CN113215029B - 一种约氏乳杆菌及其应用 - Google Patents
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- CN113215029B CN113215029B CN202110391013.6A CN202110391013A CN113215029B CN 113215029 B CN113215029 B CN 113215029B CN 202110391013 A CN202110391013 A CN 202110391013A CN 113215029 B CN113215029 B CN 113215029B
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- lactobacillus johnsonii
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Abstract
本发明属于微生物技术领域,公开了一种约氏乳杆菌(Lactobacillus johnsonii EU03)及其应用。本发明所述约氏乳杆菌可提高肠道菌群中乳杆菌(Lactobacillus),Muribaculaceae(p<0.05),阿克曼菌(Akkermansia),Allobaculum,拟杆菌(Bacteroides)(p<0.05)和Blautia等肠道优势菌群的丰度,降低Romboutsia,Lachnospiraceae_NK4A136等菌群的丰度,对肠道菌群结构具有正向调节作用,从而改善代谢综合征等症状;并对肠道菌群相关代谢产物的调节作用,降低氧化三甲胺(TMAO)相关代谢物胆碱浓度、同时提高甜菜碱浓度,提高肠道菌群中提高短链脂肪酸(SCFAs)的浓度,起到缓解心血管疾病的作用。
Description
技术领域
本发明属于微生物技术领域,具体涉及一种约氏乳杆菌(Lactobacillusjohnsonii EU03)及其应用,尤其是一种能够对肠道菌群及其代谢产物起到调节作用的约氏乳杆菌及其应用。
背景技术
肠道菌群与许多疾病密切相关,主要包括胃肠道疾病、免疫性疾病、骨质疏松症、心血管疾病、代谢性疾病、恶性肿瘤及神经疾病。肠道菌群影响宿主的微生物生态系统主要是通过增强免疫功能,预防或治疗腹泻、便秘、腹胀气等肠道疾病,有助于营养吸收、调节因肠道菌群失调造成的代谢异常如骨代谢和糖脂代谢等。在肠道益生菌中,乳杆菌属(Lactobacillus sp.)是革兰氏阳性菌,被归类为乳酸菌。各种乳杆菌菌株通过影响脂质胆固醇代谢、免疫炎症反应、氧化应激反应以及涉及的肠道代谢物在骨质疏松、肥胖、糖尿病和心血管相关疾病预防和治疗中得到广泛研究。
用罗伊氏乳杆菌(Lactobacillus reuteri)喂养去势雌激素缺乏小鼠模型,对比未进行益生菌干预的去势小鼠,其皮质骨的骨量明显增加,其机制可能与调节模型小鼠的肠道菌群,抑制破骨细胞对骨质的影响,降低炎症因子的表达水平,促进骨钙的吸收,成骨标志物表达明显提高有关,因此认为益生菌可通过调节激素及免疫功能来改善骨质代谢。最新研究发现,鼠李糖乳杆菌(Lactobacillus rhamnosus)可使肠道中梭菌纲丰度上升,产生更多丁酸,进而使小鼠肠道和骨髓中调节性T细胞增多,活化成骨细胞相关的信号通路,最终刺激骨生成。加氏乳杆菌(Lactobacillus gasseri)是目前科学界有确实临床试验证实过的,可以有效减轻体重的一个神奇菌种,有科学家发现,“高脂饮食”会减少小肠中的加氏乳杆菌,并破坏ACSL3-脂肪酸感受过程,从而影响血糖代谢平衡,这也是体重增加的一个原因。而补充加氏乳杆菌,可以发挥帮助糖尿病人进行血糖调控的作用。