CN109965282B - 利用黑茶纳米聚集体联用纳米硒构建功能强化型Pickering乳液及制备方法和应用 - Google Patents
利用黑茶纳米聚集体联用纳米硒构建功能强化型Pickering乳液及制备方法和应用 Download PDFInfo
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Abstract
本发明属于材料技术领域，公开了一种利用黑茶纳米聚集体联用纳米硒构建功能强化型Pickering乳液及其制备方法和应用。该方法包括以下步骤：(1)通过超滤离心分离黑茶纳米聚集体，平均直径＞10nm和平均直径≤10nm两类胶粒。(2)利用黑茶纳米聚集体联用纳米硒构建Pickering乳液：以黑茶纳米聚集体联用纳米硒为Pickering粒子，调节水分散相pH，与油相混合，进行剪切均质和高压均质后得到稳定的Pickering乳液。本发明采用黑茶纳米聚集体联用纳米硒复合体系为Pickering稳定剂，共同构建功能强化型Pickering乳液，拓宽纳米硒的应用，强化Pickering乳液的健康功效。
Description
技术领域
本发明属于材料技术领域，特别涉及一种利用黑茶纳米聚集体联用纳米硒构建功能强化型Pickering乳液及其制备方法和应用。
背景技术
与传统乳状液相比，Pickering乳液是利用具有一定亲/疏水性的固体颗粒替代传统表面活性剂稳定的新型乳液，固体颗粒在油水界面发生不可逆吸附，使乳液热力学稳定性高，其乳化剂用量少，成本低，毒性小，在食品、化妆品等领域具有广泛应用。
黑茶水提取物中含有茶褐素、茶多酚、茶多糖、蛋白质、生物碱等活性组分，各物质能自发聚集形成粒径约1~100 nm的聚集体，可通过超滤离心分离获得。黑茶纳米聚集体含羟基、羧基、氨基等亲水性基团，以及疏水性区域，具有一定的亲/疏水性。
硒（Se）是8种人体必需微量元素之一。纳米硒是平均直径在1~100 nm范围内的无定形零价元素硒，与其他形式的硒相比，纳米硒具有高抗氧化、抗癌、低毒性等优势。目前，已有专利成功将茶水提取物（CN201710770329）和茶纳米聚集体（CN201710771056）用于制备纳米硒，茶水提取物和茶纳米聚集体含茶多酚、生物碱、茶氨酸等物质，各物质是茶叶发挥保健功效的物质基础，更能强化纳米硒功能特性。在应用方面，纳米硒一般作为硒补充剂，近年来，已有专利（CN201110215019）利用纳米硒、模板和药物的复合制备多功能载体，促进了纳米硒在癌症治疗等医药方面的应用，这些研究为构建食物活性组分的多功能载体提供了新思路。
发明内容
为了克服现有技术中存在的缺点和不足，本发明的首要目的在于提供一种利用黑茶纳米聚集体联用纳米硒构建功能强化型Pickering乳液的制备方法；以黑茶纳米聚集体联用纳米硒为Pickering稳定剂，制备功能强化的Pickering乳液。
本发明的另一目的在于提供一种上述制备方法制备得到的Pickering乳液。
本发明的再一目的在于提供一种上述Pickering乳液的应用。
本发明的目的通过下述技术方案实现：
一种利用黑茶纳米聚集体联用纳米硒构建功能强化型Pickering乳液的制备方法，包括以下步骤：
（1）分离黑茶纳米聚集体；
（2）利用黑茶纳米聚集体联用纳米硒制备Pickering乳液。
步骤（1）所述分离黑茶纳米聚集体具体按照以下步骤：向黑茶原料加入温度为25~100 ℃、体积百分比浓度为70%~100%的溶剂，所述溶剂的用量按照每g黑茶原料加入10~250mL溶剂来计；然后在25~100 ℃水浴条件下浸提2~120 min得到黑茶提取液，再使用截留分子量为3~100 kDa的离心超滤管于温度4 ℃、离心力1000~4000 g条件下离心10~40 min，收集内管和外管得到不同粒径范围的黑茶纳米聚集体的溶液，经冷冻干燥，于-20 ℃保存，得到黑茶纳米聚集体。
