CN102016005A - 鼠李糖乳杆菌的菌株 - Google Patents
鼠李糖乳杆菌的菌株 Download PDFInfo
- Publication number
- CN102016005A CN102016005A CN2009801097571A CN200980109757A CN102016005A CN 102016005 A CN102016005 A CN 102016005A CN 2009801097571 A CN2009801097571 A CN 2009801097571A CN 200980109757 A CN200980109757 A CN 200980109757A CN 102016005 A CN102016005 A CN 102016005A
- Authority
- CN
- China
- Prior art keywords
- bacterial strain
- lactobacillus rhamnosus
- strain
- cncm
- bacterium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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Abstract
本发明涉及鼠李糖乳杆菌(Lactobacillus rhamnosus)的新菌株,其具有抗微生物及免疫调节特性,还涉及含有所述菌株的组合物。
Description
本发明涉及具有抗微生物及免疫调节特性的鼠李糖乳杆菌(Lactobacillus rhamnosus)新菌株。
大量的科学研究已经报道了发酵食品(尤其是乳制品)中的某些微生物对健康的有益作用。这些微生物通常被称为“益生菌”。根据目前公认的定义,益生菌为:“在适量摄入时对寄主健康具有有益作用的活微生物”(FAO/WHO关于益生菌在食品(包括含有活乳酸菌的乳粉)中的健康及营养特性的评估报告;Cordoba,阿根廷;2001年10月1-4日)。
已经证明,食用含有益生菌的食品能够对健康产生有利作用,尤其是通过使肠道菌群再平衡、提高对感染的抵抗力和调节免疫应答来实现。
用于人类食品的益生微生物通常是乳酸菌,主要属于乳杆菌属(Lactobacillus)和双歧杆菌属(Bifidobacterium)。
然而,对健康的有益作用通常并不是同一属的所有细菌所共有的,甚至同一种的细菌也是这样。它们通常只在某些菌株中存在;此外,所观察到的作用在益生菌株之间(包括同一种的菌株之间)可存在定性和/或定量的差异。
如果微生物被认为可能用作益生菌,其必须满足至少一个(理想的是多个)以下标准:
-对可存在于肠道菌群中的致病微生物表现出抑制活性,该活性可以由于能够附着至肠道细胞从而由此排除或降低病原体的附着,或是由于能够产生抑制病原体的物质,或是由于这两种特征的组合;
-表现出免疫调节特性,尤其是免疫调节和/或抗炎特性。
此外,如果意图将该微生物加入乳制品中,则其应当优选在乳中表现出令人满意的生长。
最后,它应该在其所掺入食品的生产和储存期间以及在消费者摄入该食品之后保持良好的生存力,以使其能够到达肠道并在肠道环境中存活。
然而,应当注意,尽管为了对应目前的“益生”定义,生存力是必要的,但是已经证明,与益生菌株相关的一些有益作用即便在不存在活菌的情况下也能够获得,这些有益作用归因于某些细菌级分,或归因于其培养上清液中的活性级分。例如,PCT申请WO2004093898描述了一种通过对CNCM I-2219菌株的培养上清液进行分级分离而获得的免疫调节制备物。
现在,本发明人已成功分离出满足上述标准的鼠李糖乳杆菌。
本发明的一个主题是该菌株,其根据布达佩斯条约于2006年11月9日在CNCM(Collection Nationale de Cultures de Microorganismes[国家微生物培养物保藏中心])保藏,保藏号为I-3690。