动物实验显示,乳杆菌干预后脂肪组织质量减少,且通过调节肝脏中的生脂和脂解基因的表达、降低肝脏脂肪变性、改善血脂和葡萄糖耐受性、降低内毒素血症以及调节炎症等途径来缓解肥胖。临床研究显示,人体在摄入益生菌或益生元后,益生菌会在人体的肠道定植,参与到人体的消化、吸收、代谢过程中,从而发挥出分解脂肪等功效,肥胖及代谢性疾病患者采取益生菌治疗可降低体重。向小鼠注射鼠乳杆菌(Lactobacillus murinus)会减少TH17细胞,防止主动诱导的实验性自身免疫性脑脊髓炎(脑炎小鼠模型)和盐敏感性高血压发生盐诱导型恶化。在健康人类身上进行的小型前导性研究发现,增加盐摄入会降低多种乳杆菌属细菌的存活,而且伴有TH17细胞增加和血压上升,这与上述研究结果一致。关于益生菌降压的机制,有产生肽类等降压物质或短链脂肪酸的作用,改善血管氧化和炎症水平等。通过添加益生菌改变肠道菌群的组成也能够影响动脉粥样硬化的发展,研究表明,给载脂蛋白E(ApoE)基因敲除小鼠注射了嗜酸乳杆菌(Lactobacillus acidophilus)ATCC4356后能够通过减少氧化应激和炎症反应减缓的动脉粥样硬化病变的发展。此外,乳杆菌有益菌能降低胆固醇及有害物质氧化三甲胺(TMAO)的水平,继而降低心血管疾病的发生。胆碱(Choline)、甜菜碱(Betaine)和肉碱(Carnitine)循环水平也被证实与心血管疾病的发生有关,并可预测重大不良心脏事件。然而,乳杆菌对TMAO前体化合物胆碱、甜菜碱和肉碱的影响作用尚不明确。
中国公布的细菌名录包括13种乳酸菌,它们被公认为安全成分,广泛用于食品生产。国家食品药品监督管理局以公告的形式对名单进行了补充(http://samr.cfda.gov.cn/WS01/CL1975/228275.html,[2010]65号)包括L.acidophilus,L.casei,L.crispatus,L.delbrueckii,L.fermentum,L.gasseri,L.helveticus,L.johnsonii,L.paracasei,L.plantarum,L.reuteri,L.rhamnosus和L.salivarius。乳杆菌的肠道菌群依赖机制也引起了广泛的关注。它们对菌群结构的调控特点通常表现为门水平上拟杆菌/厚壁菌比率增加,属水平上拟杆菌、乳杆菌和双歧杆菌的丰度增加。然而,约氏乳杆菌对肠道菌群的调节作用研究较少。
TMA-TMAO通路是饮食、肠道菌群、心血管事件形成风险的交叉因素。TMAO循环水平升高被证明可独立预测重大心脏不良事件的风险,包括心肌梗死等危险因素引起的死亡。胆碱、甜菜碱和肉碱循环水平也被证明与心血管疾病的发生有关,并可预测重大不良心脏事件的风险。不利的心血管风险因素与血液中高胆碱和低甜菜碱浓度有关。还有研究显示血清甜菜碱浓度与体质指数(body mass index,BMI)、腰围(waist circumference,WC)、吸烟、血清甘油三酯呈负相关,与血清高密度脂蛋白胆固醇(high-densitylipoproteincholesterol,HDLC)呈正相关;而血清胆碱浓度与BMI、WC、血清甘油三酯呈正相关,与血清HDL-C呈负相关。目前关于人体需要多少胆碱还没有达成共识,现在医学和营养学共同建立了关系数据分析系统进行平行研究也没能得出明确的需求值,最后不得已建立了一个“足够摄入量”,这是根据大数据推算出的“最佳数值”来满足大多人对胆碱的需求。然而补充胆碱会导致血小板聚集增加,因此现在目前通过服用阿司匹林抑制这一风险。通过益生菌降低高胆碱饮食导致的血液中高胆碱水平并同时升高甜菜碱水平还未有报道。
因此,本研究从健康小鼠肠道中分离得到一株约氏乳杆菌,它可以定植于心肌梗死(MI)大鼠对肠道中,且对改变肠道菌群组成,降低胆碱,提高丙酸、丁酸和甜菜碱在循环中的水平,具有开发成功能性食品的潜力,且对疾病的临床治疗具有重要的指导意义。
