CN116515676A - 一种调节肠道炎性反应的副干酪乳杆菌e10 - Google Patents
一种调节肠道炎性反应的副干酪乳杆菌e10 Download PDFInfo
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Abstract
本发明涉及一种调节肠道炎性反应的副干酪乳杆菌E10,该菌株于2021年06月21日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC No.22744。本发明提供的菌株Lactobacillus paracasei E10可以有效保护体内炎性反应以及肠道菌群紊乱,显著降低体内硫酸盐还原菌的丰度。因此,针对结肠炎Lactobacillus paracasei E10在作为食品或微生态制剂方向具有广泛应用的前景。
Description
技术领域
本发明涉及生物技术领域,尤其涉及一种调节肠道炎性反应的副干酪乳杆菌E10。
背景技术
炎症性肠病(Inflammatory bowel disease,IBD)是一种慢性非特异性肠道炎症性疾病,主要累及结肠粘膜及粘膜下层,并表现为反复发作的慢性肠道炎症。炎症性肠病分为两种亚型,包括克罗恩病和溃疡性结肠炎。最新数据表明,全球炎症性肠病的发病率和患病率仍在上升,大约有0.2%的欧洲人口患有IBD,已经成为严重的全球健康负担。然而,药物治疗炎症性肠病往往会带来一定的副作用。随着研究的进展,补充传统药物治疗存在的不足的一些替代治疗方法如微生物制剂、益生菌等,逐渐进入人们的视野。
副干酪乳杆菌是一种具有调节肠道菌群平衡、抗肿瘤和增强人体免疫等功能的益生菌,近年来引起国内外的广泛关注。副干酪乳酸菌是一种革兰氏阳性异型发酵乳酸菌,可以从人体肠道、口腔以及各种乳制品当中分离出来。是一种兼性厌氧、不运动、无芽孢的杆菌或长杆菌。已有研究表明,副干酪乳杆菌可以通过抑制促炎细胞因子分泌、改善肠道上皮细胞屏障、提高结肠组织中总抗氧化能力、调节肠道菌群平衡来保护人体健康。此外还有研究发现,副干酪乳酸菌的代谢产物如有机酸、细菌素和苯乳酸等,能够有效地抑制致病菌和腐败菌的生长。
专利CN201811008074.4公开了一种副干酪乳杆菌N1115,该菌株可用于预防结肠炎;专利CN202011369708.6公开了一种副干酪乳杆菌ET-22在提升肠道细菌感染抗性和肠道免疫力中的应用,其可抑制肠道病原菌的黏附和肠道炎症因子产生,缓解由于ETECH10407引起的炎症反应;专利CN201911139707.X公开了一种副干酪乳杆菌K56在缓解肠道炎症方面的应用,K56能够降低炎症因子IL-6和/或TNF-α,促进抑炎因子IL-10,降低结肠炎组织损伤;CN202111386682.0公开了一种副干酪乳杆菌Jn1及其应用,Jn1在胃肠液中模拟消化存活率达到82%,且耐胆盐特性强,在胆盐浓度达到0.6%时,ABS能够达到14.08。但目前公开的具有缓解肠炎作用的菌株在实际治疗中的效果并不理想,因此提出本发明。
发明内容
为解决上述技术问题,本发明提供的菌株Lactobacillus paracasei E10在制备缓解炎症性疾病的食品和微生态制剂方向具有广泛的应用前景。
本发明的第一个目的是提供一种调节肠道炎性反应的副干酪乳杆菌Lactobacillus paracasei E10,于2021年06月21日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCCNo.22744。
本发明的第二个目的是提供上述副干酪乳杆菌Lactobacillus paracasei E10在制备缓解肠道炎性反应产品中的应用。
进一步地,所述缓解肠道炎性反应产品中,副干酪乳杆菌Lactobacillusparacasei E10的活菌数不低于1×106CFU/mL或不低于1×106CFU/g。
进一步地,所述缓解肠道炎性反应产品为食品、药品或保健品。
进一步地,所述食品为发酵果蔬、发酵乳、乳酪、含乳饮料或乳粉。
进一步地,所述药品中含有药物载体和/或药用辅料。
进一步地,所述药物载体为微囊、微球、纳米粒和/或脂质体。
进一步地,所述药用辅料包含赋形剂和/或附加剂。
进一步地,所述赋形剂包含溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、吸收剂、稀释剂、絮凝剂、反絮凝剂、助滤剂和/或释放阻滞剂。
