CN111264870A - 一种高环境抗性的益生菌微胶囊及其制备方法 - Google Patents
一种高环境抗性的益生菌微胶囊及其制备方法 Download PDFInfo
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Abstract
本发明提供一种高环境抗逆性且能够改善结肠炎大鼠肠道菌群的益生菌微胶囊，其中壁材是由乳清分离蛋白、结冷胶和邻苯二甲酸乙酸纤维素组成的，芯材为益生菌；其中，乳清分离蛋白、结冷胶和邻苯二甲酸乙酸纤维素在壁材中的质量百分比分别为10％、0.3％和2％；益生菌为干酪乳杆菌BNCC 134415或枯草芽孢杆菌BNCC 189983。本发明得到的益生菌微胶囊环境抗逆性强，在经过120min的模拟胃肠液处理后仍然具有较高的存活率。经过19周4℃储存和68℃热处理25min后与未包被的益生菌相比，存活率明显提高。且两种益生菌微胶囊联合对结肠炎大鼠的肠道菌群结构有一定影响，具有显著修复效果。
Description
技术领域
本发明属于食品加工技术领域，具体涉及一种益生菌微胶囊及其制备方法。
背景技术
益生菌是人体肠道内重要的有益菌，其具有多种生理功能，包括调节人体肠道功能、减少肠道疾病、促进营养物质吸收、缓解乳糖不耐症、降低胆固醇、调节免疫系统、预防癌症等。
益生菌想要发挥益生功能，其到达肠道并发挥作用的活菌数必须达到一定数量。世界卫生组织中要求益生菌产品中活菌数不少于106CFU/ml。在益生菌进入人体后，受低pH环境以及胆汁、胰液内的一些蛋白酶的影响，从而无法达到益生菌起作用的阈值。同时在食品的生产、加工、储存和销售过程中的一些不利环境也会降低益生菌活性。因此，寻找一种增强益生菌环境抗逆性的方法至关重要。
微胶囊技术在生产方面成本低，适用性高，生物相容性高；所以作为一种能有效保护益生菌抵抗不良环境的技术手段已被广泛应用和研究。微胶囊的原理是在益生菌周围形成一个致密的物理屏障，有利于抵御或者延缓空气、胃酸、胆盐等有害物质的渗透，从而降低不利环境对细胞的损害作用。
目前，利用微胶囊保护益生菌的现有技术中，对益生菌在通过胃肠道时的保护作用不是很明显，许多技术不够成熟有待于创新，且在益生菌微胶囊的制作过程中程序较为复杂，导致成本较高，这就为益生菌微胶囊的大规模生产，储存和应用提出了更高的要求，极大地限制了益生菌微胶囊产品的工业生产、储存、运输和销售环节的实际应用。
发明内容
本发明的目的在于提供一种高环境抗逆性的益生菌微胶囊及其制备方法；从而弥补现有技术的不足。
本发明首先提供一种益生菌微胶囊，其中壁材由乳清分离蛋白、结冷胶和邻苯二甲酸乙酸纤维素制成，芯材为益生菌；
所述的乳清分离蛋白、结冷胶和邻苯二甲酸乙酸纤维素，在壁材中质量百分比含量分别为8-12％和0.1-0.5％和1-3％，余量为溶液；
优选的，乳清分离蛋白、结冷胶和邻苯二甲酸乙酸纤维素，在壁材中质量百分比含量分别为10％和0.3％和2％；
所述的溶液，优选为PBS溶液；
所述的益生菌为干酪乳杆菌BNCC 134415和/或枯草芽孢杆菌BNCC 189983。
所述的益生菌微胶囊的制备方法，包括以下步骤：
1)制备壁材溶液
A液制备：将结冷胶GG溶于PBS溶液中，煮沸搅拌使其溶解为透明溶液，80℃保温备用；然后将邻苯二甲酸乙酸纤维素CAP边加热搅拌边调节pH至7.0，继续搅拌至完全溶解为透明溶液；
B液制备：再乳清分离蛋白WPI边搅拌边加热至90℃使其中的酶变性，继续搅拌至完全溶解；
将A液与B液混合，边搅拌混匀边降温，制成壁材溶液备用；
2)将干酪乳杆菌和枯草芽孢杆菌的菌悬液与步骤1)制备的壁材溶液混匀，-20℃冷冻过夜，再进行低温冷冻干燥制成益生菌微胶囊；
所述低温冷冻干燥的条件如下：温度为-57℃、9.7Pa·s；
其中干酪乳杆菌和枯草芽孢杆菌菌悬液的制备方法如下：将干酪乳杆菌以2％的接种量接种于MRS培养基中，37℃厌氧静置培养48h后将菌液4℃、6000rmp条件下离心15min，得到离心菌体；用无菌生理盐水清洗离心菌体两次。确定菌悬液的菌含量为5×108CFU/mL；
