CN111938157B - 一种叶黄素纳米乳液的制备方法 - Google Patents
一种叶黄素纳米乳液的制备方法 Download PDFInfo
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Abstract
一种叶黄素纳米乳液制备方法，涉及食品营养健康领域。本发明以天然安全的鹰嘴豆分离蛋白和甜菊苷为原料，利用纳米化鹰嘴豆分离蛋白颗粒与甜菊苷分子之间的强相互作用结合形成鹰嘴豆分离蛋白‑甜菊苷稳定复合体系，辅以反溶剂沉淀与探针式超声耦合分散技术，制备出富含叶黄素的纳米尺度乳液。本发明制备的叶黄素纳米乳液改善了叶黄素的水溶性，提高了叶黄素的包埋率，乳液平均粒径小于200nm，叶黄素乳化产率大于90％；同时，鹰嘴豆分离蛋白‑甜菊苷复合体系具备直接用作叶黄素功能性食品配料的能力。
Description
技术领域
本发明涉及一种叶黄素纳米乳液制备方法，属于食品营养健康领域。
技术背景
叶黄素是一种重要含氧类胡萝卜素，在人体胚胎发育与婴幼儿成长中扮演着重要的角色，它还可保护视网膜免受光损伤，对老年性黄斑变性病、白内障、青光眼和糖尿病视网膜病变、色素性视网膜炎等眼疾有预防和缓解作用，此外，叶黄素具有抗氧化、抑制炎症、抗癌的功效。
由于叶黄素含有羟基和多个共轭不饱和双键结构，其水溶性和稳定性差，口服生物利用度低，极大影响了生理功效的发挥，因此，开发合适的叶黄素负载和输送体系对此类脂溶性色素物质的高效利用具有重要现实意义。纳米乳液包埋技术作为纳米科技的核心技术之一，在功能性食品组分的运输载体构建方面显示出极大的潜力，目前的研究表明采用乳化包埋技术在一定程度上提高了叶黄素的溶解性、稳定性和生物利用率。Jo等(Characterization of β-carotene nanoemulsions prepared by microfluidizationtechnique.Food Science and Biotechnology，2014，23(1)：107-113)分别用吐温20、乳清分离蛋白和酪蛋白酸钠作为乳化剂，通过微射流均质制得β-胡萝卜素纳米乳液，发现乳清分离蛋白制得的乳液稳定性最好。Tan等(Effect of polyglycerol esters of fattyacids on physicochemical properties and stability of beta-carotenenanodispersions prepared by emulsification/evaporation method.Journal of theScience of Food and Agriculture，2005，85(1)：121-126)通过反溶剂蒸发法制备β-胡萝卜素纳米分散体系，体系中β-胡萝卜素含量为0.03％(w/w)，并且研究比较了6种不同聚甘油酯对乳液的理化性质及稳定性的影响，探讨了制备工艺条件、两相比例与颗粒大小以及色素含量之间的关系。Mora-Gutierrez等(Complexes of lutein with bovine andcaprine caseins and their impact on lutein chemical stability in emulsionsystems：Effect of arabinogalactan.Journal of Dairy Science，2017，101(1)：18-27)研究了酪蛋白对叶黄素稳定性和水溶性的影响，结果表明酪蛋白可以提高叶黄素的稳定性，其作用强度：山羊αS1-II-酪蛋白＞山羊αS1-I-酪蛋白＞牛酪蛋白。