进一步地,所述附加剂包含微晶纤维素、羟丙基甲基纤维素和/或精制卵磷脂。
进一步地,所述药品的剂型为粉剂、颗粒剂、胶囊剂、片剂、丸剂或口服液。
进一步地,所述缓解肠道炎性反应产品为预防或治疗炎症性肠病的药物。
本发明的第三个目的是提供一种含有上述副干酪乳杆菌Lactobacillusparacasei E10的菌剂。
进一步地,所述的菌剂为固态菌剂,可为粉末或者颗粒状。
进一步地,所述的菌剂为液态菌剂。
借由上述方案,本发明至少具有以下优点:
(1)本发明的菌株Lactobacillus paracasei E10显著将因葡聚糖硫酸钠(DSS)感染而造成的小鼠体重下降、疾病活动指数评分、结肠长度变短、结肠组织病理损伤恢复至接近正常小鼠水平。因此,本发明所述具有缓解溃疡性结肠炎的菌株E10在食品和微生态制剂方向具有广泛的应用前景。
(2)本发明所提供的菌株Lactobacillus paracasei E10可显著降低因葡聚糖硫酸钠(DSS)感染以及补充高硫饮食后加重的结肠炎所造成的促炎细胞因子IL-6、IL-1β、TNF-α的含量升高,且与Lgg菌剂和Jn1菌剂干预组相比,E10组对促炎细胞因子IL-6、IL-1β、TNF-α的含量降低效果更好。
(3)本发明所提供的菌株Lactobacillus paracasei E10可显著降低因葡聚糖硫酸钠(DSS)感染以及补充高硫饮食后所造成的硫酸盐还原菌(SRB)的比例增加,并降低硫酸盐还原菌代谢硫酸盐后产生的代谢产物硫化氢的含量。
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例说明如后。
附图说明
图1为菌株Lactobacillus paracasei E10对结肠炎小鼠体重的影响,以及小鼠疾病活动指数(disease activity index,DAI)评分,**表示P<0.01,*表示P<0.05;
图2为菌株Lactobacillus paracasei E10对结肠炎小鼠结肠长度的影响,**表示P<0.01,*表示P<0.05;
图3为小鼠结肠组织形态学观察及小鼠结肠组织病理学评分,**表示P<0.01,*表示P<0.05;
图4为菌株Lactobacillus paracasei E10对小鼠血清中细胞因子IL-6、IL-1β、TNF-α的含量影响,**表示P<0.01,*表示P<0.05;
图5为菌株Lactobacillus paracasei E10对小鼠肠道微生物硫酸盐还原菌(SRB)的比例及其代谢产物硫化氢的影响,**表示P<0.01,*表示P<0.05。
生物材料保藏
副干酪乳杆菌Lactobacillus paracasei E10,于2021年06月21日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC No.22744。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
下述实施例中涉及的菌株如下:
鼠李糖乳杆菌(Lactobacillus rhamnosus,Lgg),购买于ATCC,产品编号ATCC53103。
副干酪乳杆菌Jn1(Lactobacillus paracasei Jn1),于2021年06月21日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC No.22745,记载于专利CN202111386682.0中。
下述实施例中涉及的培养基如下:
MRS培养基配方:将下述物质溶于1L蒸馏水中。
物质名称 | 物质含量 |
蛋白胨 | 10g |
牛肉膏 | 10g |
酵母粉 | 5g |
葡萄糖(一水葡萄糖) | 20g |
柠檬酸氢二胺 | 2g |
(无水)乙酸钠 | 5g |
(三水)磷酸氢二钾 | 2.6g |
(七水)硫酸镁 | 0.2g |
硫酸锰 | 0.05g |
吐温80 | 1mL |
MRS固体培养基:向1L的MRS液体培养基中加入15g琼脂,加热灭菌后待其凝固。
下述实施例中涉及的菌株Lactobacillus paracasei E10菌悬液的制备方法如下:
