CN110959865B - 可调节胃肠道菌群平衡的副干酪乳杆菌k56的新应用 - Google Patents
可调节胃肠道菌群平衡的副干酪乳杆菌k56的新应用 Download PDFInfo
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Abstract
本发明提供了可调节胃肠道菌群平衡的副干酪乳杆菌K56的新应用。本发明的副干酪乳酸杆菌(Lactobacillus paracasei subsp.paracasei)，其保藏编号为DSM27447。本发明发现该菌株单菌即具有显著促进肠道双歧杆菌和乳酸菌增长的能力，能够抑制肠道中脱硫弧菌和/或肠杆菌属，可抑制幽门螺旋杆菌和/或埃希氏菌‑志贺菌属，可以耐受体外模拟胃肠液胁迫环境，小鼠实验表明该菌株无口服急性毒性，无抗生素耐受，安全可以用于食品加工。
Description
技术领域
本发明涉及微生物技术领域，尤其是涉及一种副干酪乳杆菌(Lactobacillusparacasei subsp.paracasei)K56(保藏编号DSM27447)在调节胃肠道菌群平衡方面的新用途。
背景技术
肠道菌群的平衡与人体健康息息相关，肠道菌群包括有益菌、中性菌和有害菌。健康的人体肠道中有益菌占据优势地位，并且与有害菌不断相互作用，维护人体健康。如果由于各种因素影响使肠道中有益菌减少，有害菌便会大量繁殖，肠内微生态平衡被打破，引起肠炎、腹泻等多种临床症状。同时，临床上通常使用大量的抗生素，虽然能有效的杀害有害菌，但同时杀灭有益菌，造成肠道菌群失调，引起疾病。
对于肠道菌群失调者，经口补充益生菌是调节肠道菌群直接有效的办法。经口补充益生菌制剂或含益生菌产品可以直接或间接调节肠道菌群组成，激活宿主内源性微生物群或者免疫系统活性来实现益生作用。
世界卫生组织对益生菌产品的定义为食品中含有充足数量的活的微生物，经过食品加工的各个过程，以及进入人体肠道以后，仍能保持适当的活菌数量和菌活性。因此菌株在菌粉的制作、产品的生产加工以及经人体胃肠道的胃酸、胆盐胁迫后，菌株应能够保持较为稳定的活菌数量是有必要的。此外，即便乳酸菌是公认的安全菌株，但近年来的研究表明，很多乳酸菌，尤其是食源性乳酸菌和肠道乳酸菌，也出现了毒性因子与抗生素抗性，对人类的健康造成了潜在风险。因此，在评价菌株益生特性的同时，也应全面考量其在食用过程的安全性与生产加工过程中的稳定性。
CN107916236A公开了一种副干酪乳酸杆菌(Lactobacillus paracaseisubsp.paracasei)K56，该菌株已于2013年6月27日保存于德国微生物及细胞培养物收集中心(German Collection of Microorganisms and Cell Cultures)，保藏编号DSM27447。CN107916236A还公开了副干酪乳酸杆菌K56在调节免疫反应功效方面的用途，但并没有关于副干酪乳酸杆菌K56是否具有胃肠道菌群平衡功效的任何记载或启示。
发明内容
本发明的一个目的在于提供副干酪乳杆菌K56的新用途。
副干酪乳酸杆菌(Lactobacillus paracasei subsp.paracasei)K56菌株已于2013年6月27日保存于德国微生物及细胞培养物收集中心(German Collection ofMicroorganisms and Cell Cultures)，保藏编号DSM27447。
本发明发现副干酪乳酸杆菌(Lactobacillus paracasei subsp.paracasei)K56菌株单菌即具有显著促进肠道双歧杆菌和乳酸菌增长的能力，能够抑制肠道中脱硫弧菌和/或肠杆菌属，可抑制幽门螺旋杆菌和/或埃希氏菌-志贺菌属，可以耐受体外模拟胃肠液胁迫环境，小鼠实验表明该菌株无口服急性毒性，无抗生素耐受，安全可以用于食品加工。
