CN113892650A - 一种益生菌脂质体及其制备方法 - Google Patents

一种益生菌脂质体及其制备方法 Download PDF

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CN113892650A
CN113892650A CN202010557068.5A CN202010557068A CN113892650A CN 113892650 A CN113892650 A CN 113892650A CN 202010557068 A CN202010557068 A CN 202010557068A CN 113892650 A CN113892650 A CN 113892650A
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liposome
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罗洁
李媛
沈清武
刘成国
周辉
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Hunan Agricultural University
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Abstract

本发明提供了一种益生菌脂质体及其制备方法，所述益生菌脂质体包括益生菌和脂肪球膜磷脂脂质体，以脂肪球膜磷脂、胆固醇、吐温‑80和维生素E，和/或壳聚糖为制备脂质体的原料。制备时，首先将脂肪球膜磷脂、胆固醇、吐温‑80和维生素E混合，混合物在无水乙醇中充分溶解，利用真空旋转蒸发去除有机溶剂，再通过真空干燥获得磷脂薄膜；利用PBS磷酸缓冲液稀释益生菌浓缩液，而后与上述磷脂薄膜在超声的作用下水合，从而获得所述益生菌脂质体。本发明通过制备脂肪球膜磷脂脂质体包埋益生菌，增强了益生菌对胃环境的耐受性，使其得以顺利到达肠道，发挥其益生功能；利用壳聚糖包被所述益生菌脂质体，可防止磷脂被氧化，进一步增加脂质体的贮藏稳定性。

