CN114668152A - 一种稳定性高、在小肠定向释放的ace抑制肽脂质体及其制备方法 - Google Patents
一种稳定性高、在小肠定向释放的ace抑制肽脂质体及其制备方法 Download PDFInfo
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Abstract
本发明提供了一种稳定性高、在小肠定向释放的ACE抑制肽脂质体及其制备方法，该脂质体在体外模拟胃肠消化试验中，处理后ACE抑制保留率为90％，说明制备的ACE抑制肽脂质体可以抵抗胃液的消化，在小肠定向释放。本发明通过在ACE抑制肽脂质体表面复合三层修饰层，解决了ACE抑制肽会被胃蛋白酶酶解破坏造成活性低的难题。
Description
技术领域
本发明涉及保健食品生产的技术领域，特别涉及一种稳定性高、在小肠定向释放的ACE抑制肽脂质体及其制备方法。
背景技术
ACE抑制肽是一类具有抑制ACE(血管紧张素)活性的多肽物质，这些多肽的氨基酸序列和肽链长度各不相同，但都具有类似功能。一些研究表明ACE抑制肽的活性依赖于其对ACE活性区域亲和力较强的竞争性抑制，因此ACE抑制肽的结构对于其抑制活性至关重要，但消化道中的消化酶会破坏其结构，使其抑制活性消失。如何保证ACE抑制肽在通过消化道的同时保留其活性已成为保健食品领域关注的热点。
现有技术中CN104522291A公开了一种蛋清源ACE抑制肽微胶囊的制备的方法，采用喷雾冷凝法制备蛋清源ACE抑制肽微胶囊，掩盖了蛋清蛋白经酶解后，由于疏水性结构暴露而产生的苦味；另外，壁材的包埋效果也降低了环境因素对于蛋清源ACE抑制肽在贮藏过程中的影响，增加其贮藏的稳定性。
CN109602913A公开了一种增强蛋清ACE抑制肽稳定性的方法，利用氧化石墨烯修饰蛋清ACE抑制肽，制备得到氧化石墨烯-活性肽复合材料，该复合材料具有良好的生物兼容性，同时发挥氧化石墨烯作为递送载体特性进而提高活性肽的稳定性，而且克服了活性肽在胃肠道不稳定的缺点。
为保证ACE抑制肽的抑制活性，需要既保证其抵抗胃液消化的同时，又能够在小肠特异性定向释放，且释放出的ACE抑制肽仍有较高的ACE抑制活性。
发明内容
有鉴于此，本发明目的在于提供一种稳定性高、在小肠定向释放的ACE抑制肽脂质体及其制备方法，本发明提供的ACE抑制肽脂质体在磷脂双分子层壁材外构建三层修饰层，这些修饰层可以抵抗胃液的消化的同时，可以被肠液中的胆盐和酶分解，并且释放后的ACE抑制肽仍能保持原有抑制率的95％。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种稳定性高、在小肠定向释放的ACE抑制肽脂质体，ACE抑制肽被包覆于卵磷脂脂质体内，脂质体外附着从内到外依次为壳聚糖、海藻酸钠和羧乙基纤维素修饰层。
本发明提供了上述方案所述ACE抑制肽脂质体的制备方法，包括以下步骤：
1)脂质体制备：
将大豆卵磷脂、胆固醇、吐温-80溶解于无水乙醇溶液，四者比例依次为6:1:1:100(m:m:v:v)60℃旋转蒸发形成薄膜。
向旋蒸瓶中加入含有浓度为0.1％-0.3％(g/mL)的ACE抑制肽溶液，加入量与所述无水乙醇的体积比为1:1，于60℃水合1h，得到粗脂质体混悬液。将所述粗脂质体混悬液超声处理得到纳米脂质体。
2)壳聚糖修饰：
将脂质体逐滴加入到等体积的浓度为1-2％(g/mL)的壳聚糖溶液，边加边搅拌，得到壳聚糖-ACE抑制肽脂质体，4℃储存。
3)海藻酸钠修饰：
将所述壳聚糖-ACE抑制肽脂质体溶液逐滴加入到等体积的浓度为1-2％(g/mL)的海藻酸钠溶液中，边加边搅拌，得到海藻酸钠-壳聚糖-ACE抑制肽脂质体，4℃储存。
4)羧乙基纤维素修饰：
所述海藻酸钠-壳聚糖-ACE抑制肽脂质体溶液逐滴加入到等体积的浓度为1-2％(g/mL)的羧乙基纤维素溶液中，边加边搅拌，得到三层修饰的羧乙基纤维素-海藻酸钠-壳聚糖-ACE抑制肽脂质体，4℃储存。
