CN107723327A - 蜂乳蛋白活性多肽的制备方法 - Google Patents
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Abstract
本发明公开了一种蜂乳蛋白活性多肽的制备方法,包括浸提、分步酶解、纳滤、凝胶层析和高效液相色谱纯化。采用胰蛋白酶和枯草芽孢杆菌中性蛋白酶分步酶解,将浸提提取的蜂乳蛋白酶解成分子量较小的活性物质。采用氨基酸序列为SCASRSCKCFRC的活性短肽来激活枯草芽孢杆菌中性蛋白酶。有益效果为:得到的蜂乳蛋白活性多肽纯度非常高,质量好,体外活性显示具有较好的血管紧张素转化酶(ACE)抑制活性。
Description
技术领域
本发明涉及生物活性肽制备技术领域,具体是一种蜂乳蛋白活性多肽的制备方法。
背景技术
蜂王浆(royal jelly, RJ),又称蜂皇浆、蜂乳,是幼龄(5~15日龄)工蜂头部的咽下腺和上颚腺共同分泌的一种乳白色至浅黄色浆状物,略带甜、酸、涩、辛辣味、并具有特殊香气。蜂王浆是蜜蜂把从各种花朵上采集到的花蜜和花粉酿造成蜂蜜或蜂粮后,被幼蜂取食、消化分解和经过体内一系列极其复杂的生理生化过程再从腺体中分泌出来的微量物质,是蜂王整个生命周期的唯一食物。营养学家认为,蜂王浆是唯一可供人类直接服用的含高活性成分的超级营养食品,具有多种生物活性,如抗氧化,调节免疫,增强记忆力,抗菌、抗肿瘤等。
发明内容
本发明的目的在于提供一种蜂乳蛋白活性多肽的制备方法,采用分步酶解蜂乳蛋白,酶解效率高。制备得到的蜂乳蛋白活性多肽,体外活性显示具有较好的血管紧张素转化酶(ACE)抑制活性。
本发明针对背景技术中提到的问题,采取的技术方案为:蜂乳蛋白活性多肽的制备方法,包括浸提、分步酶解、纳滤、凝胶层析和高效液相色谱纯化。得到的蜂乳蛋白多肽C端氨基酸为脯氨酸;N 端为具支链的疏水氨基酸,具有较好的抑制ACE活性作用。其中,分步酶解步骤为将蜂乳蛋白溶液置于25~40℃的恒温环境中,加入胰蛋白酶酶解,酶解结束后灭酶,加入枯草芽孢杆菌中性蛋白酶和活性短肽酶解,酶解结束后灭酶,过滤取滤液。采用胰蛋白酶和枯草芽孢杆菌中性蛋白酶分步酶解,将浸提提取的蜂乳蛋白酶解成分子量较小的活性物质。胰蛋白酶和枯草芽孢杆菌中性蛋白酶的酶解条件不同,采用分步酶解的方式使得两种蛋白酶的酶解效率均处于较高状态。
作为优选,胰蛋白酶的加入量为蜂乳蛋白溶液质量的0.2~0.7%,酶解pH为7.5~9.0,酶解温度为25~40℃,酶解时间为2~4h;枯草芽孢杆菌中性蛋白酶的加入量为蜂乳蛋白溶液质量的0.1~0.5%,活性短肽的加入量为枯草芽孢杆菌中性蛋白酶质量的1~5%,酶解pH为7.0~7.8,酶解温度为35~50℃,酶解时间为10~30min。上述酶解条件下,蛋白酶酶解效率高,所耗费的材料成本、人工成本和时间成本低。
作为优选,活性短肽的氨基酸序列为SCASRSCKCFRC。上述活性短肽对枯草芽孢杆菌中性蛋白酶有激活作用,即在活性短肽存在条件下,枯草芽孢杆菌中性蛋白酶的酶解效率明显提高,降低了枯草芽孢杆菌中性蛋白酶的加入量并缩短了酶解时间。
作为优选,浸提步骤为在蜂乳脱脂粉末中按料液比1:10~1:25加入0.5~2.0%NaCl溶液,调节pH为7.0~9.0,0~8℃下浸提2~5h,在0~8℃下离心取上清液,上清液即为蜂乳蛋白溶液。上述操作可去除蜂乳中的非盐溶性蛋白和酸溶性蛋白,在提取的同时初步纯化目标蛋白。
作为优选,纳滤步骤为将分步酶解得到的滤液置于蒸馏水中浓缩纳滤24~36h。纳滤可有效除去500道尔顿以下的杂质和溶液中的无机盐,对活性多肽进一步进行纯化。
作为优选,凝胶层析步骤为用蒸馏水将Sephadex G-15凝胶溶胀后装柱,检查柱均匀性后上样,用自动取样器收集洗脱液,在280nm下测紫外吸光度,做曲线;重复上样,根据吸光度曲线收集分离的溶液;上样溶液为纳滤浓缩液用蒸馏水稀释10~20倍得到。先采用Sephadex G-15凝胶色谱柱层析初步纯化降压肽粗品,减少后续高效液相色谱分离纯化的时间成本、原料成本和人工成本。