Description

一种益生菌脂质体及其制备方法
技术领域
本发明属于生物技术领域，特别涉及一种益生菌脂质体及其制备方法，以脂肪球膜磷脂脂质体装载益生菌。
背景技术
伴随我国经济社会快速发展，居民生活节奏加快、生活方式改变，居民胃肠道疾病和代谢性疾病等慢性疾病发病率逐年增高，严重危害居民健康。以改善人体微生态营养为目的的益生菌是公认的改善代谢综合征和慢性胃肠道疾病的有效手段。大量研究显示，益生菌作为人体肠道重要的生理菌，具有多种生理功能，包括改善人体肠道功能、减少肠道疾病、促进营养物质吸收、缓解乳糖不耐症、降胆固醇、调节免疫系统、预防癌症等，因此被广泛青睐。为获得这些功效，必须使益生菌在产品和宿主中都保持活性和代谢稳定，且达到一定数量（≥107 cfu /mL）。然而，由于益生菌很难耐受胃酸pH 1.2的强酸性环境，在胃环境中菌的数量和活性显著下降，使最终定植于人体肠道中的活菌数低于理论上能够发挥益生功效的最小阈值。因此，益生菌的微胶囊包埋技术越来越受到人们的关注。
乳脂肪球膜是乳脂肪球特有的膜组分，它包裹在乳脂肪球表面，在乳中充当乳化剂和稳定剂，防止乳脂肪絮凝与聚合，也阻止乳中脂肪与酶的接触。乳脂肪球膜是由极性脂质、胆固醇和膜特异性蛋白等组成的复杂体系，其中极性脂质约占膜的40%，由磷脂酰乙醇胺及乳脂肪特有的鞘磷脂和神经节甘脂等构成。近年来，脂肪球膜磷脂被发现具有改善婴幼儿大脑和认知发育，增强免疫力，促进肠道发育、调节脂代谢等益生功能，因此受到广泛关注。
发明内容
本发明的目的是提供一种益生菌脂质体及其制备方法，通过富集脂肪球膜磷脂作为脂质体材料包埋益生菌，增强益生菌对胃环境的耐受性，使其得以顺利到达肠道，发挥其益生功能。
本发明的目的是通过以下技术方案实现的：
一种益生菌脂质体，包括益生菌和脂质体，所述脂质体为脂肪球膜磷脂脂质体，以脂肪球膜磷脂、胆固醇、吐温-80和维生素E为原料制备所述脂质体。
进一步的，制备所述益生菌脂质体的原料还包括壳聚糖。
进一步的，所述益生菌为选自乳杆菌属、双歧杆菌属、肠球菌属、大肠杆菌属、芽孢杆菌属、丁酸梭菌属和酵母菌属中的一种或多种菌株。
所述益生菌脂质体的制备方法，包括以下步骤：
步骤1，磷脂薄膜的制备：将脂肪球膜磷脂、胆固醇、吐温-80和维生素E以一定的比例混合，混合物在无水乙醇中充分溶解，而后利用真空旋转蒸发去除有机溶剂，再通过真空干燥获得磷脂薄膜；
步骤2，益生菌的包埋：利用PBS磷酸缓冲液稀释益生菌浓缩液，而后与步骤1得到的磷脂薄膜在超声的作用下水合，从而获得所述益生菌脂质体。
进一步的，所述制备方法还包括以下步骤：
将步骤2制备好的益生菌脂质体与等体积的壳聚糖溶液混合，持续搅拌，并在高压匀浆器中进行匀浆处理，得到壳聚糖包被的益生菌脂质体。
进一步的，所述脂肪球膜磷脂、胆固醇、吐温-80和维生素E的质量比为6：1：18：0.12。
本发明益生菌脂质体的用途，包括以下方面：
本发明所述的益生菌脂质体用于制备食品添加剂。
本发明所述的益生菌脂质体用于制备保健品添加剂。
本发明所述的益生菌脂质体用于制备饲料添加剂。
本发明所述的益生菌脂质体用于制备药物添加剂。
本发明相比现有技术的有益效果为：
（1）本发明利用脂肪球膜磷脂作为制备脂质体的原料，脂肪球膜磷脂本身具有改善婴幼儿大脑和认知发育，增强免疫力，促进肠道发育、调节脂代谢等多种益生功能，有利于增加脂质体的益生功能；
（2）脂肪球膜磷脂与疏水性表面的细菌具有特异性粘附的作用，可提高益生菌的包封率，与由大豆卵磷脂脂质体相比，脂肪球膜磷脂脂质体膜较厚、膜渗透性较低且环境压力抵抗能力强，可耐受胃酸环境，从而更有效地在肠道内靶向释放益生菌；
（3）本发明制备得到的益生菌脂质体，可作为食品添加剂、保健品添加剂、饲料添加剂及药物添加剂，尤其适用于婴幼儿配方食品中。
附图说明
下面结合附图和实施例对本发明作进一步说明。
图1为本发明所述益生菌脂质体的制备流程示意图；
图2为胃肠阶段益生菌存活率示意图。
具体实施方式
实施例1
本实施例提供了一种益生菌脂质体，包括益生菌和脂质体，所述脂质体为脂肪球膜磷脂脂质体，制备所述脂质体的材料包括脂肪球膜磷脂、胆固醇、吐温-80和维生素E，制备方法包括以下步骤：
步骤1，薄层分散法制备磷脂薄膜悬浮液：将脂肪球膜磷脂、胆固醇、吐温-80和维生素E以6:1:18:0.12的质量比混合，混合物在无水乙醇中充分溶解，在45℃下真空旋转蒸发除去有机溶剂，并将圆底烧瓶放入真空干燥箱中真空干燥12h，得到干燥后的磷脂薄膜；
所述脂肪球膜磷脂选用脂肪球膜磷脂富集物Phospholac 600，所有用到的玻璃器皿在121℃下灭菌15分钟。
步骤2，益生菌的包埋：2 g动物双歧杆菌A6的浓缩液用PBS磷酸缓冲液稀释，后与干燥后的磷脂薄膜在超声的作用下水合。在100W的超声作用10分钟后，在45℃水浴锅水合1h，获得浓度约为8 mg/mL的益生菌脂质体。
制备好的益生菌脂质体储存在4 ℃，以待后续研究。同时，以不加益生菌的空脂质体悬液作为对照。
实施例2
本实施例是实施例1基础上的改进，为了进一步增加脂质体的稳定性，防止磷脂被氧化，提高肠溶性，本实施例采用壳聚糖包被实施例1制得的益生菌脂质体。
具体方法为：将实施例1步骤2制备得到的益生菌脂质体与等体积的壳聚糖溶液混合，持续搅拌（700 rpm，2分钟），并在9000 psi的高压匀浆器中进行三次，得到壳聚糖包被的脂质体，再利用壳聚糖包被的脂质体，包埋益生菌。通过实验测定发现，采用壳聚糖包备的益生菌脂质体，进一步提高了益生菌粉的冻干存活率及贮藏稳定性。
所述壳聚糖溶液的浓度为0.2%（w/v）。
实施例3——益生菌脂质体的表征
取适量实施例1制备得到的益生菌脂质体，以及不加益生菌的空脂质体悬液，通过离心测定活菌数法，测定益生菌脂质体的包封率；用PBS缓冲液将脂质体稀释，采用动态光散射粒度仪测定微胶囊的平均粒径，用电位移测定所述脂质体的表面电位。
表1 载益生菌的益生菌脂质体表征
Figure DEST_PATH_IMAGE001
如上表1所示，通过优化超声条件，实施例1所制备得到的益生菌脂质体，其粒径为4.26μm，Zeta电势为-15.4mV，包埋率可达78.63%。
实施例4——模拟胃肠消化
将3g实施例1制备得到的益生菌脂质体加入含有27mL预热至37℃的模拟胃液中，涡流5s，用1M的HCl将模拟胃液调至pH值为2.0，加入胃蛋白酶及胃脂肪酶，消化2h。模拟胃消化后，再转入模拟含胆盐和胰消化酶的肠液中，用1M的NaOH将模拟胃液调至pH值为7.4，消化2h。每隔60min取样，加入10 mL 0.1 mol/L 磷酸盐缓冲液，涡旋振荡5 min，使胶粒完全溶解，吸取1 mL 样液进行梯度稀释，根据平板计数方法测得活菌数量。同时，以无脂质体包封的益生菌作为对照。
如图2所示，采用脂肪球膜磷脂制备的脂质体包埋益生菌后，益生菌在胃环境的存活率大幅提升。在胃消化终点（120min）时，所述益生菌脂质体中的益生菌活菌数为5.4×109 cfu /mL，而未胶囊化的益生菌的活菌数几近于零。在肠阶段，益生菌从脂质体中释放，作用浓度约为5.0×109 cfu /mL，超过益生菌在肠道有效作用的阈值（≥107 cfu /mL）。
脂质体想要在人体内发挥益生作用，必须在肠道内尽快崩解，释放出益生菌。结果表明，本发明制备的益生菌脂质体在模拟肠液消化 30 min 左右就已经完成崩解，基本观察不到固体颗粒，可达到肠溶的目的，以发挥益生菌相应的益生效果。

