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CN113559072B - 一种京尼平交联的蛋白-壳寡糖纳米粒、Pickering乳液、制备方法及应用 - Google Patents

一种京尼平交联的蛋白-壳寡糖纳米粒、Pickering乳液、制备方法及应用 Download PDF

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CN113559072B
CN113559072B CN202111017469.2A CN202111017469A CN113559072B CN 113559072 B CN113559072 B CN 113559072B CN 202111017469 A CN202111017469 A CN 202111017469A CN 113559072 B CN113559072 B CN 113559072B
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续旭
焦博文
毕德成
曹珏
李文灏
姚丽君
李梅婷
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Shenzhen University
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Abstract

本发明公开了一种京尼平交联的蛋白‑壳寡糖纳米粒、Pickering乳液、制备方法及应用，涉及化学制剂技术领域。本发明提供的京尼平交联的蛋白‑壳寡糖纳米粒的具有良好的生物相容性，且大豆分离蛋白和COS均营养丰富、来源广泛，有益于人类健康；可将其用作稳定剂制备Pickering乳液。本发明提供的Pickering乳液，以食用油、水、京尼平交联的蛋白‑壳寡糖纳米粒为原料，通过高能乳化法制备得到的；具有优异的抗氧化特性、热稳定性和储存稳定性，能够保护类似岩藻黄质脂溶性物质的抗氧化性。本发明提供的制备方法具有操作简单、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的优点，获得的产物在食品、保健品、日化用品、药品行业均具有广阔的应用前景。

