CN113005049A - 一株可缓解腹泻的短双歧杆菌及其应用 - Google Patents
一株可缓解腹泻的短双歧杆菌及其应用 Download PDFInfo
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- CN113005049A CN113005049A CN202011608651.0A CN202011608651A CN113005049A CN 113005049 A CN113005049 A CN 113005049A CN 202011608651 A CN202011608651 A CN 202011608651A CN 113005049 A CN113005049 A CN 113005049A
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Abstract
本发明公开了一株可缓解腹泻的短双歧杆菌及其应用,属于微生物技术领域。本发明筛选出了一株短双歧杆菌CCFM1151,此短双歧杆菌CCFM1151具有缓解由ETEC引起的腹泻的作用,具体体现在:(1)显著降低腹泻小鼠的体重损失;(2)显著降低腹泻小鼠的粪便含水量;(3)显著降低腹泻小鼠血清中的促炎因子水平;(4)显著提高腹泻小鼠粪便中短链脂肪酸的含量;(5)显著提高腹泻小鼠肠道菌群多样性,因此,短双歧杆菌CCFM1151在制备预防和/或治疗由ETEC引起的腹泻的产品(如食品或药品等)中,具有巨大的应用前景。
Description
技术领域
本发明涉及一株可缓解腹泻的短双歧杆菌及其应用,属于微生物技术领域。
背景技术
根据流行病学分析,腹泻是最常见的消化系统疾病之一,细菌腹泻是占据主要地位,其中最常见的细菌性痢疾病原体包括大肠杆菌、沙门氏菌、志贺氏菌和金黄色葡萄球菌。产肠毒素大肠杆菌(ETEC)是旅行者腹泻以及发展中国家儿童特有的腹泻和发育迟缓的主要原因。 ETEC产生热不稳定(LT)和热稳定(ST)肠毒素,与抗霍乱毒素的结构和功能相似,刺激细胞中cAMP的合成,并通过在小肠中定殖而引起人和其他动物的腹泻。
对于治疗腹泻,使用抗生素是一种有效的手段。在确定以ETEC为最常见病因的旅行者腹泻的治疗中,红霉素、诺氟沙星、环丙沙星和氧氟沙星等抗生素均十分有效。然而,由于儿童腹泻不仅是由ETEC引起,还有可能由其他细菌和病毒因子引起,并且,临床表现不足以区分它们,因此,很难研究抗生素对ETEC病患儿童的作用,同时,抗生素也并不是一种治疗儿童腹泻的常规方法。另外,服用抗生素还存在副作用大、成本高、ETEC易出现耐药性等问题。上述缺陷均使得这些抗生素的使用受到限制,而替代方法的有效性还在研究中。
除此之外,对于预防腹泻,常使用灭活的全细胞疫苗全细胞疫苗主要是为预防霍乱而设计,该疫苗包含霍乱毒素的重组B亚基,其抗原性类似于ETEC的热不稳定毒素。但是,目前的实验结果不足以支持口服霍乱疫苗可以预防由ETEC引起的腹泻,这可能是因为ETEC血清型的O和H抗原多样性导致。
益生菌是通过定殖在人体内,改变宿主肠道菌群组成,进而代谢产生如短链脂肪酸等有益代谢物,以对宿主产生有益影响的一类菌。与普通的药物相比,益生菌具有安全性高、无副作用、不会出现耐药性和成本低等优势。并且,研究表明,少数益生菌确实可对一些特殊的疾病起到预防和/或治疗作用,例如,公开号为CN108220206A的专利申请文本中,长双歧杆菌YS108R就可很好的预防和/或治疗结肠炎。因此,急需找到一株可缓解由ETEC引起的腹泻的益生菌。
发明内容
[技术问题]
本发明要解决的技术问题是提供一株可缓解由ETEC引起的腹泻的短双歧杆菌(Bifidobacterium breve)。
[技术方案]
为解决本发明的技术问题,本发明提供了一株短双歧杆菌(Bifidobacteriumbreve) CCFM1151,分类命名为Bifidobacterium breve,已于2020年12月10日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61199,保藏地址为广州市先烈中路100号大院59 号楼5楼。
所述短双歧杆菌CCFM1151是从来源于河南封丘地区的桦甸泡菜样本中分离得到的,该菌株经测序分析,其16S rDNA序列如SEQ ID NO.1所示,将测序得到的序列在NCBI中进行核酸序列比对,结果显示菌株为短双歧杆菌,命名为短双歧杆菌(Bifidobacteriumbreve) CCFM1151。
所述短双歧杆菌CCFM1151在MRS培养基上的菌落呈现乳白色、表面光滑、圆形凸起、有光泽。
本发明还提供了所述短双歧杆菌CCFM1151制备预防和/或治疗腹泻的产品中的应用。
在本发明的一种实施方式中,所述产品中,上述短双歧杆菌CCFM1151的活菌数为不低于1×109CFU/mL或1×109CFU/g。