从而，本发明提供了副干酪乳酸杆菌(Lactobacillus paracaseisubsp.paracasei)在制备用于调节胃肠道菌群的组合物中的应用，所述副干酪乳酸杆菌的保藏编号为DSM27447。
根据本发明的具体实施方案，所述副干酪乳酸杆菌以固态或液态菌制剂的形式用于制备所述组合物。
根据本发明的具体实施方案，所述组合物可以包括食品组合物、饲料组合物或药品组合物。
根据本发明的具体实施方案，所述组合物可使用于动物或是人类。所述组合物还可包括所属领域中的常规用料组分。例如，对于药物组合物，可包括适量的辅料，所述辅料可以为赋型剂、稀释剂、填充剂、吸收促进剂等。对于食品组合物，本发明的乳双歧杆菌可以按照现有技术中含乳双歧杆菌的食品进行生产，所述组合物可根据受施予者的需要，而采用不同形态。例如粉剂、锭剂、造粒、微胶囊、液体制剂等。
根据本发明的具体实施方案，所述组合物是用于增加肠道中双歧杆菌和/或乳酸菌的数量。具体应用时，所述副干酪乳酸杆菌的应用量为1.0×103CFU～1.0×1010CFU/kg体重/天，优选为1.0×104CFU～1.0×109CFU/kg体重/天。
根据本发明的具体实施方案，所述组合物是用于能够抑制肠道中脱硫弧菌和/或肠杆菌属的数量。具体应用时，所述副干酪乳酸杆菌的应用量为1.0×103CFU～1.0×1010CFU/kg体重/天，优选为1.0×104CFU～1.0×109CFU/kg体重/天。
根据本发明的具体实施方案，所述组合物是用于能够抑制幽门螺旋杆菌和/或埃希氏菌-志贺菌属的数量。具体应用时，所述副干酪乳酸杆菌的应用量为1.0×103CFU～1.0×1010CFU/kg体重/天，优选为1.0×104CFU～1.0×109CFU/kg体重/天。
在本发明的一具体实施方案中，所述组合物为食品组合物，所述食品为发酵乳制品(例如发酵乳、风味发酵乳、发酵乳饮料等)、乳酪、含乳饮料、固体饮料或乳粉。
综上所述，本发明提供了副干酪乳杆菌K56的新用途，该菌具有调节胃肠道菌群功效，特别是能够抑制肠道中脱硫弧菌和/或肠杆菌属，抑制幽门螺旋杆菌和/或埃希氏菌-志贺菌属，可以用于制备具有调节肠道菌群的食品、药物及饲料等，具有广泛的应用前景。
附图说明
图1为副干酪乳杆菌K56肠道黏附性示意显微镜照图。
图2显示副干酪乳杆菌K56肠道黏附性对比测试结果。
图3显示副干酪乳杆菌K56调节肠道菌群测试结果。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现结合具体实施例及对本发明的技术方案进行以下详细说明，应理解这些实例仅用于说明本发明而不用于限制本发明的范围。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
除非另外专门定义，本文使用的所有技术和科学术语都与相关领域普通技术人员的通常理解具有相同的含义。除非另有说明，本发明中使用的所有表示成分、细胞培养、处理条件等的量的数字应当理解为在所有条件下受到术语“约”的修饰。因此，除非另有相反的说明，数值参数为近似值，并且可以根据通过本发明试图获得的期望特性而变化。除非另有说明，一系列元素之前的术语“至少”应当理解为指该系列中的每个元素。
本发明的各实施例中，除特别说明外，实验数据表示为Mean±S.E.M.数据采用PRISM version 5.0(GraphPad,San Diego,CA,USA)进行统计。组间差异采用one-wayANOVA跟随Tukery’s multiple comparison test的方法进行统计。P<0.05时具有显著的统计学差异。
实施例1：耐胃酸实验
