CN114532541A - 一种包埋益生菌的乳脂肪球膜复合微胶囊制备方法 - Google Patents
一种包埋益生菌的乳脂肪球膜复合微胶囊制备方法 Download PDFInfo
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- CN114532541A CN114532541A CN202210086315.7A CN202210086315A CN114532541A CN 114532541 A CN114532541 A CN 114532541A CN 202210086315 A CN202210086315 A CN 202210086315A CN 114532541 A CN114532541 A CN 114532541A
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- A—HUMAN NECESSITIES
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Abstract
本发明公开了一种包埋益生菌的乳脂肪球膜复合微胶囊制备方法，属于微生物技术领域。本发明采用乳脂肪球膜、角瓜多糖、水苏糖和卡拉胶作为壁材，分别利用喷雾、冷冻和流化床三种干燥方法进行包埋，喷雾干燥得到的微胶囊包埋率可以达到71.64％，干燥存活率22.97％；流化床干燥包埋率72.54％，干燥存活率18.86％；冷冻干燥效果最好，包埋率达到76.6％，冻干存活率29.2％。使用包埋后的微胶囊在体外模拟胃液环境下进行测试，发现三种包埋方法制得的微胶囊处理60min益生菌的存活率均在65％以上；处理120min存活率均在43％以上，说明本发明对益生菌的保护效果显著，有助于益生菌顺利定植肠道发挥益生作用。
Description
技术领域
本发明涉及一种包埋益生菌的乳脂肪球膜复合微胶囊制备方法，属于微生物技术领域。
背景技术
乳脂肪球膜的成分包括磷脂、鞘脂、唾液酸和糖蛋白。近几年来，从牛奶中分离和浓缩乳脂肪球膜的新技术的发展使研究乳脂肪球膜的生理功能和特性成为可能。已经证明，乳脂肪球膜成分具有多种有益健康的特性，包括支持大脑发育、防止病原体附着和宿主免疫调节。
益生菌已被纳入一系列产品，包括儿科营养产品和补充剂。然而，在肠道条件和益生菌生存能力方面仍有许多挑战需要克服。例如，益生菌的生存能力受到一系列因素的影响，包括pH值、储存期间的酸化、暴露于胃肠系统中的胆汁和储存温度。一些食品成分已经被研究作为益生菌的载体，并被证明在体外消化过程中提高了它们的生存能力。
目前，关于乳脂肪球膜对益生菌生长和存活的影响还缺乏相关信息。乳脂肪球膜在硒、脂溶性维生素、有机磷酸盐、茶多酚等微量元素的传递中起着重要作用，并能包裹乳酸菌。
多糖在水中表现出良好的溶解性，并且在高浓度下具有较低的粘度。一些多糖具有良好的阻氧性和阻湿性，对被包裹的生物活性材料也有很好的保护作用，如今有许多由蔬菜水果中提取的中性多糖已经被证实作为微胶囊的壁材有更好的保护作用，角瓜(又名西葫芦)作为家庭中常见的蔬菜之一，其含有丰富的多糖，且因为成本低廉，满足工业微胶囊壁材的需要。