在本发明的一种实施方式中,所述产品包含食品或药品。
在本发明的一种实施方式中,所述药品含有上述短双歧杆菌CCFM1151、药物载体和/或药用辅料。
本发明的一种实施方式中,所述食品包含含有上述短双歧杆菌CCFM1151的保健食品;或所述食品包含使用含有上述短双歧杆菌CCFM1151的发酵剂生产得到的乳制品、豆制品、肉制品或果蔬制品。
在本发明的一种实施方式中,所述发酵剂的制备方法为将上述短双歧杆菌CCFM1151按照占培养基总质量2~4%的接种量接种到培养基中,于37℃下培养24~48h,得到培养液;将培养液离心,得到菌体;将菌体用生理盐水重悬,得到发酵剂。
在本发明的一种实施方式中,所述培养基为MRS培养基。
在本发明的一种实施方式中,所述腹泻为由ETEC引起的旅行者腹泻。
本发明还提供了一种用于预防和/或治疗腹泻的产品,所述产品含有上述短双歧杆菌 CCFM1151。
在本发明的一种实施方式中,所述产品中,上述短双歧杆菌CCFM1151的活菌数为不低于1×109CFU/mL或1×109CFU/g。
在本发明的一种实施方式中,所述产品包含食品或药品。
在本发明的一种实施方式中,所述药品含有上述短双歧杆菌CCFM1151、药物载体和/或药用辅料。
在本发明的一种实施方式中,所述食品包含含有上述短双歧杆菌CCFM1151的保健食品;或所述食品包含使用上述短双歧杆菌CCFM1151的发酵剂生产得到的乳制品、豆制品、肉制品或果蔬制品。
在本发明的一种实施方式中,所述发酵剂的制备方法为将上述短双歧杆菌CCFM1151按照占培养基总质量2~4%的接种量接种到培养基中,于37℃下培养24~48h,得到培养液;将培养液离心,得到菌体;将菌体用生理盐水重悬,得到发酵剂。
在本发明的一种实施方式中,所述培养基为MRS培养基。
在本发明的一种实施方式中,所述腹泻为由ETEC引起的旅行者腹泻。
[有益效果]
1、本发明筛选出了一株短双歧杆菌(Bifidobacterium breve)CCFM1151,此短双歧杆菌 CCFM1151具有缓解由ETEC引起的腹泻的作用,具体体现在:
(1)显著降低腹泻小鼠的体重损失;
(2)显著降低腹泻小鼠的粪便含水量;
(3)显著降低腹泻小鼠血清中的促炎因子水平;
(4)显著提高腹泻小鼠粪便中短链脂肪酸的含量;
(5)显著提高腹泻小鼠肠道菌群多样性,
因此,短双歧杆菌CCFM1151在制备预防和/或治疗由ETEC引起的腹泻的产品(如食品或药品等)中,具有巨大的应用前景。
2、短双歧杆菌是益生菌的一种,目前已被纳入卫生部下发的《可用于食品的菌种名单》,因此,本发明筛选得到的短双歧杆菌CCFM1151不会给腹泻患者带来任何潜在的安全隐患。
3、短双歧杆菌的培育过程仅需培养基以及一些培养条件的控制,成本相对低廉,不会给腹泻患者带来太大的经济负担。
生物材料保藏
一株短双歧杆菌(Bifidobacterium breve)CCFM1151,分类学命名为Bifidobacterium breve,已于2020年12月10日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61199,保藏地址为广州市先烈中路100号大院59号楼5楼。
附图说明
图1:不同组小鼠的体重变化情况。
图2:不同组小鼠粪便含水量的变化情况。
图3:不同组小鼠血清中IFN-γ的含量。
图4:不同组小鼠血清中TNF-α的含量。
图5:不同组小鼠粪便中乙酸的含量。
图6:不同组小鼠粪便中丙酸的含量。
图7:不同组小鼠粪便中丁酸的含量。
图8:不同组小鼠粪便中戊酸的含量。
图9:不同组小鼠肠道菌群的α多样性。
图10:不同组小鼠肠道菌群的β多样性。
具体实施方式
下面结合具体实施例和附图对本发明进行进一步的阐述。
下述实施例中涉及的BALB/c小鼠购自浙江维通利华公司;下述实施例中涉及的链霉素购自上海生工生物工程(上海)股份有限公司;下述实施例中涉及的ETEC O78:K80购自中国工业微生物菌种保藏管理中心;下述实施例中涉及的鼠李糖乳杆菌(Lactobacillusrhamnosus) GG由江南大学食品学院生物技术中心分离得到;下述实施例中涉及的环丙沙星购自生工生物工程(上海)股份有限公司;下述实施例中涉及的检测IFN-γ(货号:ML720140-2)和TNF-α (货号:ML720852-2)的ELISA试剂盒购自上海酶联生物科技有限公司。
下述实施例中涉及的培养基如下:
MRS固体培养基:蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4 0.05g/L、吐温80 1mL/L、琼脂15g/L、半胱氨酸氨酸盐0.5g/L。
