CN111420064B - 一种蛋白-EGCG复合纳米颗粒和抗氧化Pickering高内相乳液 - Google Patents

一种蛋白-EGCG复合纳米颗粒和抗氧化Pickering高内相乳液 Download PDF

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CN111420064B
CN111420064B CN202010315392.6A CN202010315392A CN111420064B CN 111420064 B CN111420064 B CN 111420064B CN 202010315392 A CN202010315392 A CN 202010315392A CN 111420064 B CN111420064 B CN 111420064B
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唐传核
彭丽萍
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South China University of Technology SCUT
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Abstract

本发明公开了一种蛋白‑EGCG复合纳米颗粒和抗氧化Pickering高内相乳液。本发明的蛋白‑EGCG复合纳米颗粒的制备方法包括以下步骤：1)将大豆β‑伴球蛋白加水分散，进行水化，得到蛋白溶液；2)将EGCG加水分散，得到EGCG溶液；3)将蛋白溶液加入极性有机溶剂中使蛋白质变性，再加入EGCG溶液，得到混合液；4)将混合液透析去除极性有机溶剂和游离的EGCG，得到蛋白‑EGCG复合纳米颗粒。本发明的抗氧化Pickering高内相乳液中的稳定剂为上述蛋白‑EGCG复合纳米颗粒。本发明的蛋白‑EGCG复合纳米颗粒具有良好的生物相容性，将其用作稳定剂制备的Pickering高内相乳液具有优异的抗氧化特性、热稳定性和储存稳定性。

Description

一种蛋白-EGCG复合纳米颗粒和抗氧化Pickering高内相乳液
技术领域
本发明涉及一种蛋白-EGCG复合纳米颗粒和抗氧化Pickering高内相乳液。
背景技术
表没食子儿茶素没食子酸酯(EGCG)是从茶叶中分离得到的儿茶素类单体，具有抗菌、抗病毒、抗氧化、抗动脉硬化、抗血栓形成、抗血管增生、抗炎、抗肿瘤等作用，可以预防血栓、癌症、糖尿病、心血管疾病、神经退行性疾病等疾病。因此，利用其优良特性来开发一些功能食品是发展健康产业的内在需求。然而，EGCG的稳定性较差，易受温度、pH和氧的影响，而且其口服的生物利用度很低，这也使得这类多酚活性物质在临床应用上缺乏显著的疗效。
研究发现，纳米包埋技术(即指将活性物质包埋于纳米粒子内部或吸附于纳米粒子表面)可以有效提高EGCG的生物利用率。食品蛋白基纳米颗粒具有无毒、可降解、价格低廉、生物相容性好等优点，因此常被用作纳米载体以改善活性物质(包括多酚)的稳定性和生物利用度。但是，现有的蛋白基纳米载体制备方法如共价交联、纳米复合、乳化-蒸发等普遍存在荷载量低、药物突释、交联剂使用等问题，例如：CN 110393683 A公开了采用经多重物理诱导改性的食品蛋白为载体在特定条件下封装载运茶叶多酚，然后结合壳聚糖构建三元复合稳态体系来提高其稳定性和活性功效，但是其制备过程繁琐复杂、耗时耗力、成本高，难以推广应用。因此，如何通过简便工艺制备出成本低、载药量高、稳定性好、生物毒性小的载体是目前食品和药品领域亟待解决的问题。