制备微胶囊最常见的方法是挤压法，具有制备工艺简单，反应条件温和，成本低廉等优点，但这种方法耗时耗力，颗粒大小难以控制，且由于微珠的缓慢形成，难以用于大规模生产。乳化法是将细胞悬液添加到油相中，经过匀质形成油包水(W/O)乳液，水溶性聚合物分散到油相中,形成不溶性的微小胶粒，因此乳化法制作的微胶囊，粒径分布范围更窄，呈球形更好，细菌存活率高。相比于液态益生菌微胶囊，干燥形式的微胶囊具有保存时间长、处理、储存、销售方便以及随后在功能食品开发中的应用等优点，但不同的干燥方式对益生菌也会有不同的影响，本发明包括乳脂肪球膜微胶囊的冷冻干燥、喷雾干燥和流化床干燥三种干燥方式。
目前市场上的益生菌微胶囊技术比较成熟，但不同微胶囊壁材在不同干燥方法下对益生菌的保护效果差异很大，无法应对不同的包埋需求，因此市场上急需一种在不同干燥方法下对益生菌保护效果稳定的微囊化方法。
发明内容
本发明目的是提供一种新的益生菌微胶囊包埋方法，提高益生菌存活率，有助于益生菌定植肠道、发挥益生功能，同时为生产益生菌微胶囊提供更加稳定的方法，让益生菌微胶囊的生产更加稳定、高效，提高益生菌质量。
为实现本发明目的，本发明的内容如下：
一种乳脂肪球膜复合微胶囊制备方法与应用，按照如下步骤进行：一、将角瓜洗净，用捣钵捣碎，称量捣碎的角瓜1.0～2.0g，加入50～100mL的70～85％乙醇溶液，水浴回流提取45～90min(75～85℃)，趁热过滤，用85％热乙醇冲洗2次，等到溶剂挥干后，将残渣和滤纸一同放入锥形瓶中，加蒸馏水100mL，水浴(75℃)回流45min，取出冷却至室温后，精密量取10mL的回流液于离心管中，以3000～10000r/min离心5～10min，之后再精密吸取离心后的上清液1mL，至10mL量瓶中加蒸馏水定容，振摇均匀，备用。二、接种环取冻存管中的益生菌于MRS固体培养基中划板，34～37℃厌氧培养18～24h；之后挑取单个菌落在MRS液体培养基中34～37℃厌氧培养18～24h，作为初始菌液。三、将初始菌液以1～2％体积比接种至MRS液体培养基中34～37℃培养15～18h作为待微囊化的菌液，用离心机将菌液在5000～10000g下离心10～20min，撇去上清液，加入与沉淀等质量的生理盐水，通过加入3％NaOH溶液调节菌液pH至6.5～6.8待用。四、配置2.5～30g/L的乳脂肪球膜、10～50g/L角瓜多糖、5～20g/L阴离子多糖和20～200g/L冻干保护剂作为微胶囊壁材待用，将菌液与微胶囊壁材以体积比1～3:2～9混合，以未混合菌液为空白对照；在与微胶囊壁材混合后的菌液中以体积比1～2:3～5加入植物油，使用搅拌器搅拌10-15min；之后添加冰醋酸至pH为4.6，搅拌5min；以体积比1～2:1～2加入冰箱中4℃下冷藏的生理盐水，搅拌5～10min；而后将菌液冷藏静置20～30min后在5000～10000g下离心10～20min；撇去上清液，以质量比1～2:1～2添加生理盐水，得到微胶囊溶液。五、用平皿盛放微胶囊溶液于-18～-20℃冰箱中预冻8～12h，之后将平皿放入冻干机，设置冻干温度-78～-80℃，真空度4.0～4.2Pa，干燥15～24h后得到微胶囊冻干样品。或者向混合物中加入约2～3％吐温80。然后使用均质机以8000～11000rpm的速度乳化10～15min。用喷雾干燥机进行喷雾干燥，在喷雾干燥机上喷雾干燥乳状液的工艺参数如下：进料速度3～5ml/min，吸气器100％，雾化压力0.2～0.3bar，进风温度150～180℃，出风温度50～60℃，雾化器嘴直径1.4～1.6mm。或者设置流化床进风温度为78-88℃，出风温度为38-42℃，倒入颗粒，将包衣锅转速调至2-3rpm，把喷枪位置调至离距离床约30-40cm处，开始预热物料。至物料温度32-38℃，喷雾空气压力在0.5-0.6MPa，开始包衣，包.衣20-30min后逐渐提高包衣锅转速和加大流量(以颗粒间不黏连为度)。在包衣过程中，持续搅拌包衣液，尽快喷完全部浆液，至要求增重量，此时保持热风风机的运转状态，干燥几分钟后再关闭热风风机，开启冷却风冷至室温，出料。