此外，现有专利技术中，中国发明专利(申请号：201710019164.2，公开号：106690271A)公开一种提高叶黄素生物利用度的纳米乳液制备方法，先制备玉米纤维胶，然后将玉米纤维胶、柠檬酸、苯甲酸钠溶解于去离子水中，缓慢搅拌2-3h，制得水相；然后将叶黄素添加到玉米油中，超声处理使其完全溶解，制得油相；将油相与水相混合，添加去离子水定量至100％，采用高速剪切均质机进行均质，制备粗乳液，然后进行高压均质，制备出叶黄素纳米稳定乳液，粒径在100-200nm范围，具有较好的储存稳定性，叶黄素包埋率达到86％，乳液中叶黄素的生物利用度由10％增加至55％。中国发明专利(申请号：201810030481.9，公开号：108308615A)公开了一种玉米肽为载体的叶黄素纳米粒的制备方法，对玉米蛋白粉进行酶解改性，然后采用超滤膜分离目标玉米蛋白肽，再采用高压均质制备玉米肽负载叶黄素纳米粒，是一种营养价值高、稳定性和溶解性好以及体内利用率高的玉米肽负载叶黄素纳米粒的制备方法。中国发明专利(申请号：201810457216.9，公开号：108634169A)公开了一种叶黄素纳米乳液的制备方法，选用食品级酪蛋白酸钠作为乳化剂对叶黄素进行乳化，经过高速剪切和高压均质后得到叶黄素纳米乳液，平均粒径在200-300nm范围，安全性高且能显著改善叶黄素的储存稳定性，减少了储存期间叶黄素的化学降解。
上述研究与技术方案均采用了一些存在安全隐患的乳化剂如苯甲酸钠、吐温20、聚甘油酯等，而以蛋白质作为乳化剂尚主要集中于乳清分离蛋白、乳铁蛋白、酪蛋白酸钠等动物蛋白，未见植物蛋白在叶黄素乳化制剂中的应用。未来，叶黄素纳米乳液将向着平均粒径更小、包封率高、稳定性好、包埋材料更安全健康的方向发展，运用新技术和研发新原料，对提升叶黄素乳液的稳定性、功效性及安全性极具应用前景。
发明内容
技术问题：
为了克服现有技术的不足与缺陷，本发明的目的在于提供一种叶黄素纳米乳液制备方法，通过纳米化鹰嘴豆分离蛋白颗粒与甜菊糖苷分子之间的强相互作用形成天然复合体系，进而稳定负载高量叶黄素，制得叶黄素纳米乳液。
技术方案：
本发明采用以下技术方案来实现本发明的目的：
一种叶黄素纳米乳液制备方法，包括如下步骤：
(1)鹰嘴豆分离蛋白纳米颗粒制备：称取一定量的鹰嘴豆分离蛋白样品，溶于10mMpH 7.0磷酸盐缓冲液，在室温下于500rpm磁力搅拌2h，再将搅拌后的鹰嘴豆分离蛋白液置于探针式超声波破碎仪进行超声处理10min，超声处理后的鹰嘴豆分离蛋白液于8000rpm离心10min，获取鹰嘴豆分离蛋白纳米上清液；
(2)鹰嘴豆分离蛋白-甜菊苷复合体系制备：称取一定量的甜菊苷样品，溶于10mMpH 7.0磷酸盐缓冲液，配制甜菊苷溶液。将50mL含质量百分比0.25％-1.0％的甜菊苷溶液与步骤(1)所述50mL含质量百分比1％的鹰嘴豆分离蛋白纳米上清液混合，于500-800rpm磁力搅拌5-10min，形成鹰嘴豆分离蛋白-甜菊苷复合体系；
(3)含叶黄素的纳米乳液制备：将含5-15mg/mL的叶黄素无水乙醇溶液加入步骤(2)所述处于磁力搅拌过程的100mL鹰嘴豆分离蛋白-甜菊苷复合体系中，再置于探针式超声波破碎仪进行超声处理6-10min，而后挥干乙醇，获得稳定性良好、乳化产率高的叶黄素纳米乳液。
步骤(1)所述鹰嘴豆分离蛋白纯度≥90％，超声处理条件为：超声频率20kHz，超声振幅37.5μm，脉冲比0.5。
步骤(3)所述叶黄素纯度≥80％，叶黄素无水乙醇溶液体积控制在5-10mL，采用注射器逐滴加入方式。
步骤(3)所述超声处理条件为：超声频率20kHz，超声振幅70μm，脉冲比0.65。
所制备的叶黄素纳米乳液体系平均粒径小于200nm，叶黄素乳化产率大于90％。
有益效果：
采用单一载体包埋，叶黄素乳液稳定性仍然较差，且叶黄素易损失，利用两种及以上的天然材料构建复合输送体系成为解决此类问题的有效途径。鹰嘴豆分离蛋白-甜菊糖苷复合体系中，不仅含有甜菊苷功能活性物质，药理试验证明甜菊苷有较强而持久的降压作用，显著改善降血糖、尿糖及改善“三多”症状的效果，而且鹰嘴豆分离蛋白具有良好的生物兼容性、乳化性及表面活性，甜菊苷分子共价结合到鹰嘴豆分离蛋白纳米颗粒表面，使蛋白质发生舒展，无规则卷曲结构增加，提高了其负载叶黄素的能力，进而改善鹰嘴豆分离蛋白-甜菊糖苷复合体系的物理稳定性。本专利制得的鹰嘴豆分离蛋白-甜菊糖苷-叶黄素三元复合体系在胃肠液中稳定性明显高于鹰嘴豆分离蛋白-叶黄素二元复合体系，能更加有效的负载叶黄素，不被胃肠液中的消化酶所降解。
