CN114806965B - 一种提高菌种贮存稳定性的益生菌剂及其制备方法与应用 - Google Patents
一种提高菌种贮存稳定性的益生菌剂及其制备方法与应用 Download PDFInfo
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Abstract
本发明提供了一种提高菌种贮存稳定性的益生菌剂及其制备方法与应用,所述提高菌种贮存稳定性的益生菌剂的制备原料包括益生菌、保护剂和包埋剂;所述保护剂包括灭活乳酸菌和细胞膜稳态剂;所述细胞膜稳态剂包括硒代半胱氨酸、甲硫氨酸、甘露醇或磷脂酰丝氨酸中的任意一种或至少两种的组合;所述包埋剂包括聚乳酸‑羟基乙酸共聚物。所述保护剂重点为保护益生菌细胞内源性稳态,包埋剂是为形成微囊结构,隔绝不利的外源性条件,进一步保护益生菌,提高其贮存稳定性。相较于现有技术而言,本发明方案提高菌种贮存稳定性的效果更好,不仅适用于乳酸菌等常见益生菌,还适用于阿克曼氏菌等严格厌氧类菌种,具有重要的应用价值。
Description
技术领域
本发明属于微生物技术领域,涉及一种提高菌种贮存稳定性的益生菌剂及其制备方法与应用。
背景技术
乳酸菌等益生菌产品在不同温度下的贮存稳定性是长期以来困扰行业发展的核心问题,导致无论在产品执行标准,法规认证及宿主服用过程中均因乳酸菌活性的衰减影响其功效表达,目前行业内已有诸多专利文献声称具备有效保障乳酸菌产品在常温或更恶劣条件下对于乳酸菌活性的保障方法,通常的是通过降低环境中的水分活度(aw)或增强抗氧化体系、微包埋等手段来满足其目的。
CN102325873A公开了以抗坏血酸盐或抗坏血酸作为抗氧化剂进行乳酸菌浓缩物制备用于提高长期贮存活菌率。
CN109464425B公开了添加HPMC,滑石粉,多孔淀粉等物质进行微包埋提升乳酸菌产品耐受性。
CN108048349B公开了在保护剂中添加明胶,壳聚糖,海藻酸钠等材质所形成凝胶结构进行加工存活率及贮存稳定性的提升方法。
CN112795558A公开了通过液氮深冷干燥,以脱脂乳,β-环糊精,抗性糊精,海藻糖,海藻酸钠,阿拉伯胶等物质进行组合多层包埋所形成的乳酸菌颗粒可提升乳酸菌稳定性。
但从实际产业化应用及数据结果反馈上来看,诸多的组合物菌剂并没有达到预期效果,特别是在针对不同程度,不同环境条件下的活性胁迫干预难以保障其基本活性,特别针对严格厌氧的肠道微生物菌种,常规理论上的“封装”手段难以隔绝外源性胁迫,以及进一步提升或保护不同类型活性细胞物质在长期室温(或高温)条件下存活率。
综上所述,亟待开发一种具备规模产业化效应,能够有效提升不同特性菌种(特别是严格厌氧类菌种)的贮存稳定性的制备技术。
发明内容
针对现有技术的不足,本发明的目的在于提供一种提高菌种贮存稳定性的益生菌剂及其制备方法与应用。尤其针对于那些对氧敏感的益生菌,本发明的目的在于提高其活性氧耐受性,从而提高其在有氧常温(15-40℃)环境下的贮存稳定性。
为达到此发明目的,本发明采用以下技术方案:
第一方面,本发明提供一种提高菌种贮存稳定性的益生菌剂,所述提高菌种贮存稳定性的益生菌剂的制备原料包括益生菌、保护剂和包埋剂;
所述保护剂包括灭活乳酸菌和细胞膜稳态剂;
所述细胞膜稳态剂包括硒代半胱氨酸、甲硫氨酸、甘露醇、海藻糖、脱脂乳粉或磷脂酰丝氨酸中的任意一种或至少两种的组合,所述至少两种的组合例如硒代半胱氨酸与甲硫氨酸的组合、甲硫氨酸与甘露醇的组合、甘露醇与磷脂酰丝氨酸的组合等,其他任意的组合方式均可;
