CN109568566B - 多酚-胶原肽-多糖纳米胶囊及其制备方法 - Google Patents
多酚-胶原肽-多糖纳米胶囊及其制备方法 Download PDFInfo
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Abstract
本发明涉及一种多酚‑胶原肽‑多糖纳米胶囊的制备方法，包括以下步骤：将植物多酚和多糖分别溶于溶剂中，然后在500～1000rpm的搅拌速度下，按照100:1～1:100的重量比将植物多酚溶液滴加至多糖溶液中，搅拌12～15h，得到植物多酚‑多糖复合体；将胶原肽溶于溶剂中，然后在500～1000rpm的搅拌速度下，将植物多酚‑多糖复合体以0.5d/s～5d/s的速度滴加至胶原肽溶液中，离心，收集沉淀，超声分散。本发明的方法单易行，安全可靠，适合大规模生产。
Description
技术领域
本发明涉及食品保健和医药领域，尤其涉及一种多酚-胶原肽-多糖纳米胶囊及其制备方法。
背景技术
多酚是存在于植物体内的一种次生代谢产物，其以苯酚为基本结构，以苯环的多羟基取代为特征，既包括了含有1个酚环分子的简单酚类，如酚酸和酚醇，还包括具有多个酚环分子的复杂酚类化合物。目前报道较多的的植物多酚包括了酚酸、黄酮、花青素、香豆素类、单宁等。研究已证实,植物多酚具有清除自由基、防止DNA受损、调节细胞内抗氧化防御系统及抗肿瘤、抗炎、抗肥胖等生物学功能。尽管多酚类化合物具有多种优异的生物功能，但是容易被消化系统分解破坏而失活，因此需要制备包载多酚的载体，以提高多酚稳定性和活性。目前公开文献中所报道的载体主要为合成材料，如合成高分子材料PLGA-PEG等，但是这些技术应用于食品和药品领域仍然存在很多局限性，如纳米载体的稳定性、包封率较低、制备过程复杂繁琐，甚至可能涉及到有机溶剂的使用。
胶原蛋白富含甘氨酸、脯氨酸和羟脯氨酸，其降解物，即胶原肽，由于分子量小，不仅具有更好的消化性，还具有抗氧化、促角膜上皮生产等生理活性。多糖是自然界含量最丰富的天然聚合物，其普遍存在于动植物中，研究表明多糖具有免疫调节、抗感染、抗肿瘤等多种生物学功能。因此也有少量文献资料探索了将这些生物天然材料应用于多酚纳米载体的制备中。如专利CN201210375599.8公开了一种包封多酚类活性物质的纳米粒子及其制备方法，其制备方法步骤为(1)美拉德反应制备蛋白-多糖共价结合物；(2)室温下，水溶液直接混合多酚类与蛋白-多糖共价结合物，并调节pH值为3-8使得多酚类化合物与蛋白-多糖共价结合物通过非共价作用力进行自组装形成多酚类化合物-蛋白-多糖纳米粒子。但是美拉德反应是一个十分复杂的反应过程，中间产物众多，终产物结构十分复杂，完全抑制美拉德反应相当困难。此外，蛋白质与糖的结合产物不易被酶利用，营养成分不被消化，甚至还可能会产生有毒物质。专利CN201711403972.5公开了一种蛋白多酚复合微球的制备方法，其步骤为(1)用去离子水将蛋白提取物配制成浓度为10mg/mL～0.2mg/mL的蛋白提取物水溶液；(2)用去离子水将多酚类化合物配制成浓度为50mg/mL～0.2mg/mL的多酚类化合物水溶液；(3)用去离子水将醛基化合物配制成体积百分比为10％～0.001％的醛基化合物水溶液；(4)将步骤(1)得到的蛋白提取物水溶液和步骤(2)得到的多酚类化合物水溶液按照体积比1：(1～2)计量并混合得到第一混合液；将第一混合液和步骤(3)得到的醛基化合物水溶液按照体积比100：(1～4)计量并混合得到第二混合液；将第二混合液于室温搅拌反应，搅拌反应时间的控制以第二混合液转变为呈现乳光或乳白色的混悬液即可；(5)将步骤(4)所得混悬液离固液分离，再将分离所得固体粒子洗涤、冷冻干燥即得到蛋白-多酚复合微球。而实际上，本领域技术人员在实际实验中发现，蛋白与多酚溶液混合时通常会由于两者的共价作用力，产生不可逆的凝聚沉淀反应。
发明内容
为解决上述技术问题，本发明的目的是提供一种多酚-胶原肽-多糖纳米胶囊及其制备方法，本发明的方法简单易行，安全可靠，绿色环保，适合大规模生产，所制备的纳米胶囊粒径均一、稳定性良好。
本发明的第一个目的是提供一种多酚-胶原肽-多糖纳米胶囊的制备方法，包括以下步骤：