高内相乳液(油相体积不低于74％)目前已经广泛应用于化妆品、食品、医药及化工等领域。近年来，将具有成本低、用量少、环境友好、安全性高等特点的蛋白基颗粒用作皮克林高内相乳液(Pickering HIPEs)的稳定剂成为了研究的热点方向。然而，高内相乳液由于含油体积高而油相易氧化变质，限制了其在食品中的应用，而且众多营养物质为脂溶性活性物质，不易被人体吸收或者在人体中过早失效，很难缓释起到靶向作用。此外，食品在加工储藏运输过程中易受到各种外界因素的影响，例如：温度、湿度等。因此，如何开发一种具有良好稳定性、强抗氧化性的皮克林稳定剂，进而制备得到抗氧化Pickering高内相乳液是目前食品和药品领域亟待解决的问题。
发明内容
本发明的目的在于提供一种蛋白-EGCG复合纳米颗粒和抗氧化Pickering高内相乳液。
本发明所采取的技术方案是：
一种蛋白-EGCG复合纳米颗粒的制备方法，包括以下步骤：
1)将大豆β-伴球蛋白加水分散，进行水化，得到蛋白溶液；
2)将EGCG加水分散，得到EGCG溶液；
3)将蛋白溶液加入极性有机溶剂中使蛋白质变性，再加入EGCG溶液，得到混合液；
4)将混合液透析去除极性有机溶剂和游离的EGCG，得到蛋白-EGCG复合纳米颗粒。
优选的，所述大豆β-伴球蛋白、EGCG的质量比为1：(0.01～0.3)。
优选的，步骤1)所述蛋白溶液的浓度为0.5wt％～10wt％。
优选的，步骤1)中进行水化前还进行了以下操作：加入质量分数0.08％的叠氮钠溶液，再将溶液的pH调节至7，搅拌均匀后离心去除不溶性杂质。
优选的，步骤2)所述EGCG溶液的浓度为0.2wt％～2wt％。
优选的，步骤3)所述混合液中大豆β-伴球蛋白的浓度为2～20mg/mL。
优选的，步骤3)所述混合液中极性有机溶剂的体积百分含量为30％～40％。
优选的，步骤3)所述极性有机溶剂为乙醇、甲醇、丙酮中的一种。
优选的，步骤4)所述透析的温度为2～6℃，时间为64～72h。
优选的，步骤4)中进行透析所采用的透析液为去离子水，每隔8h更换一次。
一种蛋白-EGCG复合纳米颗粒，由上述方法制备得到。
一种抗氧化Pickering高内相乳液，稳定剂为上述蛋白-EGCG复合纳米颗粒。
优选的，所述抗氧化Pickering高内相乳液中分散相和连续相的体积比为4:1。
本发明的原理如下：在极性有机试剂的作用下，天然蛋白质结构的构象会发生改变，蛋白质结构展开发生变性，从而将埋藏于蛋白质内部的疏水位点暴露于表面，接着在透析去除极性有机试剂和游离EGCG的过程中，暴露的蛋白疏水位点牵引着EGCG富集于蛋白自组装载体内核，从而提高了活性物质的稳定性以及赋予颗粒新的功能特性。
本发明的有益效果是：本发明的蛋白-EGCG复合纳米颗粒具有良好的生物相容性，将其用作稳定剂制备的Pickering高内相乳液具有优异的抗氧化特性、热稳定性和储存稳定性。
具体来说：
1)本发明的蛋白-EGCG复合纳米颗粒具有良好的生物相容性，且大豆β-伴球蛋白(β-CG)和EGCG均营养丰富、来源广泛，有益于人类健康；
2)本发明的蛋白-EGCG复合纳米颗粒可以高效地稳定Pickering高内相乳液，而且可以赋予其优越的抗氧化特性，不仅可以抑制油脂的氧化，还能作为脂溶性活性物质(例如：β-胡萝卜素)潜在的保护容器和输送体系，同时该Pickering高内相乳液具有良好的热稳定性和储存稳定性；
3)本发明的蛋白-EGCG复合纳米颗粒的制备方法简单、安全、成本低、能耗低，操作可控，适合大规模工业化生产与加工，在食品、保健品、日化用品、药品行业具有广阔的应用前景。