在本发明的实施方式中，所述阴离子多糖为羧甲基纤维素钠、海藻酸钠、黄原胶或阿拉伯胶中的一种或一种以上。
在本发明的实施方式中，所述冻干保护剂为低聚果糖、低聚半乳糖、棉籽糖、异麦芽酮糖、乳酮糖、低聚木糖、低聚异麦芽酮糖、水苏糖、低聚龙胆糖、大豆低聚糖、低聚异麦芽糖、低聚壳聚糖中的一种或一种以上。
在本发明的实施方式中，所述益生菌包括罗伊氏乳杆菌、青春双歧杆菌、短双歧杆菌、长双歧杆菌、乳双歧杆菌、两歧双岐杆菌、婴儿双岐杆菌、鼠李糖乳杆菌、嗜酸乳杆菌、约氏乳杆菌、格氏乳杆菌、干酪乳杆菌、副干酪乳杆菌、植物乳杆菌、发酵乳杆菌、卷曲乳杆菌、唾液乳杆菌、清酒乳杆菌、德氏乳杆菌。
本发明提供了一种可显著提高益生菌微胶囊中益生菌存活率的微胶囊壁材，此微胶囊壁材的成分包含乳脂肪球膜、角瓜多糖、阴离子多糖以及冻干保护剂；使用此微胶囊壁材制备益生菌微胶囊时，益生菌的包埋率高达78.11％，将使用此微胶囊壁材制备得到的益生菌微胶囊进行冷冻干燥后，益生菌的冻干存活率高达30.14％，并且，将使用此微胶囊壁材制备得到的益生菌微胶囊用pH为2.5的模拟胃液处理1h后，益生菌存活率高达68.55％；处理2h后，益生菌的存活率高达47.56％。
下面用具体实施方式进一步说明本发明，可以理解的是，本发明的具体实施方式不会构成对本发明保护范围的任何限制。
具体实施方式
下面结合具体实施例对本发明进行进一步阐述。
以下实施例中涉及的乳脂肪球膜购自美赞臣营养公司；水苏糖、海藻酸钠、氯化钠、卡拉胶、乙醇、MRS肉汤培养基购自哈尔滨超峰生物科技有限公司。
以下实施步骤为：
实施例1：
步骤1：角瓜多糖的提取
将角瓜洗净，用捣钵捣碎，称量捣碎的角瓜2.0g，加入50mL的80％乙醇溶液，水浴回流提取45min(75℃)，趁热过滤，用85％热乙醇冲洗2次，等到溶剂挥干后，将残渣和滤纸一同放入锥形瓶中，加蒸馏水100mL，水浴(75℃)回流45min，取出冷却至室温后，精密量取10mL的回流液于离心管中，以10000r/min离心5min，之后再精密吸取离心后的上清液1mL，至10mL量瓶中加蒸馏水定容，振摇均匀，备用。
步骤2：益生菌的活化
接种环取冻存管中的益生菌于MRS固体培养基中划板，37℃厌氧培养24h；之后挑取单个菌落在MRS液体培养基中37℃厌氧培养24h，作为初始菌液。
步骤3：婴儿双歧杆菌的扩容培养和洗涤
将初始菌液以2％体积比接种至MRS液体培养基中37℃培养18h作为待微囊化的菌液，用离心机将菌液在10000xg下离心15min，撇去上清液，加入与沉淀等质量的生理盐水，通过加入3％NaOH溶液调节菌液pH至6.5待用。
步骤4：乳脂肪球膜-角瓜多糖微胶囊的制备
首先配置10g/L的乳脂肪球膜、20g/L角瓜多糖、10g/L卡拉胶和100g/L水苏糖作为微胶囊壁材待用，将菌液与微胶囊壁材以体积比2:5混合，以未混合菌液为空白对照；在与微胶囊壁材混合后的菌液中以体积比1:3加入植物油，使用搅拌器搅拌10-15min；之后添加冰醋酸至pH为4.6，搅拌5min；以体积比1:1加入冰箱中4℃下冷藏的生理盐水，搅拌5min；而后将菌液冷藏静置20min后在10000xg下离心15min；撇去上清液，以质量比1:1添加生理盐水，得到微胶囊溶液。