附图说明
图1模拟胃肠液中纳米乳液叶黄素的保留率
图2叶黄素纳米乳液照片
具体实施方式
下面结合实施例对本发明做进一步阐述，但不因此而限制本发明。
实施例1
称取一定量的纯度为90.8％的鹰嘴豆分离蛋白样品，溶于10mM pH 7.0磷酸盐缓冲液，在室温下于500rpm磁力搅拌2h，再将搅拌后的鹰嘴豆分离蛋白液置于探针式超声波破碎仪进行超声处理10min，其中超声频率20kHz，超声振幅37.5μm，脉冲比为0.5，超声处理后的鹰嘴豆分离蛋白液于8000rpm离心10min，获取鹰嘴豆分离蛋白纳米上清液。将50mL含质量百分比为0.25％的甜菊苷溶液与50mL含质量百分比1％的鹰嘴豆分离蛋白纳米上清液混合，于500rpm磁力搅拌5min，形成鹰嘴豆分离蛋白-甜菊苷复合体系。将含5mg/mL的叶黄素无水乙醇溶液加入处于磁力搅拌过程的100mL鹰嘴豆分离蛋白-甜菊苷复合体系中，再置于探针式超声波破碎仪进行超声处理6min，其中超声频率20kHz，超声振幅80μm，脉冲比0.6，而后真空旋转挥干乙醇，获得稳定性良好、乳化产率高的叶黄素纳米乳液。
实施例2
称取一定量的纯度为90.8％的鹰嘴豆分离蛋白样品，溶于10mM pH 7.0磷酸盐缓冲液，在室温下于500rpm磁力搅拌2h，再将搅拌后的鹰嘴豆分离蛋白液置于探针式超声波破碎仪进行超声处理10min，其中超声频率20kHz，超声振幅37.5μm，脉冲比为0.5，超声处理后的鹰嘴豆分离蛋白液于8000rpm离心10min，获取鹰嘴豆分离蛋白纳米上清液。将50mL含质量百分比0.5％的甜菊苷溶液与50mL含质量百分比1％的鹰嘴豆分离蛋白纳米上清液混合，于800rpm磁力搅拌10min，形成鹰嘴豆分离蛋白-甜菊苷复合体系。将含7.5mg/mL的叶黄素无水乙醇溶液加入处于磁力搅拌过程的100mL鹰嘴豆分离蛋白-甜菊苷复合体系中，再置于探针式超声波破碎仪进行超声处理10min，其中超声频率20kHz，超声振幅60μm，脉冲比0.7，而后真空旋转挥干乙醇，获得稳定性良好、乳化产率高的叶黄素纳米乳液。
实施例3
进一步采用响应曲面方法对超声辅助反溶剂沉淀法制备鹰嘴豆分离蛋白-甜菊苷-叶黄素复合体系工艺优化，以单因素分析所得到的理想值超声脉冲比0.7、超声振幅80μm、超声时间7min及甜菊苷配比0.50％为中心实验值，设定平均粒径和叶黄素乳化产率为响应值。响应面优化实验安排及结果见表1。
表1响应面分析试验设计及结果
通过统计分析软件Design-Expert进行数据分析，对平均粒径建立回归模型如下：
Y1＝199.97-6.25A-2.62B-1.88C+5.88D-3.66AB+4.15AC+4.63BC-2.82BD+4.77A2+6.97B2+2.62C2+8.38D2，其中均方值：0.9295；Adj R-Squared：0.8637，对模型方程进行方差分析，结果如表2所示。
表2平均粒径回归模型方差分析结果
由表2知，响应面优化拟合出的方程回归项极显著，且失拟项0.3017＞0.05，拟合方程的各因素指标与响应值之间线性关系显著，拟合模型方程的均方值R2＝0.9295，R2 Adj＝0.8637，表明该模型方程可靠。由F值检验可知，F值大小为：D＞B＞A＞C，即甜菊苷配比＞超声振幅＞超声脉冲比＞超声时间，所以甜菊苷配比对体系的平均粒径影响最大。
应用响应面寻优分析方法对回归模型进行分析，超声脉冲比A，超声振幅B，超声时间C，甜菊苷配比D所对应的编码值为0.7、77.4μm、5min、0.45％，估计其条件下的理论粒度为199.28nm，通过实验验证其实际粒度为198.95nm，与理论值接近，证明模型具有可靠性。
乳化产率Y2通过统计分析软件Design-Expert进行数据分析，建立二次响应面回归模型如下：
Y2＝89.73-0.91A-0.46B-1.30C+2.04D-0.81AB+1.83AD+0.92BC+0.65BD-0.83CD-0.47A2-0.75B2-1.60C2-2.19D2，其中均方值：0.9309；Adj R-Squared：0.8664。