它具有有如下特征:
-形态学:小体积,有时为球菌样、杆菌样,孤立的或为小链。
-发酵以下糖(在37℃下,在api 50CH strip-API MRS培养基中48小时后获得的结果):核糖、半乳糖、D-葡萄糖、D-果糖、D-甘露糖、L-山梨糖、鼠李糖、甘露醇、山梨醇、甲基-D葡糖苷、N-乙酰氨基葡糖胺、熊果苷、七叶苷、水杨苷、麦芽糖、乳糖、海藻糖、松三糖、β-龙胆二糖、D-松二糖、D-塔格糖和葡糖酸。
此外,它还有抗微生物特性,这是由于其抑制培养的致病微生物生长的强大能力。
它还有附着至甘露糖的特性。已知存在于肠上皮细胞表面的富甘露糖糖缀合物对致病细菌(例如具有肠毒性的大肠杆菌、沙门氏菌(salmonellae)、霍乱弧菌(Vibrio cholerae)或绿脓假单胞菌(Pseudomonas aeruginosa))的附着起作用,并且已经报道了某些益生菌的甘露糖附着性使其能够与这病原体竞争,以抑制它们对肠粘膜的附着,从而赋予针对它们的抗感染特性(Michail和Abernathy,Pediatr.Gastroenterol.Nutr.,35,350-355 2002;Mangell等,Dig Dis Sci.,47,511-506,2002)。然而,这些特性主要在植物乳杆菌(Lactobacillus plantarum)种的细菌(尤其是植物乳杆菌菌株WCFS1和申请EP0817640中记载的植物乳杆菌菌株229v)中观察到,而不是在鼠李糖乳杆菌种的细菌中。
CNCM I-3690菌株还有免疫调节特性,尤其是抗炎特性。
作为主题,本发明还包括可通过CNCM I-3690菌株的诱变或基因转化获得的鼠李糖乳杆菌菌株。优选地,这些菌株至少保留CNCM I-3690菌株的抗微生物特性或免疫调节特性。它们可以是其中CNCM I-3690菌株的一个或多个内源性基因已被突变的菌株,例如用于改变它的一些代谢特性(例如,该菌株代谢糖的能力,它对肠道转运的抗性,它对酸性的抗性,其后酸化或其代谢物产生)。它们也可以是用一种或多种目的基因对CNCM I-3690菌株进行基因转化所得的菌株,例如,用于赋予所述菌株以额外的生理特征,或者用于表达期望通过所述菌株施用的治疗或疫苗目的蛋白质。
这些菌株可以通过用于乳杆菌的随机或定点诱变和基因转化的常规技术从CNCM I-3690菌株获得,例如,Gury等人(Arch Microbiol.,182,337-45,2004)或Velez等人(Appl Environ Microbiol.,73,3595-3604,2007)的技术,或通过称为“基因组改组(genome shuffling)”的技术获得(Patnaik等Nat Biotechnol,20,707-12,2002;Wang Y.等,J Biotechnol.,129,510-15,2007)。
本发明的一个主题还包括可从本发明鼠李糖乳杆菌菌株获得的细胞级分。它们尤其是从所述菌株的培养物获得的DNA制备物或细菌壁制备物。它们还可以是培养上清液或这些上清液的级分。
本发明的一个主题还包括包含本发明鼠李糖乳杆菌菌株或从所述菌株获得的细胞级分的组合物。
这些组合物尤其可以是乳酵素(lactic ferment),其将本发明的鼠李糖乳杆菌菌株与一种或多种其他乳酸菌(任选地为益生菌)菌株组合。
它们还可以是包含本发明鼠李糖乳杆菌菌株或得自所述菌株的细胞级分的食品(尤其是乳制品)或者药物或化妆品。
当所述菌株以活细菌形式存在时,它们优选以所述产品中至少105cfu/g、有利地至少106cfu/g、更有利地至少107cfu/g和甚至更有利地至少108cfu/g的比例存在。
参考实施例,从以下说明中将更清楚地理解本发明,所述实施例举例说明了CNCM I-3690菌株的抗微生物、免疫调节及抗感染特性。
实施例1:CNCM I-3690菌株与已知益生菌菌株的特性比较
将CNCM I-3690菌株的特性与现有技术的益生特性已知的多种菌株进行比较。