优选地，步骤1)中ACE抑制肽的浓度为0.2％；
优选地，步骤1)中ACE抑制肽溶液的溶剂使用PBS缓冲液(pH 7.4；0.01M)；
优选地，步骤1)中所述ACE抑制肽的制备方法包括：核桃脱脂粉与水按照1:7(v:v)比例混合，使用非蛋白复合酶进行第一次酶解，得脱脂核桃粕破壁悬浮液。使用碱性蛋白酶对所述脱脂核桃粕破壁悬浮液进行第二次酶解，得到核桃粕酶解液。对所述核桃粕酶解液进行超滤，5kDa分子量以下的滤出物为ACE抑制肽。
优选地，步骤1)中所述超声处理的条件为：超声频率20kHz，功率为500W，间隔2s循环工作；超声温度为4℃，超声时间8min。
优选地，步骤2)中所述壳聚糖溶液的制备方法包括：壳聚糖溶解于1％乙酸溶液中，离心取上清液，上清液过0.22μm滤膜，调节pH为5.5。
优选地，步骤3)中所述海藻酸钠溶液的制备方法包括：海藻酸钠溶解于蒸馏水中，离心取上清液，上清液过0.22μm滤膜，调节pH为5.5。
优选地，步骤4)中所述羧乙基纤维素溶液的制备方法包括：羧乙基纤维素溶解于蒸馏水中，离心取上清液，上清液过0.22μm滤膜，调节pH为5.5。
得到的ACE抑制肽脂质体的修饰层——海藻酸钠与羧乙基纤维素在低pH条件下(pH 1.5左右)可以收缩转换为不溶性的皮壳结构，这层皮壳结构可以阻挡胃蛋白酶的进入，避免胃蛋白酶对ACE抑制肽的酶解作用，保护了ACE抑制肽的活性。这层皮壳结构进入到小肠，小肠的环境变为中性到弱碱性条件，随着pH值增加，皮壳的网格结构变得松散，在肠道中的酶和胆盐作用下易乳化、水解，壁材结构破坏，ACE抑制肽释放，以此保证了ACE抑制肽的活性。
酶解制备得到的ACE抑制肽的抑制率为75.29％，将其包覆于卵磷脂脂质体中，由于ACE抑制肽被壁材阻挡，脂质体的ACE抑制率为55.03％。对脂质体进行体外模拟消化实验，经模拟胃液消化后，由于壁材不能阻挡胃液的消化作用，ACE抑制肽结构被破坏，再经肠液消化后抑制率为22.02％。
制备羧乙基纤维素-海藻酸钠-壳聚糖-ACE抑制肽脂质体，带修饰层的脂质体ACE抑制率为41.26％，说明单独的卵磷脂脂质体对ACE抑制肽的包埋率不高，缓冲液会残留一部分肽，修饰层的多糖有凝胶网络结构，能吸附溶液中的肽，增加包埋率，因而ACE抑制肽被包覆，缓冲液不显现抑制活性。对三层修饰层脂质体进行体外模拟消化实验，经模拟胃液消化，再经肠液消化后，ACE抑制率为68.95％，保留了原始抑制率的90％。
具体实施方式
本发明提供了一种稳定性高、在小肠定向释放的ACE抑制肽脂质体及其制备方法，下面结合具体实施例对本发明做进一步说明，以下实施方式旨在说明本产品而不是对本发明的进一步限定，任何在本发明基础上所做的修改、等同替换等均在本发明的保护范围内。“所有的设备和原料等均可从市场购得或是本行业常用的”，本发明所用技术方案或表达方式，如未特别说明，均为本领域技术人员所公知的。
(1)按照大豆卵磷脂：胆固醇：吐温-80：无水乙醇6:1:1:100(m:m:v:v)，称取上述原料，溶解于无水乙醇；
(2)将步骤(1)中的溶液置于旋转蒸发仪上除去无水乙醇，形成均匀薄膜；
(3)加入浓度为0.1％-0.3％(g/mL)的ACE抑制肽的PBS缓冲液(pH 7.4；0.01M)，得到粗脂质体混悬液。
ACE抑制肽的制备方法为：核桃脱脂粉与水按照1:7(v:v)比例混合，使用非蛋白复合酶进行第一次酶解，得脱脂核桃粕破壁悬浮液。使用碱性蛋白酶对所述脱脂核桃粕破壁悬浮液进行第二次酶解，得到核桃粕酶解液。对所述核桃粕酶解液进行超滤，5kDa分子量以下的滤出物为ACE抑制肽
(4)将步骤(3)得到的粗脂质体混悬液于4℃下超声处理得到纳米脂质体溶液。
(5)将步骤(4)得到的纳米脂质体逐滴加入到等体积的浓度为1-2％的壳聚糖溶液中，持续搅拌1h后，得到第一层修饰的壳聚糖-ACE抑制肽脂质体溶液。
