的测定图;
图3:为本发明实施例3中大鼠血清中总胆固醇的测定图;
图4:为本发明实施例3中大鼠血清中高密度脂蛋白的测定图;
图5:为本发明实施例3中大鼠血清中低密度脂蛋白的测定图。
具体实施方式
下面结合实施案例对本发明做进一步详细说明。
实施例1南极磷虾肽制备方法的筛选
1)蛋白酶的选择:以水解度为指标,在酶添加量为100u/g、料液比1:1g/ml和酶解时间6h条件下,比较六种不同蛋白酶在其最合适酶解条件下(见表1)下对南极磷虾蛋白的酶解效果。最终确定胃蛋白酶、胰蛋白酶和木瓜蛋白酶为最适用酶。
表1:不同蛋白酶的最适酶解温度和pH值信息表
2)以水解度为指标,分析温度、pH值、料液比、酶添加量和酶解时间对制备的多肽的影响。
将南极磷虾加水进行匀浆,得到南极磷虾匀浆液,调节所选定的蛋白酶对应的pH值,添加蛋白酶,酶解一定的时间;待反应结束后,将酶解液置于沸水浴中加热使蛋白酶失活,10000g离心20min,取上清多肽液进行水解度测定。并在单因素实验基础上,用Design-Expert8.0软件进行响应面优化分析。
表2:木瓜蛋白酶酶解工艺响应面实验设计因素水平编码表
表3:木瓜蛋白酶酶解工艺响应面实验方案及结果表
表4:胰蛋白酶酶解工艺响应面实验设计因素水平编码表
表5:胰蛋白酶酶解工艺响应面实验方案及结果表
表6:胃蛋白酶酶解工艺响应面实验设计因素水平编码表
表7:胃蛋白酶酶解工艺响应面实验方案及结果表
结果显示,木瓜蛋白酶的最优酶解工艺为:温度为55℃、pH值为7.0、料液比为1.0g/ml、酶添加量为100u/g和酶解时间为6h;胰蛋白酶的最优酶解工艺为50℃、pH值为7.0、料液比为1.54g/ml、酶添加量为100u/g和酶解时间7h,胃蛋白酶最优酶解工艺为50℃、pH值为7.0、料液比为1.58g/ml、酶添加量为100u/g和酶解时间7h。
表8:不同蛋白酶组合方式对南极磷虾蛋白酶解效果的影响表
备注:A-木瓜蛋白酶B-胰蛋白酶C-胃蛋白酶。
由表8结果确定的最佳组合方式如下:先添加胰蛋白酶,在温度为50℃、pH值为7.0、料液比为1.54g/ml、酶添加量为100u/g和酶解时间为7h;再添加胃蛋白酶,在温度50℃、pH值为7.0、料液比为1.58g/ml、酶添加量为100u/g条件下继续酶解7h,最后添加木瓜蛋白酶,在温度为55℃、pH值为7.0、料液比为1.0g/ml、酶添加量为100u/g和酶解时间为6h;所获得的水解产物降胆固醇的效果能够达到降低73.64%。在此酶解条件下获得的南极磷虾肽的降胆固醇效果最好。
实施例2:对制备的多肽进行过滤浓缩
(1)原料的处理:称取50kg南极磷虾的鲜虾肉为原料,用超纯水清洗,将清洗后的鲜虾肉放入粉碎机中进行粉碎,并控制南极磷虾浆颗粒大小在140μm;
(2)第一步酶解:测定步骤(1)所得南极磷虾的水分和蛋白质含量,根据测定结果,加水调整蛋白质的质量浓度为3%;调整pH值为7.0,控制温度为50℃,加入胰蛋白酶,胰蛋白酶的用量为每克蛋白质加入100u的胰蛋白酶,保温酶解7h,得到南极磷虾胰蛋白酶水解液;
(3)第二步酶解:将步骤(2)得到的南极磷虾胰蛋白酶水解液的pH值调整至7.0,保持温度为50℃,加入胃蛋白酶,胃蛋白酶的用量为每克蛋白质加入100u的胃蛋白酶,酶解7h,得到南极磷虾双酶酶解液;
(4)第三步酶解:将步骤(3)得到的南极磷虾双酶酶解液的pH值调整至7.0,并调整温度为55℃,加入木瓜蛋白酶,木瓜蛋白酶的用量为每克蛋白质加入100u的木瓜蛋白酶,酶解4h,得到南极磷虾复合酶解液;