所述黑茶原料是以茶树[
Camellia sinensis(L.)O.Kuntze]鲜叶和嫩梢为原料，经杀青、揉捻、渥堆、干燥工艺加工制成的产品，优选为茯砖茶、青砖茶、六堡茶、康砖茶、普洱茶或千两茶。
所述溶剂为水、甲醇或乙醇。
所述溶剂的用量按照每g黑茶原料加入50mL溶剂来计；所述水浴的温度为100 ℃；所述浸提的时间为30 min；所述离心超滤管截留分子量为100 kD；所述离心力为4000g；所述离心时间为20 min；所得黑茶纳米聚集体为平均直径＞10 nm和平均直径≤10 nm两类胶粒。
步骤（2）所述利用黑茶纳米聚集体联用纳米硒制备Pickering乳液具体按照以下步骤：制备5~20倍纳米硒稀释液，用纳米硒稀释液溶解黑茶纳米聚集体，使得黑茶纳米聚集体的质量百分数为0.5%~6.0%；加入质量分数0.02%的叠氮钠，调节黑茶纳米聚集体分散相pH为2~11，按油和水的体积比为1:9~6:4加入油相，10000~20000 r/min高速剪切均质1~2min，40MPa高压均质1~3次，得Pickering乳液。
所述纳米硒为软模板法制备所得的液相纳米硒；所述油相与水不相溶。
所述纳米硒稀释液的稀释倍数为20倍；所述黑茶纳米聚集体的质量百分数为2%；所述黑茶纳米聚集体分散相pH为7；所述油和水的体积比为4:6；所述高速剪切均质的转速为20000 r/min，时间为2 min；所述高压均质的压力为40 MPa，均质次数为3次。
一种根据上述的制备方法制备得到的Pickering乳液。
上述的Pickering乳液在食品、化妆品和生物医药领域中的应用。
通过激光光散射技术测定Pickering乳液液滴粒径，评价本发明所得Pickering乳液液滴大小；通过激光多普勒测速技术测定Pickering乳液体系Zeta电位，评价本发明所得Pickering乳液静电稳定性，通过光学显微镜观察乳液液滴微观结构。
通过MTT法检测肿瘤细胞存活率，评价本发明所得纳米硒的抗癌活性。
上述肿瘤细胞模型包括：HCT 116细胞系、Hepa1c1c7细胞系、MDA-MB-231细胞系、HepG2细胞系、Hela细胞系等，优选HCT116细胞系。
本发明相对于现有技术具有如下的优点及有益效果：本发明采用黑茶纳米聚集体联用纳米硒为Pickering稳定剂，共同构建功能强化型Pickering乳液，拓宽纳米硒的应用，强化Pickering乳液的健康功效。
附图说明
图1为黑茶纳米聚集体（平均直径>10 nm）联用纳米硒Pickering乳液液滴的粒径分布。
图2为黑茶纳米聚集体（平均直径≤10 nm）联用纳米硒Pickering乳液液滴的粒径分布。
图3为黑茶纳米聚集体（平均直径>10 nm和平均直径≤10 nm）联用纳米硒Pickering乳液体系的Zeta电位。
图4为黑茶纳米聚集体（平均直径>10 nm和平均直径≤10 nm）联用纳米硒Pickering乳液的微观结构。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
为了使本发明的目的、技术方案更加清晰明确，下面以黑茶纳米聚集体联用茶-纳米硒作为稳定剂举例，对本发明进行进一步详细说明。本发明所用设备仪器和试剂均为本领域所常用。应当理解，此处所描述的举例仅仅用以解释本发明，并不用于限定本发明。
实施例1：黑茶纳米聚集体联用纳米硒制备Pickering乳液
（1）准确称取4.5 g磨碎茶叶，加入225mL温度为100 ℃的超纯水，于100 ℃水浴浸提30min，每隔10 min摇一次，浸提结束后用定量滤纸抽滤，定容，得到茶水提取液；茶水提取液经冷冻干燥得到冻干粉，即为茶水提取物（黑茶提取物以黑茶作为浸提原料）。