Description

一种京尼平交联的蛋白-壳寡糖纳米粒、Pickering乳液、制备 方法及应用
技术领域
本发明涉及化学制剂技术领域，尤其涉及一种京尼平交联的蛋白-壳寡糖纳米粒、Pickering乳液、制备方法及应用。
背景技术
Pickering乳液是一种由超细固体粒子作为乳化剂代替传统有机表面活性剂稳定体系的乳液，其具有多种类型，如o/w(水包油)型、w/o(油包水)型，甚至复合型。其稳定机理主要为固体颗粒吸附于油水界面并形成固体颗粒单层或多层膜从而得到稳定乳液。Pickering乳液拥有与传统表面活性剂稳定的乳液相同的性质特征。在一定程度上可作为传统乳液的替代者应用于各领域中。传统乳液所使用的表面活性剂往往有一定程度的副作用，使其在化妆品、药品、食品等领域的使用收到了限制。而由固体颗粒稳定的Pickering乳液相对安全、低毒。因此Pickering乳液在化妆品、食品、制药、石油和废水处理等行业己有广泛应用。
壳寡糖(COS)是壳聚糖/甲壳素通过化学水解或酶促降解(包括脱乙酰和解聚过程)的主要降解产物。COS在生物医学、制药和农业等行业具有很大的作用。COS在化妆品、药品和营养制品以及废水处理等行业具有宽泛的应用。相对于其壳聚糖和其他可用的多糖，COS具有较低的分子量、较高的脱乙酰度、较高的聚合度、较低的粘性和完全的水溶性，这赋予了它显著的生物特性，如抗菌、抗氧化、抗炎和抗高血压，以及药物递送能力。此外，它还显示出抗糖尿病、抗肥胖、抗HIV-1、抗阿尔茨海默病、低胆固醇、钙吸收和止血的作用。
然而，目前尚未有关于将壳寡糖制成超细纳米颗粒得到Pickering乳液的报道，以扩大壳寡糖的普及应用。
发明内容
本发明所要解决的技术问题是如何将壳寡糖制成超细纳米颗粒得到Pickering乳液，扩大壳寡糖的普及应用。
为了解决上述问题，本发明提出以下技术方案：
第一方面，本发明提供一种京尼平交联的蛋白-壳寡糖纳米粒制备方法，包括以下步骤：
S1、用去离子水将壳寡糖配成溶液，得到壳寡糖溶液；用去离子水将大豆分离蛋白配成溶液，得到大豆分离蛋白溶液；用无水乙醇将京尼平配成溶液，得到京尼平溶液；
S2、将壳寡糖溶液与大豆分离蛋白溶液混合，并加入京尼平溶液，进行磁力搅拌反应，获得包含纳米粒子的体系，并将体系冷冻干燥，获得京尼平交联的蛋白-壳寡糖纳米粒；
其进一步地技术方案为，所述壳寡糖溶液中，壳寡糖的用量为去离子水质量的0.1％-10％；
所述大豆分离蛋白溶液中，大豆分离蛋白的用量为去离子水质量的1％-10％；
所述京尼平溶液中，京尼平的用量为无水乙醇质量的0.01-0.1％。
其进一步地技术方案为，所述步骤S2中，混合时，大豆分离蛋白溶液、壳寡糖溶液与京尼平溶液的体积比为1-3:1-2:0.2-0.4。
本发明还提供由第一方面所述的京尼平交联的蛋白-壳寡糖纳米粒制备方法制得的纳米粒在制备Pickering乳液中的应用；或者，制得的纳米粒在制备食品、保健品、日化用品、药品行业中的应用。
具体地，所述的纳米粒在Pickering乳液中作为稳定剂。
第二方面，本发明提供一种含京尼平交联的蛋白-壳寡糖纳米粒的Pickering乳液的制备方法，将纳米粒子均匀分散在去离子水中得到水相，再与油相混合，均质，获得含有大豆分离蛋白-壳寡糖纳米颗粒的Pickering乳液；所述纳米粒子由第一方面所述的京尼平交联的蛋白-壳寡糖纳米粒制备方法制得。
其进一步地技术方案为，所述步骤S3中所述油相占乳液的体积分数为25-75％；所述水相中，纳米粒子的质量分数为0.5％-3％。
其进一步地技术方案为，所述油相包括油脂、油脂与脂溶性活性物质的混合物；所述油脂包括葵花籽油、亚麻籽油、大豆油、玉米油、橄榄油；所述脂溶性活性物质包括岩藻黄质。
其进一步地技术方案为，步骤S2中，所述磁力搅拌反应的时间为12h-108h；
步骤S3中，均质时，转速为9000rpm-13000rpm，均质时间为1min-3min。
第三方面，本发明提供一种含京尼平交联的蛋白-壳寡糖纳米粒的Pickering乳液，由第二方面所述的含京尼平交联的蛋白-壳寡糖纳米粒的Pickering乳液的制备方法制得。
本发明还提供所述的含京尼平交联的蛋白-壳寡糖纳米粒的Pickering乳液在制备食品、保健品、日化用品、药品行业中的应用。
与现有技术相比，本发明所能达到的技术效果包括：
由本发明提供的方法制得的京尼平交联的蛋白-壳寡糖纳米粒的具有良好的生物相容性，且大豆分离蛋白和COS均营养丰富、来源广泛，有益于人类健康；可将其用作稳定剂制备Pickering乳液。
由本发明提供的方法制得的Pickering乳液，以京尼平交联的蛋白-壳寡糖纳米粒作为稳定剂，无添加任何表面活性剂，乳液能稳定保持长达一个月以上：在不同温度下储存以及促氧化实验中，发现本发明提供的含有京尼平交联的蛋白-壳寡糖纳米粒的Pickering乳液具有良好的抗氧化性能。
本发明提供的Pickering乳液，具有优异的抗氧化特性、热稳定性和储存稳定性。
本发明提供的京尼平交联的蛋白-壳寡糖纳米粒的制备方法、含京尼平交联的蛋白-壳寡糖纳米粒的Pickering乳液的制备方法，具有操作简单、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的优点，获得的产物在食品、保健品、日化用品、药品行业均具有广阔的应用前景。
附图说明
图1为本发明实施例1制得的京尼平交联的蛋白-壳寡糖纳米粒粒径分布图；
图2为本发明实施例1制得的京尼平交联的蛋白-壳寡糖纳米粒的扫描电镜图；
图3为本发明实施例3的含京尼平交联的蛋白-壳寡糖纳米粒的Pickering乳液的储存稳定性图；
图4为本发明实施例4的含京尼平交联的蛋白-壳寡糖纳米粒的Pickering乳液的热稳定性结果图；
图5为本发明实施例5的含京尼平交联的蛋白-壳寡糖纳米粒的Pickering乳液的、含蛋白的Pickering乳液的抗氧化性结果对比图。
具体实施方式