所述包埋剂包括聚乳酸-羟基乙酸共聚物(PLGA)。
优选地,所述细胞膜稳态剂包括硒代半胱氨酸和甘露醇。
优选地,所述硒代半胱氨酸与甘露醇的质量比为(0.01-1):(1-5),优选为 0.1:(2.5-5.5)。
上述(0.01-1)中的具体数值例如0.01、0.05、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1等。
上述(1-5)中的具体数值例如1、1.5、2、2.5、3、3.5、4、4.5、5等。
上述(2.5-5.5)中的具体数值例如2.5、2.7、3、3.2、3.5、3.7、4、4.2、4.5、 4.7、5、5.2、5.5等。
优选地,所述保护剂以重量份数计包括灭活乳酸菌10-70份和细胞膜稳态剂1-5份。
上述10-70份中的具体数值例如10份、15份、20份、25份、30份、35份、 40份、45份、50份、55份、60份、65份、70份等。
上述1-5份中的具体数值例如1份、1.5份、2份、2.5份、3份、3.5份、4 份、4.5份、5份等。
本发明采用PLGA进行益生菌活细胞囊化,不仅能够隔绝外环境,保护益生菌,而且其作为可降解载体结构能够实现控释。PLGA可以在水相条件下进行降解,降解过程通过酯键断裂来完成,同时在降解过程中产生乳酸等酸性物质促进此反应。此外,试验证明,PLGA黏度越低、分子量越小,其形成囊化后的空隙率高且孔径大,导致突释大,同时其添加含量也会影响其突释速率,因此通过可以通过调节其粘度、分子量及加入量来实现定位控释效果。
优选地,本发明所述的PLGA中,丙交酯与乙交脂的摩尔比为 (50-90):(10-50),分子量范围10000-20000,特性粘度为0.2-2.0dl/g。
上述(50-90)中的具体数值例如50、55、65、70、75、80、85、90等。
上述(10-50)中的具体数值例如10、15、20、25、30、35、40、45、50等。
上述(10000-20000)中的具体数值例如10000、11000、12000、13000、14000、15000、16000、17000、18000、19000、20000等。
优选地,所述益生菌包括乳酸菌、阿克曼氏菌、脆弱拟杆菌或柔嫩梭菌中的任意一种或至少两种的组合,所述至少两种的组合例如乳酸菌与阿克曼氏菌的组合、阿克曼氏菌与脆弱拟杆菌的组合、乳酸菌与柔嫩梭菌的组合等,其他任意的组合方式均可。
优选地,在所述益生菌中,所述乳酸菌包括乳杆菌属的乳酸菌、双歧杆菌属的乳酸菌、链球菌属的乳酸菌、肠球菌属的乳酸菌、乳球菌属的乳酸菌或片球菌属的乳酸菌中的任意一种或至少两种的组合,所述至少两种的组合例如乳杆菌属的乳酸菌和双歧杆菌属的乳酸菌的组合、双歧杆菌属的乳酸菌和链球菌属的乳酸菌的组合、乳杆菌属的乳酸菌和链球菌属的乳酸菌的组合等,其他任意的组合方式均可。
优选地,所述灭活乳酸菌包括灭活乳杆菌属的乳酸菌、灭活链球菌属的乳酸菌、灭活肠球菌属的乳酸菌、灭活乳球菌属的乳酸菌或灭活片球菌属的乳酸菌中的任意一种或至少两种的组合,不包含灭活双歧杆菌属的乳酸菌,所述至少两种的组合例如灭活乳杆菌属的乳酸菌和灭活链球菌属的乳酸菌的组合、灭活肠球菌属的乳酸菌和灭活乳球菌属的乳酸菌的组合、灭活乳球菌属的乳酸菌和灭活片球菌属的乳酸菌的组合,其他任意的组合方式均可。