将菌株Lactobacillus paracasei E10划线于MRS固体培养基上,37℃条件下培养24h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下培养24h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于MRS液体培养基中,37℃条件下培养24h,得到菌液;将菌液经5000g离心10min,得到菌株Lactobacillus paracasei E10菌体;将菌株Lactobacillus paracasei E10菌体经PBS洗涤后重悬于50%的甘油溶液中至菌终浓度为1×1010CFU/mL,得到菌悬液,将菌悬液于-80℃下保存待用。
下述实施例中涉及的菌株Lactobacillus paracasei Jn1菌悬液的制备方法如下:
将菌株Lactobacillus paracasei Jn1划线于MRS固体培养基上,37℃条件下培养24h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下培养24h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于MRS液体培养基中,37℃条件下培养24h,得到菌液;将菌液经5000g离心10min,得到菌株Lactobacillus paracasei Jn1菌体;将菌株Lactobacillus paracasei Jn1菌体经PBS洗涤后重悬于50%的甘油溶液中至菌终浓度为1×1010CFU/mL,得到菌悬液,将菌悬液于-80℃下保存待用。
下述实施例中涉及的菌株Lactobacillus rhamnosus Lgg菌悬液的制备方法如下:
将菌株Lactobacillus rhamnosus Lgg划线于MRS固体培养基上,37℃条件下培养24h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下培养24h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于MRS液体培养基中,37℃条件下培养24h,得到菌液;将菌液经5000g离心10min,得到菌株Lactobacillus rhamnosus Lgg菌体;将菌株Lactobacillus rhamnosus Lgg菌体经PBS洗涤后重悬于50%的甘油溶液中至菌终浓度为1×1010CFU/mL,得到菌悬液,将菌悬液于-80℃下保存待用。
下述实施例中所涉及的检测方法如下:
疾病活动指数(Disease activity index,DAI)的检测方法:
疾病活动指数结合患病动物的体重下降百分率、粪便形状、大便隐血/肉眼血便评分,评分标准如下表:
正常粪便=形成良好的颗粒;松散粪便=不粘在肛门的糊状、半成形粪便;腹
泻稀便=粘在肛门的稀水样粪便
即①体重下降(不变为0,1-5为1分,5-10为2分,10-15为3分,大于15为4分);②大便形状(正常为0,松散的大便为2分,腹泻为4分);③大便出血(正常0分,隐血阳性为2分,肉眼血便为4分)三种情况进行综合评分,将3项结果的总分除以3即得到DAI值。即DAI=(体重指数+大便形状+出血情况)/3。
结肠长度的检测方法:
造模成功后,将小鼠进行处死后解剖,取小鼠结肠组织,用直尺对每只小鼠结肠全长进行测定(单位:厘米)。
结肠组织病理学特征的检测方法及组织损伤评分标准:组织损伤的程度根据炎症细胞浸润(0-3分)和组织损伤(0-3分)的程度进行评分。
结肠组织病理学特征的检测方法及组织损伤评分标准参见论文:Propolis fromDifferent Geographic Origins Decreases Intestinal Inflammation andBacteroides spp.Populations in a Model of DSS-Induced Colitis.
结肠组织生化指标的测定:
取小鼠血液,室温静置半小时后4℃,1500g,离心15min得到血清,使用ELISA试剂盒(Thermo Fisher Scientific Inc,Waltham,USA)测定血清中促炎细胞因子IL-6、IL-1β、TNF-α的含量。
16S rDNA测序:收集小鼠造模结束后的粪便于1.5mL离心管中,送测华大科技公司进行16S rDNA测序。
小鼠粪便样品中的H2S用内源性硫化氢(H2S)测定试剂盒(南京建成生物,中国南京)根据制造商的说明书进行测定。
实施例1菌株Lactobacillus paracasei E10的分离筛选
菌株的分离
菌株Lactobacillus paracasei E10为从醋醅当中分离提取得到的纯化菌株。750μL的50%甘油加750μL菌液,-80℃条件下保存。