(6)将步骤(5)得到的壳聚糖-ACE抑制肽脂质体逐滴加入到等体积的浓度为1-2％的海藻酸钠溶液中，持续搅拌1h后，得到第二层修饰的海藻酸钠-壳聚糖-ACE抑制肽脂质体溶液。
(7)将步骤(6)得到的海藻酸钠-壳聚糖-ACE抑制肽脂质体逐滴加入到等体积的浓度为1-2％的羧乙基纤维素溶液，持续搅拌1h后，得到第三层修饰的羧乙基纤维素-海藻酸钠-壳聚糖-ACE抑制肽脂质体溶液。
实施例1
1)称取0.6g大豆卵磷脂，0.1g胆固醇和0.1mL吐温-80，完全溶解于10mL无水乙醇中，在60℃下条件下旋转蒸发除去无水乙醇，形成均匀薄膜。
2)核桃脱脂粉与水按照1:7(v:v)比例混合，搅拌均匀后加入非蛋白复合酶(纤维素酶、α-淀粉酶与果胶酶按照5：2：3组成)，非蛋白复合酶的添加量为脱脂粉质量的0.1％，反应温度为35℃，反应时间3h，以300r/min的速率全程搅拌，得到破壁的悬浮液。调节温度50℃，加入1mol/L NaOH溶液调节pH为8.0，加入脱脂粉质量0.3％的碱性蛋白酶，以600r/min的速率全程搅拌。通过滴加氢氧化钠溶液维持pH不变，反应4h后得到核桃粕酶解液。将水浴锅温度升至90℃，高温灭酶10min，冷却至室温后调pH为7。酶解液4000r/min离心15min，再8000r/min低温高速离心20min，搜集上清液。
3)加入含有0.02g ACE抑制肽的10mL PBS缓冲液(pH 7.4；0.01M)，得到粗脂质体混悬液。
4)将粗脂质体混悬液于4℃下超声处理得到纳米脂质体溶液。
5)1g壳聚糖溶解于100mL 1％乙酸溶液，离心取上清液，上清液过0.22μm滤膜，调节pH为5.5。将纳米脂质体按照等体积比1:1，逐滴加入壳聚糖溶液中，持续搅拌1h后，得到壳聚糖-ACE抑制肽脂质体溶液。
6)1g海藻酸钠溶解于100mL蒸馏水中，离心取上清液，上清液过0.22μm滤膜，调节pH为5.5。将壳聚糖-ACE抑制肽脂质体按照等体积比1:1，逐滴加入至海藻酸钠溶液中，持续搅拌1h后，得到海藻酸钠-壳聚糖-ACE抑制肽脂质体溶液。
7)1g羧乙基纤维素溶解于100mL蒸馏水中，离心取上清液，上清液过0.22μm滤膜，调节pH为5.5。将海藻酸钠-壳聚糖-ACE抑制肽脂质体按照等体积比1:1，逐滴加入至羧乙基纤维素溶液中，持续搅拌1h后，得到羧乙基纤维素-海藻酸钠-壳聚糖-ACE抑制肽脂质体溶液。
制备的ACE抑制肽的抑制率为75.29％，羧乙基纤维素-海藻酸钠-壳聚糖-ACE抑制肽脂质体的粒径为288.45nm，ζ电位为+21.5mV，包埋率为92.6％，包埋后ACE抑制率为41.26％，经模拟胃肠消化实验后，ACE抑制率为68.95％。
对比例1
1)称取0.6g大豆卵磷脂，0.1g胆固醇和0.1mL吐温-80，完全溶解于10mL无水乙醇中，在60℃下条件下旋转蒸发除去无水乙醇，形成均匀薄膜。
2)加入含有0.02g ACE抑制肽(制备方法同实施例1)的20mL PBS缓冲液(pH 7.4；0.01M)，得到粗脂质体混悬液。
3)将粗脂质体混悬液于4℃下超声处理得到纳米脂质体溶液。
制备的ACE抑制肽的抑制率为75.29％，脂质体的粒径为69.33nm，ζ电位为-7.5mV，包埋率为63.09％，包埋后抑制率为55.03％，模拟胃肠消化实验，ACE抑制率为22.02％。
对比例2
1)称取0.6g大豆卵磷脂，0.1g胆固醇和0.1mL吐温-80，完全溶解于10mL无水乙醇中，在60℃下条件下旋转蒸发除去无水乙醇，形成均匀薄膜。
2)加入含有0.02g ACE抑制肽(制备方法同实施例1)的20mL PBS缓冲液(pH 7.4；0.01M)，得到粗脂质体混悬液。
3)将粗脂质体混悬液于4℃下超声处理得到纳米脂质体溶液。
4)1g壳聚糖溶解于100mL 1％乙酸溶液，超声离心取上清液，上清液过0.22μm滤膜，调节pH为5.5。将纳米脂质体按照等体积比1:1，逐滴加入壳聚糖溶液中，持续搅拌1h后，得到壳聚糖-ACE抑制肽脂质体溶液。