下表1给出这些菌株的列表。
表1
材料和方法
1-抗微生物活性
对三种靶标致病菌进行抗微生物活性研究:大肠杆菌E1392-75-2A、肠沙门氏菌(Salmonella enteritidis)NIZO B1241和单核细胞增生李斯特菌(Listeria monocytogenes)4B。在多种培养基中在培养皿上培养乳酸菌:补充有1%乳糖的LM17(M17培养基,Terzaghi & Sandine,Appl.Microbiol.29,807-813,1975)、Elliker培养基(Elliker等,J.Dairy Sci.,39,1611-1612,1956)和TGE培养基(胰蛋白胨-葡萄糖-肉提取物)。
将培养皿在37℃下孵育,直至出现细菌菌落。双歧杆菌的培养在厌氧条件下进行。然后将含有BHI(脑心浸出液)培养基和病原体的琼脂层倾倒在培养皿的表面。再次将培养皿在37℃孵育24小时。然后测量每个乳酸菌菌落周围的病原体抑制区域的直径。评分1对应于1-3mm的直径。评分2对应于4-6mm的直径。评分3对应于大于6mm的直径。每个实验对每个菌株独立地进行3次。
将每个实验中对靶标病原体获得的评分进行加和,以获得每种乳酸菌的抗微生物活性总分。
结果示于下表II中。
这些结果显示,在试验的菌株当中,CNCM I-3690菌株以及ATCC55544菌株具有最高的抗微生物活性。
2-免疫调节
通过检测由这些细菌对结肠上皮细胞(HT-29)的炎性应答所引发的调节来评价多种乳酸菌的免疫调节特性,这通过在模拟炎症攻击条件的TNFα、IL-1β和IFNγ混合物(Cytomix)的存在下测量这些细菌对于转录调控因子NF-κB的活化和HT-29细胞之促炎细胞因子IL-8分泌的影响来实现。
乳酸菌的培养
乳酸菌的培养在MRS培养基(De Man等,J.Appl.Bacteriol.23,130-135,1960)或补充L-半胱氨酸(终浓度为1%)的MRS培养基或在Elliker培养基中进行,这取决于所试验的细菌种类。在将细菌接种到10mL第一预培养物中并在37℃下培养16小时,然后在37℃下在10mL第二培养物中再培养16小时,然后在稳定期结束时收获。
细胞的培养和制备
在37℃和5%的CO2下,将HT29细胞维持在补充有4.5g/L的D-葡萄糖、L-谷氨酰胺(终浓度为1mM)、非必需氨基酸(AA)(终浓度为1%)、青霉素/链霉素(终浓度为1%)和胎牛血清(FCS)(终浓度为10%)的Dulbecco改进的Eagle培养基(DMEM)培养基中。
在进行与细菌相互作用和用Cytomix刺激的实验前两天,将HT-29细胞接种到12孔板中,比例为每个孔的2mL培养基中具有2×105个细胞。
为了测量转录调控因子NF-κB的活化,在接种后第二天用Ig-κB-萤光素酶报告基因质粒转染细胞,如Tien等人(J.Immunol.,176,1228-37,2006)所述。在转染之前,清洗细胞,并将培养基更换为不含抗生素的相同培养基。通过在每个孔中放入含有75ng pIg-κB-萤光素酶质粒的100μL混合物和99μL OptiPro培养基中的1μL Lipofectamine 2000来进行转染。然后将板在37℃和5%CO2的条件下孵育24小时。
与细菌相互作用和用Cytomix进行刺激
在稳定期末期回收细菌,并且用PBS清洗两次,然后与细胞相互作用。
然后将其与细菌一起孵育2小时,感染倍数为每个细胞100个细菌。在与细菌相互作用2小时后,将细胞在细菌和Cytomix存在下在孵育培养基中孵育6小时,Cytomix的比例为50ng/mL的TNFα、2.5ng/mL的IL-1β和7.5ng/mL的IFNγ。所有的孵育都在37℃和5%CO2的条件下进行。
为了确定不存在乳酸菌时NF-B活化和IL-8分泌的基础水平,在无细菌情况下将细胞孵育2小时后,利用Cytomix刺激进行相同的实验,该实验进行6小时。