菌种保藏与鉴定
(1)菌种保藏:将纯化的Lactobacillus paracasei E10培养液750μL混匀,再加入750μL的50%甘油,放入-80℃冰箱保存。并备份一管菌种鉴定,5000r/min离心3min,弃上清得菌体。
(2)菌种鉴定:取步骤(1)用于菌种鉴定的保藏管,按照试剂商所提供的细菌基因组DNA提取试剂盒(上海捷瑞生物工程有限公司,Shanghai Generay Biotech Co.,Ltd)说明进行操作。具体操如下:
取步骤(1)用于菌种鉴定的保藏管,8000rpm/min离心1min,弃上清得菌体,加入150μL TE(pH8.0)悬浮细菌,加入5μL溶菌酶至上述150μLTE溶液中,室温下酶解5-10min。再向其中加入300μL消化液混匀;再加入4μL Rnase A混匀,55℃保温10min;再加入4μL蛋白酶K,55℃保温10-30min。再向其中加入300μL PB溶液,充分摇动混匀,12000rpm室温离心5min,得上清液。准备gDNA recovery column,向柱子中加入200μL Buffer CBS,10000rpm离心1min,弃去透过液,备用。将前述所获得的上清液转移到套放于2mL收集管内的gDNArecovery column柱子中。室温8000rpm离心1min,弃去收集管内的废液。将gDNA recoverycolumn放回收集管内,加入500μL的洗涤液,8000rpm离心1min,取下gDNA recovery column柱子,弃去收集管中的废液,重复此步骤一次。将gDNA recovery column放回收集管中,12000rpm离心1min,以去除残留的洗涤液。将gDNA recovery column放入新的洁净的1.5mL离心管中,在gDNA recovery column柱子中央加入50-100μL的Elution buffer,室温或37℃静置2min,12000rpm室温离心1min,离心管中的液体即为细菌组DNA。
测定提取的基因组浓度及其OD260/280比值,用于后续的PCR反应。
PCR反应体系(20μL):
Forward primer | 1μL |
Reverse primer | 1μL |
2X PCR | 10μL |
DNA模板 | <100ng |
ddH2O | 补至20μL |
PCR反应模板为27F(5’-AGAGTTTGATCCTGGCCTCA-3’),1492R(GGTTACCTTGTTACGACTT);反应条件为:95℃,3min,1个循环;95℃,30s,55℃,30s,72℃,90s,35个循环;72℃,5min。
PCR产物经核酸电泳分析确认后,送华大基因测序;将测序返回序列正在NCBI进行种属确认;通过在NCBI数据库中比对,结果发现菌株Lactobacillus paracasei E10归属于Lactobacillus属,将其命名为菌株Lactobacillus paracasei E10。
实施例2菌株Lactobacillus paracasei E10对DSS诱导的溃疡性结肠炎的作用
将36只7周龄的雄性C57BL/6J小鼠按平均体重分为6只/组,共6组,如表1所示,分别为①空白组(Control组)、②模型组(DSS组)、③DSS+高硫饮食组(简称为高硫组,即DSS+GL组)、④DSS+高硫饮食+菌株Lgg组(简称为Lgg组,即DSS+GL+Lgg组)、⑤DSS+高硫饮食+菌株E10组(简称为E10组,即DSS+GL+E10组)和⑥DSS+高硫饮食+菌株Jn1组(简称为Jn1组,即DSS+GL+Jn1组)。
小鼠经适应性喂养一周后,Lgg组、E10组和Jn1组分别预先连续灌胃Lgg、E10和Jn1菌悬液14天,同时空白组、模型组、高硫组灌胃无菌PBS缓冲液。从第8天开始,模型组、高硫组、Lgg组、E10组和Jn1组给予2.5%DSS溶液供小鼠自由饮用。
其中,2.5%DSS的水溶液的配制方法为:称取2.5g DSS固体粉末溶于100mL无菌水中,轻轻摇晃至完全溶解,每天更换新鲜的2.5%DSS溶液。
正常对照组:第1~14天,连续灌胃200μL无菌PBS缓冲液14天,自由饮水,食用普通纯化饲料。
模型组:第1~14天,连续灌胃200μL无菌PBS缓冲液14天,第8-14天,自由饮用含2.5%DSS的水溶液,食用普通纯化饲料。