表3叶黄素乳化产率回归模型方差分析结果
由表3知，响应面优化拟合出的方程回归项极显著，失拟项0.6514＞0.05，拟合方程的各因素指标与响应值之间线性关系显著，拟合模型方程的均方值R2＝0.9309，R2 Adj＝0.8664，表明该模型方程可靠。由F值检验可知，F值大小为：D＞C＞A＞B，即甜菊苷配比＞超声时间＞超声脉冲比＞超声振幅。因此，甜菊苷配比对体系的叶黄素乳化产率影响最大，振幅对其影响最小。
应用响应面寻优分析方法对回归模型进行分析，超声脉冲比A，超声振幅B，超声时间C，甜菊苷配比D所对应的编码值为0.80、60.00μm、5.05min、0.73％，估计该条件下的理论叶黄素乳化产率为91.50％，通过实验验证其实际乳化产率为90.96％，与理论值接近，证明模型具有可靠性。
为了同步获取小平均粒径和高叶黄素乳化产率的复合体系，采用联合求解法确定平均粒径及乳化产率均优的工艺条件下，超声脉冲比A、超声振幅B、超声时间C与甜菊苷配比D所对应的编码值为0.66、72.42μm、6.30min、0.52％，模拟预计在此处理条件下理论平均粒径为197.2nm，叶黄素乳化产率为90.87％，通过实验验证，在此处理条件下其平均粒径为195.1nm，实际乳化产率为91.04％，与理论值接近。
Claims (1)
1.一种叶黄素纳米乳液制备方法，其特征在于，包括如下步骤：
(1)鹰嘴豆分离蛋白纳米颗粒制备：称取一定量纯度≥90％的鹰嘴豆分离蛋白样品，溶于10mM pH 7磷酸盐缓冲液，在室温下于500rpm磁力搅拌2h，再将搅拌后的鹰嘴豆分离蛋白液置于探头式超声波破碎仪进行超声处理10min，超声处理条件：超声频率20kHz，超声振幅37.5μm，脉冲比0.5，超声处理后的鹰嘴豆分离蛋白液于8000rpm离心10min，获取鹰嘴豆分离蛋白纳米上清液；
(2)鹰嘴豆分离蛋白甜菊苷复合体系制备：称取一定量的甜菊苷样品，溶于10mM pH7.0磷酸盐缓冲液，配制甜菊苷溶液；将50mL含质量百分比0.25％-1.0％的甜菊苷溶液与步骤(1)所述50mL含质量百分比1％的鹰嘴豆分离蛋白纳米上清液混合，于500-800rpm磁力搅拌5-10min，形成鹰嘴豆分离蛋白-甜菊苷复合体系；
(3)含叶黄素的纳米乳液制备：将5-10mL含5-15mg/mL纯度≥80％的叶黄素无水乙醇溶液，用注射器逐滴加入步骤(2)所述处于磁力搅拌过程的100mL鹰嘴豆分离蛋白-甜菊苷复合体系中，再置于探头式超声波破碎仪进行超声处理6-10min，超声处理条件为超声频率20kHz，超声振幅70μm，脉冲比0.65，而后挥干乙醇，获得稳定性良好、乳化产率高的叶黄素纳米乳液；
乳液体系平均粒径小于200nm，叶黄素乳化产率大于90％。
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| CN108618146A (zh) * | 2018-04-12 | 2018-10-09 | 华南理工大学 | 由大豆蛋白-甜菊糖苷复合稳定的植物甾醇纳米乳液及其制备方法与应用 |
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| CN107189117A (zh) * | 2017-06-05 | 2017-09-22 | 江苏大学 | 大豆蛋白‑多糖复合物的超声制备方法及功能食品的应用 |
| CN107897647A (zh) * | 2017-11-21 | 2018-04-13 | 江苏省农业科学院 | 一种水溶性叶黄素的绿色制备方法 |
| CN108618146A (zh) * | 2018-04-12 | 2018-10-09 | 华南理工大学 | 由大豆蛋白-甜菊糖苷复合稳定的植物甾醇纳米乳液及其制备方法与应用 |
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