（2）移取4 mL步骤（1）茶水提取液于100 KDa超滤管内管，于4℃，4000g，离心20min；收集内/外管溶液得不同粒径范围（平均直径＞10 nm和平均直径≤10 nm）的茶纳米聚集体溶液，冷冻干燥得冻干粉，即为茶纳米聚集体，待用（黑茶纳米聚集体以黑茶作为原料）。
（3）分别配制100 mM的***钠溶液、500 mM的维生素C溶液。固定反应体系中Na2SeO3浓度为20 mmol/L，Vc和Na2SeO3配比为8:1（20 mM:160 mM），以步骤（1）同样方法制备得到的茶水提取物或以步骤（2）同样方法制备得到的茶纳米聚集体为模板，其添加量为500 mg/L，于40 ℃水浴条件下静置反应1 h，4 ℃、11000 r/min离心30 min，重复3次，重悬浮等体积超纯水中，得纳米硒胶体溶液。
（4）固定纳米硒胶体溶液稀释倍数为20倍体积，黑茶纳米聚集体添加量为质量分数2%，加入质量分数0.02%的叠氮钠，用1 mol/L HCl或1 mol/L NaOH分别调节水相pH为7，避光静置过夜（12~14 h），按油和水的体积比为4:6加入大豆油（20 mL:30 mL），20000r/min高速乳化2 min，40 MPa高压均质3次，测定Pickering乳液液滴粒径、体系Zeta电位值，观察乳液液滴微观结构，结果如图1~4所示，黑茶纳米聚集体（平均直径＞10 nm）联用纳米硒Pickering乳液稳定性高于黑茶纳米聚集体（平均直径≤10 nm）联用纳米硒Pickering乳液。
实施例2：乳液液滴粒径及粒径分布测定
将本发明新配制或储藏后乳液的粒径及粒径分布利用马尔文3000激光粒度仪测量，分别用去离子水和1% SDS作为分散剂。乳液的相对折光系数设为1.095，系大豆油折光系数（1.456）与水折光系数（1.33）的比值。乳液的粒度以d4,3（体积加权平均粒径）表示。结果表明：黑茶纳米聚集体（平均直径＞10 nm）联用纳米硒Pickering乳液滴粒径为2.93±0.02 μm，粒子分布集中，以小粒子液滴为主；黑茶纳米聚集体（平均直径≤10 nm）联用纳米硒Pickering乳液滴粒径为8.56±0.06 μm，粒子分布广，以大粒子液滴为主。
实施例3：乳液体系静电稳定性测定
采用LDV技术测定乳液Zeta电位，研究本发明利用黑茶纳米聚集体联用纳米硒构建功能强化型Pickering乳液的静电稳定性。结果显示：黑茶纳米聚集体（平均直径＞10 nm和平均直径≤10 nm）联用纳米硒Pickering乳液体系Zeta电位值大于40 mV，体系静电稳定性高。其中，黑茶纳米聚集体（平均直径＞10 nm）联用纳米硒Pickering乳液体系Zeta电位值为48.1±1.3 mV，黑茶纳米聚集体（平均直径≤10 nm）联用纳米硒Pickering乳液体系Zeta电位值为57.5±0.6 mV。
实施例4：乳液液滴微观结构观察
将本发明利用黑茶纳米聚集体联用纳米硒构建功能强化型Pickering乳液分别用超纯水和1% SDS将乳液稀释10倍，利用光学显微镜观察乳液液滴大小和形态（调节物镜倍数，通过计算机采集数据并进行拍照）。结果表明：黑茶纳米聚集体（平均直径>10 nm和平均直径≤10 nm）联用纳米硒Pickering乳液液滴呈球型。其中，黑茶纳米聚集体（平均直径>10nm）联用纳米硒Pickering乳液液滴大小相近，液滴小、数目多；而黑茶纳米聚集体（平均直径≤10 nm）联用纳米硒Pickering乳液液滴大小相差大，液滴大、数目少。与激光粒度仪结果一致。
实施例5：乳液抗癌活性测定