发明内容
有鉴于此,本发明旨在克服上述现有技术中存在的缺陷,提供一种从肠道中分离的乳杆菌,该乳杆菌可定植于人体肠道,提高乳杆菌,Muribaculaceae,阿克曼菌,Allobaculum,拟杆菌和Blautia等肠道优势菌群的丰度,降低Romboutsia,Lachnospiraceae_NK4A136等菌群的丰度;并进一步降低TMAO相关代谢物胆碱浓度、同时提高甜菜碱浓度,提高肠道菌群中提高短链脂肪酸的浓度。
为达到上述目的,本发明的技术方案是这样实现的:
本发明提供了一种约氏乳杆菌EU03(Lactobacillusjohnsonii EU03),于2020年10月9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(CGMCC),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.20845,状态为存活。
在一个实施方案中,本案考察了约氏乳杆菌EU03对肠道菌群的调节作用,结果显示,约氏乳杆菌EU03的摄入可提高肠道菌群中乳杆菌,Muribaculaceae,阿克曼菌,Allobaculum,拟杆菌和Blautia等肠道优势菌群的丰度,降低Romboutsia,Lachnospiraceae_NK4A136等菌群的丰度,对肠道菌群结构具有正向调节作用,从而改善代谢综合征,缓解糖尿病及肥胖胰岛素抵抗等症状。
在一个实施方案中,本案考察了约氏乳杆菌EU03对肠道菌群相关代谢产物的调节作用,结果显示,约氏乳杆菌EU03的摄入可降低TMAO相关代谢物胆碱浓度、同时提高甜菜碱浓度,提高肠道菌群中提高短链脂肪酸的浓度,表明约氏乳杆菌EU03可以起到缓解心血管疾病的作用。
因此本发明提供了约氏乳杆菌EU03在制备调节肠道菌群和相关代谢产物的产品中的应用;
其中,所述调节肠道菌群为提高乳杆菌,Muribaculaceae,阿克曼菌,Allobaculum,拟杆菌和Blautia等肠道优势菌群的丰度,降低Romboutsia,Lachnospiraceae_NK4A136等菌群的丰度;所述调节相关代谢产物为降低TMAO相关代谢物胆碱浓度、同时提高甜菜碱浓度,提高肠道菌群中提高短链脂肪酸的浓度。
本发明所述产品包括但不限于药物。
进一步地,本发明提供了含有所述约氏乳杆菌EU03的菌剂。
优选的,所述菌剂中约氏乳杆菌EU03的活菌数大于108CFU/mL。
本发明所书菌剂可以按常规方法制备。
在一些实施方案中,所述菌剂的制备方法为,将约氏乳杆菌EU03按照体积分数2%的接种量接种至MRS液体培养基中,在37℃兼性厌氧条件下培养24h,取出离心,弃去上清,用灭菌水洗两次后,用灭菌水调整菌液浓度为108CFU/mL。
本发明所书制备方法中,所述离心为在4℃的条件下进行离心操作,离心机转速为10000r/min,离心的时间为10min。
本发明中,菌落的计数方法为:将冻存的约氏乳杆菌转移至灭菌的脱脂乳培养基中,42℃培养4h,反复活化3代,使菌种活力恢复,置于4℃保存备用。将活化的约氏乳杆菌分别转种至MRS液体培养基中,37℃静置培养16h,以此作为种子液。用灭菌水依次10倍稀释至106倍稀释液。取100~106倍稀释液50μL分别涂布于MRS琼脂平皿上,每个稀释度做3个平行,37℃恒温培养24h后,对菌落数在30~300个的平板计数,求平均值,以CFU/mL为单位,确定其中1μL乳杆菌培养液相当于105CFU活菌数。
在一些实施方案中,所述约氏乳杆菌调节受试者肠道微生物是通过增加肠道中细菌的数量,所述细菌选自:乳杆菌(Lactobacillus),Muribaculaceae,阿克曼菌(Akkermansia),Allobaculum,拟杆菌(Bacteroides),Blautia或其任何组合。
