CN110801021A - 一种利用改性果胶包埋肠道复合益生菌的方法 - Google Patents
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Abstract
本发明公开了一种利用改性果胶包埋肠道复合益生菌的方法，包括如下步骤：1)果胶改性将果胶分散液与NaCl溶液混合均匀，调整pH值为7‑7.5，加入果胶甲酯酶反应，随后将PH降到4‑4.5，并乙醇沉淀，得改性果胶，最后风干干燥；2)益生菌包埋：将上述改性果胶与复合益生菌在纯水进行混合，静置5～10min后，加入蛋白胨水再静置5～10min后，随后通过挤压喷雾至氯化钙溶液中实现离子凝胶包封；3)回收冻干低转速离心获得复合益生菌微胶囊，后经冷冻干燥获得复合益生菌粉制剂；该方法获得的微胶囊益生菌在模拟胃酸环境下更大的存活率，肠道中释放率更低，存活量更大。
Description
技术领域
本发明涉及益生菌产品技术领域，具体涉及一种利用改性果胶包埋肠道复合益生菌的方法。
背景技术
益生菌(Probiotics)是一类对宿主有益的活性微生物，是定植于肠道内，能产生确切健康功效从而改善宿主微生态平衡、发挥有益作用的活性有益微生物的总称。人体、动物体内有益的细菌或真菌主要有：酪酸梭菌、乳酸菌、双歧杆菌、嗜酸乳杆菌、放线菌、酵母菌等。
为了发挥其益生菌的功能，高浓度的益生菌必须能够到达肠道的远端，在那里它们可以与共生微生物相互作用。益生菌在加工和生产食品时暴露于环境条件中，这些细胞减少细胞数量和活力，包括暴露于氧、热、湿度、光和剪切，破坏细胞壁、细胞膜或导致与氧化有关的损伤。此外，口服益生菌的递送可能会受到胃肠道恶劣环境条件(例如胃酸、消化酶和小肠胆盐)的限制；因此，可能需要开发提高益生菌细胞活力的方法，直到它们到达下胃肠道。
在这方面，微胶囊技术是提高益生菌生存能力的潜在解决方案。包埋技术包括物理方法和生物聚合物包埋技术，这些技术对于生物活性物质的靶向传递非常有效。
发明内容
发明目的：为了解决以上现有技术的不足，提供一种利用改性果胶包埋肠道复合益生菌的方法，包括如下步骤：
1)果胶改性
将果胶分散液与NaCl溶液混合均匀，用NaOH调整pH值为7-7.5，加入果胶甲酯酶反应10～30min，随后利用盐酸将PH降到4-4.5，并在95％乙醇中煮沸10～20min，停止反应，冷却至室温后，用乙醇和丙酮洗涤沉淀的改性果胶，最后风干干燥；
2)益生菌包埋
上述改性果胶与复合益生菌在纯水进行混合，静置5～10min后，加入蛋白胨水再静置5～10min后，随后通过挤压喷雾至氯化钙溶液中实现离子凝胶包封；
3)回收冻干
低转速离心获得复合益生菌微胶囊，后经冷冻干燥获得复合益生菌粉制剂。
优选地，果胶分散液的浓度为30-35mg/mL；NaCl溶液的浓度为1-1.2M，果胶分散液与NaCl溶液的体积比10:1-15:1。
优选地，果胶甲酯酶酶活为4.5-5PEU/mL，加入果胶甲酯酶体积为果胶溶液体积的3/100-7/100。
优选地，改性果胶与复合益生菌质量比为2:1-5:1。
优选地，加入的蛋白胨水的浓度为0.1wt％，加入蛋白胨水的体积为改性果胶-益生菌复合溶液体积的1/5-1/10。
优选地，挤压喷雾喷口直径为0.4-0.5mm。
优选地，氯化钙溶液浓度为300-400mM
有益效果：
本发明基于一种基于碳水化合物的生物高聚物，果胶，是一种可溶纤维，在结肠细菌发酵前通过胃肠系统存活下来，常用作药物的生物载体。离子性凝胶化和生物活性物质的包封归因于果胶的带电高半乳糖醛酸结构域。与真菌果胶甲基酯酶或化学皂化相比，植物果胶甲基酯酶对果胶的去酯化作用在相同的总电荷和较高的凝胶强度下产生高度反应性的电荷分布块，并且稳定性更强、包埋效果更好，内包物质释放率较低，去酯化果胶可与钙离子反应形成“鸡蛋盒”复合物进而控制内包物质的释放，而普通果胶的这种特性很差。因此，利用去酯化果胶块包裹的益生菌在储存期间表现出更大的存活率，在模拟胃肠道转运下表现出更大的稳定性。
附图说明
图1是益生菌与包埋形成微胶囊复合益生菌在模拟胃液中的存活率比较图；
图2是益生菌与包埋形成微胶囊复合益生菌在模拟肠液中的释放试验图；
图3为本发明流程图。
具体实施方式
为了加深对本发明的理解，下面将结合实施例和附图对本发明作进一步详述，该实施例仅用于解释本发明，并不构成对本发明保护范围的限定。
一种利用改性果胶包埋肠道复合益生菌的方法，如图2所示，包括如下步骤：
1)果胶改性
将果胶分散液(30mg/mL，w/v)与0.1M NaCl按体积比10:1混合均匀，形成果胶溶液，用NaOH调整pH值为7.5，再加入果胶溶液4/100体积的果胶甲酯酶(4.8PEU/mL)反应10～30min，随后利用盐酸将pH降到4.3，并在95％乙醇中煮沸10～20min，停止反应，冷却至室温后，用乙醇和丙酮洗涤沉淀的改性果胶，最后风干干燥；
