CN114159409A - 一种糖基化玉米蛋白多肽载运叶黄素纳米粒的制备方法 - Google Patents
一种糖基化玉米蛋白多肽载运叶黄素纳米粒的制备方法 Download PDFInfo
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- CN114159409A CN114159409A CN202110811200.5A CN202110811200A CN114159409A CN 114159409 A CN114159409 A CN 114159409A CN 202110811200 A CN202110811200 A CN 202110811200A CN 114159409 A CN114159409 A CN 114159409A
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- lutein
- zein
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Abstract
本发明解决了玉米醇溶蛋白在水中易聚集、溶解性差，对叶黄素负载效率低和溶解控释效果差等问题。本发明一种糖基化玉米蛋白多肽载运叶黄素纳米粒的制备方法按照如下步骤进行：1.将玉米醇溶蛋白进行酶水解获得特定分子量大小的玉米蛋白多肽；2.制备糖基化修饰的玉米蛋白水解多肽；3.以糖基化玉米肽为载体制备高负载叶黄素纳米粒。本发明的优点是：一、提高了玉米蛋白对叶黄素的载运控释性能；二、改善了叶黄素的结构稳定性和消化释放性；三、营养安全性高，能够提高脂溶性叶黄素成分的营养价值。是一种对脂溶性叶黄素等活性成分包埋效率高、溶解分散性好以及营养功能性强的改性玉米多肽‑叶黄素纳米粒的制备方法。
Description
技术领域：
本发明涉及一种糖基化玉米蛋白多肽载运叶黄素纳米粒的制备方法。
背景技术：
玉米蛋白中大部分为醇溶蛋白，主要来源于玉米加工副产物玉米黄粉中，是一种安全无毒、成本低、生物相容性高和降解性较好的玉米蛋白。但由于玉米蛋白溶解性低、吸收利用率差，目前主要用于饲料中代替部分饲料蛋白，开发利用率较低。近年来研究发现，玉米醇溶蛋白自身的非极性氨基酸(约占玉米醇溶蛋白总氨基酸残基的50％，包括亮氨酸、丙氨酸和脯氨酸)，可以自组装成球形颗粒，使其为生物活性化合物、药物以及其他营养食品和食品成分的理想传递载体。如采用玉米醇溶蛋白/可溶性大豆多糖复合物通过一种简便的反溶剂沉淀法封装叶黄素，制备出尺寸均匀，包封效果较好的纳米颗粒，且制备的复合纳米粒子显示出优异的酸碱稳定性和高盐稳定性；采用抗溶剂沉淀和静电沉积相结合的方法制备了白藜芦醇-玉米醇溶蛋白/果胶纳米粒，提高了白藜芦醇的体外抗氧化能力和生物利用度；采用抗溶剂沉淀技术制备玉米醇溶蛋白-卵磷脂纳米粒，提高了姜黄素的耐热、紫外辐照和高离子强度稳定性。因此，玉米蛋白可作为活性成分负载和包埋的有效载体。
叶黄素是一种重要的类胡萝卜素，具有清除自由基和抗氧化作用，能够起到提高机体免疫力、预防心脑血管疾病和多种癌症以及保护视力等多种生理功能，尤其是叶黄素的护眼功能被人们广泛认可。现代医学证明，叶黄素可减缓视力退化和近视等症状，有效预防老年黄斑变性这一不可逆的致盲性眼病。但是，由于叶黄素分子结构中碳链较长且含有较多的疏水基团，在水中溶解性差，降低了体内吸收和生物利用率；叶黄素分子中含有多个不饱和双键，导致易受氧、光辐射、高温、pH值以及体内酶、抗体和胃酸等因素的影响而发生分解和失活，失去原有的生理活性，稳定性较差。这两个因素严重限制了叶黄素进入体内到达有效部位和被较好的吸收，难以有效地发挥其生理功能。同时也极大地影响了叶黄素在食品和医药领域的应用。
