CN104605228B - 一种EGCG壳聚糖/β‑乳球蛋白复合纳米粒及其制备方法 - Google Patents
一种EGCG壳聚糖/β‑乳球蛋白复合纳米粒及其制备方法 Download PDFInfo
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Abstract
一种EGCG壳聚糖/β‑乳球蛋白复合纳米颗粒,其特征是所述复合纳米颗粒由EGCG、壳聚糖以及β‑乳球蛋白组成,所述EGCG、壳聚糖以及β‑乳球蛋白的质量比为1:3‑6:1‑3,所述壳聚糖选自羧甲基壳聚糖和壳聚糖盐酸盐的混合,其质量比为1:1‑1.5;所述复合纳米颗粒的粒径范围为100‑300nm,Zeta电位为20‑30mV。本发明利用壳聚糖羧甲基壳聚糖和壳聚糖盐酸盐以及β‑乳球蛋白制备了包封EGCG的壳聚糖/β‑乳球蛋白复合纳米颗粒。通过热处理的β‑乳球蛋白与壳聚糖相互交联与组装,进而形成运输EGCG的“芯‑壳”复合纳米颗粒结构。该壳聚糖/β‑乳球蛋白复合纳米颗粒在体内能够避免酸和酶的降解而具有很强的稳定性,具有一定的靶向和缓释性能,解决了目前壳聚糖‑三聚磷酸钠纳米颗粒作为口服输送载体容易被胃酸降解的问题,提高了EGCG口服吸收的生物利用效率。
Description
技术领域
本发明专利涉及一种可用于食品工业中的EGCG壳聚糖/β-乳球蛋白复合纳米颗粒及其制备方法,属于茶叶及食品深加工技术领域。
背景技术
表没食子儿茶素没食子酸酯(Epigallocatechin gallate,EGCG)是绿茶多酚中含量较高的一种重要儿茶素类组分。因其具有突出的抗氧化等生物学活性,而被广泛应用在食品工业领域中。然而,EGCG的口服吸收较差,体内生物利用度较低,需要一种良好的递送载体来增强其肠道的有效吸收。壳聚糖具有良好的生物相容性和生物可降解性,使得其非常适合作为食品营养素的良好递送载体。壳聚糖纳米载体通过调节其包埋营养素的释放速度,使其在预定时间内缓慢释放,并在较长时间内维持有效浓度,延长其作用时间,进而提高营养素的生物利用效率。近年来,有关壳聚糖作为EGCG的包封载体而将其高效递送到肠细胞,进而增强其机体生物利用率的研究,国内外均有相关报道。然而,作为口服输送载体,此类壳聚糖纳米颗粒一般不耐强酸,进而导致被输送的营养物在胃酸中易遭破坏而难以被靶向输送至肠道吸收。此外,壳聚糖能溶于酸性溶液的性能缺陷,也使得其在食品营养素缓释领域的应用受限。因此,利用水溶性壳聚糖衍生物制备包埋EGCG的新型壳聚糖纳米载体,对探索绿茶EGCG口服生物学活性的有效开发与广泛应用尤为关键。
羧甲基壳聚糖(Carboxymethyl chitosan)和壳聚糖盐酸盐(Chitosanhydrochloride)是两种不同的水溶性壳聚糖,因其结构中分别具有特殊的羧甲基和氨基,而能够在水溶液中自组装形成壳聚糖纳米粒。申请人在前期研究中(详见“一种包封茶多酚的壳聚糖纳米粒的制备方法”专利号:ZL 201010176543.0),以这两种水溶性壳聚糖衍生物为纳米粒基质,制备了包封茶多酚的壳聚糖纳米粒,体外缓释实验显示,该纳米粒对茶多酚具有良好的缓释效应。然而,该壳聚糖纳米粒载体在体内易受胃酸的影响而难以将营养物有效输送至肠道吸收。