下面将结合本发明实施例中的附图，对实施例中的技术方案进行清楚、完整地描述。显然，以下将描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
应当理解，实验中未详述的试验操作均为本领域技术人员所熟知的常规试验操作。
实施例1
制备京尼平交联的蛋白-壳寡糖纳米粒：
将0.05g大豆分离蛋白分散于5mL去离子水中，得到大豆分离蛋白溶液；将0.1g壳寡糖溶于5mL去离子水中，得到壳寡糖溶液；将0.03g京尼平分散于1mL无水乙醇中，得到京尼平溶液；
将5mL大豆分离蛋白溶液、5mL壳寡糖溶液，1mL京尼平溶液混合，磁力搅拌反应72h，获得包含纳米粒子的体系。将得到的纳米粒子溶液透析4h(透析液为去离子水，透析袋截留分子量为500Da)，将透析得到的纳米粒子溶液放置于-80℃，冷冻干燥得到京尼平交联的蛋白-壳寡糖纳米粒。
通过纳米激光粒度分析仪测量获得的京尼平交联的蛋白-壳寡糖纳米粒的粒径，结果如图1所示，平均粒径和ζ-电势如表1所示。
表1京尼平交联的蛋白-壳寡糖纳米粒ζ-电势与平均粒径
京尼平交联的蛋白-壳寡糖纳米粒
ζ-电势(mV) 28.60±1.55
平均粒径(nm) 279±9.33
结果表明，本发明实施例制得的京尼平交联的蛋白-COS纳米颗粒呈现稳定的纳米体系。
实施例2
对实施例1制备得到的京尼平交联的蛋白-壳寡糖纳米粒进行扫描电镜观察：
将冻干的京尼平交联的蛋白-壳寡糖纳米粒放置于样品台上；
对京尼平交联的蛋白-壳寡糖纳米粒喷金；
用扫描电子显微镜观察京尼平交联的蛋白-壳寡糖纳米粒，结果见附图2。可见，本发明实施例制得的京尼平交联的蛋白-壳寡糖纳米粒呈现表面粗糙的球状结构。
实施例3
制备含京尼平交联的蛋白-壳寡糖纳米粒的Pickering乳液：
将实施例1制得的冻干的京尼平交联的蛋白-壳寡糖纳米粒分散在水中作为水相；
将大豆油作为油相；
将水相与油相混合，经高速分散器均质(13000rpm,1min)，获得含有大豆分离蛋白-壳寡糖纳米颗粒的Pickering乳液。
其中，水相中冻干的京尼平交联的蛋白-壳寡糖纳米粒的质量百分数分别为(0.5，1，2，3％wt)；
大豆油占乳液总体系的体积含量分别为(30％，40％，50％，60％，70％)。
分别将制得的具有不同水相和油相的含京尼平交联的蛋白-壳寡糖纳米粒的Pickering乳液装入西林瓶，在4℃下进行储存稳定性实验，并在第七天以及第三十天拍照，结果如图3所示。
结果表明，本发明提供的含京尼平交联的蛋白-壳寡糖纳米粒的Pickering乳液：含不同质量百分比的京尼平交联的蛋白-壳寡糖纳米粒、不同比例的水相和油相，通过均质制得的Pickering乳液在4℃下稳定储存时间长达30天，没有出现分层。
实施例4
含京尼平交联的蛋白-壳寡糖纳米粒的Pickering乳液的热稳定性实验：
将实施例1制得的冻干的京尼平交联的蛋白-壳寡糖纳米粒分散在水中作为水相，质量分数为3％wt；将大豆油作为油相，大豆油占乳液总体系的体积含量为60％，经高速分散器均质(13000rpm,1min)后，制备得到乳液，即为本发明提供的含京尼平交联的蛋白-壳寡糖纳米粒的Pickering乳液。将其分别放置4℃、25℃、37℃、60℃保存，在第1天和第3天取样观察并测定其粒径，评价其稳定性。结果见图4，表明本发明实施例制得的乳液在不同温度下都比较稳定没有出现分层，具有良好的热稳定性。
实施例5
含京尼平交联的蛋白-壳寡糖纳米粒的Pickering乳液的抗氧化性实验：
将1mg岩藻黄质通过超声仪超声溶于2mL大豆油中作为油相；3％wt京尼平交联的蛋白-壳寡糖纳米粒分散在2mL的去离子水中作为水相；将3％wt大豆分离蛋白分散在2mL的去离子水中作为对比的水相；将油相和水相通过13000rpm，1min的条件均质制备成为两种Pickering乳液。分别将两种乳液放置于紫外灯下，选择特定的时间间隔(0，30，60，90，120min)，各取200μL的乳液，加入800μL的无水乙醇与1.5mLEP管中，通过离心机以10000rpm，15min的条件进行离心，取200μL离心之后的上清于96孔板中，在450nm的条件下，使用酶标仪进行测量。
结果见图5，表明本发明提供的含京尼平交联的蛋白-壳寡糖纳米粒的Pickering乳液比使用大豆分离蛋白作为稳定剂的Pickering乳液对岩藻黄质的保留率高，具有更好的抗氧化性。
可见，通过将脂溶性物质(例如：岩藻黄质)包裹京尼平交联的蛋白-壳寡糖纳米粒制得的Pickering乳液中，脂溶性物质的抗氧化能力得到提升。即，本发明提供的京尼平交联的蛋白-壳寡糖纳米粒可以高效地稳定Pickering乳液；而且得到的含京尼平交联的蛋白-壳寡糖纳米粒的Pickering乳液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:岩藻黄质)潜在的保护容器和输送体系,
小结：
本发明实施例制得的京尼平交联的蛋白-壳寡糖纳米粒，其平均粒径为279nm，均为粒度分布均匀的球形粒子。通过比较不同油相水相的乳化指数，获得最优的含京尼平交联的蛋白-壳寡糖纳米粒的Pickering乳液，该乳液具有优异的储存稳定性、热稳定性。
实施例5的抗氧化性实验表明，与大豆分离蛋白作为稳定剂的Pickering乳液相比，本发明提供的含京尼平交联的蛋白-壳寡糖纳米粒的Pickering乳液具有较强的抗氧化性，这对易氧化的脂溶性物质功效性的提高具有重要意义，同时本发明利用Pickering乳液载体实现了对易氧化的脂溶性物质抗氧化性的保护。
在上述实施例中，对各个实施例的描述都各有侧重，某个实施例中没有详细描述的部分，可以参见其他实施例的相关描述。
以上所述，为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到各种等效的修改或替换，这些修改或替换都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以权利要求的保护范围为准。