将枯草芽孢杆菌以2％的接种量接种于液体培养基中，37℃180rmp摇床培养48h后将菌液4℃、6000rmp条件下离心15min，得到离心菌体。用无菌生理盐水清洗离心菌体两次。确定菌悬液的菌含量为5×108CFU/mL。
本发明的有益效果如下：
1)本发明得到的益生菌微胶囊环境抗逆性强，在经过120min的模拟胃肠液处理后仍然具有较高的存活率。经过19周4℃储存和68℃热处理25min后与未包被的益生菌相比，存活率明显提高。且本发明得到的益生菌能够改善结肠炎大鼠的肠道菌群结构，提高肠道中乳酸菌等有益菌的丰度。
2)本发明以乳清分离蛋白、结冷胶和邻苯二甲酸乙酸纤维素的组合混合物为壁材冷冻干燥制备益生菌微胶囊，冷冻干燥后直接得到粉末状产品，与现有的技术相比明显缩短了制备流程且操作过程简单。冷冻干燥后的益生菌活菌数高，环境抗逆性强，可以广泛的应用于保健食品领域。
附图说明
图1：益生菌微胶囊的扫描电镜图，其中A为冻干的游离干酪乳杆菌细胞，B为乳清分离蛋白WPI微胶囊，C为WPI+CAP微胶囊，D为WPI+GG微胶囊，E为WPI+GG+CAP微胶囊。
图2：储存稳定性试验结果图。
图3：热处理试验结果图。
图4：模拟胃液试验结果图。
图5：模拟肠液试验结果图。
图6:物种分布柱状图(门水平)。
图7：物种分布柱状图(科水平)。
具体实施方式
本发明提供了一种益生菌微胶囊，相比益生菌初始数量值1×108CFU/mL，本发明制备的益生菌微胶囊在4℃储存19周后，活菌数仅下降了0.45～0.6个数量级，且在65℃条件下处理25min后与未包被的益生菌相比后仍有7.37～7.45log CFU/g的活菌数，且WPI+GG微胶囊的耐热效果更好好。
在模拟体外胃液120min后，活菌数比初始活菌数仅下降了0.6～0.7个数量级，相同条件下，未包被的益生菌在模拟pH 2.0的胃液120min后，未检测到任何活菌。
在模拟肠液120min后，包被的益生菌出现了明显的增殖现象，且WPI和WPI+GG微胶囊的保护效果较为明显。在WPI+GG益生菌微胶囊对结肠炎大鼠肠道菌群结构的影响试验发现，结肠炎大鼠服用益生菌微胶后，肠道中乳酸菌属试验组比对照组的数量显著增高。
下面结合实施例和附图对本发明做详细的说明。
实施例1：干酪乳杆菌微胶囊的制备
一种干酪乳杆菌微胶囊的制备工艺，菌种购买于北纳创联生物技术有限公司。
该实施例的实施步骤如下：
(1)菌株的活化：
将购买的菌种在斜面上传代活化1～2代，将活化后的斜面菌种在MRS平板培养基上做划线分离，培养并挑选最佳的单菌落移接斜面保藏与4℃冰箱保藏。将保藏的干酪乳杆菌以2％的接种量接种于MRS液体培养基中，37℃厌氧静置培养48h后将菌液4℃、6000rmp条件下离心15min，得到离心菌体。用无菌生理盐水清洗离心菌体两次。确定菌悬液的菌含量为5×108CFU/mL。
(2)干酪乳杆菌微胶囊壁材的制备
10％乳清分离蛋白(WPI)：准确称取10g乳清分离蛋白溶于100ml PBS中，加热到90℃搅拌使其中的酶变性，继续搅拌至乳清分离蛋白完全溶解，备用。
10％乳清分离蛋白+0.3％结冷胶(WPI+GG)：首先将0.3g结冷胶与100mlPBS中煮沸搅匀，使其完全溶解。再准确称取10g乳清分离蛋白加热到90℃搅拌30min，使乳清分离蛋白中的酶变性，继续搅拌至完全溶解，冷却备用。
8％乳清分离蛋白+0.3％结冷胶+2％邻苯二甲酸乙酸纤维素(WPI+GG+CAP)：A液:将0.3g结冷胶溶于20ml PBS中，煮沸搅拌使其溶解为透明溶液，80℃保温备用；B液：将2g的CAP边加热搅拌边调节pH至7.0，继续搅拌至完全溶解为透明溶液，再加入8％的乳清分离蛋白边搅拌边加热至90℃使其中的酶变性，继续搅拌至完全溶解。将A液趁热与B液混合，边搅拌混匀边降温备用。
8％乳清分离蛋白+2％CAP(WPI+CAP)：将2g的CAP加入到100ml PBS中，边加热搅拌边调节pH至7.0继续搅拌至完全溶解为透明溶液，再加入8g的乳清分离蛋白边搅拌边加热至90℃使其中的酶变性，继续搅拌至乳浊液完全溶解，备用.