Claims (10)

1.一种益生菌脂质体，包括益生菌和脂质体，其特征在于，所述脂质体为脂肪球膜磷脂脂质体，以脂肪球膜磷脂、胆固醇、吐温-80和维生素E为原料制备所述脂质体。
2.根据权利要求1所述的益生菌脂质体，其特征在于，制备所述益生菌脂质体的原料还包括壳聚糖。
3.根据权利要求1或2所述的益生菌脂质体，其特征在于，所述益生菌为选自乳杆菌属、双歧杆菌属、肠球菌属、大肠杆菌属、芽孢杆菌属、丁酸梭菌属和酵母菌属中的一种或多种菌株。
4.一种如权利要求1-3任一项所述益生菌脂质体的制备方法，其特征在于，所述制备方法包括以下步骤：
步骤1，磷脂薄膜的制备：将脂肪球膜磷脂、胆固醇、吐温-80和维生素E以一定的比例混合，混合物在无水乙醇中充分溶解，而后利用真空旋转蒸发去除有机溶剂，再通过真空干燥获得磷脂薄膜；
步骤2，益生菌的包埋：利用PBS磷酸缓冲液稀释益生菌浓缩液，而后与步骤1得到的磷脂薄膜在超声的作用下水合，从而获得所述益生菌脂质体。
5.根据权利要求4所述的制备方法，其特征在于，所述制备方法还包括以下步骤：
将步骤2制备好的益生菌脂质体与等体积的壳聚糖溶液混合，持续搅拌，并在高压匀浆器中进行匀浆处理，得到壳聚糖包被的益生菌脂质体。
6.根据权利要求4所述的制备方法，其特征在于，所述脂肪球膜磷脂、胆固醇、吐温-80和维生素E的质量比为6：1：18：0.12。
7.一种如权利要求1-3任一项所述的益生菌脂质体用于制备食品添加剂。
8.一种如权利要求1-3任一项所述的益生菌脂质体用于制备保健品添加剂。
9.一种如权利要求1-3任一项所述的益生菌脂质体用于制备饲料添加剂。
10.一种如权利要求1-3任一项所述的益生菌脂质体用于制备药物添加剂。
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