2)益生菌包埋
上述改性果胶与复合益生菌按质量比4:1在纯水中溶解形成改性果胶-益生菌复合溶液，静置5～10min后，加入改性果胶-益生菌复合溶液1/5体积的蛋白胨水(0.1wt％)再静置5～10min后，随后通过挤压喷雾(0.40mm内喷嘴)至300mM氯化钙溶液中实现离子凝胶包封；
3)回收冻干
低转速离心获得复合益生菌微胶囊，后经冷冻干燥获得复合益生菌粉制剂。
所述的复合益生菌由以下质量份的组份组成：鼠李糖乳杆菌40-60份、植物乳杆菌15-30份、干酪乳杆菌15-30份、乳双歧杆菌5-15份、低聚木糖0.5-5份、抗性糊精1-10份、聚葡萄糖5-15份。
在模拟胃液中的存活率是确保菌体通过胃部定植肠道中发挥益生功能的保障，其在模拟胃液中的存活率如图1所示；由图1可知，自由菌在模拟胃液中的存活率非常低，60min后的存活率不足一半，经过120min后，检测不到活菌；改性果胶包埋复合菌微胶囊组在模拟胃液处理120min后益生菌存活率仍有一半左右；果胶包埋复合菌微胶囊组模拟胃液处理120min后，检测不到活菌；结果表明，由于改性果胶微胶囊相对于普通果胶微胶囊结构致密，孔径更少，机械强度高，能较好地抵御胃液渗透，保持微胶囊的完整性，从而显著提高益生菌的存活率。
在肠道中的有效释放是发挥益生作用的首要条件，其在模拟肠液中的释放试验如图2所示，从图2可知，改性果胶包埋复合菌微胶囊组经模拟肠液处理60min后，活菌数达到(9.8±0.24)Logcfu/g，而果胶包埋复合菌微胶囊组活菌数更低，表明改性果胶包埋复合菌所制备的微胶囊具有较好的肠溶性，这主要由于改性果胶所制备的微胶囊在肠液中的溶胀性明显要大于在胃液中的溶胀性，在胃液中，改性果胶所制备的微胶囊呈收缩趋势，能有效保护益生菌免受酸性损伤，而在肠液中，由于磷酸根离子的作用，改性果胶所制备的微胶囊明显膨胀，最终导致破裂释放。
益生菌要对人体发挥其益生作用，除能抵抗胃酸的环境外，还需在到达肠道时保持一定存活量。由表1可看出，自由菌在模拟肠液中处理60min后，几乎未检测到活菌，而改性果胶包埋形成微胶囊复合益生菌活菌数仍达到足量的益生菌，明显比果胶包埋形成微胶囊复合益生菌活菌数多，说明改性果胶微胶囊既能抵抗胃酸的环境，还对胆盐有一定的耐受性。
表1
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (9)
1.一种利用改性果胶包埋肠道复合益生菌的方法，其特征在于，包括如下步骤：
1)果胶改性
将果胶分散液与NaCl溶液混合均匀形成果胶溶液，用NaOH调整pH值为7-7.5，加入果胶甲酯酶反应10～30min，随后利用盐酸将pH降到4-4.5，并在95％乙醇中煮沸10～20min，停止反应，冷却至室温后，用乙醇和丙酮洗涤沉淀的改性果胶，最后风干干燥；
2)益生菌包埋
上述改性果胶与复合益生菌在纯水混合形成改性果胶-益生菌复合溶液，静置5～10min后，加入蛋白胨水再静置5～10min后，随后通过挤压喷雾至氯化钙溶液中实现离子凝胶包封；
3)回收冻干
低转速离心获得复合益生菌微胶囊，后经冷冻干燥获得复合益生菌粉制剂。
2.根据权利要求1所述的一种利用改性果胶包埋肠道复合益生菌的方法，其特征在于，果胶分散液的浓度为30-35mg/mL。
3.根据权利要求1所述的一种利用改性果胶包埋肠道复合益生菌的方法，其特征在于，NaCl溶液的浓度为1-1.2M。
4.根据权利要求1所述的一种利用改性果胶包埋肠道复合益生菌的方法，其特征在于，果胶分散液与NaCl溶液的体积比10:1-15:1。
5.根据权利要求1所述的一种利用改性果胶包埋肠道复合益生菌的方法，其特征在于，果胶甲酯酶酶活为4.5-5PEU/mL，加入果胶甲酯酶体积为果胶溶液体积的3/100-7/100。
6.根据权利要求1所述的一种利用改性果胶包埋肠道复合益生菌的方法，其特征在于，改性果胶与复合益生菌质量比为2:1-5:1。
7.根据权利要求1所述的一种利用改性果胶包埋肠道复合益生菌的方法，其特征在于，加入的蛋白胨水的浓度为0.1wt％，加入蛋白胨水的体积为改性果胶-益生菌复合溶液体积的1/5-1/10。
8.根据权利要求1所述的一种利用改性果胶包埋肠道复合益生菌的方法，其特征在于，挤压喷雾喷口直径为0.4-0.5mm。
9.根据权利要求1所述的一种利用改性果胶包埋肠道复合益生菌的方法，其特征在于，氯化钙溶液浓度为300-400mM。
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