CN117327612A - 一种能够增加肠道蠕动性的益生菌及其应用 - Google Patents
一种能够增加肠道蠕动性的益生菌及其应用 Download PDFInfo
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Abstract
本发明属于微生物技术领域,具体涉及一种能够增加肠道蠕动性的益生菌及其应用。该益生菌命名为WCFM1001,分类学命名为长双歧杆菌长亚种(Bifidobacterium longum subsp.longum),2023年7月24日保藏在中国典型培养物保藏中心,保藏编号为CCTCC M 20231366。WCFM1001在小鼠肠道中具有较强的定植能力,能够显著增强小鼠肠道蠕动性,缓解便秘症状;WCFM1001不影响小鼠体重变化,不影响小鼠血清中谷丙转氨酶和谷草转氨酶的水平,生物安全性好。本发明筛选得到的益生菌有潜力发展成为治疗便秘的药物。
Description
技术领域
本发明属于微生物技术领域,具体涉及一种能够增加肠道蠕动性的益生菌及其应用。
背景技术
便秘是一类常见的胃肠道疾病,其临床症状主要表现为:排便困难,排便不尽感,排便费时或需手法辅助排便,自发排便次数少于3次/周。目前,全球便秘的患病率约为4-10%(中国慢性便秘专家共识意见(2019,广州),且呈逐年上升趋势,许多儿童与青少年也同样面临者便秘的风险;随着年龄的增长,患病率不断上升,老年人患病率甚至高达20%(柯美云,王英凯.老年人慢性便秘的流行病学和研究进展[J].实用老年医学,2010,24(2):92-94)。国外的一项流行病学调查结果显示,便秘的症状谱以排便费力最为常见(81.0%),其他症状依次为粪便干硬(71.5%)、排便不尽(54.2%)、直肠堵塞感(38.8%)、腹胀(36.7%)、排便次数减少(35.6%)和需辅助排便(28.4%)(An epidemiological surveyof constipation in Canada:definitions,rates,demographics,and predictors ofhealth care seeking[J].Am J Gastroenterol,2001,96(11):3130-3137)。
长期的排便困难轻则引起痔疮、胀气、失眠、内分泌失调、肥胖和失血性贫血等,重则可能诱发或者加重高血脂、心脑血管疾病、动脉硬化、肿瘤等疾病及其并发症。因便秘发病率高、病因复杂,患者常有许多苦恼,长期便秘严重影响患者健康和生活质量。
目前便秘的治疗方式分为两种,通过改变生活方式的保守治疗和药物治疗。保守治疗包括调整膳食结构,多吃粗纤维,形成良好的生活习惯,培养正常的肠道习惯,但见效慢。药物治疗包括使用粪便软化剂、刺激性泻剂、渗透性泻剂和膨账剂等,这些药物短期内十分有效,但是长期使用会产生耐药性、药效减弱,同时常会伴随腹部绞痛,账气,电解质素乱等副作用。
有研究表明便秘患者的肠道菌群紊乱,有益菌群减少,而潜在致病菌和真菌增加,从属水平水看,一些潜在的致病菌(肠球菌、梭杆菌等)丰度升高,而拟杆菌丰度降低。这个现象提示改变肠道菌可能成为一个治疗便秘的有效治疗手段。专利CN115287207A公开了一种有助于缓解便秘的乳双歧杆菌,专利CN114703102A公开了一种有助于缓解便秘的两双歧杆菌,专利CN114350577A公开了一种改善便秘的动物双歧杆菌乳亚种BLa36。
但由于每个人肠道菌群存在差异,某种特定的益生菌并不能对所有人都有效,所以扩大能缓解便秘的益生菌的种属多样性十分必要,这将能提高患者可选的多样性和治疗的有效性。