MRS液体培养基:蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4 0.05g/L、吐温80 1mL/L、半胱氨酸氨酸盐0.5g/L。
实施例1:短双歧杆菌CCFM1151的筛选及菌种鉴定
1、筛选
以来源于河南封丘地区的健康人体粪便样本,取0.5g保存于30%(v/v)甘油中的样本在无菌环境下加入装有4.5mL生理盐水(含有0.5g/L半胱氨酸)的10mL离心管中,得到10-1稀释液,重复上述稀释步骤,依次得到10-2、10-3、10-4、10-5、10-6稀释液;分别吸取100 μL不同梯度的梯度稀释液涂布于MRS固体培养基(含有0.5g/L半胱氨酸)上,37℃培养72h,得到稀释涂布平板;挑取稀释涂布平板上的典型菌落分别在MRS固体培养基(含有0.5 g/L半胱氨酸)上划线,37℃培养48h,得到纯化菌落;挑取纯化菌落接种至MRS液体培养基(含有0.5g/L半胱氨酸)中,37℃培养48h,得到菌株CCFM1151。
2、鉴定
提取CCFM1151的基因组,将CCFM1151的16S rDNA进行扩增和测序(由上海生工生物工程股份有限公司完成),将测序分析得到的CCFM1151的16S rDNA序列(CCFM1151 的16SrDNA序列如SEQ ID NO.1所示)在GenBank中进行比对,结果显示此菌株均为短双歧杆菌,命名为短双歧杆菌(Bifidobacterium breve)CCFM1151。
实施例2~5中,乳酸菌菌液的制备方法如下:
蘸取乳酸菌菌液在MRS固体培养基上划线,37℃培养48h,得到单菌落;挑取单菌落接入MRS液体培养基中,于37℃培养24h,得到活化液;将活化液按照1%(v/v)的接种量接入MRS液体培养基中,于37℃培养24h,得到一级种子液;将一级种子液按照1%(v/v) 的接种量接入MRS液体培养基中,于37℃培养24h,得到二级种子液;将二级种子液按照 1%(v/v)的接种量接入MRS液体培养基中,于37℃培养24h,得到菌液;将菌液6000g 离心15min,收集沉淀;将沉淀用pH为7.4的PBS缓冲液洗涤两次后,6000g再次离心10min,得到菌体;用含有130g/L脱脂乳、20g/L海藻糖和20g/L蔗糖的保护剂溶液将乳酸菌菌体重悬至细胞浓度为5×109CFU/mL,得到乳酸菌菌液。
研究证明鼠李糖LGG菌株是存在于健康人体内的益生菌类型(主要存在于人类的消化道环境中)。自分离出鼠李糖LGG菌株以来,已进行了广泛的研究,它在调整和改善胃肠道功能、预防和减少腹泻、提高机体免疫力、预防龋齿等诸多方面都具有作用。鼠李糖LGG已成为商业化菌株并且已有文献报道鼠李糖LGG在缓解ETEC引起的腹泻的有益作用。同时,环丙沙星用于治疗由细菌感染引起的感染性的腹泻已被人们所熟知,比如治疗由志贺菌属,大肠埃希菌等引起的感染性腹泻有一定的效果。因此,下述实施例中以鼠李糖LGG和环丙沙星作为菌株的阳性对照。
实施例2:短双歧杆菌CCFM1151对腹泻小鼠体重和粪便含水量的影响
具体步骤如下:
取3~4周龄的雌性无病原体(SPF)BALB/c小鼠48只,于饲养室温为22~24℃,湿度为40~60%,12h/12h昼夜交替,自由进食及饮水的条件下饲养1周后,随机分为6组,每组 8只,6组分别为:对照组、模型组、灌胃环丙沙星的药物组、灌胃鼠李糖乳杆菌(Lactobacillus rhamnosus)GG的LGG组、灌胃短双歧杆菌(Bifidobacterium breve)CCFM1151菌液的 CCFM1151组、灌胃短双歧杆菌(Bifidobacterium breve)FJSWX38M7菌液的FJSWX38M7 组,其中短双歧杆菌FJSWX38M7按照实施例1相同的筛选方法筛选自江苏无锡的婴幼儿粪便样品,其16S rDNA序列如SEQ ID NO.2所示,菌液的制备方法同实施例1。
实验共3周:动物适应性饲养1周后开始实验。从造模前7天开始,一直持续到实验结束(不包括抗生素处理的那3天),对照组和模型组小鼠每只每天灌胃0.2mL含有130g/L脱脂乳、20g/L海藻糖和20g/L蔗糖的保护剂溶液,药物组每只每天灌胃0.2mL浓度为1g/L的环丙沙星水溶液(无菌),LGG组每只每天灌胃0.2mL鼠李糖乳杆菌(Lactobacillusrhamnosus)GG菌液,CCFM1151组每只每天灌胃0.2mL短双歧杆菌(Bifidobacterium breve)CCFM1151菌液,FJSWX38M7组每只每天灌胃0.2mL短双歧杆菌(Bifidobacterium breve)FJSWX38M7菌液;第2周到第3周为造模期,造模第1~3天,在小鼠饮用水中加入5g/L的链霉素,以消除BALB/c小鼠肠道正常菌群,造模第4~7天,用不含抗生素的无菌水代替含有链霉素的水作为小鼠饮用水,并且,对小鼠禁食12h,12h后,给每组小鼠每只灌胃0.2mL 浓度为1.2×1011CFU/mL的ETEC O78:K80悬液(通过将ETEC O78:K80菌体溶于浓度为 8.5g/L的生理盐水而得),连续灌胃4天,1天2次,每次间隔2h。