高硫组:第1~14天,连续灌胃200μL无菌PBS缓冲液14天,第8-14天,自由饮用含2.5%DSS的水溶液,食用高硫饲料。
Lgg组:第1~14天,连续灌胃200μL Lgg菌液14天,第8-14天,自由饮用含2.5%DSS的水溶液,食用高硫饲料。
E10组:第1~14天,连续灌胃200μL E10菌液14天,第8-14天,自由饮用含2.5%DSS的水溶液,食用高硫饲料。
Jn1组:第1~14天,连续灌胃200μL Jn1菌液14天,第8-14天,自由饮用含2.5%DSS的水溶液,食用高硫饲料。
结肠炎造模期间,隔天定时称量小鼠体重,计算小鼠体重变化的百分比。第15天处死小鼠,取样用于后续实验。
表1动物实验设计如下
普通纯化饲料和高硫饲料具体成分表如下表:
注:无菌PBS和菌悬液每日灌胃剂量为200μL,菌悬液的活菌数为1×109CFU/mL。
实施例3菌株Lactobacillus paracasei E10对DSS诱导的溃疡性结肠炎的小鼠体重和结肠长度变化
DSS诱导小鼠结肠炎会导致小鼠体重下降和结肠缩短,因此,体重和结肠长度是评价结肠炎小鼠炎症严重程度的重要指标。造模成功后,将小鼠进行处死后解剖,取小鼠结肠组织,用直尺对每只小鼠结肠全长进行测定。
体重变化如图1A所示,与正常组比较,DSS组小鼠体重显著下降,说明溃疡性结肠炎模型构造成功;与DSS组相比,在补充高硫饮食之后,小鼠体重下降加重,而在DSS和高硫饮食的双重干预造模下,再补充E10菌液,可以显著缓解小鼠的体重下降,且E10菌液对体重下降的减缓趋势要优于Lgg干预组。在补充另一株副干酪乳杆菌Jn1后,其体重下降趋势与DSS+GL组存在显著差异,且E10组对小鼠的体重下降缓解情况要优于补充Jn1菌剂之后。在疾病活动指数(DAI)评分中,如图1B所示,与DSS组相比,在补充高硫饮食后,小鼠疾病活动指数评分增加。而在DSS和高硫的基础上再补充E10菌剂,则可以显著降低小鼠的疾病活动指数评分;此外,补充Lgg菌剂和Jn1菌剂也可以降低小鼠的疾病活动指数评分,但它们的疾病活动指数评分要高于E10组,其中补充Jn1菌剂组小鼠在三组补充益生菌组当中的疾病病理学评分最高。说明E10菌剂对小鼠的临床表现恢复情况可能要优于补充Lgg菌剂或Jn1菌剂。
结肠长度变化如图2所示,正常组小鼠的结肠长度最长,其他组小鼠的结肠长度都有不同程度的下降;与Control组相比,DSS组小鼠结肠长度明显缩短,说明小鼠体内的炎症发生,而在DSS和高硫的双重影响下,再补充E10菌剂,可以显著恢复小鼠的结肠长度;补充Lgg菌剂或Jn1菌剂也可以恢复小鼠结肠部分长度,但其改善效果不如补充E10菌剂明显。
取约1cm的小鼠远端结肠置于4%多聚甲醛溶液中固定12h,再通过洗涤、乙醇溶液梯度脱水、透明、浸蜡、包埋和切片等步骤制备切片,使用苏木精-伊红染色液对切片进行H&E染色,接着使用Pannoramic MIDI数字切片扫描仪对H&E染色切片进行扫描、观察和拍照,并根据炎症细胞浸润(0-3分)和组织损伤(0-3分)情况对小鼠结肠组织进行组织病理学评分。小鼠结肠组织病理图及其组织病理学评分如图3所示。
从H&E染色结果可以看出,空白组小鼠的结肠组织各层结构清晰,粘膜上皮完整,无明显上皮细胞变性、坏死和脱落,固有层腺体数量丰富,排列整齐,无明显炎性细胞浸润,而DSS组和DSS+GL组的小鼠的结肠组织出现粘膜损伤,粘膜上皮和腺体结构缺失,杯状细胞大量减少,粘膜下层和固有层水肿以及大量的淋巴细胞和中性粒细胞浸润,这表明DSS和高硫造成了小鼠结肠组织的严重损伤,从而造成其结肠组织病理评分明显升高(如图3B)。与DSS+GL组小鼠相比,E10明显增加了结肠隐窝结构完整性,减少杯状细胞消耗。因此,E10菌株显著降低了结肠组织病理评分。综上,DSS和高硫对小鼠结肠组织造成了严重的损伤,E10对结肠组织具有一定的保护作用。
菌株Lactobacillus paracasei E10对DSS及高硫饮食双重干预下的小鼠模型的结肠组织切片观察及组织病理学评分,在DSS干预造模的结肠炎当中,小鼠结肠组织粘膜屏障出现明显损伤,存在大量炎细胞浸润灶;在DSS和高硫饮食双重影响下,也出现了相似对的组织病理学表现,且DSS+GL组的组织病理学评分要高于DSS组,但在补充E10菌剂之后,小鼠结肠组织屏障显著恢复至健康表型,且其组织病理学评分与DSS+GL组相比,也显著下降。补充Lgg菌剂或Jn1菌剂小鼠肠道屏障损伤也有所恢复,但其组织病理学评分要高于E10组,说明补充E10菌剂对肠道屏障损伤的恢复情况要优于补充Lgg菌剂或Jn1菌剂。
