CN110432496A - 双壁多孔微胶囊益生球及其制备方法 - Google Patents
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Abstract
本发明涉及一种双壁多孔微胶囊益生球及其制备方法，采用双壁层层自组装表面修饰芯材。内壁壁材以纳米碳酸钙为基料联合超声振荡制备多孔水凝胶增容，使少部分接触的碳酸钙进行分散疏松，避免团聚，显著提高益生菌载荷；外壁采用蛋白‑多糖复合凝胶包膜，以静电力为驱动力电荷中和而产生凝聚，形成比蛋白质和多糖更具优良功能特性的复合物，从而改变蛋白质在界面上的吸附行为及界面吸附膜的流变特性，包裹芯材微囊化，改善微胶囊的稳定性。
Description
技术领域
本发明属于微胶囊技术领域,尤其是一种双壁多孔微胶囊益生球及其制备方法。
背景技术
双岐杆菌是人体肠道最重要的有益菌群，对维持人体肠道的微生态平衡和正常生理功能、促进人体健康有着重要的意义。但是，双岐杆菌是厌氧菌，又不耐受强酸环境，在经口服进人体肠胃时，绝大多数受到胃酸一、胆汁和消化酶等的作用而失活，难以对人体起到有效的治疗或保健作用。
微囊化益生菌包埋技术是现阶段比较实用，但是依旧存在很多难以攻克的难题。市面上大部分微胶囊益生菌载荷量小，菌群包埋环境不易控制而导致存活量低，以及壁材稳定性差等。
本发明针对胃肠功能弱及肠胃菌群紊乱的人群，制备双壁多孔微胶囊益生球，调整肠道菌群，改善胃肠道功能，强化营养，增强免疫调节作用。此球分两部分，内壁为多孔水凝胶制成的多孔小球用于结合双歧杆菌及营养强化物质，外壁采用改性大豆蛋白-卡拉胶复合凝胶进行涂膜包裹。芯材内包括双歧杆菌及其双岐因子乳酸链球菌、大豆低聚糖等，以及多种维生素，多种氨基酸，蛋白质，膳食纤维等营养强化物质。本发明制造工艺简单，适合大规模生产。
根据检索，与本申请相关的专利文献公开内容及评价如下：
1、双岐杆菌微胶囊的制备方法(CN104324056A)，2018年08月31日授权。该专利包括以下步骤：(1)将双岐杆菌依次经种子活化、种子培养和厌氧罐培养后，将菌液的离心分离，菌体冷冻干燥；(2)按配比称取明胶、低聚麦芽，干燥杀菌后无菌操作溶于热水中，配成浓度为20％的无菌溶液；(3)将菌体加入无菌溶液中分散均匀，并进行喷雾干燥，制成微胶囊产品即双岐杆菌微胶囊。该发明所述的双岐杆菌微胶囊的制备方法，出粉率达到87.35％，包埋率达到92.14％，每克双岐杆菌微胶囊含活菌大于109个以上。优点：较好的提高益生菌存活率和包封率。缺点：①其芯材营养环境差，双岐杆菌繁殖率低；②单层壁在胃部低酸性环境中易被破坏，益生菌大量失活；③益生菌载荷小。
2、一种外用益生菌微胶囊的制备方法(CN104856924A)，2018年05月04日授权，该发明公开了一种外用益生菌微胶囊的制备方法：(1)将双歧杆菌、嗜酸乳杆菌、鼠李糖乳杆菌的冻干菌粉，分别接种于三角瓶中活化处理，扩大发酵培养，将三种菌的发酵液按照体积比3：3：4进行混合，得到菌悬液；(2)制备葛仙米提取液；(3)将菌悬液，葛仙米提取液和海藻酸钠溶液加入吐温80乳化后，固化，待湿胶囊形成后，用纱布过滤，然后再用生理盐水冲洗3次。优点：充分利用植物提取和益生菌的协同作用，一方面协同性提高存活率和包封率，另外一方面，植物提取液还对皮肤护理有进一步的效果。缺点：①其芯材营养环境差，双岐杆菌繁殖率低；②单层壁在胃部低酸性环境中易被破坏，益生菌大量失活；③益生菌载荷小。