(1)将植物多酚和多糖分别溶于溶剂中，分别得到植物多酚溶液和多糖溶液，然后在500～1000rpm的搅拌速度下，按照100:1～1:100的重量比将植物多酚溶液滴加至多糖溶液中，搅拌12～15h，得到植物多酚-多糖复合体；
(2)将胶原肽溶于溶剂中，得到胶原肽溶液，然后在500～1000rpm的搅拌速度下，将所述植物多酚-多糖复合体以0.5d/s～5d/s(优选为1d/s)的速度滴加至所述胶原肽溶液中，离心，收集沉淀，超声分散后得到所述多酚-胶原肽-多糖纳米胶囊。
进一步地，在步骤(1)中，所述植物多酚为苹果多酚、花青素类多酚和茶多酚等多种植物来源的多酚中的一种或几种。
进一步地，在步骤(1)中，所述植物多酚溶液的浓度为0.01～1g/L。
进一步地，在步骤(1)中，所述多糖为枸杞多糖和/或香菇多糖等植物来源的多糖。
进一步地，在步骤(1)中，所述多糖溶液的浓度为2～20g/L。
进一步地，在步骤(2)中，所述胶原肽的分子量为0.3～3kDa。优选地，胶原肽的分子量为2～3kDa。胶原肽可以来源于牛、羊等动物的皮或骨。
进一步地，在步骤(2)中，所述胶原肽溶液的浓度为10～50g/L。
进一步地，步骤(1)或步骤(2)在pH为6.8～7.2的条件下进行。
进一步地，在步骤(1)或步骤(2)中，所述溶剂为50％～100％的乙醇水溶液或水。
在本发明中，发明人通过实验发现，通过改变多糖、多酚、胶原肽的滴加顺序与组合，简单地控制搅拌，即可获得稳定的多酚-多糖-胶原肽纳米微胶囊。
在步骤(1)中，植物多酚溶液滴加至多糖溶液中时，二者之间在非共价键相互作用力的影响下发生相互缠结，通过自组装形成植物多酚-多糖复合体，然后将复合体滴加至胶原肽溶液中，胶原肽在多酚-多糖复合体表面形成包裹层从而自发形成纳米粒。
本发明的第二个目的是要求保护一种采用上述制备方法所制备的多酚-胶原肽-多糖纳米胶囊，其粒径为10～300nm。
本发明的方法所制备的多酚-胶原肽-多糖纳米胶囊，其中包括植物多酚、胶原肽和多糖，三者的分子链相互缠结，形成纳米粒。
借由上述方案，本发明至少具有以下优点：
本发明方法简单易行，避免了有机溶剂的使用，使用的原料天然安全，同时提高了植物多酚的稳定性和生物活性，还引入了多糖、胶原肽这两种活性物质，适合大规模生产与应用，显示了多酚在食品、药品及化妆品领域的应用具有良好的前景。
本发明所制备的纳米胶囊粒径均一，稳定性良好。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合附图详细说明如后。
附图说明
图1是透射电镜下不同方法制得的多酚-胶原肽-多糖纳米胶囊微观结构图。
具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1多酚-多糖-胶原肽纳米胶囊的制备及理化性质评价
为了作为对照，采用不同方法制备多酚纳米粒，其中，方法1如下：
参照专利CN201210375599.8方法，利用美拉德反应制备蛋白-多糖共价结合物；室温下，水溶液直接混合多酚类与蛋白-多糖共价结合物，并调节pH值为3使得多酚类化合物与蛋白-多糖共价结合物通过非共价作用力进行自组装形成多酚类化合物-蛋白-多糖纳米粒子。
方法2如下：
参照专利CN201711403972.5方法用去离子水将蛋白提取物配制成浓度为10mg/mL的蛋白提取物水溶液；用去离子水将多酚类化合物配制成浓度为50mg/mL的多酚类化合物水溶液；用去离子水将醛基化合物配制成体积百分比为10％的醛基化合物水溶液；将蛋白提取物水溶液和多酚类化合物水溶液按照体积比1：1计量并混合得到第一混合液；将第一混合液和醛基化合物水溶液按照体积比100：1计量并混合得到第二混合液；将第二混合液于室温搅拌反应,搅拌反应时间的控制以使第二混合液转变为呈现乳光的混悬液，固液分离后再将分离所得固体粒子洗涤、冷冻干燥，即得到蛋白-多酚复合微球。
此外，采用本发明的方法制备纳米胶囊，方法如下：
将苹果多酚溶解于50％的乙醇水溶液中，得到多酚溶液，其浓度为1.0g/L。将枸杞多糖溶解在水中，得到多糖溶液，其浓度为20g/L。在500rpm的搅拌速度下，按照1:50重量比，将多酚溶液滴加至多糖溶液中，搅拌12h，得到多酚-多糖复合体。将胶原肽(分子量为1～3kDa)溶解于水中，得到胶原肽溶液，其浓度为50g/L，在500rpm的搅拌速度下，将多酚-多糖复合体以1d/s的滴加速度滴加至胶原肽溶液中。滴加完毕后，高速离心，收集沉淀，超声分散，即得多酚-胶原肽-多糖纳米胶囊。