以0.1N HCl溶液分别调整MRS培养液至pH 2.0、pH 2.5及pH 3.0，取100μL(109CFU/ml)活化菌液接种于10mL的不同pH值的测试液中，活菌初始浓度为约107CFU/mL，置于37℃下1小时取样测残存菌数。取出1mL菌液，以0.85％食塩水进行连续稀释，涂于MRSagar上，在37℃条件下，培养24-48小时，计算所生成的菌落数。另取乳酸菌菌液100μL(109CFU/ml)，加入未调pH的MRS培养液(pH 6.8)中，作为对照组。
菌株的酸耐受性(％)＝(含pH 2.0或pH 2.5或pH 3.0的测试液残存菌数/未调pH的MRS培养液的菌数)×100％
酸耐受性被认为是乳酸菌能在胃部酸性环境中存活的必要特性之一。K56于不同酸性环境中的耐酸能力结果如下表1所示，结果显示由初活菌数由2.96×107CFU/mL，在酸性环境1小时后，虽然在pH 2.0处理1小时后几乎完全死灭(0％)，菌株K56于pH 2.5及pH3.0耐受条件中仍有菌体存活，pH 2.5有6.39％的存活率，且对于pH 3的酸性环境下较不敏感保有84.31％的存活率。
表1、K56耐酸性试验结果
实施例2.耐胆盐实验
配制MRS培养液含有不同浓度的牛胆盐(oxgall bile)分别为0.1％,0.5％及1％的测试液。取100μL(109CFU/ml)活化菌液分别接种于10mL含有不同浓度牛胆盐的测试液中，活菌初始浓度为约107CFU/mL，置于37℃下1小时取样测残存菌数。取出1mL菌液，以PBS(0.1M,pH 6.2)进行连续稀释，涂于MRS agar上，在37℃条件下，培养24-48小时，计算所生成的菌落数。另取乳酸菌菌液100μL(109CFU/ml)，加与不含牛胆盐的MRS的MRS培养液(pH6.8)中，作为对照组。
菌株的胆盐耐受性(％)＝(含0.1％或0.5％或1％的牛胆盐测试液残存菌数/不含牛胆盐测试液之菌数)×100％
胆盐耐受性被认为是乳酸菌能在小肠中存活的必要特性之一。菌株K56于不同浓度胆盐耐受性1小时的结果如表2所示，可知随着胆盐浓度的增加，菌株对于胆盐的敏感性增加致使死亡率提升；初活菌数为4.34×107CFU/mL，菌株K56于培养1小时的0.1％胆盐耐受性89.60％，于0.5％胆盐1小时后耐受性仍有82.73％存活，而在1％胆盐浓度下仍然保有69.44％存活率。综合以上，显示K56对于酸性环境及含胆盐环境中具有高度的耐受性。
表2、K56耐胆盐试验结果
实施例3：肠道细胞吸附效果
Caco-2细胞用培养瓶培养，在瓶中加入含10％热灭活的胎牛血清和双抗(100U/mL青霉素和100μg/mL链霉素)的DMEM细胞培养液，置于37℃、5％CO2的培养箱中培养，每2d换1次培养液。待细胞贴壁生长为单层细胞后(5～7d)，用0.25％胰酶消化传代，并用0.4％的台盼蓝染色液染色，用血细胞计数器在显微镜下检测细胞的数量和活性，保证细胞活性在95％以上。
通过黏附实验对菌株对Caco-2细胞的黏附性进行检测，结果如图1(其中图片B为图片A的放大图)、图2显示，K56的肠道黏附能力与LcS及LcA相近，且强于LGG和NCFM。
实施例4：肠道菌群调节效果
本实施例意图证实本发明的副干酪乳杆菌在肠道调节方面的效果，其原理和步骤参见“保健食品检验与评价技术规范-调节肠道菌群功能判定标准”。
取36只健康SPF级BABL/c小鼠，体重18-22g(由北京华阜康生物科技有限责任公司提供)。适应性饲养3天后，将其随机分为3组，每组12只，即空白对照组，样品组。向每组动物分别灌胃溶解了乳双歧杆菌K56菌粉的无菌水(灌胃体积0.2mL/10g)，空白对照组灌胃相同体积的无菌水。每天1次，连续饲喂或灌胃14天。灌胃计量：1.3×107CFU/ml(按人体需要量为2×109CFU/d，人体和小鼠换算系数0.0026进行换算)。取灭菌离心管，编号，适应性喂养后于无菌条件下采集小鼠粪便，每只2-3粒，约100mg，低温条件转移至无菌操作间进行菌群的检测。实验结束时，再次采集小鼠粪便。使用苦味酸对小鼠进行分组编号后，分别于给予受试物第8天、第14天称重，计算小鼠灌胃量，实验结束时称重1次。菌落计数：根据待鉴定菌种配制选择性培养基，待测菌种及相应培养基如表3，灭菌，摇匀，冷至45℃-50℃倾注平板，备用。