步骤5：乳脂肪球膜-角瓜多糖微胶囊冻干菌粉的制备:
用平皿盛放微胶囊溶液于-20℃冰箱中预冻8h，之后将平皿放入冻干机，设置冻干温度-80℃，真空度4.0Pa，冷冻干燥15h后得到微胶囊冻干样品。
步骤6：微胶囊保护作用的测试
益生菌活菌数的检测方法：采用国标《GB 4789.35-2016食品安全国家标准食品微生物学检测乳酸菌检测》。益生菌微胶囊包埋率的检测方法：测定微胶囊悬浮液中的总活菌数和2mL菌悬液中的总活菌数，并计算益生菌的包埋率；其中，益生菌微胶囊包埋率的计算公式如下：
包埋率＝微胶囊悬浮液中总活菌数/菌悬液中总活菌数100％
步骤7：益生菌微胶囊冻干存活率的检测方法
在冷冻真空干燥前测定微胶囊悬浮液中益生菌的活菌数作为微胶囊冻干前活菌浓度，在真空冷冻干燥后，将冻干菌粉用无菌水复溶至真空冷冻干燥前体积，得到复溶液，测定复溶液中益生菌活菌数作为微胶囊冻干后活菌浓度，根据测定结果计算益生菌微胶囊冻干存活率；其中，益生菌微胶囊冻干存活率的计算公式如下：
冻干存活率＝冻干后活菌浓度\冻干前活菌浓度100％
测得本实施例方法对益生菌的包埋率为76.6±1.51％；未包埋的益生菌冻干存活率为0，冻干存活率为29.2±0.94％。
步骤7：益生菌微胶囊体外模拟测试
本实施例进一步对制备得到的益生菌微胶囊进行了体外模拟。模拟胃液测试：将2g的NaCl、7mL的浓HCl(浓度为36％～38％)和0.26g的胃蛋白酶加入到1L去离子水中并调节pH＝2以制备出模拟胃液，将1g待测样品(S3得到的微胶囊菌粉)加入到模拟胃液中并在37℃、180rpm的水浴摇床中恒温震荡孵育120min，并分别在60min和120min时从消化液中取出1mL的模拟胃液，梯度稀释后利用上述的平板菌落计数法测定益生菌的存活率，将未经包埋的自由菌作为对照样品，采用同样的处理方法进行实验并测定相应的益生菌存活率。
结果发现处理60min存活率为48±3.66％；微胶囊化的细菌60min存活率为67.3±1.25％，120min存活率为45.4±2.16％，有显著的保护效果。
实施例2：益生菌微胶囊和喷雾干燥菌粉的制备
本实施例与实施例1的菌体制备、测试方法相同，区别在于干燥方式选择喷雾干燥，将壁材与菌液使用磁力搅拌器以1200rpm的转速搅拌30分钟。然后将分散液在冰箱中保存约18小时(4℃)，以使聚合物充分水化。
向混合物中加入约2％吐温80。然后使用均质机以11000rpm的速度乳化15min。用喷雾干燥机进行喷雾干燥，在喷雾干燥机上喷雾干燥乳状液的工艺参数如下：进料速度5ml/min，吸气器100％，雾化压力0.3bar，进风温度150℃，出风温度50℃，雾化器嘴直径1.4mm。
按实施例1测试方法测得本实施例对益生菌的包埋率为70.2±1.44％；未包埋的益生菌存活率为0，干燥存活率为19.7±3.27％。本实施例进一步对制备得到的益生菌微胶囊进行了体外模拟。
在模拟胃液下未包埋的益生菌处理60min全部死亡，存活率为0；微胶囊化的细菌60min存活率为65.9±0.66％，120min存活率为43.1±2.47％。
实施例3：益生菌微胶囊和流化床干燥菌粉的制备