附图说明
图1为实施例1中大豆β-伴球蛋白浓度为1wt％、乙醇体积分数为40％、EGCG浓度依次为0～0.3wt％时得到的纳米颗粒溶液进行透析前后的外观图。
图2为实施例1中大豆β-伴球蛋白浓度为1wt％、乙醇体积分数为40％、EGCG浓度依次为0～0.3wt％时得到的纳米颗粒的水合粒径图。
图3为实施例1中大豆β-伴球蛋白浓度为1wt％、乙醇体积分数为40％、EGCG浓度依次为0.05wt％～0.3wt％时得到的蛋白-EGCG复合纳米颗粒中β-CG对EGCG的荷载量图。
图4为实施例1中大豆β-伴球蛋白浓度为1wt％、乙醇体积分数为40％、EGCG浓度依次为0～0.3wt％时，纳米颗粒的形成机理图。
图5为实施例2中的Pickering高内相乳液的外观图和光学显微镜图。
图6为实施例2中的Pickering高内相乳液的弹性模量(G′，实心)和粘性模量(G″，空心)随频率的变化情况图。
图7为实施例2中的Pickering高内相乳液室温储藏50天前后的外观图和光学显微镜图。
图8为实施例2中的Pickering高内相乳液热处理前后的外观图和光学显微镜图以及冻融处理一次后的外观图。
图9为实施例2中的Pickering高内相乳液于50℃电热恒温箱中储藏21天内产生的初级氧化产物氢过氧化物和次级氧化产物丙二醛的含量随时间变化图。
图10为实施例3中的Pickering高内相乳液经热处理后的外观图。
图11为实施例3中的Pickering高内相乳液经热处理后的β-胡萝卜素的保留率图。
具体实施方式
下面结合具体实施例对本发明作进一步的解释和说明。
实施例1：
一种纳米颗粒，其制备方法包括以下步骤：
1)将5g的大豆β-伴球蛋白粉末分散在100g的蒸馏水中，室温搅拌2h，再加入1滴质量分数0.08％的叠氮化钠溶液(防止微生物的生长)，用浓度0.2mol/L的HCl溶液和浓度0.2mol/L的NaOH溶液调节溶液的pH＝7，搅拌2h后置于4℃冰箱中过夜，再从冰箱中取出恢复至室温，离心(8000rpm，15min)去除不溶性杂质，得到浓度5wt％的蛋白溶液；
2)将0.4g的EGCG粉末分散在20g的蒸馏水中，室温搅拌2h后置于4℃冰箱中过夜，得到浓度2wt％的EGCG溶液；
3)取出5个容量50mL的高脚烧杯分别编号为1、2、3、4、5号样品，先向所有烧杯中加入20mL的无水乙醇，再向1、2、3、4、5号样品中依次添加20mL、18.75mL、17.5mL、15mL、12.5mL去离子水，搅拌均匀，再各加入10mL的蛋白溶液，搅拌均匀，再边搅拌边逐滴加入EGCG溶液，依次向5个样品中添加0mL、1.25mL、2.5mL、5mL、7.5mL的EGCG溶液，使最终体系中蛋白浓度为10mg/mL，乙醇体积分数为40％，EGCG浓度依次为0mg/mL、0.5mg/mL、0.1mg/mL、0.2mg/mL、0.3mg/mL，继续搅拌3h，得到混合液；
4)将混合液置于4℃冰箱中透析三天(透析液为蒸馏水，透析袋截留分子量为3500Da，每隔8h更换一次透析液)，调节pH至7，得到纳米颗粒溶液(1号样品为蛋白颗粒，2～5号样品为蛋白-EGCG复合纳米颗粒)。
性能测试：
大豆β-伴球蛋白浓度为1wt％、乙醇体积分数为40％、EGCG浓度依次为0～0.3wt％时得到的纳米颗粒溶液进行透析前后的外观图如图1所示。
由图1可知：1号样品的蛋白颗粒溶液和2～5号样品的蛋白-EGCG复合纳米颗粒溶液透析前后外观和浊度并没有显著变化，都是透明状的，表明形成了纳米级别的颗粒。