发明内容
为了解决现有技术中存在的问题,本发明的目的之一在于一种能够增加肠道蠕动性的益生菌。
该益生菌命名为WCFM1001,分类学命名为长双歧杆菌长亚种(Bifidobacteriumlongum subsp.longum),2023年7月24日保藏在中国典型培养物保藏中心,保藏地址为湖北省武汉市武昌区八一路299号,保藏编号为CCTCC M 20231366。
优选的,所述益生菌WCFM1001的16s rDNA的序列如SEQ ID No.1所示。
本发明的目的之二在于提供一种如上所述的能够增加肠道蠕动性的益生菌WCFM1001的培养方法,所述培养方法包括将益生菌WCFM1001接种于MRS固体培养基上进行培养。
本发明的目的之三在于提供一种如上所述的能够增加肠道蠕动性的益生菌WCFM1001在制备治疗和/或预防便秘的药物中的应用。
本发明还提供一种治疗和/或预防便秘的药物,所述药物包括如上所述的能够增加肠道蠕动性的益生菌WCFM1001。
优选的,所述药物中益生菌WCFM1001含量大于1×108CFU/g或大于1×108CFU/mL。
优选的,所述药物还包括药学上可接受的载体,所述载体为赋形剂、稳定剂、崩解剂、着色剂、矫味剂中的任意一种或几种。
优选的,所述药物为颗粒剂、胶囊剂、片剂、丸剂或口服液剂型。
本发明的目的之四在于提供一种如上所述的能够增加肠道蠕动性的益生菌WCFM1001在制备食品中的应用。
本发明还提供一种食品组合物,所述食品组合物包含如上所述的能够增加肠道蠕动性的益生菌WCFM1001。
本发明的有益效果在于:
本发明提供的益生菌从中国健康人体内分离得到,具有较好的安全性。
试验证明,该益生菌在小鼠肠道中具有较强的定植能力,能够显著增强小鼠肠道蠕动性,缓解便秘症状。该益生菌不影响小鼠体重变化,不影响小鼠血清中谷丙转氨酶和谷草转氨酶的水平,生物安全性好。
本发明筛选得到的益生菌能够作为食品成分在制备食品中使用,并且该益生菌有潜力发展成为治疗和/或预防便秘的药物。
附图说明
图1为本发明提供的长双歧杆菌长亚种在RMS培养基上的菌落形态;
图2为本发明提供的长双歧杆菌长亚种对小鼠体重变化的影响;
图3为本发明提供的长双歧杆菌长亚种对小鼠血清中谷丙转氨酶含量的影响;
图4为本发明提供的长双歧杆菌长亚种对小鼠血清中谷草转氨酶含量的影响;
图5为本发明提供的长双歧杆菌长亚种对小鼠肠道蠕动性的影响;
图6为本发明提供的长双歧杆菌长亚种对小鼠粪便含水量的影响。
具体实施方式
下面结合实施例对本发明技术方案做出更为具体的说明。
如无特殊说明,本文中用到的各种原材料、试剂、仪器和设备等均可通过市场购买得到或者可通过现有方法制备得到。
实施例1
菌种分离
该株功能菌从年龄0-6月的中国健康志愿者的粪便中分离得到。
具体地:取粪便1g,用PBS缓冲液将粪便重悬并匀浆,接着用PBS缓冲液将其进行梯度稀释,取100ul粪便悬浊液加入900μl PBS中,制备成10-1混合液,并取部分分别稀释至10-5,10-6,10-7,每个梯度取100μl均匀涂布于MRS固体培养基中,放入厌氧培养箱中进行培养,培养48-56小时,直至有明显菌落出现。通过16s rDNA通用引物进行PCR扩增,扩增产物送测序,通用生物公司测序16s rDNA序列,使用EzBioCloud比对确定种属。
上述MRS固体培养基的制备:大豆蛋白胨10g、牛肉膏5g、酵母粉5g、葡萄糖20g、Tween80 1ml、磷酸二氢钠2g、无水乙酸钠5g、柠檬酸三胺2g、硫酸锰0.02g、硫酸镁0.1g,蒸馏水1L,调节pH至6.2左右,琼脂15g,在121℃下,灭菌15min,倒平板冷却备用。