(3)干酪乳杆菌微胶囊的制备
将步骤(1)制备的菌悬液与步骤(2)制备的壁材以1：4的体积比混匀后倒入提前灭菌的平板中，-20℃冷冻过夜，进行-57℃、9.7Pa·s冷冻干燥38～68h。收集冷冻干燥后的益生菌菌粉即为益生菌微胶囊。
实施例2：枯草芽孢杆菌微胶囊的制备
一种枯草芽孢杆菌微胶囊的制备工艺，菌种购买与北纳创联生物技术有限公司。
该实施例的实施步骤如下：
(1)菌株的活化：
将购买的菌种在斜面上传代活化1～2代，将活化后的斜面菌种在固体平板培养基上做划线分离，培养并挑选最佳的单菌落移接斜面保藏与4℃冰箱保藏。将保藏的枯草芽孢杆菌以2％的接种量接种于液体培养基中，37℃，180rmp摇床培养48h后将菌液4℃、6000rmp条件下离心15min，得到离心菌体。用无菌生理盐水清洗离心菌体两次。确定菌悬液的菌含量为1×108CFU/mL。
上述液体培养基包括：蛋白胨5g，牛肉浸取物3.0g，氯化钠5.0g，蒸馏水1升，pH7.0。
上述固体培养基包括：蛋白胨5g，牛肉浸取物3.0g，氯化钠5.0g，琼脂15g，蒸馏水1升，pH7.0。
(2)枯草芽孢杆菌微胶囊壁材的制备
10％乳清分离蛋白(WPI)：准确称取10g乳清分离蛋白溶于100ml PBS中，加热到90℃搅拌使其中的酶变性，继续搅拌至乳清分离蛋白完全溶解，备用。
10％乳清分离蛋白+0.3％结冷胶(WPI+GG)：首先将0.3g结冷胶与100mlPBS中煮沸搅匀，使其完全溶解。再准确称取10g乳清分离蛋白加热到90℃搅拌30min，使乳清分离蛋白中的酶变性，继续搅拌至完全溶解，冷却备用。
8％乳清分离蛋白+0.3％结冷胶+2％邻苯二甲酸乙酸纤维素(WPI+GG+CAP)：A液:将0.3g结冷胶溶于20ml PBS中，煮沸搅拌使其溶解为透明溶液，80℃保温备用；B液：将2g的CAP边加热搅拌边调节pH至7.0，继续搅拌至完全溶解为透明溶液，再加入8％的乳清分离蛋白边搅拌边加热至90℃使其中的酶变性，继续搅拌至完全溶解。将A液趁热与B液混合，边搅拌混匀边降温备用。
8％乳清分离蛋白+2％CAP(WPI+CAP)：将2g的CAP加入到100ml PBS中，边加热搅拌边调节pH至7.0继续搅拌至完全溶解为透明溶液，再加入8g的乳清分离蛋白边搅拌边加热至90℃使其中的酶变性，继续搅拌至乳浊液完全溶解，备用。
(3)枯草芽孢杆菌微胶囊的制备
将步骤(1)制备的菌悬液与步骤(2)制备的壁材以1:4的体积比混匀后倒入提前灭菌的平板中，-20℃冷冻过夜，进行-57℃、9.7Pa·s冷冻干燥38-68h。收集冷冻干燥后的益生菌菌粉即为益生菌微胶囊。
实施例3：微胶囊抗逆效果
对本发明制备的益生菌微胶囊进行了在耐热性，储存稳定性和模拟胃液和肠液活菌数测定。以实施案例1制备的干酪乳杆菌微胶囊为例进行详细说明。
以未包埋的干酪乳杆菌冻干菌粉作为对照组，测试上述微胶囊益生菌存活率：
(1)4℃储存：将上述制备的干酪乳杆菌微胶囊和对照组未包埋的冻干菌粉置于4℃条件下储藏，分别测定0，1，4，8，12和19周微胶囊和未包埋的冻干菌粉中的活菌数，以评价其保藏特性。经过19周的储存后，干酪乳杆菌微胶囊的活菌数仅下降了0.48～0.64个数量级，且WPI和WPI+GG微胶囊的活菌数最高。而对照组的冻干菌粉在储存8周时活菌数成直线下降趋势，到19时下降了1.62个数量级。