表3、待测菌种及对应选择性培养基
| 待测菌种 | 选择性培养基 |
| 肠杆菌 | 伊红美蓝(EMB)琼脂 |
| 肠球菌 | 叠氮钠-结晶紫-七叶苷琼脂 |
| 双歧杆菌 | BBL琼脂 |
| 乳酸杆菌 | LBS琼脂 |
| 产气荚膜梭菌 | 胰胨-亚硫酸盐-环丝氨酸(TSC)琼脂 |
将采集的小鼠粪便，置于装有0.5mL生理盐水的灭菌管中，配制成菌悬液，使用前震荡1min。使用0.1mL微量移液器吸取0.1mL菌悬液，缓慢注于0.9mL灭菌生理盐水中，震荡或反复吹打使其混匀，制成1:10的菌悬液。另取0.1mL微量移液器吸头，依此方法，进行10倍梯度稀释，直至10-7g/ml。根据待鉴定菌种的活菌数，选择两个连续的适宜稀释度，每个稀释度使用10μL微量移液器吸取10μL菌悬液，在选择性琼脂平板上进行表面涂布，按表2所示培养条件进行培养。菌落计数方法参考《GB 4789.2-2010食品安全国家标准食品微生物学检验菌落总数测定》进行。
表4、肠道菌群检验用培养基及鉴定方法
| 项目 | 培养基 | 培养条件 |
| 肠杆菌 | 伊红美蓝琼脂 | 36℃±1℃培养24h |
| 肠球菌 | 叠氮钠-结晶紫-七叶苷琼脂 | 36℃±1℃培养48h |
| 双歧杆菌 | BBL琼脂 | 36℃±1℃培养48h，厌氧培养 |
| 乳酸菌 | LBs琼脂 | 36℃±1℃培养48h |
| 产气荚膜梭菌 | TSC琼脂 | 36℃±1℃培养24h，厌氧培养 |
采用SPSS17.0进行数据统计。比较实验前后自身及组间双歧杆菌、乳酸菌、肠球菌、肠杆菌的变化情况，试验组实验前后自身比较变化有显著性，符合以下任意一项，可以判定该受试样品动物实验结果阳性。第一、粪便中双歧杆菌或者乳酸菌明显增加，梭菌减少或无明显变化，肠球菌、肠杆菌无明显变化。第二、粪便中双歧杆菌或者乳酸菌明显增加，梭菌减少或无明显变化，肠球菌、肠杆菌明显增加，但增加的幅度低于双歧杆菌或者乳酸菌增加的幅度。
实验期间动物的体重变化结果如表5所示。在实验期内，动物表征正常，给予受试物后未出现任何不良反应，实验周期内，两组动物体重并未出现显著差异。从表6～10可以看出，副干酪乳杆菌K56能显著促进双歧杆菌和乳酸菌的生长，对于肠杆菌、肠球菌、产气荚膜梭菌无显著影响，根据保健食品检验与评价技术规范-调节肠道菌群功能判定标准可以判定，本研究中副干酪乳杆菌K56具有调节肠道菌群的功效(见图3)。
表5、动物的体重变化
| 分组 | 动物数 | 初始体重(g) | 中期体重(g) | 终末体重(g) |
| 对照组 | 14 | 21.89±1.25 | 22.14±0.87 | 21.24±0.87 |
| L.paracasei K56 | 14 | 22.11±1.08 | 21.79±1.17 | 20.95±0.22 |
表6、受试前后动物肠道双歧杆菌的变化(LgCFU/g)
表7、受试前后动物肠道乳杆菌的变化(LgCFU/g)
表8、受试前后动物肠道肠杆菌的变化(LgCFU/g)
表9、受试前后动物肠道肠球菌的变化(LgCFU/g)
表10、受试前后动物肠道荚膜梭菌的变化(LgCFU/g)
实施例5：不同剂量K56的肠道菌群调节效果比较
本实施例中，分别检测了不同剂量K56的肠道菌群调节效果。
活菌样品：依据样品规格，称取1gK56活菌样品，使用PBS溶液悬浮至40ml，即活菌浓度均为2.5x109CFU/ml。
高剂量组：按照小鼠0.2ml/10g灌胃量计算，20g小鼠灌胃量为0.4ml，高剂量组小鼠灌胃剂量为109CFU/20g。