采用纳米包埋技术可有效保护叶黄素的结构和稳定性，更好的保留叶黄素的生物活性和吸收利用率。目前，以玉米醇溶蛋白及其修饰产物作为活性物质的载体已有研究，但是在叶黄素溶解释放性、体内吸收利用和营养安全等方面还存在不足，如中国专利CN103431156A和CN 106420666A公开了一种玉米醇溶蛋白纳米粒子及其制备方法及糖基化玉米醇溶蛋白纳米载体的制备方法，分别是将玉米醇溶蛋白和糖基化的醇溶蛋白经过挤压喷射设备制成纳米载体颗粒，但所制备颗粒较大，作为纳米载体不具有良好的水溶性、释放性和吸收性。再如中国专利103445282A公布了一种包埋脂溶性维生素的玉米肽糖基化产物纳米颗粒的制备方法，制备了玉米肽糖基化产物纳米颗粒负载维生素等脂溶性纳米粒，脂溶性维生素包埋率高，粒径小，溶液具有较好的透明度和稳定性，但所采用的干热法糖基化产物的结构性质不易控制，糖基化蛋白的载运亲和性能不显著，构效关系不明确，仅限于包埋液态的油溶性维生素D、E等成分。因此，综合以上现状可知，目前还没有一种采用糖基化玉米蛋白水解多肽制备高效载运叶黄素纳米粒的方法。
发明内容：
本发明的目的是提供一种对叶黄素载运性能好、溶解性高、吸收释放性能好的以糖基化改性玉米蛋白多肽为载体的叶黄素纳米粒的制备方法。
本发明的一种糖基化玉米蛋白多肽载运叶黄素纳米粒的制备方法包括以下步骤：
1、一种糖基化玉米蛋白多肽载运叶黄素纳米粒的制备方法，其特征是：该方法包括下列步骤：
(1)玉米蛋白多肽的酶解制备
将玉米蛋白粉分散到10～20倍体积的去离子水中，加入蛋白粉质量1.0％～3.0％的蛋白酶，在40～60℃条件下酶解60～120min。酶反应后在95℃条件下蒸煮5min灭酶。离心后取上清液，分别采用不同分子量的超滤膜获得一定分子量大小的玉米肽溶液，经真空浓缩和冷冻干燥后，得到玉米蛋白水解多肽粉末。
(2)糖基化修饰玉米蛋白多肽的制备
取上述玉米肽粉末，用20倍体积的蒸馏水溶解，加入1～5倍玉米肽质量的氨基葡萄糖，混匀，将反应体系调至7.0～9.0，加入玉米肽质量为10％～20％的转谷氨酰胺酶，在30～50℃下反应3～7h，反应结束后95℃条件下蒸煮5min灭酶，随后将反应液置于4℃下透析24h除去未反应的氨基葡萄糖，对其粒径大小进行测定，样品冷冻干燥后4℃下储藏备用。
(3)糖基化玉米多肽负载叶黄素纳米粒的制备
将叶黄素标准液和糖基化玉米蛋白肽溶液等体积混合均匀，加入2倍体积的PBS缓冲溶液，在一定温度下搅拌水合10～60min，旋转蒸发除去乙醇，得到糖基化玉米蛋白负载叶黄素纳米粒，对叶黄素的包封率可达90％以上，经粒度分析仪测得玉米肽负载叶黄素纳米粒的粒径范围为100～200nm，透射电子显微镜显示为分散球型纳米粒结构。抗氧化研究表明，经糖基化修饰的玉米肽负载叶黄素纳米粒的抗氧化能力显著高于未修饰的玉米肽和叶黄素。
附图说明：
图1.玉米蛋白多肽负载叶黄素纳米粒、糖基化玉米蛋白多肽负载叶黄素纳米粒粒径分析图
图2.叶黄素、玉米蛋白多肽负载叶黄素纳米粒、糖基化玉米蛋白多肽负载叶黄素纳米粒溶解性比较
图3.叶黄素、玉米蛋白多肽负载叶黄素纳米粒、糖基化玉米蛋白多肽负载叶黄素纳米粒自由基清除能力比较
本发明和现有的技术相比具有以下优点：
一、提高了玉米蛋白的溶解分散性和载运控释性能
通过玉米蛋白在较温和的条件下水解获得玉米多肽，再通过糖基化手段对玉米蛋白多肽进一步修饰，结构中引入更多的亲水性羟基，赋予玉米肽具有两亲性，提高玉米蛋白在水溶液体系中的溶解分散性，所制备的叶黄素粒径在纳米级范围，有助于包埋体系中叶黄素的溶解释放。
二、提高了叶黄素的结构稳定性和体内消化释放性