通过胰蛋白酶消化贴壁HCT 116细胞/MDA-MB-231制成单细胞悬液，以5×104个细胞/孔接种96孔培养板，置于37 ℃、5% CO2培养箱中预培养24 h，将本发明利用黑茶纳米聚集体联用纳米硒构建功能强化型Pickering乳液用培养基稀释10倍、20倍、40倍、80倍、160倍、320倍后，按100μL/孔加入培养基，继续培养48 h。往培养板加入0.25 mg/mL MTT稀释液100μL/孔，放置在37 ℃、5% CO2培养箱中继续培养2 h后弃上清，每孔加入200 μL DMSO溶液，放置在100 r/min的摇床中振荡15~20min，测定各孔OD550值，以对照组OD550值为100%，计算各组纳米硒抑制细胞增殖的半抑制浓度（IC50），如表1所示，其中，黑茶纳米聚集体（平均直径≤10 nm）联用纳米硒Pickering乳液抑制HCT 116细胞增殖强于黑茶纳米聚集体（平均直径>10 nm）联用纳米硒Pickering乳液。
表1 黑茶纳米聚集体联用纳米硒Pickering乳液抑制HCT 116细胞增殖的
半抑制浓度（IC50）
| 稀释倍数 | |
| 乳液（平均直径>10 nm） | 63.40±5.49 |
| 乳液（平均直径≤10 nm） | 46.85±4.93 |
（黑茶纳米聚集体联用纳米硒Pickering乳液在此浓度范围内，MDA-MB-231细胞存活率均大于0.5。）
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 (7)
1.一种利用黑茶纳米聚集体联用纳米硒构建功能强化型Pickering乳液的制备方法，其特征在于包括以下步骤：
（1）分离黑茶纳米聚集体：
向黑茶原料加入温度为25~100 ℃、体积百分比浓度为70%~100%的水，所述溶剂的用量按照每g黑茶原料加入50mL溶剂来计；然后在100 ℃水浴条件下浸提30min得到黑茶提取液，再使用截留分子量为100 kDa的离心超滤管于温度4 ℃、离心力4000 g条件下离心20min，收集内管和外管得到不同粒径范围的黑茶纳米聚集体的溶液，经冷冻干燥，于-20 ℃保存，得到黑茶纳米聚集体；
（2）利用黑茶纳米聚集体联用纳米硒制备Pickering乳液：
制备20倍纳米硒稀释液，用纳米硒稀释液溶解黑茶纳米聚集体，使得黑茶纳米聚集体的质量百分数为2%；加入质量分数0.02%的叠氮钠，调节黑茶纳米聚集体分散相pH为7，按油和水的体积比为4:6加入油相， 20000 r/min高速剪切均质2 min，40 MPa高压均质3次，得Pickering乳液。
2.根据权利要求1所述的一种利用黑茶纳米聚集体联用纳米硒构建功能强化型Pickering乳液的制备方法，其特征在于：所述黑茶原料是以茶树鲜叶和嫩梢为原料，经杀青、揉捻、渥堆、干燥工艺加工制成的产品。
3.根据权利要求1所述的一种利用黑茶纳米聚集体联用纳米硒构建功能强化型Pickering乳液的制备方法，其特征在于：所述黑茶原料包括茯砖茶、青砖茶、六堡茶、康砖茶、普洱茶或千两茶。
4.根据权利要求1所述的一种利用黑茶纳米聚集体联用纳米硒构建功能强化型Pickering乳液的制备方法，其特征在于：所得黑茶纳米聚集体为平均直径＞10 nm和平均直径≤10 nm两类胶粒。
5.根据权利要求1所述的一种利用黑茶纳米聚集体联用纳米硒构建功能强化型Pickering乳液的制备方法，其特征在于：所述纳米硒为软模板法制备所得的液相纳米硒；所述油相与水不相溶。
6.一种根据权利要求1-5任一项所述的方法制备得到的Pickering乳液。
7.根据权利要求6所述的Pickering乳液在化妆品和生物医药领域中的应用。
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