挑培养基上单克隆至相应的液体培养基,待菌株生长至明显浑浊,通过16s rDNA通用引物进行PCR扩增,扩增产物送测序,通用生物公司测序16s rDNA序列比对确定种属。使用终浓度为20%的甘油,保存至-80℃冰箱备用。
通过对健康人粪便的分离,得到了十几个种属的肠道菌,分别验证了它们对小鼠便秘改善作用,其中本发明长双歧杆菌长亚种效果显著,以下仅阐述长双歧杆菌长亚种的功能。
将冻存的长双歧杆菌长亚种划线于MRS平板中,厌氧培养,直至长出单克隆,挑单克隆至液体培养基进行活化,并按照上述步骤进行测序,并将菌株16S rRNA基因序列提交到NCBI数据库进行BLAST比对,确定菌株为长双歧杆菌长亚种(Bifidobacterium longumsubsp.longum),菌株16S rRNA基因序列如SEQ ID No.1所示。
如图1所示,长双歧杆菌长亚种在MRS培养基上生长形成不透明乳白色菌落,圆形,表面光滑,菌落较小。
筛选得到的长双歧杆菌长亚种命名为WCFM1001,送至中国典型培养物保藏中心保藏,保藏编号CCTCC M 20231366。
实施例2
长双歧杆菌长亚种WCFM1001(以下简称为长双歧杆菌)的安全性测试
将24只6-8周龄的SPF级C57/B6j雄性小鼠(集萃药康购入)随机分为四组,一组为正常组+生理盐水组(6只),一组为正常组+长双歧杆菌组(6只),一组为便秘组+生理盐水组(6只),一组为便秘组+长双歧杆菌(6只)。
小鼠适应环境后,从第0天,正常组给予普通小鼠饲料喂养,便秘组给予采用盐酸洛哌丁胺诱导慢传输型便秘模型:以盐酸洛哌丁胺50mg/kg的剂量连续灌胃7天,灌胃频率均为每天一次,通过结肠排钢珠时间,胭脂红染料排便时间的实验检测,该模型下小鼠在一周时出现显著的排钢珠时间延长,胭脂红染料排出时间延长现象,提示小鼠存在肠道蠕动性减慢的便秘症状。
造模成功后,通过灌胃的方式对生理盐水组给予无菌PBS,对长双歧杆菌组给予数量为1×109CFU/mL的长双歧杆菌,灌胃频率均为每天一次,每只鼠每次200μL,为期一周。
监测小鼠的体重变化,每天记录小鼠的体重数据,绘制体重曲线。结果如图2中所示的。实验结果显示,四组小鼠的体重变化均无明显差异,证实长双歧杆菌不影响小鼠的正常体重增长。
小鼠摘眼球取全血,室温静置10分钟,3000rpm离心10分钟,取上层血清,利用谷丙转氨酶(ALP/GPT南京建成货号C009-2-1)和谷草转氨酶(AST/GPT南京建成货号C008-2-1)测试盒,检测小鼠血清中谷丙转氨酶和谷草转氨酶的水平。实验结果表明,四组小鼠的血清中谷丙转氨酶(图3)和谷草转氨酶(图4)的水平均无显著差异,证实本长双歧杆菌具有生物安全性。
实施例3
长双歧杆菌长亚种WCFM1001(以下简称为长双歧杆菌)对小鼠肠道蠕动性的影响
将24只6-8周龄的SPF级C57/B6j雄性小鼠(集萃药康购入)随机分为四组,一组为正常组+生理盐水组(6只),一组为正常组+长双歧杆菌组(6只),一组为便秘组+生理盐水组(6只),一组为便秘组+长双歧杆菌(6只)。
小鼠适应环境后,开始构建小鼠便秘模型,小鼠便秘模型同实施例2,造模成功后,通过灌胃的方式对生理盐水组给予无菌PBS,对长双歧杆菌组给予数量为1×108CFU/mL的长双歧杆菌,灌胃频率均为每天一次,持续2周。第2周进行小鼠的肠道蠕动性测试。具体地,小鼠禁食12小时,配制胭脂红染料(6%胭脂红+0.5%甲基纤维素溶于无菌水中),每只小鼠灌胃200μL该染料。然后将小鼠放在洁净笼盒中,观察并记录小鼠排出第一颗红色粪便的时间,比较各组小鼠直接排出红色粪便的时间。