造模期间以及造模结束后,通过体重计测定每组小鼠体重;实验结束后,收集小鼠粪便,通过Lyobeta 5ps冻干机(西班牙泰事达公司)测定小鼠粪便的含水量,测定结果分别见图1~2。
由图1可知,在第4天造模前,由于禁食12h,各组小鼠的体重均明显下降,恢复饮食后,各组小鼠的体重均明显上升;第3周时,模型组小鼠的体重显著低于对照组小鼠(体重比降低了5%),CCFM1151组小鼠的体重均显著高于模型组小鼠(体重比提高了7%),其中,对照组、药物组、LGG组、CCFM1151组和FJSWX38M7组和模型组小鼠的体重比(第 21天与第14天的体重之比)分别为1.01、0.98、0.97、1.03和1.00。
由图2可知,对照组和CCFM1151组小鼠的粪便含水量显著低于模型组小鼠(分别为58%和52%),药物组、LGG组和FJSWX38M7组小鼠的粪便含水量分别为61%、65%、59%,与模型组无显著性差异。
可见,短双歧杆菌CCFM1151可有效缓解腹泻小鼠体重降低、粪便含水量升高的症状,且效果优于环丙沙星、鼠李糖乳杆菌(Lactobacillus rhamnosus)GG和短双歧杆菌(Bifidobacterium breve)FJSWX38M7。
实施例3:短双歧杆菌CCFM1151对腹泻小鼠血清中促炎因子水平的影响
具体步骤如下:
取3~4周龄的雌性无病原体(SPF)BALB/c小鼠48只,于饲养室温为22~24℃,湿度为40~60%,12h/12h昼夜交替,自由进食及饮水的条件下饲养1周后,随机分为6组,每组 8只,6组分别为:对照组、模型组、灌胃环丙沙星的药物组、灌胃鼠李糖乳杆菌(Lactobacillus rhamnosus)GG的LGG组、灌胃短双歧杆菌(Bifidobacterium breve)CCFM1151菌液的 CCFM1151组、灌胃短双歧杆菌(Bifidobacterium breve)FJSWX38M7菌液的FJSWX38M7 组。
实验共3周:动物适应性饲养1周后开始实验。从造模前7天开始,一直持续到实验结束(不包括抗生素处理的那3天),对照组和模型组小鼠每只每天灌胃0.2mL含有130g/L脱脂乳、20g/L海藻糖和20g/L蔗糖的保护剂溶液,药物组每只每天灌胃0.2mL浓度为1g/L的环丙沙星水溶液(无菌),LGG组每只每天灌胃0.2mL鼠李糖乳杆菌(Lactobacillusrhamnosus)GG菌液,CCFM1151组每只每天灌胃0.2mL短双歧杆菌(Bifidobacterium breve)CCFM1151菌液,FJSWX38M7组每只每天灌胃0.2mL短双歧杆菌(Bifidobacterium breve)FJSWX38M7菌液;第2周到第3周为造模期,造模第1~3天,在小鼠饮用水中加入5g/L的链霉素,以消除BALB/c小鼠肠道正常菌群,造模第4~7天,用不含抗生素的无菌水代替含有链霉素的水作为小鼠饮用水,并且,对小鼠禁食12h,12h后,给每组小鼠每只灌胃0.2mL 浓度为1.2×1011CFU/mL的ETEC O78:K80悬液(通过将ETEC O78:K80菌体溶于浓度为 8.5g/L的生理盐水而得),连续灌胃4天,1天2次,每次间隔2h。
实验结束后,取血并处死小鼠,取小鼠血清,通过ELISA试剂盒测定每组小鼠血清中IFN-γ和TNF-α的含量,检测结果见图3~4。
如图3所示,模型组小鼠血清中IFN-γ的浓度为150.59pg/mL,和对照组(73.42pg/mL) 相比具有显著性变化;相比于模型组小鼠,LGG组和CCFM1151组小鼠血清中IFN-γ水平显著降低,分别为87.07pg/mL,86.71pg/mL,药物组和FJSWX38M7组则无显著性变化。
如图4所示,结果与IFN-γ水平类似,对照组、LGG组、CCFM1151组和FJSWX38M7 组小鼠血清中TNF-α的含量分别为118.42pg/mL,98.51pg/mL,110.17pg/mL和127.26pg/mL 均较模型组小鼠(188.26ng/L)显著性降低,其中,LGG组和CCFM1151组小鼠血清中TNF-α的含量降低的更为明显。
可见,短双歧杆菌(Bifidobacterium breve)CCFM1151和鼠李糖乳杆菌(Lactobacillus rhamnosus)GG均可降低由ETEC导致的腹泻小鼠血清中促炎因子IFN-γ和TNF-α的水平。
实施例4:短双歧杆菌CCFM1151对腹泻小鼠粪便中短链脂肪酸含量的影响
具体步骤如下:
取3~4周龄的雌性无病原体(SPF)BALB/c小鼠48只,于饲养室温为22~24℃,湿度为40~60%,12h/12h昼夜交替,自由进食及饮水的条件下饲养1周后,随机分为6组,每组 8只,6组分别为:对照组、模型组、灌胃环丙沙星的药物组、灌胃鼠李糖乳杆菌(Lactobacillus rhamnosus)GG的LGG组、灌胃短双歧杆菌(Bifidobacterium breve)CCFM1151菌液的 CCFM1151组、灌胃短双歧杆菌(Bifidobacterium breve)FJSWX38M7菌液的FJSWX38M7 组。
实验共3周:动物适应性饲养1周后开始实验。从造模前7天开始,一直持续到实验结束(不包括抗生素处理的那3天),对照组和模型组小鼠每只每天灌胃0.2mL含有130g/L脱脂乳、20g/L海藻糖和20g/L蔗糖的保护剂溶液,药物组每只每天灌胃0.2mL浓度为1g/L的环丙沙星水溶液(无菌),LGG组每只每天灌胃0.2mL鼠李糖乳杆菌(Lactobacillusrhamnosus)GG菌液,CCFM1151组每只每天灌胃0.2mL短双歧杆菌(Bifidobacterium breve)CCFM1151菌液,FJSWX38M7组每只每天灌胃0.2mL短双歧杆菌(Bifidobacterium breve)FJSWX38M7菌液;第2周到第3周为造模期,造模第1~3天,在小鼠饮用水中加入5g/L的链霉素,以消除BALB/c小鼠肠道正常菌群,造模第4~7天,用不含抗生素的无菌水代替含有链霉素的水作为小鼠饮用水,并且,对小鼠禁食12h,12h后,给每组小鼠每只灌胃0.2mL 浓度为1.2×1011CFU/mL的ETEC O78:K80悬液(通过将ETEC O78:K80菌体溶于浓度为 8.5g/L的生理盐水而得),连续灌胃4天,1天2次,每次间隔2h。
实验结束后,收集小鼠粪便置于液氮中,后转移至-80℃冰箱,在进行短链脂肪酸含量的检测前取出,进行真空冷冻干燥,准确称取0.05g冻干后的粪便样品溶解于0.5mL饱和氯化钠溶液中,浸泡30min,组织匀浆机匀浆,加入0.02mL浓度为10%的硫酸,震荡30s,在通风橱内向粪便溶液中准确加入0.8mL乙醚溶液,震荡30s后离心15min(8000g、4℃),移取上清液至含有0.25g无水硫酸钠的离心管中,震荡均匀,离心15min(8000g、4℃),取上清至气质容量瓶中,通过GCMS检测短链脂肪酸含量,检测结果见图5~8。
如图5~8所示,模型组小鼠粪便中乙酸、丙酸、丁酸和戊酸含量(分别为127.59g/mol, 10.32g/mol,3.32g/mol,2.25g/mol)较对照组小鼠(分别为199.32g/mol,19.03g/mol,4.45g/mol, 2.47g/mol)下降,其中,乙酸、丙酸、丁酸含量下降最为显著;CCFM1151组小鼠粪便中乙酸的含量(185.42g/mol)与模型组相比显著上调;药物组、LGG组和FJSWX38M7组小鼠粪便中乙酸、丙酸、丁酸和戊酸的含量则与模型组无显著差异。
可见,短双歧杆菌CCFM1151可显著提高腹泻小鼠粪便中短链脂肪酸的含量,环丙沙星、鼠李糖乳杆菌(Lactobacillus rhamnosus)GG和短双歧杆菌(Bifidobacteriumbreve)FJSWX38M7 则无此效果。
实施例5:短双歧杆菌CCFM1151对腹泻小鼠肠道菌群多样性的影响
具体步骤如下:
取3~4周龄的雌性无病原体(SPF)BALB/c小鼠48只,于饲养室温为22~24℃,湿度为40~60%,12h/12h昼夜交替,自由进食及饮水的条件下饲养1周后,随机分为6组,每组 8只,6组分别为:对照组、模型组、灌胃环丙沙星的药物组、灌胃鼠李糖乳杆菌(Lactobacillus rhamnosus)GG的LGG组、灌胃短双歧杆菌(Bifidobacterium breve)CCFM1151菌液的 CCFM1151组、灌胃短双歧杆菌(Bifidobacterium breve)FJSWX38M7菌液的FJSWX38M7 组。。
实验共3周:动物适应性饲养1周后开始实验。从造模前7天开始,一直持续到实验结束(不包括抗生素处理的那3天),对照组和模型组小鼠每只每天灌胃0.2mL含有130g/L脱脂乳、20g/L海藻糖和20g/L蔗糖的保护剂溶液,药物组每只每天灌胃0.2mL浓度为1g/L的环丙沙星水溶液(无菌),LGG组每只每天灌胃0.2mL鼠李糖乳杆菌(Lactobacillusrhamnosus)GG菌液,CCFM1151组每只每天灌胃0.2mL短双歧杆菌(Bifidobacterium breve)CCFM1151菌液,FJSWX38M7组每只每天灌胃0.2mL短双歧杆菌(Bifidobacterium breve)FJSWX38M7菌液;第2周到第3周为造模期,造模第1~3天,在小鼠饮用水中加入5g/L的链霉素,以消除BALB/c小鼠肠道正常菌群,造模第4~7天,用不含抗生素的无菌水代替含有链霉素的水作为小鼠饮用水,并且,对小鼠禁食12h,12h后,给每组小鼠每只灌胃0.2mL 浓度为1.2×1011CFU/mL的ETEC O78:K80悬液(通过将ETEC O78:K80菌体溶于浓度为 8.5g/L的生理盐水而得),连续灌胃4天,1天2次,每次间隔2h。