(5)分离浓缩:将步骤(4)得到的南极磷虾复合酶解液在10000g下离心20min,取上清液用1000Da的超滤膜进行过滤,得到分子量小于1000Da的南极磷虾活性肽过滤浓缩液;并进行浓缩。该过滤浓缩液在300mg/kg剂量时,其降胆固醇效果优于步骤(4)的南极磷虾复合酶解液500mg/kg剂量的效果。
实施例3:多肽的纯化
1)第一步提纯:将实施例2的步骤5)中制备得到的南极磷虾活性肽浓缩液用SehadexG-25凝胶柱(1.6cm×30cm)分离提纯,其中洗脱液为双蒸水,洗脱速度为0.5mL/min,收集活性最高的组分;
2)第二步提纯:将步骤1)得到的活性最高的组分用SP Sephadex C-25阳离子交换柱(2.6cm×30cm)进一步分离纯化。其中平衡液为20mM的醋酸缓冲液(pH=4.0),用含有NaCl的醋酸缓冲液进行线梯度洗脱,洗脱速度为0.5mL/min,收集活性最高的组分;
3)第三步纯化:将步骤2)中得到的组分用反相高效液相色谱C18半制备柱(9.4cm×250cm)进一步分离纯化,用10-20%的乙腈梯度洗脱40min,洗脱速度为0.7mM/min,柱温为30℃,在220nm下收集主峰,得到南极磷虾活性肽P液(图1)。
通过LC-MS技术对南极磷虾活性肽P液组分进行分析鉴定,得到了3条多肽序列,其信息如表9所示。
表9:南极磷虾活性肽P液的多肽信息表
将上述的p-1、p-2和p-3多肽送到生物公司进行合成,合成的多肽进行动物实验来验证降胆固醇效果。
建立高血脂动物模型:实验采用200只SPF级别雄性SD成年大鼠,体重约为91-112g,随机选取30只SD大鼠作为空白组,给予普通饲料喂养,其余的SD大鼠均给予D12492高脂饲料喂养,即造模组,所有动物均自由饮水,每周分别从空白组和造模组随机抽取5只SD大鼠,尾静脉取血,检测TC(总胆固醇)、TG(甘油三酯)判断造模是否成功。6周后造模组动物TC水平约升高为空白组的2倍,视为造模成功。
降胆固醇南极磷虾活性肽肽P动物实验:筛选100只造模成功的高血脂SD大鼠,随机分为5组,分别为F1组、F2组、F3组、F4组和F5,每组20只SD大鼠。F1组给予样品(实施例2所制备的南极磷虾活性肽过滤浓缩液)水溶液400mg/kg剂量灌胃;F2组给予(p-1合成多肽)300mg/kg剂量灌胃;F3组给予(p-2合成多肽)300mg/kg剂量灌胃;F4组给予(p-3合成多肽)300mg/kg剂量灌胃,F5组给予等体积0.9%的生理盐水灌胃;各组均给予D12492高脂饲料喂养。
所有SD大鼠均自由饮水,每天灌胃一次,连续灌胃8周,断食12h,眼球取血制备血清。测定血清中总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)和高密度脂蛋白胆固醇(HDL-C),实验结果如表10所示。
表10:SD大鼠血清指标检测结果表(n=20,mean±SD)
注:与F5组相比*P<0.05,**P<0.01;
由表10和图2-图5,SD大鼠灌胃8周后,F1至F4组的实验大鼠的血清中甘油三酯、总胆固醇、高密度脂蛋白和低密度脂蛋白均有所下降,与F5组相比有显著性的差异(**P<0.01)。F2、F3和F4组与F1组相比,血清中甘油三酯、总胆固醇、高密度脂蛋白和低密度脂蛋白均有所下降。结果显示p-1、p-2和p-3多肽的降降胆固醇效果优于实施例2中未纯化的多肽。
Claims (2)
1.一种海洋南极磷虾肽,其特征在于,所述的海洋南极磷虾肽的氨基酸序列为EIEVDENDEH或NTGDPVIREDE或SYVGQKDAQGED。
2.权利要求1所述的南极磷虾肽在制备降胆固醇的制品中的应用。
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