(2)耐热性试验测定：将上述制备的干酪乳杆菌微胶囊和对照组未包埋的冻干菌粉从室温条件下升温至68℃，并保持在该温度下25min后降至室温，平板计数测定活菌数。试验平行三次取平均值。结果表明，干酪乳杆菌微胶囊在68℃下热处理25min后仍有7.37～7.45log CFU/g的活菌数，且WPI和WPI+GG微胶囊的活菌数最高，而对照组已检测不到活菌数。
(3)模拟胃肠液试验：
A模拟胃液配置：制备含有氯化钠(2g)，盐酸(2ml)和去离子水1L的储备胃液，再加入胃蛋白酶(0.064g)到20ml的储备胃液中，调pH至2.0，最后用0.22μm滤膜过滤除菌。
B模拟肠液制备：称取10g胰酶，8.5g氯化钠，3g胆汁盐和10g胰蛋白酶至1L去离子水中，用1.0M的HCL或NaOH将pH调节至6.5，最后用0.22μm滤膜过滤除菌。
分别称取0.1g实施例1制备的干酪乳杆菌微胶囊和对照组干酪乳杆菌冻干菌粉，将其分别置于模拟胃液和肠液中，37℃、50rmp在摇床上温育0、60和120min后取样进行活菌计数，三次平行取平均值。
结果表明，益生菌微胶囊在经过120min的模拟胃液活菌数仅下降0.64～0.76个数量级，且WPI和WPI+GG微胶囊的保护效果较好，而对照组已检测不到活菌数。同时在模拟肠液后干酪乳杆菌微胶囊有明显的生殖现象，且WPI和WPI+GG微胶囊的增值效果较为明显，表明干酪乳杆菌在肠液也能够较好的释放且能够在肠液中繁殖。
对本发明制备的实施案例2制备的枯草芽孢杆菌微胶囊进行了耐热性，储存稳定性和模拟胃液和肠液活菌数测定试验。结果与上述实施案例1制备的干酪乳杆菌的结果一致。表明本发明所述的微胶囊的制备方法能够有效地提高益生菌的环境抗逆性。
实施例4:干酪乳杆菌微胶囊和枯草芽孢杆菌微胶囊联合对结肠炎大鼠肠道菌群的影响
利用制备的益生菌微胶囊对大鼠结肠炎进行修复试验，观察肠道菌群结果变化。所述的益生菌微胶囊为实施案例1和实施案例2制备的WPI+GG干酪乳杆菌微胶囊和WPI+GG枯草芽孢杆菌微胶囊。所述的大鼠结肠炎模型为葡聚糖硫酸钠(DSS)诱导的大鼠产生的结肠炎。
该案例具体实施如下：
将54只7周龄的SD大鼠(雄性)随机分为3个处理组，每个处理组3个重复，每个重复6只老鼠。NC组为正常组，1～14天灌胃PBS；CC组为炎症组，1～7天饮水中加入5％的DSS诱导溃疡性结肠炎，1～14天灌胃PBS；WC组为壁材对照组，1～7天饮水中加入5％的DSS诱导溃疡性结肠炎，1～14天灌胃WPI+GG；BSLC为实验组，1～7天饮水中加入5％的DSS诱导溃疡性结肠炎，1-14天灌胃枯草芽孢杆菌和干酪乳杆菌为胶囊的混合物，其中枯草芽孢杆菌和干酪乳杆菌为胶囊的活菌数为5×108CFU/ml。
每组大鼠灌胃14天后脱颈处死取盲肠，采用16SrRNA测序技术，基于IlluminaHiSeq测序平台，利用双末端测序(Paired-End)的方法对盲肠微生物菌群结构和菌群数量进行测定。得出如下试验结果：
在门水平上，厚壁菌门占主导地位，其次是拟杆菌门。与对照组相比，试验组的厚壁菌门和拟杆菌门的丰度更高，而益生菌组的有益微生物(放线菌)高于其他组。在科水平上，添加枯草杆菌和干酪乳杆菌微胶囊给药降低了脱硫弧菌科和肠杆菌科的丰度，而增加了乳酸菌科的丰度。表明，添加本发明制备的益生菌微胶囊对结肠炎大鼠肠道菌群结构具有一定影响，具有明显修复作用。