在另一些实施方案中,所述约氏乳杆菌调节受试者肠道微生物是通过抑制肠道中细菌的数量,所述细菌选自:Romboutsia,Lachnospiraceae_NK4A136或其任何组合。
在某些实施方案中,所述受试者是指哺乳动物,包括但不限于,人,啮齿类动物(小鼠,大鼠,豚鼠),狗,马,牛,猫,猪,猴,黑猩猩。
在某些优选的实施方案中,所述受试者是人。
本发明还提供了一种发酵食品,以所述约氏乳杆菌EU03作为发酵剂发酵生产的食品。
所述发酵食品为发酵乳制品、发酵豆制品或发酵果蔬制品。
本发明还提供了一种药物制剂,包括有效剂量的约氏乳杆菌EU03和药学上可接受的辅料。
术语解释:
如本文中所使用的,术语“16S rDNA序列”是指编码16S rRNA的DNA序列,其存在于所有细菌基因组中,一般由保守区和可变区组成。保守区在细菌间无显著差异,可用于构建所有生命的统一进化树。可变区在不同细菌中存在一定差异,对16S rDNA可变区进行测序,可对细菌菌群进行精细鉴定至种的级别。
本发明的有益效果在于:
本发明所述约氏乳杆菌EU03可提高肠道菌群中乳杆菌,Muribaculaceae,阿克曼菌,Allobaculum,拟杆菌和Blautia等肠道优势菌群的丰度,降低Romboutsia,Lachnospiraceae_NK4A136等菌群的丰度,对MI大鼠肠道菌群结构具有正向调节作用,从而改善代谢综合征等症状;且对肠道菌群相关代谢产物的调节作用,可降低TMAO相关代谢物胆碱浓度、同时提高甜菜碱浓度,提高肠道菌群中提高短链脂肪酸的浓度,从而起到缓解心血管疾病的作用。本发明所述的约氏乳杆菌EU03可用于制备改善代谢综合征以及缓解心血管疾病的药物,具有广泛的应用前景。
生物保藏说明:
EU03,分类命名:约氏乳杆菌EU03(Lactobacillusjohnsonii EU03),其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,有保藏号为CGMCC No.20845,保藏日期:2020年10月9日,状态为存活。
附图说明
构成本发明的一部分的附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。在附图中:
图1A为将约氏乳杆菌EU03进行16S rDNA测序出的序列与GenBank中细菌的序列进行比对后的系统进化树;
图1B为约氏乳杆菌EU03的菌落特征;
图1C为约氏乳杆菌EU03的革兰氏染色图;
图2A-E为菌群结构Alpha多样性分析结果(包括(A)Chao1指数、(B)Shannon指数、(C)Simpson指数、(D)组间差异ANOSIM分析;(E)Beta多样性分析PCoA分析,*p<0.05,**p<0.01,***p<0.001);
图3A-C为菌群物种组成相对丰度及差异分析结果(包括(A)门水平、(B)属水平、(C)种水平,纵坐标为各菌的相对丰度);
图4为大鼠血清中短链脂肪酸含量测定结果,其中vs Sham#p<0.05,##p<0.01;vsModel*p<0.05,**p<0.01;
图5为大鼠血清中TMAO相关代谢产物含量测定结果,其中vs Sham#p<0.05,##p<0.01;vs Model*p<0.05,**p<0.01。
具体实施方式
下面将结合实施例对本申请的实施方案进行详细描述,但是本领域技术人员将理解,下列实施例仅用于说明本申请,而不是对本申请的范围的限定。根据优选实施方案的下列详细描述,本申请的各种目的和有利方面对于本领域技术人员来说将变得显然。
为了进一步理解本发明,下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1:C57B6/J小鼠肠道中乳杆菌的分离与鉴定