将上述三种方法所制备的纳米载体采用粒度仪进行粒径测试，并在透射电镜下进行结构表征，结果分别如表1和图1所示，图1a-c分别为方法1、方法2和本发明方法制备的纳米载体的结构图。表1和图1表明，采用本发明方法制备的多酚纳米载体与现有技术的粒径相当，分散程度接近，其粒径较小，分布均匀。
表1不同方法制备的纳米载体的粒径
“+”表示有；“-”表示没有
实施例2多酚-多糖-胶原肽纳米胶囊的稳定性的评价
采用实施例1中的方法制备三种不同的纳米粒的溶液，将所得的纳米载体在微波下灭菌，测试其稳定性，结果如表2所示，结果表明，经过微波灭菌后，不同方法制备的纳米载体稳定性有差异，本发明的方法所制备的纳米载体稳定性高于现有技术，本发明所得的纳米载体溶液稳定性良好，方法1和方法2都产生了少量的絮凝。
表2不同方法制备的纳米载体的稳定性
实施例3多酚-多糖-胶原肽纳米胶囊的体外抗氧化评价
采用实施例1中的方法制备三种不同的纳米粒溶液，采用总抗氧化能力试剂盒检测抗氧化能力。结果如表3所示，数据表明，采用本发明的方法制得的纳米载体抗氧化性能最强，其次是方法2，方法1所制备的纳米载体的抗氧化性能最低。
表3不同方法制备的纳米载体的抗氧化性
实施例4多酚-多糖-胶原肽纳米胶囊的制备
采用本发明的方法制备纳米胶囊，方法如下：
将花青素类多酚溶解于70％的乙醇水溶液中，得到多酚溶液，其浓度为0.01g/L。将香菇多糖溶解在水中，得到多糖溶液，其浓度为10g/L。在500rpm的搅拌速度下，按照1:100重量比，将多酚溶液滴加至多糖溶液中，搅拌12h，得到多酚-多糖复合体。将胶原肽(分子量为1～3kDa)溶解于水中，得到胶原肽溶液，其浓度为10g/L，在500rpm的搅拌速度下，将多酚-多糖复合体以0.5d/s的滴加速度滴加至胶原肽溶液中。滴加完毕后，高速离心，收集沉淀，超声分散，即得多酚-胶原肽-多糖纳米胶囊。
实施例5多酚-多糖-胶原肽纳米胶囊的制备
采用本发明的方法制备纳米胶囊，方法如下：
将茶多酚溶解于100％的乙醇水溶液中，得到多酚溶液，其浓度为0.08g/L。将香菇多糖溶解在水中，得到多糖溶液，其浓度为5g/L。在500rpm的搅拌速度下，按照100:10重量比，将多酚溶液滴加至多糖溶液中，搅拌12h，得到多酚-多糖复合体。将胶原肽(分子量为1～3kDa)溶解于水中，得到胶原肽溶液，其浓度为30g/L，在500rpm的搅拌速度下，将多酚-多糖复合体以5d/s的滴加速度滴加至胶原肽溶液中。滴加完毕后，高速离心，收集沉淀，超声分散，即得多酚-胶原肽-多糖纳米胶囊。
以上所述仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。
Claims (3)
1.一种多酚-胶原肽-多糖纳米胶囊的制备方法，其特征在于，包括以下步骤：
（1）将植物多酚和多糖分别溶于溶剂中，分别得到植物多酚溶液和多糖溶液，然后在500~1000rpm的搅拌速度下，按照100:1~1:100的重量比将植物多酚溶液滴加至多糖溶液中，搅拌12~15 h，得到植物多酚-多糖复合体；所述植物多酚为苹果多酚、花青素类多酚或茶多酚；所述多糖为枸杞多糖或香菇多糖；所述植物多酚溶液的浓度为0.01~1g/L；所述多糖溶液的浓度为2~20g/L；所述胶原肽溶液的浓度为10~50 g/L；
（2）将胶原肽溶于溶剂中，得到胶原肽溶液，然后在500~1000rpm的搅拌速度下，将所述植物多酚-多糖复合体以0.5滴/s~ 5滴/s的速度滴加至所述胶原肽溶液中，离心，收集沉淀，超声分散后得到所述多酚-胶原肽-多糖纳米胶囊；所述胶原肽的分子量为0.3~3kDa；
步骤（1）或步骤（2）在pH为6.8~7.2的条件下进行。
2.根据权利要求1所述的制备方法，其特征在于：在步骤（1）或步骤（2）中，所述溶剂为50%~100%的乙醇水溶液或水。
3.一种权利要求1或2所述的制备方法所制备的多酚-胶原肽-多糖纳米胶囊，其特征在于：其粒径为10~300nm。
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