Claims (4)

1.一种京尼平交联的蛋白-壳寡糖纳米粒制备方法，其特征在于，包括以下步骤：
S1、用去离子水将壳寡糖配成溶液，得到壳寡糖溶液；用去离子水将大豆分离蛋白配成溶液，得到大豆分离蛋白溶液；用无水乙醇将京尼平配成溶液，得到京尼平溶液；
S2、将壳寡糖溶液与大豆分离蛋白溶液混合，并加入京尼平溶液，进行磁力搅拌反应，获得包含纳米粒子的体系，并将体系冷冻干燥，获得京尼平交联的蛋白-壳寡糖纳米粒；
所述壳寡糖溶液中，壳寡糖的用量为去离子水质量的0.1%-10%；
所述大豆分离蛋白溶液中，大豆分离蛋白的用量为去离子水质量的1%-10%；
所述京尼平溶液中，京尼平的用量为无水乙醇质量的0.01-0.1%；
所述步骤S2中，混合时，大豆分离蛋白溶液、壳寡糖溶液与京尼平溶液的体积比为1-3:1-2:0.2-0.4。
2.一种Pickering乳液的制备方法，其特征在于，将由权利要求1所述方法制得的京尼平交联的蛋白-壳寡糖纳米粒均匀分散在去离子水中得到水相，再与油相混合，均质，获得含有大豆分离蛋白-壳寡糖纳米颗粒的Pickering乳液；所述油相占乳液的体积分数为25-75%；所述水相中，纳米粒的质量分数为0.5%-3%；所述油相选自大豆油。
3.如权利要求2所述的Pickering乳液的制备方法，其特征在于，步骤S2中，所述磁力搅拌反应的时间为12h-108h；
均质时，转速为9000rpm-13000rpm，均质时间为1min-3min。
4.一种Pickering乳液，其特征在于，由权利要求2-3任一项所述制备方法制得。
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