另外，肠道修复试验结果还表明，利用乳清分离蛋白和结冷胶(WPI+GG)作为壁材制备的益生菌微胶囊优于单一利用乳清分离蛋白(WPI)或结冷胶GG)包被的效果。且乳清分离蛋白+结冷胶(WPI+GG)制备的益生菌微胶囊与乳清分离蛋白+结冷胶+邻苯二甲酸乙酸纤维素(WPI+GG+CAP)制备的益生菌微胶囊效果差异不显著。因此，优选WPI+GG作为干酪乳杆菌微和枯草芽孢杆菌微胶囊壁材。
说明利用乳清分离蛋白和结冷胶(WPI+GG)双壁材制备的益生菌微胶囊或利用乳清分离蛋白+结冷胶+邻苯二甲酸乙酸纤维素(WPI+GG+CAP)三壁材制备的益生菌微胶囊，能够较好的保护干酪乳杆菌微和枯草芽孢杆菌顺利通过消化道进入肠道后段，以较高活力对肠道菌群结构产生影响。
Claims (7)
1.一种益生菌微胶囊，其特征在于，所述的益生菌微胶囊，其中壁材由乳清分离蛋白、结冷胶和邻苯二甲酸乙酸纤维素制成，芯材为益生菌。
2.如权利要求1所述的益生菌微胶囊，其特征在于，所述的乳清分离蛋白、结冷胶和邻苯二甲酸乙酸纤维素在壁材中质量百分比含量分别为8-12％和0.1-0.5％和1-3％，余量为溶液。
3.如权利要求1所述的益生菌微胶囊，其特征在于，所述的乳清分离蛋白、结冷胶和邻苯二甲酸乙酸纤维素在壁材中质量百分比含量分别为10％和0.3％和2％。
4.如权利要求1所述的益生菌微胶囊，其特征在于，所述的益生菌为干酪乳杆菌和/或枯草芽孢杆菌。
5.权利要求1-4任一项所述的益生菌微胶囊，其特征在于，所述的益生菌微胶囊的制备方法如下：
1)制备壁材溶液
A液制备：将结冷胶GG溶于PBS溶液中，煮沸搅拌使其溶解为透明溶液，80℃保温备用；然后将邻苯二甲酸乙酸纤维素CAP边加热搅拌边调节pH至7.0，继续搅拌至完全溶解为透明溶液；
B液制备：再乳清分离蛋白WPI边搅拌边加热至90℃使其中的酶变性，继续搅拌至完全溶解；
将A液与B液混合，边搅拌混匀边降温，制成壁材溶液备用；
2)将干酪乳杆菌和枯草芽孢杆菌的菌悬液与步骤1)制备的壁材溶液混匀，-20℃冷冻过夜，再进行低温冷冻干燥制成益生菌微胶囊。
6.如权利要求5所述的益生菌微胶囊，其特征在于，所述的干酪乳杆菌和枯草芽孢杆菌菌悬液的制备方法如下：将干酪乳杆菌以2％的接种量接种于MRS培养基中，37℃厌氧静置培养48h后将菌液4℃、6000rmp条件下离心15min，得到离心菌体；用无菌生理盐水清洗离心菌体两次；确定菌悬液的菌含量为5×108CFU/mL；
将枯草芽孢杆菌以2％的接种量接种于液体培养基中，37℃180rmp摇床培养48h后将菌液4℃、6000rmp条件下离心15min，得到离心菌体；确定菌悬液的菌含量为5×108CFU/mL。
7.如权利要求5所述的益生菌微胶囊，其特征在于，所述的低温冷冻干燥的条件如下：温度为-57℃、9.7Pa·s。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113892649A (zh) * | 2020-06-18 | 2022-01-07 | 湖南农业大学 | 一种益生菌微胶囊及其制备方法 |
| CN113881595A (zh) * | 2021-10-09 | 2022-01-04 | 湖北工业大学 | 一种包含蛋白纤维的乳酸菌发酵剂及其制备方法 |
| CN113881595B (zh) * | 2021-10-09 | 2023-08-25 | 湖北工业大学 | 一种包含蛋白纤维的乳酸菌发酵剂及其制备方法 |
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