本实施例与实施例1的菌体制备、测试方法相同，区别在于干燥方式选择流化床干燥，本实施例开启高效包衣机检查机器是否良好，设置进风温度为78-88℃，出风温度为38-42℃，倒入颗粒，将包衣锅转速调至2-3rpm，把喷枪位置调至离距离床约30-40cm处，开始预热物料。至物料温度32-38℃，喷雾空气压力在0.5-0.6MPa，开始包衣，包.衣20-30min后逐渐提高包衣锅转速和加大流量(以颗粒间不黏连为度)。在包衣过程中，持续搅拌包衣液，尽快喷完全部浆液，至要求增重量，此时保持热风风机的运转状态，干燥几分钟后再关闭热风风机，开启冷却风冷至室温，出料。
按实施例1测试方法测得本实施例方法对益生菌的包埋率为69.5±3.04％；未包埋的益生菌干燥存活率为0，干燥存活率为18.3±0.56％。本实施例进一步对制备得到的益生菌微胶囊进行了体外模拟。
在模拟胃液下未包埋的益生菌处理60min全部死亡，存活率为0；微胶囊化的细菌60min存活率为67.8±1.78％，120min存活率为45.6±1.53％。
Claims (10)
1.一种包埋益生菌的乳脂肪球膜复合微胶囊制备方法，其成分包含乳脂肪球膜、角瓜多糖、阴离子多糖以及冻干保护剂。
2.如权利要求1所述的一种乳脂肪球膜复合微胶囊制备方法，其特征在于按如下步骤进行：首先配置乳脂肪球膜、角瓜多糖、卡拉胶和水苏糖的混合溶液，灭菌后作为微胶囊壁材待用，将菌液与微胶囊壁材按比例混合，以未混合菌液为空白对照；在与微胶囊壁材混合后的菌液中按比例加入植物油，使用搅拌器搅拌10-15min；之后添加冰醋酸至pH为4.6，搅拌5min；按比例加入冰箱中4℃下冷藏的生理盐水，搅拌5min；而后将菌液冷藏静置20min后在10000×g下离心15min；撇去上清液，按比例添加生理盐水，得到微胶囊溶液。
3.如权利要求1所述的一种乳脂肪球膜复合微胶囊制备方法，其特征在于，所述微胶囊壁材的成分包含乳脂肪球膜2.5g/L～30g/L、角瓜多糖10g/L～50g/L，阴离子多糖5g/L～20g/L以及冻干保护剂20g/L～200g/L，所述的植物油为大豆油、菜籽油、椰子油、玉米油、亚麻油、芝麻油、葵花籽油、花生油、调和油一种或一种以上。
4.如权利要求1或2所述的一种乳脂肪球膜复合微胶囊制备方法，其特征在于，所述冻干保护剂为水苏糖、低聚果糖、低聚木糖、低聚半乳糖、低聚异麦芽糖、棉籽糖、异麦芽酮糖、乳酮糖、低聚异麦芽酮糖、低聚龙胆糖、大豆低聚糖、低聚壳聚糖中的一种或一种以上。
5.如权利要求1或2所述的一种乳脂肪球膜复合微胶囊制备方法，其特征在于，所述阴离子多糖为卡拉胶、羧甲基纤维素钠、海藻酸钠、黄原胶或阿拉伯胶中的一种或一种以上。
6.如权利要求1～7所述的一种乳脂肪球膜复合微胶囊制备方法，其特征在于菌液与微胶囊壁材体积比为1～3：2～9。
7.如权利要求1～8所述的一种乳脂肪球膜复合微胶囊制备方法，其干燥方式包括任何参数的冷冻干燥、喷雾干燥、流化床干燥。
8.权利要求1～9任一项所述的益生菌微胶囊方法在制备微胶囊抑菌剂保健食品或药品中的应用。
9.一种角瓜多糖的提取方法，其特征在于具体步骤按如下进行：在捣碎的1～3g角瓜中加入乙醇溶液，水浴回流提取，趁热过滤，用热乙醇冲洗2次，等到溶剂挥干后，将残渣和滤纸一同放入锥形瓶中，加蒸馏水100mL，水浴回流，取出冷却至室温后，精密量取10mL的回流液于离心管中，离心，之后再精密吸取离心后的上清液1mL，至10mL量瓶中加蒸馏水定容，振摇均匀，备用。
10.一种角瓜多糖的提取方法，其特征在于角瓜捣碎至固液比为1～2:1～2。
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