优选地,所述灭活乳酸菌的细胞数不低于1×1010个细胞/g,更优选地,不低于1×1011个细胞/g。
优选地,所述乳杆菌属的乳酸菌包括植物乳杆菌、鼠李糖乳杆菌、干酪乳杆菌、副干酪乳杆菌、罗伊氏乳杆菌、嗜酸乳杆菌、德式乳杆菌、唾液乳杆菌、发酵乳杆菌、格氏乳杆菌或约氏乳杆菌中的任意一种或至少两种的组合,所述至少两种的组合例如植物乳杆菌和鼠李糖乳杆菌的组合、罗伊氏乳杆菌和嗜酸乳杆菌的组合、格氏乳杆菌和约氏乳杆菌的组合等,其他任意的组合方式均可。
优选地,所述链球菌属的乳酸菌包括嗜热链球菌。
优选地,所述肠球菌属的乳酸菌包括粪肠球菌。
优选地,所述乳球菌属的乳酸菌包括乳酸乳球菌。
优选地,所述片球菌属的乳酸菌包括乳酸片球菌和/或戊糖片球菌。
优选地,所述双歧杆菌属的乳酸菌包括乳双歧杆菌、长双歧杆菌、短双歧杆菌、青春双歧杆菌、婴儿双歧杆菌或两歧双歧杆菌中的任意一种或至少两种的组合,所述至少两种的组合例如乳双歧杆菌和长双歧杆菌的组合、长双歧杆菌和短双歧杆菌的组合、婴儿双歧杆菌和两歧双歧杆菌的组合等,其他任意的组合方式均可。
第二方面,本发明提供一种如第一方面所述的提高菌种贮存稳定性的益生菌剂的制备方法,所述制备方法包括如下步骤:
(1)培养益生菌,收集菌体;
(2)将保护剂与菌体混合,得到活性混合物;
(3)利用包埋剂对活性混合物进行乳化包埋,得乳化液,冻干,即得。
优选地,步骤(1)所述培养在30-42℃下进行,例如30℃、32℃、34℃、 36℃、38℃、40℃、42℃等,所述培养的时间为12-24h,例如12h、14h、16h、 18h、20h、22h、24h等。
优选地,步骤(1)所述收集菌体的方式包括离心和/或膜过滤。
优选地,步骤(2)所述混合的温度不超过20℃,例如20℃、15℃、10℃、 5℃、0℃等,所述混合的时间为10-30min,例如10min、15min、20min、25min、 30min等。
优选地,步骤(2)中,所述菌体与保护剂的质量比为1:(0.1-1.5)。
上述(0.1-1.5)中的具体数值例如0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、 0.9、1、1.1、1.2、1.3、1.4、1.5等。
优选地,步骤(3)中,所述包埋剂的量占加入后体系总质量的0.1%-10%,例如0.1%、0.5%、1%、2%、3%、4%、5%、6%、7%、8%、9%、10%等。
优选地,步骤(3)中,所述乳化包埋采用膜分离工艺。
优选地,步骤(3)中,所述冻干前还包括利用液氮进行预冻。
第三方面,本发明提供一种如第一方面所述的提高菌种贮存稳定性的益生菌剂在制备食品、保健品或药品中的应用。
所述食品例如乳粉、固体饮料、含片、液体饮料、发酵乳等。
所述保健品或药品的剂型包括颗粒剂、溶液剂、片剂、胶囊剂等。
本发明所述的数值范围不仅包括上述列举的点值,还包括没有列举出的上述数值范围之间的任意的点值,限于篇幅及出于简明的考虑,本发明不再穷尽列举所述范围包括的具体点值。
本发明中,术语“益生菌”,指能够对宿主健康产生有益作用的活的微生物,包括乳酸菌等传统益生菌,还包括阿克曼氏菌、脆弱拟杆菌、柔嫩梭菌等新一代益生菌。
本发明中,术语“乳酸菌”,指发酵糖类主要产物为乳酸的一类革兰氏染色阳性细菌的总称,是利用可发酵碳水化合物产生大量乳酸的细菌。主要产酸类型为乳酸、乙酸、丙酸。常见可用于食品中的乳酸菌包括双歧杆菌、乳杆菌、链球菌、明串珠菌、乳酸乳球菌等。