β-乳球蛋白(β-lactoglobulin)是由反刍动物乳腺上皮细胞分泌的重要的水溶性乳清蛋白。近年来的研究显示,β-乳球蛋白可以作为一种优良的纳米载体与壳聚糖相结合形成运输口服营养素的“芯-壳”复合纳米颗粒,能够有效避免酸和酶的降解,以提高其包封营养素的稳定性和生物利用率(Lingyun Chen,Muriel Subirade.Chitosan/β-lactoglobulin core–shell nanoparticles as nutraceuticalcarriers.Biomaterials,2005(26)6041–6053)。
发明内容
本发明的目的是提供一种新型EGCG壳聚糖/β-乳球蛋白复合纳米粒及其制备方法,以该方法制得的纳米颗粒经口服吸收的过程中,使得儿茶素EGCG在胃肠道内能够避免酸和酶的降解而具有很强的稳定性。
本发明解决技术问题采用如下技术方案:
一种EGCG壳聚糖/β-乳球蛋白复合纳米颗粒,其特征是所述复合纳米颗粒由EGCG、壳聚糖以及β-乳球蛋白组成,所述EGCG、壳聚糖以及β-乳球蛋白的质量比为1:3-6:1-3,所述壳聚糖选自羧甲基壳聚糖和壳聚糖盐酸盐的混合,其质量比为1:1-1.5;所述复合纳米颗粒的粒径范围为100-300nm,Zeta电位为20-30mV。
一种如上所述复合纳米颗粒的制备方法,其特征是,按如下步骤进行:
(1)分别将羧甲基壳聚糖和壳聚糖盐酸盐溶于去离子水溶液中配制成质量浓度1-2%溶液;
(2)将一定量的β-乳球蛋白和EGCG加入壳聚糖盐酸盐溶液中;
(3)在搅拌条件下,将含有β-乳球蛋白和EGCG的壳聚糖盐酸盐溶液中逐滴加入羧甲基壳聚糖溶液,使羧甲基壳聚糖和壳聚糖盐酸盐相互交联,形成纳米颗粒,此时溶液呈现乳光;
(4)继续搅拌并将溶液加温至70-85℃,保持30-60min后冷却至室温,使得β-乳球蛋与EGCG形成稳定、澄清以及高载药量的复合纳米分散体;
(5)将步骤(4)所得的纳米颗粒水溶液经高速冷冻离心机离心后,弃去上清液并冷冻干燥即得复合纳米颗粒。
本发明先通过两种水溶性壳聚糖离子交联而形成包封EGCG的纳米粒,再利用热激β-乳球蛋白与壳聚糖相结合形成的“芯-壳”复合纳米颗粒能够有效运输口服营养素的良好优势,进而制备EGCG壳聚糖/β-乳球蛋白复合纳米颗粒。该“芯-壳”复合纳米颗粒在体内能够避免酸和酶的降解而具有很强的稳定性,进而使包封营养素EGCG具有一定的肠道靶向和缓释性能。
附图说明
图1是本发明纳米颗粒透射电镜图。
图2为本发明EGCG壳聚糖/β-乳球蛋白复合纳米颗粒与不含β-乳球蛋白的壳聚糖EGCG纳米粒在模拟胃肠道环境中的释放率。
以下通过具体实施方式对本发明技术方案做进一步解释说明。
具体实施方式
1.分别称取一定量的羧甲基壳聚糖和壳聚糖盐酸盐用去离子水配制成浓度为1-2%的水溶液,而两种壳聚糖的质量比为:1:1-1.5。两种壳聚糖(羧甲基壳聚糖和壳聚糖盐酸盐)均购于济南海得贝海洋生物工程有限公司。其中的羧甲基壳聚糖的分子量为60kDa,脱乙酰度为80%,壳聚糖盐酸盐的分子量为90kDa,脱乙酰度为85%。