实验结束后,收集小鼠粪便,通过FastDNA Spin Kit(美国MP生物医药公司)提取粪便 中的基因组DNA,对提取得到的基因组DNA的V3-V4区进行特异性PCR扩增,16S rDNA测序,分析粪便菌群α多样性(Chao 1、Shannon和Simpson)和β多样性的变化,分析结果 见图9~10。
由图9可知,就Chao 1指数而言,对照组、药物组、LGG组和FJSWX38M7组和模型组相比均不存在显著性差异(p>0.05),但短双歧杆菌(Bifidobacterium breve)CCFM1151 能显著提高小鼠肠道群落的多样性;就Shannon指数和Simpson指数而言,模型组能显著降低样本中微生物的多样性,而药物组、LGG组、CCFM1151组和FJSWX38M7组较模型组均不存在显著性差异(p>0.05)。
由图10可知,模型组小鼠的肠道菌群与对照组小鼠显著不同;经过短双歧杆菌(Bifidobacterium breve)CCFM1151组和短双歧杆菌(Bifidobacterium breve)FJSWX38M7组的干预后,小鼠肠道菌群向对照组发生了移动,其他组别则与造模组的肠道菌群分布没有较大差异。
可见,短双歧杆菌CCFM1151可有效提高腹泻小鼠肠道菌群多样性,且效果远优于药物组和LGG组。
实施例6:短双歧杆菌CCFM1151的应用
具体步骤如下:
短双歧杆菌CCFM1151可用于制备片剂,片剂的具体制备过程如下:
挑取实施例1获得的短双歧杆菌(Bifidobacterium breve)CCFM1151的单菌落接入MRS 液体培养基中,于37℃培养24h,得到活化液;将活化液按照1%(v/v)的接种量接入MRS 液体培养基中,于37℃培养24h,得到一级种子液;将一级种子液按照1%(v/v)的接种量接入MRS液体培养基中,于37℃培养24h,得到二级种子液;将二级种子液按照1%(v/v)的接种量接入MRS液体培养基中,于37℃培养24h,得到菌液;将菌液6000g离心15min,收集沉淀;将沉淀用pH为7.4的PBS缓冲液洗涤两次后,6000g再次离心10min,得到菌体;用含有130g/L脱脂乳、20g/L海藻糖和20g/L蔗糖的保护剂溶液将短双歧杆菌菌体重悬至细胞浓度为1×1010CFU/mL,得到短双歧杆菌菌液;将短双歧杆菌菌液冷冻干燥,得到短双歧杆菌菌粉;在短双歧杆菌菌粉中加入占短双歧杆菌菌菌粉总重计2%的硬脂酸作润滑剂、3%的CMC-Na作粘合剂后进行压片,得到片剂。
取1g上述片剂每天灌胃腹泻小鼠,连续五周,可有效缓解小鼠腹泻的症状,在预防和/ 或治疗腹泻上有极好的效果。
实施例7:短双歧杆菌CCFM1151的应用
具体步骤如下:
短双歧杆菌CCFM1151可用于制备菌粉,菌粉的具体制备过程如下:
挑取实施例1获得的短双歧杆菌(Bifidobacterium breve)CCFM1151的单菌落接入MRS 液体培养基中,于37℃培养24h,得到活化液;将活化液按照1%(v/v)的接种量接入MRS 液体培养基中,于37℃培养24h,得到一级种子液;将一级种子液按照1%(v/v)的接种量接入MRS液体培养基中,于37℃培养24h,得到二级种子液;将二级种子液按照1%(v/v)的接种量接入MRS液体培养基中,于37℃培养24h,得到菌液;将菌液6000g离心15min,收集沉淀;将沉淀用pH为7.4的PBS缓冲液洗涤两次后,6000g再次离心10min,得到菌体;用含有130g/L脱脂乳、20g/L海藻糖和20g/L蔗糖的保护剂溶液将短双歧杆菌菌体重悬至细胞浓度为1×1010CFU/mL,得到短双歧杆菌菌液;将短双歧杆菌菌液冷冻干燥,得到菌粉。
取1g上述菌粉每天灌胃腹泻小鼠,连续一周,可有效缓解小鼠腹泻的症状,在预防和/ 或治疗腹泻上有极好的效果。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 一株可缓解腹泻的短双歧杆菌及其应用
<130> BAA200980A
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1527
<212> DNA
<213> Bifidobacterium breve
<400> 1
tcacgagcct caccttagac ggctccctcc cgcaaggggt taggccaccg gcttcgggtg 60
ctgcccactt tcatgacttg acgggcggtg tgtacaaggc ccgggaacgc attcaccgcg 120