结果如图5所示。实验结果表明,给予长双歧杆菌处理的便秘组的肠道蠕动性有明显增强,证实该株功能菌能够增强小鼠肠道蠕动性。而正常组灌胃长双歧杆菌的小鼠肠道蠕动性无明显变化,证明长双歧杆菌并不会影响小鼠的正常肠道功能。
实施例4
长双歧杆菌长亚种WCFM1001(以下简称为长双歧杆菌)对小鼠粪便含水量的影响
粪便含水量反应了粪便的质量和便秘程度,含水量越低说明便秘症状越明显。对实施例3中的小鼠在第2周进行小鼠的粪便含水量测算。将小鼠单只放入垫有吸水纸的笼盒中,收集粪便,称重即为湿重,冻干后,即为干重,按照公式计算粪便含水量:
粪便含水量(%)=(粪便湿重-粪便干重)/粪便湿重。
结果如图6所示,实验结果表明,给予长双歧杆菌的便秘组小鼠的粪便含水量显著高于生理盐水组。而正常组灌胃长双歧杆菌的小鼠粪便含水量无明显变化,证明长双歧杆菌并不会影响小鼠的正常肠道功能。
以上实验表明本发明提供的长双歧杆菌长亚种WCFM1001能够促进小鼠肠道蠕动,缓解便秘,同时不会影响小鼠体重或血清中谷丙转氨酶和谷草转氨酶的水平,具有较好的生物安全性。表明本长双歧杆菌长亚种WCFM1001有潜力发展成为治疗便秘的药物。
需要强调的是,本发明提供的长双歧杆菌长亚种WCFM1001还可以与其他菌剂或具有缓解便秘或其它协同效果的成分复配使用;尽管本发明的长双歧杆菌长亚种WCFM1001以小鼠为实验对象,但将本长双歧杆菌长亚种WCFM1001或含有本长双歧杆菌长亚种WCFM1001的药物用于其他生物体上的实施均应视为本发明的一种实施方式。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。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 (10)
1.一种能够增加肠道蠕动性的益生菌,其特征在于,该益生菌命名为WCFM1001,分类学命名为长双歧杆菌长亚种(Bifidobacterium longum subsp.longum),2023年7月24日保藏在中国典型培养物保藏中心,保藏编号为CCTCC M 20231366。
2.如权利要求1所述的一种能够增加肠道蠕动性的益生菌,其特征在于,益生菌WCFM1001的16s rDNA的序列如SEQ ID No.1所示。
3.一种如权利要求1所述的能够增加肠道蠕动性的益生菌WCFM1001的培养方法,其特征在于,所述培养方法包括将益生菌WCFM1001接种于MRS固体培养基上进行培养。
4.一种如权利要求1所述的能够增加肠道蠕动性的益生菌WCFM1001在制备治疗和/或预防便秘的药物中的应用。
5.一种治疗和/或预防便秘的药物,其特征在于,所述药物包括如权利要求1所述的能够增加肠道蠕动性的益生菌WCFM1001。
6.如权利要求5所述的药物,其特征在于,所述药物中益生菌WCFM1001含量大于1×108CFU/g或大于1×108CFU/mL。
7.如权利要求5所述的药物,其特征在于,所述药物还包括药学上可接受的载体,所述载体为赋形剂、稳定剂、崩解剂、着色剂、矫味剂中的任意一种或几种。
8.如权利要求5-7任一项所述的药物,其特征在于,所述药物为颗粒剂、胶囊剂、片剂、丸剂或口服液剂型。
9.一种如权利要求1所述的能够增加肠道蠕动性的益生菌WCFM1001在制备食品中的应用。
10.一种食品组合物,其特征在于,所述食品组合物包含如权利要求1所述的能够增加肠道蠕动性的益生菌WCFM1001。
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