将新鲜的C57B6/J小鼠肠道内容物在室温下用无菌水稀释。在无菌条件下取小鼠肠道内容物100mg于试管中。加入1mL无菌水,样品以4000转/分离心1分钟。每50μL上清液涂布于MRS平板,37℃培养24小时,通过观察平板上菌落特征,从平板上筛选出菌落进行分子生物学鉴定。
采用ctab/sds法提取EU03菌株的基因组DNA,并作为PCR扩增的模板。用通用引物27f:5′-agagttttgatggctcag-3′(SEQ ID NO:1)和1492r:5′-acggttatccttgttaccgatt-3′(SEQ ID NO:2)对菌株的16s核糖体DNA序列进行扩增和测序。用聚合酶链式反应在PCR热循环仪(MyCycler;Bio-Rad Laboratories Inc.,USA)中进行扩增。PCR扩增体系为25μL,包括0.2μL taq酶(0.5u/mL)、2.5μL 10×缓冲液、1.8μL mg2+、1μL dntps混合物、1μL模板DNA、0.5μL正向引物(10μM)、0.5μL反向引物(10μM)和17.5μL ddH2O,扩增条件:95℃,3min;95℃,30s,55℃,60s,72℃,90s(30个循环);72℃,5min;4℃终止反应。扩增产物经北京华大生物科技有限公司纯化测序。根据16S rDNA测序结果,结合乳杆菌属GenBank中16SrDNA序列,使用MEGA6.0软件绘制系统进化树。
实验结果:从小鼠肠道中筛选得到的细菌进行16Sr DNA测序,其中将EU03鉴定为约氏乳杆菌,图1A为将16S rDNA测序出的序列与GenBank中细菌的序列进行比对后的系统进化树。其菌落具有白色、圆形、表面光滑半透明、边缘整齐、革兰氏染色阳性特征,菌落特征和革兰氏染色见图1B、图1C。
实施例2:约氏乳杆菌EU03对健康和MI大鼠肠道菌群多样性的调节作用
实验动物为6周龄雄性健康SD大鼠,体重为240±10g,由北京维通利华动物实验中心提供。大鼠在天津中医药大学实验动物中心(天津)饲养,室温(22±2℃),相对湿度58-65%,光周期12h交替。在这项研究中,每组测试3-6只相同处理的大鼠。
乳杆菌剂的制备:将约氏乳杆菌EU03按照2%的接种量接种至MRS液体培养基中,在37℃下培养24h后取出离心(10000r/min,10min,4℃)弃去上清,用灭菌水洗两次后,用灭菌水调整菌液浓度为108CFU/mL(菌落计数:将冻存的约氏乳杆菌转移至灭菌的脱脂乳培养基中,42℃培养4h,反复活化3代,使菌种活力恢复,置于4℃保存备用。将活化的约氏乳杆菌分别转种至MRS液体培养基中,37℃静置培养16h,以此作为种子液。用灭菌水依次10倍稀释至106倍稀释液。取100~106倍稀释液50μL分别涂布于MRS琼脂平皿上,每个稀释度做3个平行,37℃恒温培养24h后,对菌落数在30~300个的平板计数,求平均值,以CFU/mL为单位,确定其中1μL乳杆菌培养液相当于105CFU活菌数)。
为了充分保障活性,菌液每天新鲜配置。
如表1所示,实验共分四组,对照组(4周)、给菌对照组(4周)、模型组(4周)和给菌模型组(4周),分别以三组实验大鼠的粪便作为检测样品。