acgttgctga ttcgcgatta ctagcgactc cgccttcacg cagtcgagtt gcagactgcg 180
atccgaactg agaccggttt tcagggatcc gctccagctc gcactgtcgc atcccgttgt 240
accggccatt gtagcatgcg tgaagccctg gacgtaaggg gcatgatgat ctgacgtcat 300
ccccaccttc ctccgagtta accccggcgg tcccccgtga gttcccggca caatccgctg 360
gcaacacggg gcgagggttg cgctcgttgc gggacttaac ccaacatctc acgacacgag 420
ctgacgacga ccatgcacca cctgtgaacc cgccccgaag ggaaacccca tctctgggat 480
cgtcgggaac atgtcaagcc caggtaaggt tcttcgcgtt gcatcgaatt aatccgcatg 540
ctccgccgct tgtgcgggcc cccgtcaatt tctttgagtt ttagccttgc ggccgtactc 600
cccaggcggg atgcttaacg cgttagctcc gacacggaac ccgtggaacg ggccccacat 660
ccagcatcca ccgtttacgg cgtggactac cagggtatct aatcctgttc gctccccacg 720
ctttcgctcc tcagcgtcag taacggccca gagacctgcc ttcgccattg gtgttcttcc 780
cgatatctac acattccacc gttacaccgg gaattccagt ctcccctacc gcactcaagc 840
ccgcccgtac ccggcgcgga tccaccgtta agcgatggac tttcacaccg gacgcgacga 900
accgcctacg agccctttac gcccaataat tccggataac gcttgcaccc tacgtattac 960
cgcggctgct ggcacgtagt tagccggtgc ttattcgaaa ggtacactca acacaaaatg 1020
ccttgctccc taacaaaaga ggtttacaac ccgaaggcct ccatccctca cgcggcgtcg 1080
ctgcatcagg cttgcgccca ttgtgcaata ttccccactg ctgcctcccg taggagtctg 1140
ggccgtatct cagtcccaat gtggccggtc gccctctcag gccggctacc cgtcgaagcc 1200
atggtgggcc gttaccccgc catcaagctg ataggacgcg accccatccc atgccgcaaa 1260
ggctttccca acacaccatg cagtgtgatg gagcatccgg cattaccacc cgtttccagg 1320
agctattccg gtgcatgggg caggtcggtc acgcattact cacccgttcg ccactctcac 1380
caccaagcaa gcttgatgga tcccgttcga cttgcatgtg ttaagcacgc cgccagcgtt 1440
catcctgagc cagaatcgaa ccctccacaa aaaaacttca tgaaaagcat ccgaacagat 1500
gactcacaaa acaaaaattg acgagca 1527
<210> 2
<211> 1053
<212> DNA
<213> Bifidobacterium breve
<400> 2
atgattgatg tgacgaatat ccaccagcca gtgctgcttg atgactgtgt gaatcttgtg 60
gcgccggcat tacaacacga aggtgccgtg gccgtggact gcacgctggg tcttgccggt 120
cattccaccg ctttcctgaa agcggcgccg caagctcgtc ttatcggtat tgaccgtgat 180
agtgaggcgt tggccttggc gactgagcgt atggttcagg aggggctttc cgaccgattc 240
acgccggttc atgcggcctt tgatcagctt gatcaggtgc ttgccgatca aggcgtggaa 300