3、益生菌微胶囊制剂及其制备方法(CN105567669A)，2019年01月04日授权，该发明涉及一种益生菌微胶囊制剂及其制备方法，微胶囊制剂包括囊材和壁材，壁材由大豆油和Span 80组成，囊材中含有屎肠球菌和干酪乳杆菌，通过采用发酵前包被微胶囊技术，将低密度微生物细胞包裹在囊材中，继续发酵获得更高的菌体浓度和细胞活性，通过简单的分离，直接得到含有高密度屎肠球菌和干酪乳杆菌共存的双乳酸菌微胶囊制剂。优点：利用发酵前包被技术，将两种不同的乳酸菌细胞同时包被到同一微胶囊内，实现不同微生物细胞的共生；能够发挥两种乳酸菌的协同益生作用；采用发酵前包被技术，两种微生物细胞获得了再次增殖的机会，从而容易获得更高活性。缺点：①菌体培养液元素及种类单一，菌群抗逆性差，存活量较低；②胶囊内菌体载荷小，有效性降低；③环境pH发生变化时，壁凝胶与胃肠液间的解离平衡被破坏。对策：①芯材增添多种双岐因子及营养强化元素；②多孔水凝胶增容；③甲基丙烯酸酐改性大豆分离蛋白形成pH响应智能型凝胶。
本申请根据现有技术及相关专利的缺陷，进行针对性研发，获得效果更好的双壁多孔微胶囊益生球及其制备方法。
发明内容
本发明的目的在于克服现有技术的不足之处，提供一种双壁多孔微胶囊益生球及其制备方法，本发明采用芯材增添多种双岐因子及营养强化元素、甲基丙烯酸酐改性大豆分离蛋白形成pH响应智能型凝胶；采用多孔水凝胶增容。
本发明解决其技术问题是采取以下技术方案实现的：
一种双壁多孔微胶囊益生球及其制备方法，步骤如下：
⑴双岐菌培养液制备：制备菌体浓度为1-3×109CFU ml的双岐菌培养液。
⑵菌体复合液制备：
双岐菌培养液6份，乳酸链球菌1份，大豆低聚糖2份，魔芋胶水解物1份，肝浸汁1份，椰汁1份，维生素2份，氨基酸粉2份，蛋白质粉1.5份，膳食纤维粉1.5份，电磁搅拌10min，形成均匀的复合体，备用；
⑶内壁多孔水凝胶微球制备：
称取羟丙基甲基纤维素和纳米CaCO3粉末，600W超声振荡环境下，用NaOH溶液配成纤维素CaCO3混合液，避免发生团聚现象；
再用蒸馏水稀释交联剂聚乙二醇，在装有搅拌装置的四口瓶中加入分散剂和环己烷，电磁搅拌15min，加热至分散剂完全溶解，然后将温度降至室温；
再将纤维素CaCO3混合液加入到四口瓶中，电磁搅拌，二次超声提高分散效果，滴加交联剂水溶液，反应22小时，洗涤，得到纤维素水凝胶微球，向所得到的微球中滴加0.05mol/L稀盐酸，微球颗粒逐渐变为透明状，再用蒸馏水洗涤至中性，干燥处理，得到孔径5-30nm多孔水凝胶微球；
⑷将步骤⑵的复合体加入到步骤⑶的多孔水凝胶微球中，使凝胶对其进行包裹荷载，形成复合体多孔水凝胶，低温冷冻干燥备用；
⑸琥珀酰化大豆蛋白的制备：