大豆β-伴球蛋白浓度为1wt％、乙醇体积分数为40％、EGCG浓度依次为0～0.3wt％时得到的纳米颗粒的水合粒径图如图2所示(图中a～e为显著性差异标注)。
由图2可知：1号样品的蛋白颗粒和2～5号样品的蛋白-EGCG复合纳米颗粒的平均粒径依次为25.7nm、34.43nm、41.16nm、47.46nm和62.12nm。
大豆β-伴球蛋白浓度为1wt％、乙醇体积分数为40％、EGCG浓度依次为0.05wt％～0.3wt％时得到的蛋白-EGCG复合纳米颗粒中β-CG对EGCG的荷载量图如图3所示。
由图3可知：2～5号样品的蛋白-EGCG复合纳米颗粒中β-CG对EGCG的荷载量依次为3.32％、4.92％、7.02％和8.99％。
大豆β-伴球蛋白浓度为1wt％、乙醇体积分数为40％、EGCG浓度依次为0～0.3wt％时，纳米颗粒的形成机理图如图4所示。
实施例2：
一种Pickering高内相乳液，其制备方法包括以下步骤：
将实施例1中1号样品的蛋白颗粒溶液和2～5号样品的蛋白-EGCG复合纳米颗粒溶液分别与亚麻籽油按照体积比1:4混合加入均质机中，5000rpm均质1min，得到Pickering高内相乳液(1号样品对应的Pickering高内相乳液不具备抗氧化功能，2～5号样品对应的Pickering高内相乳液具备抗氧化功能)。
性能测试：
Pickering高内相乳液的外观图和光学显微镜图如图5所示。
Pickering高内相乳液的弹性模量(G′，实心)和粘性模量(G″，空心)随频率的变化情况图如图6所示。
由图5和图6可知：Pickering高内相乳液稳定、乳滴大小均匀、倒置不流动，其形成以弹性模量为主的凝胶状乳液，光学显微镜观察到乳滴大部分呈现紧密堆积且可以看到两个相邻的乳滴共享相同的界面层。
Pickering高内相乳液室温储藏50天前后的外观图和光学显微镜图如图7所示。
由图7可知：经过贮藏Pickering高内相乳液的外观和微观结构并没有发生明显变化，表现出优异的贮藏稳定性。
Pickering高内相乳液热处理(100℃加热15min→水浴15min)前后的外观图和光学显微镜图以及冻融处理(-20℃冷冻24h→25℃加热2h)一次后的外观图如图8所示。
由图8可知：热处理后Pickering高内相乳液仍然能够倒置不流动，没有发生破乳漏油现象，外观和微观结构并没太大改变，热稳定性较好；Pickering高内相乳液热处理后具有更高的冻融稳定性，冻融一次仍能保持原来状态且只发生些许破乳漏油情况，而未进行热处理的Pickering高内相乳液冻融一次即破乳漏油，表明热处理可以提高Pickering高内相乳液的冻融稳定性，充分表明本发明的Pickering高内相乳液具有优良的储藏稳定性和热稳定性。
实施例2中的Pickering高内相乳液于50℃电热恒温箱中储藏21天内产生的初级氧化产物氢过氧化物和次级氧化产物丙二醛的含量随时间变化图如图9所示(空白对照为散装亚麻籽油，样品对照为浓度2wt％的吐温-20稳定的高内相乳液)。
由图9可知：在50℃下贮藏21天后发现，1号颗粒稳定的高内相乳液、2号、3号、4号、5号颗粒(蛋白-EGCG复合纳米颗粒)稳定的高内相乳液、吐温20(2.0wt％)稳定的高内相乳液、散装亚麻籽油于50℃贮藏21天后的油脂产生的初级氧化产物——氢过氧化物含量依次为273.49mmol/kg、170.83mmol/kg、130.96mmol/kg、101.86mmol/kg、71.20mmol/kg、1057.24mmol/kg和763.07mmol/kg，次级氧化产物——丙二醛含量依次为1.51mmol/kg、1.21mmol/kg、0.78mmol/kg、0.65mmol/kg、0.47mmol/kg、4.64mmol/kg和3.79mmol/kg，可见荷载EGCG后其抑制油脂氧化能力明显增强，且随着EGCG浓度增加而增强，有利于延缓油脂在储运过程中的氧化，提高其稳定性。