实施例4菌株Lactobacillus paracasei E10对DSS诱导的溃疡性结肠炎的小鼠结肠组织细胞因子水平检测
对取得的小鼠血清进行IL-6、IL-1β、TNF-α三种促炎细胞因子含量检测。结果如图4A-C所示:
与空白组相比,模型组小鼠结肠的促炎因子IL-6、IL-1β和TNF-α水平显著升高,这表明DSS引起了小鼠严重的肠道炎症反应。
与DSS组相比,高硫饮食促进了IL-1β和TNF-α这两种细胞因子含量的增加。
与DSS+GL组相比,补充E10能下调IL-6、IL-1β和TNF-α三种促炎细胞因子,说明E10菌剂能够调节结肠炎小鼠的免疫反应失调,从而降低肠道炎症损伤。
与DSS+GL组相比,补充Lgg能够显著下调IL-6、IL-1β和TNF-α这三种促炎细胞因子。
与DSS+GL组相比,补充Jn1能够降低IL-6、IL-1β和TNF-α这三种促炎细胞因子的含量。
对补充E10菌剂和补充Lgg菌剂两组小鼠血清的炎性细胞因子进行比较,发现E10菌剂对IL-6、IL-1β和TNF-α这三种促炎细胞因子的下调作用要优于补充Lgg菌剂。
对补充E10菌剂和补充Jn1菌剂两组小鼠血清的炎性细胞因子进行比较,发现E10菌剂对IL-6、IL-1β和TNF-α这三种促炎细胞因子的下调作用要优于补充Jn1菌剂。
实施例5菌株Lactobacillus paracasei E10对DSS诱导的溃疡性结肠炎的小鼠结肠内容物当中硫酸盐还原菌比例的影响
如图5A所示,结果显示针对DSS和高硫饮食双重影响下所造成的硫酸盐还原菌比例的增加,再补充E10菌剂能够显著下降小鼠肠道内硫酸盐还原菌的含量。在补充Lgg菌剂或者Jn1菌剂之后,硫酸盐还原菌的比例也有所下降,但是其下降比例不如补充E10菌剂显著,且Lgg组和DSS+GL组之间的硫酸盐还原菌含量无显著差异,而E10组和Lgg组之间的硫酸盐还原菌含量、E10组和Jn1组之间的硫酸盐还原菌含量存在显著差异。
实施例6菌株Lactobacillus paracasei E10对DSS诱导的溃疡性结肠炎小鼠粪便中硫化氢含量的检测
取小鼠粪便,称重,按照组织质量(g):提取液体积(mL)为1:5-10的比例(建议称取约0.1g组织,加入1mL提取液)进行冰浴匀浆,然后12000rpm,4℃离心10min,取上清,置冰上待测干燥,按照试剂商提供的试剂盒说明加入试剂检测,并带入标准曲线进行计算。结果如图5B所示。如图5B所示,在模型组和高硫组当中,小鼠粪便内的硫化氢含量明显增加,而经E10灌胃处理后,小鼠粪便内的硫化氢含量显著下降。与DSS+GL组相比,灌胃Lgg菌剂组小鼠体内的硫化氢含量无明显变化;与Lgg组相比,灌胃E10菌剂组小鼠体内的硫化氢含量显著下降;与DSS+GL组相比,灌胃Jn1菌剂组小鼠体内的硫化氢有所下降,但是其降低硫化氢的效果不如补充E10菌剂明显。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
1.一种调节肠道炎性反应的副干酪乳杆菌Lactobacillus paracasei E10,其特征在于:于2021年06月21日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC No.22744。
2.权利要求1所述的副干酪乳杆菌Lactobacillus paracasei E10在制备缓解肠道炎性反应产品中的应用。
3.根据权利要求2所述的应用,其特征在于:所述缓解肠道炎性反应产品中,副干酪乳杆菌Lactobacillus paracasei E10的活菌数不低于1×106CFU/mL或不低于1×106CFU/g。
4.根据权利要求2所述的应用,其特征在于:所述缓解肠道炎性反应产品为食品、药品或保健品。
5.根据权利要求4所述的应用,其特征在于:所述食品为发酵果蔬、发酵乳、乳酪、含乳饮料或乳粉。
6.根据权利要求4所述的应用,其特征在于:所述药品中含有药物载体和/或药用辅料。
7.根据权利要求4所述的应用,其特征在于:所述缓解肠道炎性反应产品为预防或治疗炎症性肠病的药物。
8.一种含有权利要求1所述的副干酪乳杆菌Lactobacillus paracasei E10的菌剂。
9.根据权利要求8所述的菌剂,其特征在于:所述的菌剂为固态菌剂。
10.根据权利要求8所述的菌剂,其特征在于:所述的菌剂为液态菌剂。
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