作为优选,高效液相色谱分离纯化条件为:色谱柱:反相C18键合硅胶柱;流动相:0~4min,水;4~16min,60~80%水,20~40%乙腈;16~35min,乙腈;流速:0.7~1.3ml/min,柱温25~35℃,检测波长为254 nm和280 nm,收集主要色谱峰,冻干得到蜂乳蛋白活性多肽。高效液相色谱分离纯化后得到的蜂乳蛋白活性多肽纯度非常高,质量好,体外活性显示具有较好的血管紧张素转化酶(ACE)抑制活性。抑制血管紧张素转换酶(ACE)的活性,使肾素—血管紧张素系统(RAS)处于抑制状态,同时使激肽释放酶-激肽系统(KKS)处于激活状态,产生具有降低血压作用的舒缓激肽,降血压效果显著。
与现有技术相比,本发明的优点在于:
1.采用胰蛋白酶和枯草芽孢杆菌中性蛋白酶分步酶解,将浸提提取的蜂乳蛋白酶解成分子量较小的活性物质。在枯草芽孢杆菌中性蛋白酶酶解时加入氨基酸序列为SCASRSCKCFRC的活性短肽。上述活性短肽对枯草芽孢杆菌中性蛋白酶有激活作用,即在活性短肽存在条件下,枯草芽孢杆菌中性蛋白酶的酶解效率明显提高,降低了枯草芽孢杆菌中性蛋白酶的加入量并缩短了酶解时间。
2.得到的蜂乳蛋白活性多肽纯度非常高,质量好,体外活性显示具有较好的血管紧张素转化酶(ACE)抑制活性。抑制血管紧张素转换酶(ACE)的活性,使肾素—血管紧张素系统(RAS)处于抑制状态,同时使激肽释放酶-激肽系统(KKS)处于激活状态,产生具有降低血压作用的舒缓激肽,降血压效果显著。
附图标记
图1为凝胶纯化图;
图2为液相纯化图。
具体实施方式
下面通过附图和实施例对本发明方案作进一步说明:
实施例1:
蜂乳蛋白活性多肽的制备方法,包括以下步骤:
1)浸提:在蜂乳脱脂粉末中按料液比1:10~1:25加入0.5~2.0%NaCl溶液,调节pH为7.0~9.0,0~8℃下浸提2~5h,在0~8℃下离心取上清液,上清液即为蜂乳蛋白溶液;
2)分步酶解:将蜂乳蛋白溶液置于25~40℃的恒温环境中,加入胰蛋白酶酶解,酶解结束后灭酶,加入枯草芽孢杆菌中性蛋白酶和活性短肽酶解,酶解结束后灭酶,过滤取滤液。胰蛋白酶的加入量为蜂乳蛋白溶液质量的0.2~0.7%,酶解pH为7.5~9.0,酶解温度为25~40℃,酶解时间为2~4h;枯草芽孢杆菌中性蛋白酶的加入量为蜂乳蛋白溶液质量的0.1~0.5%,活性短肽的加入量为枯草芽孢杆菌中性蛋白酶质量的1~5%,酶解pH为7.0~7.8,酶解温度为35~50℃,酶解时间为10~30min。活性短肽的氨基酸序列为SCASRSCKCFRC;
3)纳滤:将分步酶解得到的滤液置于蒸馏水中浓缩纳滤24~36h;
4)凝胶层析:用蒸馏水将Sephadex G-15凝胶溶胀后装柱,检查柱均匀性后上样,用自动取样器收集洗脱液,在280nm下测紫外吸光度,做曲线;重复上样,根据吸光度曲线收集分离的溶液;上样溶液为纳滤浓缩液用蒸馏水稀释10~20倍得到;
5)高效液相色谱分离纯化条件为:色谱柱:反相C18键合硅胶柱;流动相:0~4min,水;4~16min,60~80%水,20~40%乙腈;16~35min,乙腈;流速:0.7~1.3ml/min,柱温25~35℃,检测波长为254 nm和280 nm,收集主要色谱峰,冻干得到蜂乳蛋白活性多肽。高效液相色谱分离纯化后得到的蜂乳蛋白活性多肽纯度非常高,质量好,体外活性显示具有较好的血管紧张素转化酶(ACE)抑制活性。抑制血管紧张素转换酶(ACE)的活性,使肾素—血管紧张素系统(RAS)处于抑制状态,同时使激肽释放酶-激肽系统(KKS)处于激活状态,产生具有降低血压作用的舒缓激肽,降血压效果显著。
实施例2:
蜂乳蛋白活性多肽的最优选制备方法,包括以下步骤:
1)浸提:在蜂乳脱脂粉末中按料液比1:20加入1.0%NaCl溶液,调节pH为8.0,4℃下浸提4h,在4℃下离心取上清液,上清液即为蜂乳蛋白溶液;
2)分步酶解:将蜂乳蛋白溶液置于37℃的恒温环境中,加入蜂乳蛋白溶液质量的0.6%的胰蛋白酶酶解,酶解pH为8.0,酶解温度为37℃,酶解时间为3h,酶解结束后沸水浴灭酶;调节酶解液pH为7.5,加入蜂乳蛋白溶液质量的0.2%的枯草芽孢杆菌中性蛋白酶和枯草芽孢杆菌中性蛋白酶质量3%的活性短肽酶解,酶解温度为40℃,酶解15min,酶解结束后沸水浴灭酶,过滤取滤液。活性短肽的氨基酸序列为SCASRSCKCFRC;