称取100g蛋白溶解于去离子水中，在室温下磁力搅拌1h，用2mol/LNaOH溶液调节蛋白溶液pH至8.0，向溶液中缓慢添加12.5wt％的甲基丙烯酸酸酐，维持溶液的pH在6～10范围。待溶液pH稳定后，继续反应5～7min，使酰化反应完成；
用1mol/LHCl溶液将pH调节至7.0，终止反应，将反应液在4℃透析48h，除去残余的甲基丙烯酸酐，之后冷冻干燥得到甲基丙烯酸酰化蛋白。
⑹改性大豆分离蛋白蛋白-卡拉胶复合凝胶制备：
取步骤⑸制得的改性蛋白溶解于水，依次添加改性大豆分离蛋白，卡拉胶溶液，使改性大豆分离蛋白与卡拉胶的质量比为20:1，600W超声功率处理混合溶液，超声波处理促使改性大豆分离蛋白凝胶溶解性增加。用0.05mol/L调整pH值3.5，混合均匀后置4℃反应48h以保证复凝聚反应充分，制得的高溶解度改性大豆蛋白-卡拉胶凝胶。
而且，步骤⑴的双岐菌培养液制备的制备方法为：双歧杆菌菌液接种于乳酸细菌培养基，充氮后37℃厌氧培养12h，在4℃、6000r/min下离心10min。倾出上清液后，菌体沉淀以0.85％生理盐水清洗2次并重悬于生理盐水中，菌体浓度调整至2×109CFU ml。
而且，所述交联剂：分散剂：环己烷的质量为1:0.5-5:0.5-5。
本发明的优点和积极效果是：
1、本发明芯材增添多种双岐因子及营养强化元素，芯材内包括双歧杆菌及其双岐因子乳酸链球菌、大豆低聚糖等，以及多种维生素，多种氨基酸，蛋白质，膳食纤维等营养强化物质。稳固菌体活性，强化营养。
2、本发明采用双壁层层自组装(LBL)表面修饰芯材。内壁壁材以纳米碳酸钙为基料联合超声振荡制备多孔水凝胶增容，使少部分接触的碳酸钙进行分散疏松，避免团聚，显著提高益生菌载荷；外壁采用蛋白-多糖复合凝胶包膜，以静电力为驱动力电荷中和而产生凝聚，形成比蛋白质和多糖更具优良功能特性的复合物，从而改变蛋白质在界面上的吸附行为及界面吸附膜的流变特性，包裹芯材微囊化，改善微胶囊的稳定性。
3、本发明采用甲基丙烯酸酐改性大豆分离蛋白，形成pH响应智能型凝胶，当微胶囊进入胃部，环境pH发生变化时(低pH值)，外壁复合凝胶与溶剂间的解离平衡被破坏，凝胶内外的离子浓度发生变化，凝胶与溶剂的相互作用发生改变，使得凝胶网络的交联点数量减少，加强了凝胶与溶剂的相互作用力，使凝胶的网络结构发生变化，从而引起凝胶的溶胀，有效保护芯材不溶出，稳固菌体活性，保证菌体存活数量。
附图说明
图1为大豆分离蛋白-卡拉胶＝20:1时蛋白质-多糖复合凝胶包裹的双岐菌的图片。
具体实施方式
下面通过具体实施例对本发明作进一步详述，以下实施例只是描述性的，不是限定性的，不能以此限定本发明的保护范围。
一种双壁多孔微胶囊益生球及其制备方法，步骤如下：
⑴双岐菌培养液制备：
B.Longum菌液接种于乳酸细菌培养基(MRS)，充氮后37℃厌氧培养12h，在4℃、6000r/min下离心10min。倾出上清液后，菌体沉淀以0.85％生理盐水清洗2次并重悬于生理盐水中，菌体浓度调整至2×109CFU ml。
⑵菌体复合液制备：
双岐菌培养液6份，乳酸链球菌1份大豆低聚糖(主要包括水苏糖和棉子糖)2份，魔芋胶水解物1份，肝浸汁1份，椰汁1份，多种维生素共2份(维生素A-E混合后共两份)，氨基酸粉2份，蛋白质粉1.5份，膳食纤维粉1.5份，电磁搅拌10min，形成均匀的复合体。备用。