模型组(4周):大鼠适应性饲养一周,术前禁食12h,自由饮水。造模前对大鼠称重,并以现用现配5%的水合氯醛按照0.6mL/100g体重腹腔注射麻醉大鼠。麻醉后的大鼠胸部备皮,仰卧位固定,用酒精或碘伏进行胸前皮肤的消毒,沿左侧第四肋间(或心脏搏动最明显处)剪开,用止血钳逐层分离皮下组织,少量钝性分离胸肌。在心脏搏动最明显处用止血钳撑开胸廓,暴露心脏。剥开心包膜,轻压右胸,将心脏轻轻挤压出胸壁外,持无创缝合针丝线,于肺动脉圆锥与左心耳之间,在离左冠状动脉前降支(LAD)起始部2-3mm处进针。以5/0无损伤丝线结扎冠状动脉前降支,将心脏放回胸腔,抽空胸腔内空气,关闭胸腔,以2/0无损伤丝线分别缝合肌层及皮肤,假手术组仅在相应冠脉位置穿线,在MI后24小时,使用MS-250、16.0–21.0MHZ成像传感器评估左心室功能,该传感器连接到超声心动图系统,专门设计对小动物来说。麻醉下刮除大鼠胸部,进行二维超声心动图检查。通过室间隔和左室后壁的鉴别获得图像。超声心动图自动计算左室缩短率(FS,%)和射血分数(EF,%)左心室射血分数符合(30-45%)的大鼠予以保留,不符合的条件的大鼠视为不成模,剔除。模型组给予同体积的液体培基。每天定期喂养每只大鼠,连续喂养4周。
给菌对照、给菌模型组(4周):造模后约氏乳杆菌灌胃组剂量为108CFU/mL,按每100g体重灌服1mL菌体计算,大鼠每天称重1次,根据体重变化及时调整给菌体积,连续喂养4周。
对照、模型组(4周):假手术组和模型组给予同体积的液体培基。每天定期喂养每只大鼠,连续喂养4周。饲料满足SPF级饲养和营养需要。
表1.约氏乳杆菌动物实验分组及剂量
Alpha多样性和Beta多样性分析的方法:首先对不同样品在97%一致性阈值下的Alpha多样性分析指数(Shannon、Simpson、Chao1)进行统计,分析样本物种多样性的复杂性。数据通过QIIME(V1.7.0)软件计算获得。基于距离来进行主坐标分析(PCoA,PrincipalCo-ordinates Analysis)分析,并选取贡献率最大的主坐标组合进行做图展示。样品距离越接近,表示物种组成结构越相似。使用QIIME计算Beta多样性距离矩阵,然后用R软件(Version 2.15.3)绘制PCoA。
实验结果:
Alpha多样性分析,包括Chao1(确定物种丰富度)、Shannon和Simpson指数(确定物种多样性),以描述不同处理样品中细菌的组成(图2A-C)。结果表明模型组的Alpha多样性指数比对照组强,乳杆菌剂较模型组减弱,模型组给菌后对菌群结构具有调节作用,降低了菌群多样性,但优势菌群比例较高。ANOSIM分析,即相似性分析表明组间的差异显著大于组内差异(p=0.001)(图2D)。基于UniFrac的Beta多样性PCoA显示了各个组的微生物群组成的明显聚类,约氏乳杆菌给菌后菌群结构明显改变(图2E)。对照组给菌后趋势与模型组给菌后相同。
实施例3:约氏乳杆菌EU03对健康和MI大鼠肠道菌群结构的调节作用
利用Uparse软件(Uparse v7.0.1001)对所有样品的全部Effective Tags进行聚类,默认以97%的一致性(Identity)将序列聚类成为OTUs,同时会选取OTUs的代表性序列,依据其算法原则,筛选OTUs中出现频数最高的序列作为OTUs的代表序列。用Mothur方法与SILVA(http://www.arb-silva.de/)的SSUrRNA数据库对OTUs代表序列进行物种注释分析(设定阈值为0.8~1),获得分类学信息并分别在各个分类水平:phylum(门)genus(属)统计各样本的群落组成。
实验结果:
图3A显示了大鼠肠道菌群中最丰富的门,占有效序列的98.7%,在门的水平上,厚壁菌(Firmicutes)、拟杆菌(Bacteroidetes)、疣微菌(Verrucomicrobiota)和变形菌(Proteobacteria)构成了所有样品中的四个优势门。与对照组大鼠肠道菌群相比,模型组中厚壁菌丰度较高,拟杆菌,疣微菌和变形菌丰度较低。模型组给菌后,厚壁菌减少,拟杆菌,疣微菌和变形菌增多。对照组给菌后趋势与模型组给菌后相同。