中剂量组：分别取5ml高剂量悬浮液加入PBS定容至50ml，按照小鼠0.2ml/10g灌胃量计算，20g小鼠灌胃量为0.4ml，中剂量组小鼠灌胃剂量为108CFU/20g。
低剂量组：分别取5ml中剂量组悬浮液加入PBS定容至50ml，按照小鼠0.2ml/10g灌胃量计算，20g小鼠灌胃量为0.4ml，低剂量组小鼠灌胃剂量为107CFU/20g。
6周龄BABL/c小鼠饲养于清洁级动物房，温度22℃，湿度10-60％，12小时明暗交替照明，标准饲料喂养，自由饮水。适应性喂养5天，小鼠随机分组，每组14只，分组情况见表11。
表11、调节肠道菌群实验分组
开始灌胃前，无菌条件下采集每只小鼠粪便，标记，-20℃保存，检测肠道菌群。实验按照0.2ml/10g灌胃量给予各受试物，对照组1-14天给予PBS，实验组分别依据表2灌胃给予相应剂量的受试物。小鼠每周称重一次，根据体重调整灌胃量。14天后无菌条件下采集每只小鼠粪便，标记，-20℃保存，检测肠道菌群。
实验前后，各组小鼠体重无显著差异。在门的水平，补充不同剂量的益生菌后，小鼠肠道菌群中厚壁菌门(Firmicutes)相对丰度增加而拟杆菌门(Bacteroidetes)、变形菌门(Proteobacteria)相对丰度降低。研究表明，厚壁菌门与拟杆菌门的比值与人体肠道疾病具有较强的相关性，肥胖病人倾向于二者比值降低。而肠炎、肠应激综合症患者则倾向于具有较高的变形菌门(Proteobacteria)丰度。
K56在属的水平对肠道菌群的影响参见表12。
表12、K56对肠道菌群的影响
| 菌属 | 对照组 | K56低剂量组 | K56中剂量组 | K56高剂量组 |
| 拟杆菌属 | 9.8061±2.094 | 6.0503±1.6172 | 7.4157±2.3149 | 6.5026±1.553 |
| 乳酸杆菌 | 3.0166±0.4635 | 0.5798±0.5605 | 4.0343±0.5534 | 2.158±1.2288 |
| 脱硫弧菌 | 2.1391±0.5097 | 0.853±0.3645 | 1.5655±0.9228 | 0.8367±0.4632 |
| 肠杆菌 | 0.3447±0.0971 | 0.254±0.1668 | 0.2834±0.1117 | 0.2134±0.0873 |
在属的水平，控制组(对照组)相比，益生菌组小鼠肠道菌群中K56中剂量组可显著增加小鼠肠道中的乳酸杆菌属(Lactobacillus)相对丰度。K56低剂量组、高剂量组对脱硫弧菌(Desulfovibrio)具有显著抑制作用。
K56对致病菌幽门螺杆菌、埃希氏菌-志贺氏菌属的抑制效果参见表13。
表13、K56对致病菌的抑制效果
对致病菌进行分析，结果表明，K56低剂量组对埃希氏菌-志贺菌属(Escherichia-Shigella)具有显著抑制作用，所有组别均对幽门螺旋杆菌(Helicobacter)具有显著抑制作用。
上述实验表明，K56可以调节肠道菌群平衡，促进有益菌生长，抑制有害菌甚至是致病菌。
Claims (10)
1.一种副干酪乳酸杆菌 (Lactobacillus paracasei)在制备用于调节胃肠道菌群的组合物中的应用，所述副干酪乳酸杆菌的保藏编号为DSM27447；
其中，所述调节胃肠道菌群包括抑制幽门螺旋杆菌和/或埃希氏菌-志贺菌属。
2.根据权利要求1所述的应用，其中，所述副干酪乳酸杆菌以固态或液态菌制剂的形式用于制备所述组合物。
3.根据权利要求1所述的应用，其中，所述组合物包括食品组合物、饲料组合物或药品组合物。
4.根据权利要求1所述的应用，其中，所述组合物还用于增加肠道中乳酸菌的数量。
5.根据权利要求1所述的应用，其中，所述组合物还用于增加肠道中双歧杆菌的数量。