⑶内壁多孔水凝胶微球制备：
称取一定量的羟丙基甲基纤维素(HPMC)和一定配比的纳米碳酸钙粉末(CaCO3)，600W超声振荡环境下，用NaOH溶液配成纤维素碳酸钙混合液，避免发生团聚现象，改善分散性，调高均匀性，呈现实际尺寸的纳米级分散度。
再用蒸馏水稀释交联剂聚乙二醇(5-10wt％)。在装有搅拌装置的四口瓶中加入分散剂(卵磷脂1-10wt％)和环己烷，电磁搅拌15min，加热至分散剂完全溶解，然后将温度降至室温。
交联剂：分散剂：环己烷的质量为1:0.5-5:0.5-5，看具体溶液交联情况而定，多余的会被洗涤的时候洗出，所以对用量不需严格控制。
再将含有CaCO3粉末的HPMC混合液加入到四口瓶中，电磁搅拌，二次超声提高分散效果，滴加交联剂水溶液，反应22小时。洗涤，得到纤维素水凝胶微球。向所得到的微球中滴加0.05mol/L稀盐酸，微球颗粒逐渐变为透明状。再用蒸馏水洗涤至中性，干燥处理，得到孔径10nm(可调)多孔水凝胶微球。
⑷将步骤2的复合体加入到步骤(3)的多孔水凝胶微球中，使凝胶对其进行包裹荷载，形成复合体多孔水凝胶，低温冷冻干燥备用。
⑸琥珀酰化大豆蛋白的制备(改性蛋白)：
称取100g蛋白溶解于去离子水中，在室温下磁力搅拌1h，用2mol/LNaOH溶液调节蛋白溶液pH至8.0，向溶液中缓慢添加12.5wt％的甲基丙烯酸酸酐，维持溶液的pH在6～10范围。待溶液pH稳定后，继续反应(5～7min)，使酰化反应完成，即带负电荷的甲基丙烯酸基团取代带正电的氨基基团(Lys)，此基团易水解或易质子化，胃部低pH环境下溶胀。用1mol/LHCl溶液将pH调节至7.0，终止反应。将反应液在4℃透析48h，除去残余的甲基丙烯酸酐，之后冷冻干燥得到甲基丙烯酸酰化蛋白。
⑹改性大豆分离蛋白蛋白-卡拉胶复合凝胶制备：
取步骤⑸制得的改性蛋白溶解于水，依次添加改性大豆分离蛋白(溶解后溶液浓度1wt％)，卡拉胶溶液(卡拉胶溶液浓度0.5wt％)，使改性大豆分离蛋白与卡拉胶的质量比为20:1，600W超声功率处理混合溶液，超声波处理促使改性大豆分离蛋白凝胶溶解性增加。用0.05mol/L调整pH值3.5，混合均匀后置4℃反应48h以保证复凝聚反应充分，制得的高溶解度改性大豆蛋白-卡拉胶凝胶。
大豆蛋白较高的平均分子量和卡拉胶中线性分子的长链支化度导致较大流体动力学体积，因此表现出更高的表观粘度，分散在其中的微小球沉降或上浮速率也就越小，这种传质障在一定程度上提高了混合物的稳定性，同时卡拉胶分子的三维结构(双链的螺旋分子结构)和凝胶特性会有助于提高它对双歧杆菌的保护能力。此外低渗透隔绝氧气营造厌氧环境有助于稳固双岐菌活性。
⑺把步骤⑷干燥后的复合体多孔水凝胶颗粒浸没在步骤⑹制得的改性大豆蛋白-卡拉胶复合凝胶中，电磁搅拌10min，干燥，形成不规则的包裹颗粒，大概0.5-2mm左右，即制得双层壁多孔微胶囊小球。
表1体外模拟胃液环境试验结果