5)1g海藻酸钠溶解于100mL蒸馏水中，超声离心取上清液，上清液过0.22μm滤膜，调节pH为5.5。将壳聚糖-ACE抑制肽脂质体按照等体积比1:1，逐滴加入至海藻酸钠溶液中，持续搅拌1h后，得到海藻酸钠-壳聚糖-ACE抑制肽脂质体溶液。
制备的ACE抑制肽的抑制率为75.29％，海藻酸钠-壳聚糖-ACE抑制肽脂质体的粒径为223.57nm，ζ电位为-17.1mV，包埋率为84.49％，包埋后抑制率为50.78％，模拟胃肠消化实验，抑制率为42.15％。
由以上实施例可知，本发明以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (9)
1.一种稳定性高、在小肠定向释放的ACE抑制肽脂质体，其特征在于，ACE抑制肽脂质体外附着修饰层，所述修饰层由内到外为壳聚糖、海藻酸钠和羧乙基纤维素。
2.一种稳定性高、在小肠定向释放的ACE抑制肽脂质体的制备方法，其特征在于，包括以下步骤：
1)脂质体制备：
将大豆卵磷脂、胆固醇、吐温-80溶解于无水乙醇溶液，四者比例依次为6:1:1:100(m:m:v:v)，在60℃旋转蒸发形成薄膜。
向旋蒸瓶中加入含有浓度为0.1％-0.3％(g/mL)的ACE抑制肽溶液，加入量与所述无水乙醇的体积比为1:1，于60℃水合1h，得到粗脂质体混悬液。将所述粗脂质体混悬液超声处理得到纳米脂质体。
2)壳聚糖修饰：
将脂质体逐滴加入到等体积的浓度为1-2％(g/mL)的壳聚糖溶液，边加边搅拌，得到壳聚糖-ACE抑制肽脂质体，4℃储存。
3)海藻酸钠修饰：
将所述壳聚糖-ACE抑制肽脂质体溶液逐滴加入到等体积的浓度为1-2％(g/mL)的海藻酸钠溶液中，边加边搅拌，得到海藻酸钠-壳聚糖-ACE抑制肽脂质体，4℃储存。
4)羧乙基纤维素修饰：
所述海藻酸钠-壳聚糖-ACE抑制肽脂质体溶液逐滴加入到等体积的浓度为1-2％(g/mL)的羧乙基纤维素溶液中，边加边搅拌，得到三层修饰的羧乙基纤维素-海藻酸钠-壳聚糖-ACE抑制肽脂质体，4℃储存。
3.根据权利要求2所述制备方法，其特征在于，步骤1)中所述ACE抑制肽的制备方法包括：核桃脱脂粉与水按照1:7(v:v)比例混合，使用非蛋白复合酶进行第一次酶解，得脱脂核桃粕破壁悬浮液。使用碱性蛋白酶对所述脱脂核桃粕破壁悬浮液进行第二次酶解，得到核桃粕酶解液。对所述核桃粕酶解液进行超滤，5kDa分子量以下的滤出物为ACE抑制肽。
4.根据权利要求2所述制备方法，其特征在于，步骤1)中所述超声处理的条件为：超声频率20kHz，功率为500W，间隔2s循环工作；超声温度为4℃，超声时间8min。
5.根据权利要求2所述制备方法，其特征在于，步骤2)中所述壳聚糖溶液的制备方法包括：壳聚糖溶解于1％乙酸溶液中，离心取上清液，上清液过0.22μm滤膜，调节pH为5.5。
6.根据权利要求2所述制备方法，其特征在于，步骤3)中所述海藻酸钠溶液的制备方法包括：海藻酸钠溶解于蒸馏水中，离心取上清液，上清液过0.22μm滤膜，调节pH为5.5。
7.根据权利要求2所述制备方法，其特征在于，步骤4)中所述羧乙基纤维素溶液的制备方法包括：羧乙基纤维素溶解于蒸馏水中，离心取上清液，上清液过0.22μm滤膜，调节pH为5.5。
8.根据权利要求3所述ACE抑制肽制备方法，其特征在于，所述非蛋白复合酶组成为(纤维素酶、α-淀粉酶与果胶酶按照5：2：3组成)，非蛋白复合酶的添加量为脱脂粉质量的0.1％，反应温度为35℃，反应时间3h。
9.根据权利要求3所述ACE抑制肽制备方法，其特征在于，所述第二次酶解的酶解条件为：所述碱性蛋白酶加入量为脱脂粉质量的0.1-0.4％，酶解温度为50℃，pH为8.0，酶解时间4h后得到核桃粕酶解液。
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