本发明中,术语“浓缩菌悬液”,主要指在益生菌发酵结束后,发酵液经生物分离提取技术进行固液分离所收集获得,常用离心或膜过滤进行分离收集,在本发明中用于与保护剂均匀乳化形成冻干颗粒组合物。
本发明中,术语“灭活乳酸菌”,主要指通过热处理等方式将活性乳酸菌细胞进行杀菌处理后,通过不同方式干燥所获得产品,其具备较为完整的细胞原有结构及部分胞内物质和代谢化合物。
本发明中,术语“氧化磷酸化”是一个生物化学过程,发生在真核细胞的线粒体内膜或原核生物(如乳酸菌)的细胞质中,是物质在体内氧化时释放的能量通过呼吸链供给ADP与无机磷酸合成ATP的偶联反应。对于乳酸菌来说,糖代谢中的三羧酸循环和脂肪酸β-氧化是在胞内生成NADH(还原当量),可立即通过电子传递链进行氧化磷酸化。而在细胞的胞浆中产生的NADH,如糖酵解生成的NADH则要通过穿梭系统(shuttle system)使NADH的氢进入线粒体内膜氧化。
相对于现有技术,本发明具有以下有益效果:
本发明创造性地通过对保护剂和包埋剂的合理设计,实现了提高益生菌制剂中菌种贮存稳定性的目的,相较于现有技术而言,本发明方案提高菌种贮存稳定性的效果更好,不仅适用于乳酸菌等常见益生菌,还适用于阿克曼氏菌等严格厌氧类菌种,具有重要的应用价值。本发明所述保护剂为灭活乳酸菌和细胞膜稳态剂的组合,重点为保护益生菌活菌细胞内源性稳态,包埋剂是为形成微囊结构,隔绝不利外源性条件(活性氧,水分,辐射等),进一步保护益生菌,提高其贮存稳定性,本发明技术方案的具体优势如下:
(1)将灭活乳酸菌菌体细胞作为物质结构组成高度一致的保护性载体,利用兼性好氧类、微好氧类、好氧类乳酸菌所含的活性化合物以及自身对活性氧的耐受性,降低益生菌细胞通过呼吸链供给ADP与无机磷酸合成ATP的偶联反应释放能量而产生的活性氧,大幅度缓和活性细胞常温或高温条件下呼吸作用ATP产生所带来的活性氧毒性,这种内源性干预能够保证益生菌制剂在长达数年贮存下达到要求的活菌率。
(2)细胞膜稳态剂主要用于平衡细胞在冻结及脱水浓缩干燥中的酸碱性和渗透压,保护发酵脱水浓缩后裸露的具备高度活性的益生菌细胞,同时对细胞膜稳态结构进行强化,保障其细胞结构健全,稳定性,完整性;保障细胞基础生理功能活性,对其细胞免疫力,生理活性及环境适应性等生理功能的强弱起到决定性作用。而现有技术常用的脱脂乳粉、海藻糖等冻干保护剂主要是对冻干结晶形成的细胞损伤进行保护,与本发明的细胞膜稳态剂的作用机理不同。
(3)形成微囊化封装结构是本领域较为成熟的既定工艺,现有技术中通常采用壳聚糖,海藻酸钠等进行凝胶保护,但由于这些物质在酸性条件下难以降解,同时高度脱水后极易发生结构断裂,自我修复效果较差,不适用于乳酸菌等酸性体系。本发明创造性地采用纳米PLGA作为包埋剂用于益生菌制剂中,首先,与现有技术中其他包埋剂相比,其提高益生菌贮存稳定性的效果更好,其次,其对于乳酸菌等酸性体系的生物相容性较好,其可以在水相条件下进行降解,降解过程通过酯键断裂来完成,同时在降解过程中产生乳酸等酸性物质促进此反应,安全,无毒无害。
(4)此外,细胞膜稳态剂的配方对于提高益生菌贮存稳定性的效果有一定的影响,本发明优选硒代半胱氨酸与甘露醇的组合,二者相互配合,促进了细胞膜稳态平衡,强化了细胞膜稳态结构,在提高益生菌贮存稳定性方面有预料不到的协同增效的效果。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了,所述实施例仅仅是帮助理解本发明,不应视为对本发明的具体限制。