体外模拟胃液环境试验，观察双歧杆菌活菌数，结果如图所示，双壁微小球包埋率达95％，经过在模拟胃液环境中放置2h后，仍然能够保持较好的活菌数，活菌数达到(1.6-2.4)×108CFU/g，显著高于单壁及无壁，此外，能够保持优异的耐酸性。说明本发明所提供的益生菌微胶囊具有优异的储存稳定性。
本发明属于益生菌制品技术领域，可作粉末状食品添加剂应用于奶制品，强化营养及附加保健功能。
Claims (3)
1.一种双壁多孔微胶囊益生球的制备方法，其特征在于：步骤如下：
⑴双岐菌培养液制备：制备菌体浓度为1-3×109CFU ml的双岐菌培养液；
⑵菌体复合液制备：
双岐菌培养液6份，乳酸链球菌1份，大豆低聚糖2份，魔芋胶水解物1份，肝浸汁1份，椰汁1份，维生素2份，氨基酸粉2份，蛋白质粉1.5份，膳食纤维粉1.5份，电磁搅拌10min，形成均匀的复合体，备用；
⑶内壁多孔水凝胶微球制备：
称取羟丙基甲基纤维素和纳米CaCO3粉末，600W超声振荡环境下，用NaOH溶液配成纤维素CaCO3混合液，避免发生团聚现象；
再用蒸馏水稀释交联剂聚乙二醇，在装有搅拌装置的四口瓶中加入分散剂和环己烷，电磁搅拌15min，加热至分散剂完全溶解，然后将温度降至室温；
再将纤维素CaCO3混合液加入到四口瓶中，电磁搅拌，二次超声提高分散效果，滴加交联剂水溶液，反应22小时，洗涤，得到纤维素水凝胶微球，向所得到的微球中滴加0.05mol/L稀盐酸，微球颗粒逐渐变为透明状，再用蒸馏水洗涤至中性，干燥处理，得到孔径5-30nm多孔水凝胶微球；
⑷将步骤⑵的复合体加入到步骤⑶的多孔水凝胶微球中，使凝胶对其进行包裹荷载，形成复合体多孔水凝胶，低温冷冻干燥备用；
⑸琥珀酰化大豆蛋白的制备：
称取100g蛋白溶解于去离子水中，在室温下磁力搅拌1h，用2mol/LNaOH溶液调节蛋白溶液pH至8.0，向溶液中缓慢添加12.5wt％的甲基丙烯酸酸酐，维持溶液的pH在6～10范围，待溶液pH稳定后，继续反应5～7min，使酰化反应完成；
用1mol/LHCl溶液将pH调节至7.0，终止反应，将反应液在4℃透析48h，除去残余的甲基丙烯酸酐，之后冷冻干燥得到甲基丙烯酸酰化蛋白；
⑹改性大豆分离蛋白蛋白-卡拉胶复合凝胶制备：
取步骤⑸制得的改性蛋白溶解于水，依次添加改性大豆分离蛋白，卡拉胶溶液，使改性大豆分离蛋白与卡拉胶的质量比为20:1，600 W超声功率处理混合溶液，超声波处理促使改性大豆分离蛋白凝胶溶解性增加，用0.05mol/L调整pH值3.5，混合均匀后置4℃反应48h以保证复凝聚反应充分，制得的高溶解度改性大豆蛋白-卡拉胶凝胶。
2.根据权利要求1所述的双壁多孔微胶囊益生球及其制备方法，其特征在于：步骤⑴的双岐菌培养液制备的制备方法为：双歧杆菌菌液接种于乳酸细菌培养基，充氮后37℃厌氧培养12h，在4℃、6000r/min下离心10min，倾出上清液后，菌体沉淀以0.85％生理盐水清洗2次并重悬于生理盐水中，菌体浓度调整至2×109CFU ml。
3.根据权利要求1所述的双壁多孔微胶囊益生球及其制备方法，其特征在于：所述交联剂：分散剂：环己烷的质量为1:0.5-5:0.5-5。
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