2.依照EGCG、壳聚糖总量以及β-乳球蛋白的质量比为1:3-6:1-3的添加比例,将一定量的β-乳球蛋白和EGCG加入壳聚糖盐酸盐溶液中并在室温下搅拌均匀(转速为500-800rpm,时间为20-30min)。其中的β-乳球蛋白(纯度为90%)购于上海华壹生物科技有限公司;EGCG(纯度为≥90%)购于无锡太阳绿宝科技有限公司。
3.在搅拌条件下(转速为500-800rpm),将含有β-乳球蛋白和EGCG的壳聚糖盐酸盐溶液中逐滴加入羧甲基壳聚糖溶液,使羧甲基壳聚糖和壳聚糖盐酸盐相互交联,形成纳米颗粒,此时溶液呈现乳光。
4.继续搅拌并将溶液加温至70-85℃,保持30-60min后冷却至室温,使得β-乳球蛋与EGCG形成稳定、澄清以及高载药量的复合纳米分散体。
5.将纳米颗粒的水溶液经高速冷冻离心机离心(转速15000-20000rpm,时间30-60min)后,弃去上清液并冷冻干燥,制备成冻干粉,其粒径范围为100-300nm,Zeta电位为20-30mV。
6.将包埋EGCG的壳聚糖/β-乳球蛋白复合纳米颗粒冻干粉(即本发明冻干粉)以及不含β-乳球蛋白的壳聚糖EGCG纳米粒(制备方法同本发明制备方法)对照样分别添加到pH值6.8-7.0的磷酸盐缓冲液中,再用HCl溶液调整pH值为2.0,并添加一定量的猪胃蛋白酶,使得溶液中猪胃蛋白酶浓度达到0.4-0.5%,在37℃振荡水浴中孵育150-200rpm,立即将上述样品液在0,5,10,20,30,60,90和120分钟收集并检测EGCG的释放特性。随后,利用碳酸氢钠溶液将反应液调节至pH5.3,再加入一定量的脂肪酶、胰酶和胆汁提取物配制肠液环境(具体量为:1.5毫升的20mM磷酸盐缓冲配置液中含有0.2毫克的脂肪酶,0.4mg胰酶和2.4毫克的胆汁提取物),在37℃振荡水浴中孵育150-200rpm。样品液在0,10,20,30,60,120,180和240分钟收集,检测EGCG的释放特性。释放结果如图2所示,与对照组相比,EGCG在模拟胃液中的释放量相对较低,表明壳聚糖/β-乳球蛋白复合纳米颗粒能够有效增强EGCG的稳定性,提高EGCG的口服吸收利用度。
Claims (2)
1.一种EGCG壳聚糖/β-乳球蛋白复合纳米颗粒,其特征是所述复合纳米颗粒由EGCG、壳聚糖以及β-乳球蛋白制成,所述EGCG、壳聚糖以及β-乳球蛋白的质量比为1:3-6:1-3,所述壳聚糖是羧甲基壳聚糖和壳聚糖盐酸盐,其质量比为1:1-1.5;所述复合纳米颗粒的粒径范围为100-300nm,Zeta电位为20-30mV。
2.一种权利要求1所述复合纳米颗粒的制备方法,其特征是,按如下步骤进行:
(1)分别将羧甲基壳聚糖和壳聚糖盐酸盐溶于去离子水溶液中配制成质量浓度1-2%溶液;
(2)将一定量的β-乳球蛋白和EGCG加入壳聚糖盐酸盐溶液中;
(3)在搅拌条件下,将含有β-乳球蛋白和EGCG的壳聚糖盐酸盐溶液中逐滴加入羧甲基壳聚糖溶液,使羧甲基壳聚糖和壳聚糖盐酸盐相互交联,形成纳米颗粒,此时溶液呈现乳光;
(4)继续搅拌并将溶液加温至70-85℃,保持30-60min后冷却至室温,使得β-乳球蛋与EGCG形成稳定、澄清以及高载药量的复合纳米分散体。
(5)将步骤(4)所得的纳米颗粒水溶液经高速冷冻离心机离心后,弃去上清液并冷冻干燥即得复合纳米颗粒。
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