6.根据权利要求1所述的应用，其中，所述组合物还用于能够抑制肠道中脱硫弧菌和/或肠杆菌属的数量。
7.根据权利要求1或4或5或6所述的应用，其中，所述副干酪乳酸杆菌的应用量为1.0×103CFU~1.0×1010CFU /kg体重/天。
8.根据权利要求7所述的应用，其中，所述副干酪乳酸杆菌的应用量为1.0×104CFU~1.0×109CFU /kg体重/天。
9.根据权利要求1或4或5所述的应用，其中，所述组合物为食品组合物，所述食品为发酵乳制品。
10.根据权利要求1或4或5所述的应用，其中，所述组合物为食品组合物，所述食品为乳酪、含乳饮料、固体饮料或乳粉。
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| EP19864086.4A EP3811955A4 (en) | 2018-09-30 | 2019-09-23 | NEW USE OF LACTOBACILLUS PARACASEI K56 THAT IS ABLE TO REGULATE THE REST OF THE GASTROINTESTINAL FLORA |
| PCT/CN2019/107220 WO2020063515A1 (zh) | 2018-09-30 | 2019-09-23 | 可调节胃肠道菌群平衡的副干酪乳杆菌k56的新应用 |
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| CN112869168B (zh) * | 2019-11-29 | 2023-06-06 | 内蒙古伊利实业集团股份有限公司 | 可提高胃肠道免疫能力的益生菌益生元组合物及其应用 |
| CN113712207B (zh) * | 2020-05-21 | 2023-06-06 | 内蒙古伊利实业集团股份有限公司 | 提高胃肠道免疫能力的益生菌益生元组合物及其应用 |
| CN111849810B (zh) * | 2020-07-15 | 2021-04-20 | 浙江大学 | 拮抗幽门螺旋杆菌的乳酸菌zjuids03及其应用 |
| CN114504600A (zh) * | 2020-11-16 | 2022-05-17 | 内蒙古伊利实业集团股份有限公司 | 副干酪乳杆菌k56在抗衰老、提高先天免疫方面的新应用 |
| CN113355375B (zh) * | 2020-11-26 | 2022-11-22 | 内蒙古伊利实业集团股份有限公司 | 副干酪乳杆菌k56发酵合成b族维生素的方法及应用 |
| CN114621888B (zh) * | 2020-12-10 | 2024-04-02 | 青岛蔚蓝生物股份有限公司 | 一株具有调节肠道免疫失调症状的副干酪乳酪杆菌及其应用 |
| CN112386615B (zh) * | 2021-01-19 | 2021-04-06 | 山东中科嘉亿生物工程有限公司 | 抑制幽门螺旋杆菌感染的副干酪乳杆菌jlpf-176的应用及产品 |
| CN114404458B (zh) * | 2022-01-12 | 2024-01-19 | 诺佰克（武汉）生物科技有限公司 | 副干酪乳杆菌nbk-LC16在改善幽门螺杆菌感染及制备抗炎护胃产品中的应用 |
| CN115998777B (zh) * | 2022-10-18 | 2023-11-17 | 汤臣倍健股份有限公司 | 副干酪乳杆菌207-27的用途 |
| CN116059258A (zh) * | 2022-11-22 | 2023-05-05 | 重庆市天友乳业股份有限公司 | 副干酪乳杆菌在降低血清胆固醇水平制剂中的应用 |
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