在8小时的孵育结束时,回收1mL培养基以测定分泌的IL8。该测定是利用“QuantiGlo Human IL-8 chemiluminescent ELISA”试剂盒(R&D Systems)通过ELISA法进行的。
为了测量NF-κB活化,用补充有1mM DTT的100μl缓冲液(25mM Tris,pH 7.9,8mM MgCl2,1%Triton,15%甘油)裂解细胞。为了测量萤光素酶的活性,将10μl细胞裂解物添加到补充有1mM DTT、100mM ATP和2mM萤光素/K2HPO4 pH 7.58的读数缓冲液(25mM Tris,pH 7.9,8mM MgCl2,1%Triton,15%甘油)。该测量利用光度计(Beckman Coulter)来进行。
在这一总计8小时的相互作用时间后,测量NF-κB活化和IL-8分泌。
NF-κB活化和IL-8分泌的值用相对于没有乳酸菌的情况下观察到的基础水平的百分比表示。对于每个试验菌株,通过将NF-κB活化百分比和IL-8分泌百分比相加来计算平均免疫调节评分。这些结果在下表III中给出。
表III
这些结果显示,CNCM I-3690菌株强烈抑制HT 29上皮细胞的炎性应答。在试验的其他菌株中,仅有HN001菌株具有相当的抗炎特性。3-与胃和肠应激相关的存活
使用反映胃应激和肠应激之条件的体外模型。
在补充有酵母提取物的乳中制备乳酸菌培养物。根据其种类,孵育培养物24-48小时(直至培养物的稳定期)。
肠应激:制备由猪胆汁盐(3.3g/l)和NaHCO3碳酸盐缓冲液(16.5g/l)组成的人工肠液。将pH调节至6.3。将1ml这种肠液添加到100μl细菌培养物中。然后孵育培养物5小时。接下来,评估培养皿上应激前和应激后的菌群。
胃应激:制备人工胃液。其由乳酸(9g/l)、胃蛋白酶(3.5g/l)和NaCl(2.2g/l)组成。将pH调节至3.1。将1ml这种人工液添加到100μl细菌培养物中。将培养物孵育不同时间:10分钟、30分钟和60分钟。评估培养皿上的菌群。
数值用以下方式表示:
胃应激=[log(cfu 10分钟/cfu 0)和log(cfu 0/cfu 0)]的平均值×10+[log(cfu 30分钟/cfu 0)和log(cfu 10分钟/cfu 0)]的平均值×20+[log(cfu60分钟/cfu 0)和log(cfu 30min/cfu 0)]的平均值×30。
肠应激=log(cfu 5小时/cfu 0小时)。
Cfu X分钟是用孵育X分钟后以菌落形成单位(CFU)表示的细菌浓度。
对于胃应激,当该值大于-50时,存活良好,当该值为-50至-100时,存活中度良好,当该值小于-100时,存活差。
对于肠应激,当其值大于-0.5时,存活良好,当其值为-0.5至-1.5时,存活中度良好,当其值小于-1.5时,存活差。
结果在下表IV中给出。
上文表II、III和IV中示出的结果表明,在被试验的多种菌株中,CNCM I-3690菌株是唯一一种同时具有相当好的抗微生物特性和相当好的抗炎特性的菌株,此外,其还对于胃和肠应激具有非常好的抗性。
为了举例说明该菌株的优越性,将该菌株从抗微生物角度所得各项评分加在一起,并且从免疫调节的角度计算该菌株所得结果的平均值。图1显示该菌株相对优于其他试验菌株的点。
实施例2:CNCM I-3690菌株的甘露糖附着特性
通过凝集测试来测定CNCM I-3690菌株特异性附着到甘露糖的能力。该测试是基于在酿酒酵母(Saccharomyces cerevisiae)细胞表面存在含有甘露糖的多糖。当使附着到甘露糖的细菌与酿酒酵母接触时,出现凝集现象,这可在显微镜下观察到。
该模型使得可以评价细菌的潜在抗感染特性,所述潜在抗感染特性是由于其能在附着于肠粘膜的水平上与病原体竞争。
使用已知具有强甘露糖附着能力的植物乳杆菌229v和植物乳杆菌WCFS1菌株作为阳性对照。
将乳杆菌菌株在MRS培养基中培养,温度为37℃。细菌生长在稳定期停止,然后清洗细菌并调节其浓度。在“麦芽膏”培养基(Oxoid)中进行酿酒酵母的培养。