以下实施例中,若无特殊说明,所以的试剂及耗材均购自本领域常规试剂厂商;若无特殊说明,所用的实验方法和技术手段均为本领域常规的方法和手段。
下述实施例、对比例、应用例中涉及的甘露醇购自法国罗赛洛Rousselot, 硒代半胱氨酸和甲硫氨酸购自德国默克Merck,精制去油磷脂酰丝氨酸购自德国德固赛Degussa,海藻糖购自日本林原HAYASHIBARA,脱脂乳粉购自新西兰恒天然Fonterra。
实施例1
两歧双歧杆菌BBi32冻干颗粒的制备
S1、将两歧双歧杆菌(保藏号为CGMCC No.16923)以3.0%接种量接种于 MRS培养基中进行活化扩培(温度为34℃,时间为20h),后进行发酵培养(接种量2.0%,温度34℃,时间为14h),待发酵结束后进行离心(6500rpm,30 min),倾倒排出上清液,得两歧双歧杆菌BBi32浓缩菌悬液(含两歧双歧杆菌 BBi32≥1×1011CFU/g);
S2、将保护剂与浓缩菌悬液,按浓缩菌悬液:保护剂=1:0.5的质量比进行均匀混合,在20℃下平衡20min,得到活性混合物。其中保护剂按质量百分含量计包含灭活乳酸菌菌体细胞55%,细胞膜稳态剂(硒代半胱氨酸与甘露醇的质量比为0.1:3)2.0%,其余为无菌纯化水。
其中,灭活乳酸菌菌体细胞的制备方法为:将保藏编号为CGMCC:10452 的长双歧杆菌BL21,接种于MRS培养基中,于37℃下进行常规培养18h,得培养液,离心(6500rpm,20min),收集菌体,采用高温热灭活方法进行灭活 (121℃,15min),即得。
S3、进一步将S2步骤得到的活性混合物进行PLGA乳化包埋,采用膜分离工艺,进行纳滤膜分离包埋,形成乳化液。体系中PLGA的质量百分含量为 8.5%,PLGA中丙交酯:乙交脂比例为75:25,分子量为14000,特性粘度1.0-1.4 dL/g,密度1.2g/cm3,旋光度≤50°。
S4、将S3步骤所制备而成的乳化液通过均匀压力滴管快速滴入液氮造粒机中,形成2-5mm,大小均匀的球状深冷颗粒;收集深冷颗粒进行真空冷冻干燥,冻干周期12-20小时,完全干燥后获取乳酸菌冻干颗粒,要求检测颗粒水分不高于5.0%,水分活度aw不高于0.1。
实施例2
嗜粘蛋白-阿克曼氏菌(AKK)冻干颗粒的制备
S1、将嗜粘蛋白-阿克曼氏菌(ATCC BBA-835)以5%接种量接种于培养基 (在市售的BHI脑心浸液培养基中添加0.1%粘蛋白)中进行活化扩培(温度为 37℃,时间为20h,5%CO2、95%N2、pH 6.0),后进行发酵培养(接种量2.0%,温度37℃,时间为18h),待发酵结束后进行离心(转速12000rpm,时间15min),得AKK浓缩菌悬液(含AKK≥1×108CFU/g),因菌种特性要求,需对培养全程进行厌氧保护,所采用保护气体为氮气/二氧化碳混合气体。
S2、采用灭活乳酸菌菌体细胞与细胞膜稳态剂进行复配组合,形成基础保护配方,与浓缩菌悬液按菌悬液:保护剂=1:0.8的重量比进行均匀混合,15℃下平衡15min。其中保护剂成分包含灭活乳酸菌细胞65%,细胞膜稳态剂(硒代半胱氨酸与甘露醇的质量比为0.1:5)4.0%,其余为无菌纯化水。