3)纳滤:将分步酶解得到的滤液置于蒸馏水中浓缩纳滤24;
4)凝胶层析:用蒸馏水将Sephadex G-15凝胶溶胀后装柱,检查柱均匀性后上样,用自动取样器收集洗脱液,在280nm下测紫外吸光度,做曲线如图1所示;重复上样,根据吸光度曲线收集分离的溶液。上样溶液为纳滤浓缩液用蒸馏水稀释15倍得到;
5)高效液相色谱分离纯化条件为:色谱柱:反相C18键合硅胶柱;流动相:0~4min,水;4~16min,60~80%水,20~40%乙腈;16~35min,乙腈;流速:0.7~1.3ml/min,柱温25~35℃,检测波长为254 nm和280 nm,得到液相纯化图图2,收集主要色谱峰,冻干得到蜂乳蛋白活性多肽。高效液相色谱分离纯化后得到的蜂乳蛋白活性多肽纯度非常高,质量好,体外活性显示具有较好的血管紧张素转化酶(ACE)抑制活性。抑制血管紧张素转换酶(ACE)的活性,使肾素—血管紧张素系统(RAS)处于抑制状态,同时使激肽释放酶-激肽系统(KKS)处于激活状态,产生具有降低血压作用的舒缓激肽,降血压效果显著。
本发明的操作步骤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。
序列表
<110> 兰溪市沉默生物科技有限公司
<120> 蜂乳蛋白活性多肽的制备方法
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213> 人工合成(Mytilus coruscus)
<400> 1
Ser Cys Ala Ser Arg Ser Cys Lys Cys Phe Arg Cys
1 5 10
Claims (7)
1.蜂乳蛋白活性多肽的制备方法,包括浸提、分步酶解、纳滤、凝胶层析和高效液相色谱纯化,其特征在于:所述的分步酶解步骤为将蜂乳蛋白溶液置于25~40℃的恒温环境中,加入胰蛋白酶酶解,酶解结束后灭酶,加入枯草芽孢杆菌中性蛋白酶和活性短肽酶解,酶解结束后灭酶,过滤取滤液。
2.根据权利要求1所述的蜂乳蛋白活性多肽的制备方法,其特征在于:所述的胰蛋白酶的加入量为蜂乳蛋白溶液质量的0.2~0.7%,酶解pH为7.5~9.0,酶解温度为25~40℃,酶解时间为2~4h;枯草芽孢杆菌中性蛋白酶的加入量为蜂乳蛋白溶液质量的0.1~0.5%,活性短肽的加入量为枯草芽孢杆菌中性蛋白酶质量的1~5%,酶解pH为7.0~7.8,酶解温度为35~50℃,酶解时间为10~30min。
3.根据权利要求1所述的蜂乳蛋白活性多肽的制备方法,其特征在于:所述的活性短肽的氨基酸序列为SCASRSCKCFRC。
4.根据权利要求1所述的蜂乳蛋白活性多肽的制备方法,其特征在于:所述的浸提步骤为在蜂乳脱脂粉末中按料液比1:10~1:25加入0.5~2.0%NaCl溶液,调节pH为7.0~9.0,0~8℃下浸提2~5h,在0~8℃下离心取上清液,所述上清液即为蜂乳蛋白溶液。
5.根据权利要求1所述的蜂乳蛋白活性多肽的制备方法,其特征在于:所述的纳滤步骤为将分步酶解得到的滤液置于蒸馏水中浓缩纳滤24~36h。
6.根据权利要求1所述的蜂乳蛋白活性多肽的制备方法,其特征在于:所述的凝胶层析步骤为用蒸馏水将Sephadex G-15凝胶溶胀后装柱,检查柱均匀性后上样,用自动取样器收集洗脱液,在280nm下测紫外吸光度,做曲线;重复上样,根据吸光度曲线收集分离的溶液;上样溶液为纳滤浓缩液用蒸馏水稀释10~20倍得到。
7.根据权利要求1所述的蜂乳蛋白活性多肽的制备方法,其特征在于:所述的高效液相色谱分离纯化条件为:色谱柱:反相C18键合硅胶柱;流动相:0~4min,水;4~16min,60~80%水,20~40%乙腈;16~35min,乙腈;流速:0.7~1.3ml/min,柱温25~35℃,检测波长为254 nm和280 nm,收集主要色谱峰,冻干得到蜂乳蛋白活性多肽。
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