在属水平上(图3B),丰度较高的是乳杆菌(Lactobacillus),Muribaculaceae,阿克曼菌(Akkermansia),Romboutsia,Lachnospiraceae_NK4A136,Allobaculum,拟杆菌(Bacteroides),Blautia。与对照组相比,模型组中乳杆菌,Muribaculaceae,阿克曼菌,Allobaculum,拟杆菌和Blautia丰度较低,Romboutsia,Lachnospiraceae_NK4A136丰度较高。模型组给菌后,乳杆菌,Muribaculaceae,阿克曼菌,Allobaculum,拟杆菌和Blautia增多,Romboutsia,Lachnospiraceae_NK4A136减少。对照组给菌后趋势与模型组给菌后相同。
结果表明,约氏乳杆菌对健康和MI大鼠肠道菌群结构具有正向调节作用。
图3C表明,乳杆菌中约氏乳杆菌(L.johnsonii),鼠乳杆菌(L.murimus),罗伊氏乳杆菌(L.reuteri)和肠乳杆菌(L.intestinalis)丰度较高,且与对照组相比,模型组各种乳杆菌丰度较低,约氏乳杆菌灌胃后,乳杆菌丰度升高且约氏乳杆菌增多明显,因此证明了乳杆菌在肠道中定植的作用。
表2.约氏乳杆菌施用后菌属在肠道菌群所占比例高于模型组(4周)的菌属
表3.乳杆菌施用后菌属在肠道菌群所占比例低于模型组(4周)的菌属
实施例4:约氏乳杆菌EU03对MI大鼠血清中短链脂肪酸和TMAO相关代谢物的调节作用
1.短链脂肪酸检测实验方案
量取乙酸(Acetic acid)、丙酸(Propionic acid)、丁酸(Butyric acid)、异丁酸(Isobutyric acid)、戊酸(Valeric acid)、异戊酸(Isovaleric acid)、己酸(Caproicacid)纯标准品适量,用乙醚配制成0.02μg/mL,0.05μg/mL,0.1μg/mL,0.2μg/mL,0.5μg/mL,1μg/mL,2μg/mL,5μg/mL,10μg/mL,25μg/mL,50μg/mL,100μg/mL十二个混合标准浓度梯度。
取大鼠血清样本200μL,加100μL15%磷酸,再加75μg/mL的内标(异己酸)溶液20μL和乙醚280μL匀浆1min,于4℃12000rpm离心10min,取上清上机测试。
GC-MS检测(Thermo TRACE 1310-ISQ气-质联用仪(Thermo,美国)):色谱条件:色谱柱Agilent HP-INNOWAX毛细管柱(30m*0.25mm ID*0.25μm);分流进样,进样量1μL,分流比10:1。进样口温度250℃;离子源温度230℃;传输线温度250℃,四极杆温度150℃。程序升温起始温度90℃;然后以10℃/min升温至120℃;再以5℃/min升温至150℃;最后以25℃/min升温至250℃维持2min。载气为氦气,载气流速1.0mL/min。MS条件:电子轰击电离(EI)源,SIM扫描方式,电子能量70eV。
2.TMAO相关代谢产物检测实验方案
准确称量Choline、Betaine、TMAO、Creatinine、L-Carnitine标准品适量,用1%甲酸-乙腈配制单标母液。量取各母液适量制成混合标准品,用水逐一稀释至合适浓度,制成工作标准溶液。
精确移取20μL大鼠血清样品,精确加入10μL内标溶液,然后加入750μL的1%甲酸-乙腈溶液;涡旋振荡30s,12000rpm4℃离心5min,取上清液500μL经过0.22μm膜过滤,过滤液加入到检测瓶中。