所述灭活乳酸菌菌体细胞具体为副干酪乳杆菌(保藏编号为CGMCC: 1.12731)的菌体细胞,其制备方法参照实施例1中灭活乳酸菌菌体细胞的制备方法。
S3、进一步将S2步骤的活性混合物进行PLGA乳化包埋,采用膜分离工艺,进行膜纳滤膜分离包埋,得到乳化液,其中PLGA含量为4.0%,PLGA中丙交酯:乙交脂比例为60:40,分子量为17000,特性粘度0.7-1.2dL/g,密度 1.3g/cm3,旋光度≤50°;
S4、将S3步骤所制备而成乳化液通过均匀压力滴管快速滴入液氮造粒机中,形成2-5mm,均匀颗粒大小球状深冷颗粒;收集深冷颗粒进行真空冷冻干燥,冻干周期12-20小时,完全干燥后获取AKK冻干制剂,要求检测颗粒水分不高于5.0%,水分活度aw不高于0.1。
实施例3
本实施例提供一种两歧双歧杆菌BBi32冻干颗粒的制备方法,其与实施例 1的区别仅在于将细胞膜稳态剂“硒代半胱氨酸与甘露醇”替换为等量的“甘露醇”,其它参照实施例1。
实施例4
本实施例提供一种两歧双歧杆菌BBi32冻干颗粒的制备方法,其与实施例 1的区别仅在于将细胞膜稳态剂“硒代半胱氨酸与甘露醇”替换为等量的“硒代半胱氨酸”,其它参照实施例1。
实施例5
本实施例提供一种两歧双歧杆菌BBi32冻干颗粒的制备方法,其与实施例 1的区别仅在于将细胞膜稳态剂“硒代半胱氨酸与甘露醇”替换为等量的“海藻糖与甘露醇”,其中,海藻糖的量与其替代的硒代半胱氨酸的量相等,其它参照实施例1。
实施例6
本实施例提供一种两歧双歧杆菌BBi32冻干颗粒的制备方法,其与实施例 1的区别仅在于将细胞膜稳态剂“硒代半胱氨酸与甘露醇”替换为等量的“硒代半胱氨酸与脱脂乳粉”,其中,脱脂乳粉的量与其替代的甘露醇的量相等,其它参照实施例1。
对比例1
本对比例提供一种两歧双歧杆菌BBi32冻干颗粒的制备方法,其与实施例 1的区别仅在于保护剂中不添加细胞稳态剂,缺少的量用灭活乳酸菌菌体细胞补足,其他参照实施例1。
对比例2
本对比例提供一种两歧双歧杆菌BBi32冻干颗粒的制备方法,其与实施例 1的区别仅在于保护剂中不添加灭活乳酸菌菌体细胞,缺少的量用海藻糖补足,其他参照实施例1。
对比例3
本对比例提供一种两歧双歧杆菌BBi32冻干颗粒的制备方法,其与实施例 1的区别仅在于将包埋剂“PLGA”替换为等量的“海藻酸钙凝胶”,包埋方法为:在浓缩菌悬液中添加与PLGA等量的海藻酸钠,待完全分散于体系中后添加适量氯化钙溶液,使其逐步凝胶化形成包埋结构,其它条件参照实施例1。
测试例1
贮存稳定性测试
对上述实施例及对比例制得的两歧双歧杆菌BBi32冻干颗粒在常温(25℃) 0、3、6、9、12、15、18、21,24个月条件下进行活菌率跟踪,活菌率=实际活菌数/初始活菌数,并以常规市售两歧双歧杆菌BBi32冻干粉(购自微康益生菌)作为对比进行同步跟踪,表1中展示了0、3、6、12、24个月的结果。
表1
组别 | 0个月 | 3个月 | 6个月 | 12个月 | 24个月 |
实施例1 | 100% | 98% | 84% | 77% | 67% |
实施例3 | 100% | 70% | 62% | 51% | 28% |
实施例4 | 100% | 80% | 69% | 44% | 20% |
实施例5 | 100% | 72% | 60% | 51% | 26% |
实施例6 | 100% | 88% | 75% | 61% | 38% |