糖基化玉米肽负载叶黄素纳米粒通过引入糖配基，提高了对叶黄素的包封性能，对于叶黄素的包封率由原来的82％提高到95％以上，同时，提高了叶黄素在光、热和体内酶、胃酸等条件吓的稳定性。氨基糖的修饰也能够提高叶黄素在胃肠液体系中的控释效果，进而能够有效提高叶黄素的营养价值和体内吸收利用率。
三、糖基化玉米蛋白多肽安全性高且功能性强，作为功能性成分载体能够提高叶黄素的营养价值
通过生物酶解的玉米蛋白肽是一种功能型多肽，采用功能型氨基葡萄糖修饰可进一步提高玉米肽的生物活性，通过糖基化玉米肽的载运协同作用有效保护载体叶黄素的结构和提高其抗氧化活性等功能，是一种高效安全的叶黄素纳米粒的制备方法。
四、技术先进，应用性强
本技术发明通过转谷氨酰胺酶催化作用将氨基葡萄糖修饰到玉米肽分子上，进一步改善了玉米肽的疏水性结构，使其成为一种高水溶性纳米载体对叶黄素进行有效包埋和载运，技术先进且工艺简单。本发明制备的糖基化玉米肽负载叶黄素纳米粒具有较高的抗氧化活性，对叶黄素具有较好的保护和控释作用，能提高叶黄素的吸收利用价值，并且兼有玉米肽和氨基糖的生理功能，实用性和在保健食品和医药领域具有广阔的应用前景。
具体实施方式
具体实施方式一：一种糖基化玉米蛋白多肽载运叶黄素纳米粒的制备方法通过以下步骤实现：
1、一种糖基化玉米蛋白多肽载运叶黄素纳米粒的制备方法，其特征是：该方法包括下列步骤：
(1)玉米蛋白多肽的酶解制备
将玉米蛋白粉分散到10～20倍体积的去离子水中，加入蛋白粉质量1.0％～3.0％的蛋白酶，在40～60℃条件下酶解60～120min。酶反应后在95℃条件下蒸煮5min灭酶。离心后取上清液，分别采用不同分子量的超滤膜获得一定分子量大小的玉米肽溶液，经真空浓缩和冷冻干燥后，得到玉米蛋白水解多肽粉末。
(2)糖基化修饰玉米蛋白多肽的制备
取上述玉米肽粉末，用20倍体积的蒸馏水溶解，加入1～5倍玉米肽质量的氨基葡萄糖，混匀，将反应体系调至7.0～9.0，加入玉米肽质量为10％～20％的转谷氨酰胺酶，在30～50℃下反应3～7h，反应结束后95℃条件下蒸煮5min灭酶，随后将反应液置于4℃下透析(分子量小于1000透析袋)24h除去未反应的氨基葡萄糖，对其粒径大小进行测定，样品冷冻干燥后4℃下储藏备用。
(3)糖基化玉米肽负载叶黄素纳米粒的制备
将叶黄素标准液和糖基化玉米蛋白肽溶液等体积混合均匀，加入2倍体积的PBS缓冲溶液，在一定温度下搅拌水合10～60min，旋转蒸发除去乙醇，得到糖基化玉米蛋白负载叶黄素纳米粒，对叶黄素的包封率可达90％以上，经粒度分析仪测得玉米肽负载叶黄素纳米粒的粒径范围为100～200nm，透射电子显微镜显示为分散球型纳米粒结构。抗氧化研究表明，经糖基化修饰的玉米肽负载叶黄素纳米粒的抗氧化能力显著高于未修饰的玉米肽和叶黄素。
具体实施方式二：本实施方式与具体实施方式一的不同点在于步骤(1)将纯度大于95％的玉米醇溶蛋白粉分散到10～20倍体积的去离子水中，加入蛋白粉质量1.8％～2.7％的中性蛋白酶，在50～55℃条件下酶解60～80min。其它步骤与具体实施方式一相同。
具体实施方式三：本实施方式与具体实施方式一的不同点在于步骤(1)中分别采用截留分子量10000Da和5000Da的超滤膜过滤获得分子量大小5000～10000Da的玉米肽溶液。其它步骤与具体实施方式一相同。
具体实施方式四：本实施方式与具体实施方式一的不同点在于步骤(2)中加入1～3倍玉米肽质量的氨基葡萄糖，混匀，加入转谷氨酰胺酶的活力为1000u/g，在40～50℃下反应4～6h。其它步骤与具体实施方式一相同。
具体实施方式五：本实施方式与具体实施方式一的不同点在于步骤(3)中将100μg/mL纯度为90％的叶黄素标准液和2.5mg/mL糖基化玉米蛋白肽溶液等体积混合均。其它步骤与具体实施方式一相同。