LC-MS检测(Waters UPLC液相仪(Waters ACQUITY UPLC),AB三重四极杆质谱仪):色谱条件:色谱柱:ACQUITY UPLC BEH HILIC色谱柱(2.1×100mm,1.7μm,Waters公司),进样量5μL,柱温40℃,流动相A-乙腈,B-水(含0.1%甲酸和10mM甲酸铵),流速0.4mL/min。梯度洗脱条件为0~1min,80%A;1~2min,80~70%A;2~2.5min,70%A;2.5~3min,70~50%A;3~3.5min,50%A;3.5~4min,50~80%A;4~6min,80%A。MS条件:电喷雾电离(ESI)源,正离子电离模式。离子源温度500℃,离子源电压5000V,碰撞气6psi,气帘气30psi,雾化气和辅助气均为50psi。采用多重反应监测(MRM)进行扫描。
实验结果:
图4显示了大鼠血清中短链脂肪酸的含量。与对照组相比,模型组中总短链脂肪酸、乙酸、异戊酸、戊酸和己酸含量较高,丁酸含量较低。给菌后,上述短链脂肪酸含量有向对照组调整趋势,其中丙酸和丁酸含量显著升高。
图5显示了大鼠血清中TMAO相关代谢产物的含量。与对照组相比,模型组TMAO、胆碱、肉碱水平较高,甜菜碱水平较低。给菌后,TMAO、胆碱和肉碱均降低,其中,胆碱水平显著降低,甜菜碱水平显著升高。
肠道菌群在调节胆碱、短链脂肪酸等重要物质的正常代谢中扮演重要作用,同时,这些物质的异常代谢反过来也对机体产生显著影响。胆碱通过被细菌代谢会导致血清中TMAO(氧化三甲胺)水平升高。细菌将胆碱转化为TMA,然后在肝脏中氧化成TMAO。这表明TMAO升高的更多的原因是肠道微生物发生了改变,而不是消耗多少胆碱和肉碱。如实施例4中,胆碱向甜菜碱转化的同时也降低了向TMA转化,因而TMAO水平降低。约氏乳杆菌EU03对菌群结构和多样性具有显著的调节作用,进而调节肠道菌群代谢产物,从而实现其缓解心血管疾病的作用。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,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)
1.一种约氏乳杆菌EU03(Lactobacillus johnsonii EU03),保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.20845。
2.权利要求1所述约氏乳杆菌EU03在制备调节肠道菌群和相关代谢产物的产品中的应用;
所述调节肠道菌群为提高肠道菌群中乳杆菌,Muribaculaceae,阿克曼菌,Allobaculum,拟杆菌和Blautia肠道优势菌群的丰度,降低Romboutsia,Lachnospiraceae_NK4A136菌群的丰度;
所述调节相关代谢产物为降低胆碱(Choline)浓度,同时提高甜菜碱(Betaine)浓度,提高肠道菌群中短链脂肪酸的浓度。
3.根据权利要求2所述的应用,其特征在于,所述产品为药物。
4.含有权利要求1所述的约氏乳杆菌EU03的菌剂。
5.根据权利要求4所述的菌剂,其特征在于,所述约氏乳杆菌EU03的活菌数大于108CFU/mL。
6.权利要求4所述菌剂的制备方法,其特征在于,将约氏乳杆菌EU03按照体积分数2%的接种量接种至MRS液体培养基中,在37℃兼性厌氧条件下培养24h,取出离心,弃去上清,用灭菌水洗两次后,用灭菌水调整菌液浓度为108CFU/mL。
7.根据权利要求6所述的菌剂的制备方法,其特征在于,所述离心为在4℃的条件下进行离心操作,离心机转速为10000r/min,离心的时间为10min。
8.一种发酵食品,其特征在于,以权利要求1所述约氏乳杆菌EU03作为发酵剂发酵生产的食品。
9.根据权利要求8所述的发酵食品,其特征在于,所述发酵食品为发酵乳制品、发酵豆制品或发酵果蔬制品。
10.一种药物制剂,其特征在于,包括有效剂量的权利要求1所述的约氏乳杆菌EU03和药学上可接受的辅料。
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