对比例1 | 100% | 29% | 11% | 4.2% | 1.7% |
对比例2 | 100% | 21% | 17% | 10% | 4.0% |
对比例3 | 100% | 37% | 12% | 4.2% | 1.1% |
市售冻干粉 | 100% | 79% | 56% | 41% | 17% |
测试例2
活性氧耐受性测试
AKK菌属于严格厌氧微生物,对活性氧极其敏感,为进一步评价本发明技术方案对于这种严格厌氧微生物的贮藏稳定性的提升效果,本测试例对其进行活性氧耐受性测试。
将实施例2得到的AKK冻干制剂悬浮于PBS中,高速离心(8000rpm 5 min),重复3次,调整溶液中活菌数接近109CFU/mL(要求OD600=1.5,检测活菌数),添加H2O2,要求其浓度达到1mmol/L,分别记录培养0、60min、 120min后的活菌数,并设置采用常规冻干工艺制得的AKK冻干粉作为对照组,与实施例2的产品同步进行测试。
所述常规工艺为:按照实施例2中S1步骤制得浓缩菌悬液后,按1:2的质量比加入保护剂(脱脂乳粉20%、甘油5%、低聚麦芽糖5%,余量为水),混合15min后,进行真空冷冻干燥,冻干完成后取出粉碎过筛,完成冻干菌粉的制备。
测试对比结果见表2(单位:lg CFU/mL)。
表2
组别 | 0min | 60min | 120min |
实施例2 | 10 | 9.25 | 8.1 |
对照组 | 10 | 7.2 | 3.9 |
结果显示,常规工艺制得的AKK冻干粉中AKK菌在活性氧耐受测试下快速衰亡,而经过本发明方案制备的AKK冻干制剂可在60min内保持25%以上活菌率,表现出对活性氧超强的耐受性,此结果充分证实了本发明技术方案适用于这种严格厌氧微生物,对其贮藏稳定性的提升效果显著。
应用例
产品一益生菌乳粉
制备婴儿双歧杆菌BI45(保藏编号为CGMCC:15134)冻干颗粒,制备方法参照实施例1,将得到的冻干颗粒进行粉碎,采用60-80目筛网进行过筛,后与乳粉进行混合,其中冻干颗粒与乳粉的质量比为5:95,最终益生菌乳粉中活菌数含量不低于300亿CFU/g,产品水分控制不高于5.0%,水活度控制不高于 0.2。
产品二益生菌固体饮料
制备嗜热球菌ST81(保藏编号为CGMCC:15752)冻干颗粒,制备方法参照实施例1,将得到的冻干颗粒进行粉碎,采用60-80目筛网进行过筛,后与低聚半乳糖(20份)、菊粉(50份)、脱脂乳粉(10份)、蓝莓果粉(10份) 混合均匀即得,其中冻干颗粒为10重量份,最终益生菌固体饮料中活菌数含量不低于600亿CFU/g,产品水分控制不高于5.0%,水活度控制不高于0.2。
产品三益生菌含片
制备嗜酸乳杆菌LA85(保藏编号为CGMCC:1.12735)冻干颗粒,制备方法参照实施例1,将得到的冻干颗粒进行粉碎,采用60-80目筛网进行过筛,后与山梨糖醇(40份)、低聚果糖(22份)、低聚异麦芽糖(18份)、微晶纤维素(8份)、维生素C(1.5份)、硬脂酸镁(0.4份)混合均匀,其中冻干颗粒为6.5重量份,采用压片机进行含片压制,片剂硬度控制120-140N,克重1.5 g/片,外观圆形。
对上述三种产品进行加速3个月贮存稳定性测试(具体测试方法为:按固定规格分装,采用培养箱恒定密封贮存,温度条件为37℃,湿度控制在65%RH,定期每周进行活菌数检测并计算活菌率),其加速结果可换算为长期18-24个月贮存活菌率数据,亦可快速预测评估产品在货架期的活菌率,活菌率结果见表3。