实施例3：
参照实施例2的方法，将分散相亚麻籽油换成溶有0.003wt％的β-胡萝卜素的正十二烷，再制备Pickering高内相乳液。
性能测试：
Pickering高内相乳液经热处理(70℃电热恒温箱中贮存18h)后的外观图如图10所示，β-胡萝卜素的保留率图如图11所示(图中a～f为显著性差异标注)。
由图10和图11可知：70℃下贮存18h后，1号(未加EGCG)颗粒稳定的高内相乳液、2号、3号、4号、5号颗粒(蛋白-EGCG复合纳米颗粒)稳定的高内相乳液、吐温20(2.0wt％)稳定的高内相乳液、等体积分散液正十二烷中的β-胡萝卜素保留率分别为88.8％、91.3％、93.9％、95.1％、96.8％、9.53％和67.2％。由此可见，大豆β-伴球蛋白-EGCG组装体纳米颗粒能有效保护油脂中脂溶性活性物质，防止其热降解，其保护作用与EGCG浓度成正比，即浓度越高保护作用越明显。
综上可知：利用乙醇诱导蛋白自组装荷载EGCG制备出的颗粒是纳米级别的，当EGCG浓度为0.3wt％时蛋白荷载量达到8.99wt％，样品粒径随着EGCG浓度增大而增大，用此构建的纳米颗粒稳定的高内相乳液(cβ-CG＝1wt％，φ＝0.8)呈现以弹性模量为主的凝胶状网络结构，在原有良好的热稳定性和贮藏稳定性的基础上又赋予了其优良的抗氧化功能，可以有效保护脂溶性活性物质——β-胡萝卜素的氧化降解以及有效抑制油脂初级氧化和次级氧化，具有广阔的应用前景以及潜在的应用价值。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

1.一种蛋白-EGCG复合纳米颗粒的制备方法，其特征在于：包括以下步骤：
1)将大豆β-伴球蛋白加水分散，进行水化，得到蛋白溶液；
2)将EGCG加水分散，得到EGCG溶液；
3)将蛋白溶液加入极性有机溶剂中使蛋白质变性，再加入EGCG溶液，得到混合液；
4)将混合液透析去除极性有机溶剂和游离的EGCG，得到蛋白-EGCG复合纳米颗粒。
2.根据权利要求1所述的制备方法，其特征在于：所述大豆β-伴球蛋白、EGCG的质量比为1：(0.01～0.3)。
3.根据权利要求1或2所述的制备方法，其特征在于：步骤1)所述蛋白溶液的浓度为0.5wt％～10wt％；步骤2)所述EGCG溶液的浓度为0.2wt％～2wt％。
4.根据权利要求1或2所述的制备方法，其特征在于：步骤3)所述混合液中大豆β-伴球蛋白的浓度为2～20mg/mL。
5.根据权利要求1或2所述的制备方法，其特征在于：步骤3)所述混合液中极性有机溶剂的体积百分含量为30％～40％。
6.根据权利要求1或2所述的制备方法，其特征在于：步骤3)所述极性有机溶剂为乙醇、甲醇、丙酮中的一种。
7.根据权利要求1或2所述的制备方法，其特征在于：步骤4)所述透析的温度为2～6℃，时间为64～72h。
8.一种蛋白-EGCG复合纳米颗粒，其特征在于：由权利要求1～7中任意一项所述的方法制备得到。
9.一种抗氧化Pickering高内相乳液，其特征在于：稳定剂为权利要求8的蛋白-EGCG复合纳米颗粒。
10.根据权利要求9所述的抗氧化Pickering高内相乳液，其特征在于：分散相和连续相的体积比为4:1。
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