CN113892649A - 一种益生菌微胶囊及其制备方法 - Google Patents
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Abstract
本发明提供了一种益生菌微胶囊及其制备方法，所述微胶囊以脂肪球膜蛋白为胶囊壁，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、酵母菌属中的一种或多种菌株。制备所述益生菌微胶囊的方法包括：脂肪球膜蛋白的富集，和益生菌微胶囊的制备。本发明通过富集脂肪球脂肪球膜蛋白作为胶囊壁包埋益生菌，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。
Description
技术领域
本发明属于生物技术领域，特别涉及一种益生菌微胶囊及其制备方法。
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，其中膜特异性蛋占25-70%，含500多种蛋白质，其中大多数为糖基化蛋白，包括嗜乳脂蛋白、黄嘌呤氧化还原酶、黏液素等等。近年来，脂肪球膜蛋白被发现具有免疫调节、抑菌、抗肿瘤和调节肠道菌群等益生功能，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微胶囊及其制备方法，通过富集脂肪球膜蛋白作为乳化剂包埋益生菌，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：
一种益生菌微胶囊，包括益生菌和胶囊壁，所述胶囊壁的原料为脂肪球膜蛋白。
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、酵母菌属中的一种或多种菌株。
一种益生菌微胶囊的制备方法，包括以下步骤：
步骤1，脂肪球膜蛋白的富集：
以Lacprodan MFGM-10为原料，将Lacprodan MFGM-10与三氯甲烷/甲醇混合，低温搅拌，搅拌后的液体与氯化钠混合振荡后平衡，分为上层相和下层试剂层，上层相分别用三氯甲烷/甲醇混合液、氯化钠溶液清洗，得到上层相组分；将去除脂质的下层试剂用旋转蒸发仪蒸发，提取重复2-3次，得到下层相组分，所述上层相组分和下层相组分混合，得到蛋白组分P1；
将所述蛋白组分P1溶于模拟乳超滤液中，添加凝乳酶，调节pH至6.8，凝乳后将酪凝块切成小块，收集排出的乳清液，得到蛋白浓缩液P2；
采用陶瓷膜超滤法去除所述蛋白浓缩液P2中的乳清蛋白，收集截留物，即得到脂肪球膜蛋白富集物；
步骤2，益生菌微胶囊的制备：
将脂肪球膜蛋白富集物分散至超纯水中搅拌形成脂肪球膜蛋白悬浮液，将所述悬浮液的pH调至7.0，低温下搅拌过夜，将益生菌浓缩液与悬浮液混合，制成含益生菌的蛋白-细胞混合物；
将谷氨酰转氨酶添加到所述含益生菌的蛋白-细胞混合物中，温度控制在30-50℃，并立刻加入已经预热的油中，搅拌1-3小时，使球膜蛋白胶连，再轻微离心将油从凝胶的微胶囊中分离出来，去除上清液，将沉淀物用Ringer平衡盐溶液稀释，摇匀，然后再次离心，去除上清液，沉淀物即为所述的益生菌微胶囊。
本发明益生菌微胶囊的用途，包括以下方面：
本发明所述的益生菌微胶囊用于制备食品添加剂。
本发明所述的益生菌微胶囊用于制备保健品添加剂。
本发明所述的益生菌微胶囊用于制备饲料添加剂。
本发明所述的益生菌微胶囊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蛋白作为制备益生菌微胶囊的壁材，脂肪球膜蛋白本身具有免疫调节、抑菌、抗肿瘤及调节肠道菌群等多种益生功能，有利于增加微胶囊产品的益生功能；
（2）脂肪球膜蛋白具有双亲性，是天然的乳化剂，本发明利用脂肪球膜蛋白与疏水性表面的细菌具有较高的亲和性，可提高益生菌的包封率，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微胶囊，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微胶囊的制备流程示意图；
图2为益生菌包埋率示意图；
图3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微胶囊，包括益生菌和胶囊壁，所述胶囊壁的原料为脂肪球膜蛋白，如图1所示，所述益生菌微胶囊的制备方法包括以下步骤：
步骤1，脂肪球膜蛋白的富集：
以Lacprodan MFGM-10为原料，将20g Lacprodan MFGM-10与200mL 三氯甲烷/甲醇（2:1，v/v）混合，用磁力搅拌器于4℃搅拌30min，搅拌后的液体与30mL，0.73%的氯化钠（w/w）混合振荡后平衡，分为上层相和下层试剂层，上层相分别用200mL 三氯甲烷/甲醇混合液（2:1，v/v）、0.58%的氯化钠溶液（w/w）清洗3次，得到上层相组分；将去除脂质的下层试剂用旋转蒸发仪蒸发，提取重复2-3次，得到下层相组分，所述上层相组分和下层相组分混合，得到蛋白组分P1；
为了去除蛋白组分P1中的酪蛋白胶束，采用了类似经典奶酪生产的凝乳酶诱导凝乳，具体处理为：将40g所述蛋白组分P1溶于1L模拟乳超滤液中，添加0.03%凝乳酶，调节pH至6.8，在42℃凝乳30min后，将酪凝块切成1cm3的小块，再细切至原体积的1/4，收集排出的乳清液，得到蛋白浓缩液P2；