然后将5μl体积的细菌悬液与PBS或甲基-α-D-吡喃甘露糖苷(终浓度为25mM)混合。然后添加100μl含1%酿酒酵母细胞的制备物。在室温下搅拌混合物10分钟,然后在显微镜下观察,并且利用以下等级来评分:
评分0=无凝集
评分1=弱凝集
评分3=强凝集
评分5=非常强的凝集
实验对每个菌株独立地进行三次。
结果示于下表V中。
表V
观察到,通过CNCM I-3690菌株凝集的酿酒酵母细胞的百分比与通过植物乳杆菌229v和植物乳杆菌WCFS1菌株凝集的酿酒酵母细胞百分比相当。
实施例3:CNCM I-3690菌株在乳中的生长
利用如下方法试验CNCM I-3690菌株在乳中的生长特性:
用CNCM I-3690菌株接种用水(已向其添加脱脂乳粉)重建的脱脂乳组成的培养基与一起孵育,水平为5.5×106cfu/g至3.3×107cfu/g。
通过连续监测生长培养基的pH值来测量菌株的发酵活性(与其生长相关)。
结果示于图2中。
这些结果显示,CNCM I-3690菌株能够在乳中有效生长,因此其能够用于制造发酵的乳制品。
Claims (5)
1.一种具有抗微生物及免疫调节特性的鼠李糖乳杆菌(Lactobacillus rhamnosus)菌株,其特征在于它是以保藏号I-3690保藏于CNCM的菌株。
2.一种具有抗微生物和/或免疫调节特性的鼠李糖乳杆菌菌株,其特征在于它能够通过诱变或遗传转化从权利要求1所述的菌株获得。
3.一种具有抗微生物和/或免疫调节特性的细胞级分,其特征在于它能够从权利要求1和2中任一项所述的鼠李糖乳杆菌菌株获得。
4.一种组合物,其包含权利要求1所述的鼠李糖乳杆菌菌株或权利要求3所述的细胞级分。
5.权利要求4所述的组合物,其特征在于它是食品。
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WO2014071633A1 (en) * | 2012-11-12 | 2014-05-15 | Companie Gervais Danone | Use of lactobacillus rhamnosus strain for reducing weight gain and/or insulin resistance |
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WO2007108763A1 (en) * | 2006-03-17 | 2007-09-27 | Probac Ab | Use of lactobacillus strains for promoting immunotolerance in autoimmune disease |
KR100991456B1 (ko) * | 2008-06-24 | 2010-11-04 | 목포대학교산학협력단 | 김치로부터 분리된 유산균 및 그의 용도 |
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WO2013127148A1 (zh) * | 2012-02-28 | 2013-09-06 | 江南大学 | 一种能够缓解慢性酒精性肝损伤的鼠李糖乳杆菌及其用途 |
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CN114672441A (zh) * | 2022-05-05 | 2022-06-28 | 华中农业大学 | 一种乳酸菌组合物及其应用 |
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FR2928935B1 (fr) | 2011-05-20 |
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