具体实施方式六：本实施方式与具体实施方式一的不同点在于步骤(3)中在40～50℃下搅拌水合20～30min。其它步骤与具体实施方式一相同。
Claims (4)
1.一种糖基化玉米蛋白多肽载运叶黄素纳米粒的制备方法，其特征是：该方法包括下列步骤：
(1)玉米蛋白多肽的酶解制备
将玉米蛋白粉分散到10～20倍体积的去离子水中，加入蛋白粉质量1.0％～3.0％的蛋白酶，在40～60℃条件下酶解60～120min。酶反应后在95℃条件下蒸煮5min灭酶。离心后取上清液，分别采用不同分子量的超滤膜获得一定分子量大小的玉米肽溶液，经真空浓缩和冷冻干燥后，得到玉米蛋白水解多肽粉末；
(2)糖基化修饰玉米蛋白多肽的制备
取上述玉米肽粉末，用20倍体积的蒸馏水溶解，加入1～5倍玉米肽质量的氨基葡萄糖，混匀，将反应体系调至7.0～9.0，加入玉米肽质量为10％～20％的转谷氨酰胺酶，在30～50℃下反应3～7h，反应结束后95℃条件下蒸煮5min灭酶，随后将反应液置于4℃下透析24h除去未反应的氨基葡萄糖，对其粒径大小进行测定，样品冷冻干燥后4℃下储藏备用；
(3)糖基化玉米多肽负载叶黄素纳米粒的制备
将叶黄素标准液和糖基化玉米蛋白肽溶液等体积混合均匀，加入2倍体积的PBS缓冲溶液，在一定温度下搅拌水合10～60min，旋转蒸发除去乙醇，得到糖基化玉米蛋白负载叶黄素纳米粒，对叶黄素的包封率可达90％以上，粒度分析仪和透射电子显微镜显示为分散球型纳米粒结构。抗氧化研究表明，经糖基化修饰的玉米肽负载叶黄素纳米粒的抗氧化能力显著高于未修饰的玉米肽和叶黄素。
2.根据权利要求1所述的一种糖基化玉米蛋白多肽载运叶黄素纳米粒的制备方法，其特征是：在所述的玉米蛋白多肽的酶解制备步骤中，加入蛋白粉质量1.0％～3.0％的中性蛋白酶，超滤膜截留分子量为5000～10000道尔顿。
3.根据权利要求1所述的一种糖基化玉米蛋白多肽载运叶黄素纳米粒的制备方法，其特征是：在所述的糖基化修饰玉米蛋白多肽的制备步骤中，转谷氨酰胺酶的活力为1000～2000u/g，透析采用分子量小于1000的透析袋。
4.根据权利要求1所述的一种糖基化玉米蛋白多肽载运叶黄素纳米粒的制备方法，其特征是：在所述的糖基化玉米多肽负载叶黄素纳米粒的制备步骤中，将100μg/mL纯度为90％的叶黄素标准液和2.5mg/mL糖基化玉米蛋白肽溶液等体积混合均，该纳米粒的微观结构为平均粒径100～200nm的球形微粒，该纳米粒对叶黄素溶增溶能力提高20倍以上，其羟基自由基的清除能力为游离叶黄素的4倍以上。
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| CN115462493A (zh) * | 2022-09-27 | 2022-12-13 | 青岛啤酒股份有限公司 | 基于植物多肽封装掩味的复合纳米颗粒及所得饮品 |
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2021
- 2021-07-19 CN CN202110811200.5A patent/CN114159409A/zh active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115462493A (zh) * | 2022-09-27 | 2022-12-13 | 青岛啤酒股份有限公司 | 基于植物多肽封装掩味的复合纳米颗粒及所得饮品 |
| CN115462493B (zh) * | 2022-09-27 | 2023-09-05 | 青岛啤酒股份有限公司 | 基于植物多肽封装掩味的复合纳米颗粒及所得饮品 |
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