表3
0个月 | 1个月 | 2个月 | 3个月 | |
产品一 | 100% | 79% | 51% | 33% |
产品二 | 100% | 85% | 66% | 41% |
产品三 | 100% | 70% | 42% | 25% |
结果显示,本发明方案具有显著的提高菌株贮藏稳定性的效果,在制备益乳粉、固体饮料、含片等益生菌产品中具有重要的应用价值。
申请人声明,本发明通过上述实施例、应用例来说明本发明的一种提高菌种贮存稳定性的益生菌剂及其制备方法与应用,但本发明并不局限于上述实施例、应用例,即不意味着本发明必须依赖上述实施例才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。
Claims (12)
1.一种提高菌种贮存稳定性的益生菌剂,其特征在于,所述提高菌种贮存稳定性的益生菌剂的制备原料包括益生菌、保护剂和包埋剂;
所述保护剂由灭活乳酸菌和细胞膜稳态剂组成;
所述细胞膜稳态剂为硒代半胱氨酸和甘露醇的组合;
所述包埋剂为聚乳酸-羟基乙酸共聚物;
所述益生菌为保藏编号为CGMCC No.16923的两歧双歧杆菌BBi32菌株;
所述灭活乳酸菌为保藏编号为CGMCC:10452的长双歧杆菌BL21菌株。
2.如权利要求1所述的提高菌种贮存稳定性的益生菌剂,其特征在于,所述保护剂以重量份数计包括灭活乳酸菌10-70份和细胞膜稳态剂1-5份。
3.如权利要求1所述的提高菌种贮存稳定性的益生菌剂,其特征在于,所述硒代半胱氨酸与甘露醇的质量比为0.01-1:1-5。
4.如权利要求1所述的提高菌种贮存稳定性的益生菌剂,其特征在于,所述硒代半胱氨酸与甘露醇的质量比为0.1:2.5-5.5。
5.一种如权利要求1-4中任一项所述的提高菌种贮存稳定性的益生菌剂的制备方法,其特征在于,所述制备方法包括如下步骤:
(1)培养益生菌,收集菌体;
(2)将保护剂与菌体混合,得到活性混合物;
(3)利用包埋剂对活性混合物进行乳化包埋,得乳化液,冻干,即得。
6.如权利要求5所述的提高菌种贮存稳定性的益生菌剂的制备方法,其特征在于,步骤(1)所述培养在30-42℃下进行,所述培养的时间为10-24 h。
7.如权利要求5所述的提高菌种贮存稳定性的益生菌剂的制备方法,其特征在于,步骤(1)所述收集菌体的方式包括离心和/或膜过滤。
8.如权利要求5所述的提高菌种贮存稳定性的益生菌剂的制备方法,其特征在于,步骤(2)所述混合的温度不超过20℃,所述混合的时间为10-30 min。
9.如权利要求5所述的提高菌种贮存稳定性的益生菌剂的制备方法,其特征在于,步骤(2)中,所述菌体与保护剂的质量比为1:0.1-1.5。
10.如权利要求5所述的提高菌种贮存稳定性的益生菌剂的制备方法,其特征在于,步骤(3)中,所述包埋剂的量占加入后体系总质量的0.1%-10%。
11.如权利要求5所述的提高菌种贮存稳定性的益生菌剂的制备方法,其特征在于,步骤(3)中,所述乳化包埋采用膜分离工艺。
12.一种如权利要求1-4中任一项所述的提高菌种贮存稳定性的益生菌剂在制备食品或保健品中的应用。
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