所述模拟乳超滤液：利用超滤膜（10 KDa，Sartorius）将脱脂乳超滤而得；
采用陶瓷膜超滤法去除所述蛋白浓缩液P2中的乳清蛋白，采用动态错流滤装置，孔径为80nm的陶瓷膜，跨膜压力保持在0.1MPa，温度为50℃，重复6次，收集截留物，即得到脂肪球膜蛋白富集物；
步骤2，益生菌微胶囊的制备：
将所用的玻璃器皿在121℃下灭菌15min，将脂肪球膜蛋白富集物分散至超纯水中至15%（w/w），搅拌2h形成脂肪球膜蛋白悬浮液，用5M NaOH溶液将所述悬浮液的pH调至7.0，在4℃低温下搅拌过夜后使用，将2g动物双歧杆菌A6浓缩液解冻，与10-20g步骤1制得的脂肪球膜蛋白富集物悬浮液混合，制成含1×1010 cfu g-1益生菌的蛋白-细胞混合物；
将10U谷氨酰转氨酶（TGase）添加到30g所述含益生菌的蛋白-细胞混合物中，温度控制在35℃，并立刻加入150g已经预热至40℃的菜籽油中，用磁力搅拌器以900 rpm的恒定速度搅拌2.5小时，使球膜蛋白胶连，再轻微离心（500g，1min）将油从凝胶的微胶囊中分离出来，去除上清液，将沉淀物用两倍体积的Ringer平衡盐溶液稀释，摇匀5min，然后在相同条件下再次离心，去除含水和残油组成的上清液，沉淀物即为所述的益生菌微胶囊，储存在4℃，以待后续研究。
实施例2——微胶囊包埋率
取适量实施例1制备得到的益生菌微胶囊以及不加益生菌的空白对照，通过离心测定活菌数法，测定益生菌微胶囊的包埋率。
如图2所示，随着脂肪球膜蛋白富集物的添加，益生菌微胶囊的包封率逐渐增加，添加15g膜蛋白后，益生菌微胶囊的包埋率达到87.4%，与添加20g膜蛋白的微胶囊包埋率无显著性差异。因此实验选用15g脂肪球膜蛋白作为壁材包埋益生菌。
另外，通过实验测定发现，采用脂肪球膜蛋白制备的胶囊壁包埋益生菌后，提高了益生菌粉的冻干存活率及贮藏稳定性。
实施例3——模拟胃肠消化
将3g实施例1制备得到的益生菌微胶囊加入含有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，每隔30min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，并计算其在胃消化阶段的存活率。
同时，将未胶囊化的脂肪球膜蛋白与益生菌混合物作为对照，测定其菌株存活率。
如图3所示，采用脂肪球膜蛋白包埋益生菌后，益生菌在胃环境的存活率大幅提升。在胃消化终点（120min）时，所述益生菌微胶囊中的益生菌存活率为52.1%，活菌数为4×109 cfu /mL，超过益生菌在肠道有效作用的阈值（≥107 cfu /mL），而未胶囊化的益生菌的存活率仅剩0.2%。
益生菌微胶囊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，本发明制备的微胶囊在模拟肠液消化 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 (7)
1.一种益生菌微胶囊，包括益生菌和胶囊壁，其特征在于，所述胶囊壁的原料为脂肪球膜蛋白。
2.根据权利要求1所述的益生菌微胶囊，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、酵母菌属中的一种或多种菌株。
3.一种益生菌微胶囊的制备方法，其特征在于，所述制备方法包括以下步骤：
步骤1，脂肪球膜蛋白的富集：
以Lacprodan MFGM-10为原料，将Lacprodan MFGM-10与三氯甲烷/甲醇混合，低温搅拌，搅拌后的液体与氯化钠混合振荡后平衡，分为上层相和下层试剂层，上层相分别用三氯甲烷/甲醇混合液、氯化钠溶液清洗，得到上层相组分；将去除脂质的下层试剂用旋转蒸发仪蒸发，提取重复2-3次，得到下层相组分，所述上层相组分和下层相组分混合，得到蛋白组分P1；
将所述蛋白组分P1溶于模拟乳超滤液中，添加凝乳酶，调节pH至6.8，凝乳后将酪凝块切成小块，收集排出的乳清液，得到蛋白浓缩液P2；
采用陶瓷膜超滤法去除所述蛋白浓缩液P2中的乳清蛋白，收集截留物，即得到脂肪球膜蛋白富集物；
步骤2，益生菌微胶囊的制备：
将脂肪球膜蛋白富集物分散至超纯水中搅拌形成脂肪球膜蛋白悬浮液，将所述悬浮液的pH调至7.0，低温下搅拌过夜，将益生菌浓缩液与悬浮液混合，制成含益生菌的蛋白-细胞混合物；
将谷氨酰转氨酶添加到所述含益生菌的蛋白-细胞混合物中，温度控制在30-50℃，并立刻加入已经预热的油中，搅拌1-3小时，使球膜蛋白胶连，再轻微离心将油从凝胶的微胶囊中分离出来，去除上清液，将沉淀物用Ringer平衡盐溶液稀释，摇匀，然后再次离心，去除上清液，沉淀物即为所述的益生菌微胶囊。
4.一种如权利1或2所述的益生菌微胶囊用于制备食品添加剂。
5.一种如权利要求1或2所述的益生菌微胶囊用于制备保健品添加剂。
6.一种如权利要求1或2所述的益生菌微胶囊用于制备饲料添加剂。
7.一种如权利要求1或2所述的益生菌微胶囊用于制备药物添加剂。
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| CN114532541A (zh) * | 2022-01-25 | 2022-05-27 | 东北农业大学 | 一种包埋益生菌的乳脂肪球膜复合微胶囊制备方法 |
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