cgagtggatg ccgtgttcat ggatctgggc ctgtcgagtt tgcagattga tgaaacggat 360
cgtggattct cgtactccca tgatgccccg ttggatatgc gcatggacgt gagccagaat 420
ctcacggccg agcggattct cgctgattac gatatggcat cgctgattcg cgtcttccgg 480
gaatacggtg aggagcggtt cgctcgtcag atcgcgcgtg agatcgtgtc gcgccgcacg 540
cagacgccat ttaccacaac cggccagctc aacgctctag tcgaagatgt ggtgcccaaa 600
gctcaccgtc cggccggcaa tccggcaaag cgtgtgttcc aggcgctgcg cattgaagtc 660
aacggcgagt tggacaaact gtcatccact ctgcctcagg ccgcgaatcg tcttcaggtc 720
ggaggccgtt tggtggtgga gtcctaccac tcgttggaag acaagacggt caaatccttt 780
atggcacagg gcctgaagat tgatgcgccg gcaaacctgc caatcgtgcc ggatgacgcc 840
cagccgttct tccgggaact gacccgaggc gcaatcaagg ccgatgatga ggagcggcaa 900
cgcaacccgc gatccgcttc agtgcggctc agggcggtcg agctgagcag acggattcct 960
gagaggtggc gagaacgctt cgcgcaaaca gcggcaaacc caaaggaatc gaacaccaca 1020
cgtaacaatg gcaaacacgg aaggaggggc tga 1053
Claims (10)
1.一株短双歧杆菌(Bifidobacterium breve),已于2020年12月10日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61199。
2.一种制备预防和/或治疗腹泻的产品的方法,其特征在于,使用权利要求1所述短双歧杆菌。
3.如权利要求2所述的一种制备预防和/或治疗腹泻的产品的方法,其特征在于,所述产品中,权利要求1所述短双歧杆菌的活菌数为不低于1×109CFU/mL或1×109CFU/g。
4.如权利要求2或3所述的一种制备预防和/或治疗腹泻的产品的方法,其特征在于,所述产品包含食品或药品。
5.如权利要求4所述的一种制备预防和/或治疗腹泻的产品的方法,其特征在于,所述药品含有权利要求1所述短双歧杆菌、药物载体和/或药用辅料。
6.一种用于预防和/或治疗腹泻的产品,其特征在于,所述产品含有如权利要求1所述的短双歧杆菌。
7.如权利要求6所述的一种用于预防和/或治疗腹泻的产品,其特征在于,所述产品中,权利要求1所述短双歧杆菌的活菌数为不低于1×109CFU/mL或1×109CFU/g。
8.如权利要求6或7所述的一种用于预防和/或治疗腹泻的产品,其特征在于,所述产品包含食品或药品。
9.如权利要求8所述的一种用于预防和/或治疗腹泻的产品,其特征在于,所述药品含有权利要求1所述短双歧杆菌、药物载体和/或药用辅料。
10.如权利要求8所述的一种用于预防和/或治疗腹泻的产品,其特征在于,所述食品包含含有权利要求1所述短双歧杆菌的保健食品;或所述食品包含使用权利要求1所述短双歧杆菌的发酵剂生产得到的乳制品、豆制品、肉制品或果蔬制品。
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Application publication date: 20210622 Assignee: Wuxi special food and Nutrition Health Research Institute Co.,Ltd. Assignor: Jiangnan University Contract record no.: X2023980053937 Denomination of invention: A strain of short bifidobacteria that can alleviate diarrhea and its application Granted publication date: 20221101 License type: Common License Record date: 20231225 |