CN118086137A - 一种林麝源艾克曼菌株及其用途 - Google Patents
一种林麝源艾克曼菌株及其用途 Download PDFInfo
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Abstract
本发明属于微生物技术领域,具体涉及一种林麝源艾克曼菌株及其用途。该艾克曼菌株保藏于中国典型培养物保藏中心,保藏编号为CCTCC M 2024488。体外实验表明,本发明提供的林麝源艾克曼菌株具有与人源标准菌株ATCC BAA‑835相似的益生菌功能。此外,林麝源艾克曼菌株显著改善了肠毒素大肠杆菌诱导的小鼠腹泻模型,并改善器官损伤,抑制炎症反应;同时改善肠道屏障通透性,促进肠道菌群的重建和维持稳态。
Description
技术领域
本发明属于微生物技术领域,具体涉及一种林麝源艾克曼菌株及其用途。
背景技术
林麝(Moschus spp.)是南亚和东亚的稀有动物物种,以其麝香分泌而闻名。为了获取麝香资源而捕杀麝香的行为越来越普遍,导致这一特殊属濒临灭绝。为了保护麝香资源,中国建立了许多自然保护区,加强对野生麝香种群及其栖息地的原位保护。然而,感染性肺炎、肠炎等呼吸道和肠道疾病在大量人工饲养的林麝中普遍存在,具有高发病率和致死率,这极大地挑战了林麝的长期生存能力。虽然抗生素经常用于治疗或预防传染病,但抗生素的长期和过度使用已引起日益严重的问题,包括畜禽耐药性增强、抗生素残留、超级细菌的出现和环境污染。因此,畜禽饲料和饲料添加剂无抗生素化是必然趋势。
使用益生菌制剂一方面可以减轻抗生素使用带来的不良反应,增强林麝的免疫功能;另一方面,益生菌制剂的使用可以改善动物肠道菌群的组成和微生态环境,建立以益生菌为主的微生物屏障,缓解动物肠道菌群的失衡,从而防止有害细菌的浸润和增殖。此外,益生菌可以增强小肠的潜力,并诱导肠道组织发育的变化。因此,上述影响提高了牲畜和家禽的采食量和转化效率,并降低了疾病发病率和死亡率。当务之急是通过全面筛选,找到能够适应林麝肠道环境、在定植方面具有竞争优势、对宿主肠道粘膜细胞具有高亲和力的菌株。然而,由于林麝与家畜肠道菌群的种类组成和数量存在显著差异,目前还没有能够满足林麝日粮需求的益生菌配方。
艾克曼菌(Akkermansia muciniphila,Akk)被归入疣微菌门(Verrucomicrobiota),其以降解肠道粘膜粘液而闻名。在这一降解过程中,Akk释放氨基酸和单糖,以及短链脂肪酸(SCFAs),作为肠道内共生菌群的能量来源。因此,食用Akk可能对肠道菌群的组成有调节作用。目前的因果关系研究提供了令人信服的证据,证明Akk参与了各种临床和生理过程,包括但不限于炎症性肠病(IBD)、肥胖、II型糖尿病、阿尔茨海默病和衰老。2021年,有人提议使用巴氏灭菌的Akk作为食品成分。在进行了全面的非临床安全性评估后,欧洲食品安全局(EFSA)批准将巴氏杀菌的Akk作为一种新型食品,标志着该菌株是新一代益生菌市场的先驱。
肠道益生菌对国家一级保护动物林麝免疫力和抵抗力影响的研究罕见报道。因此,开发适宜的益生菌配方饲喂林麝,对于提高林麝的免疫力,改善肠道健康,加强饲养过程中传染病的预防和控制具有重要意义,也有利于林麝资源的保护。
发明内容
鉴于以上技术问题,本发明提供一种林麝源艾克曼菌株,并验证其对肠道产毒素大肠杆菌(ETEC)引起的小鼠腹泻模型的保护作用,结果提示Akk可用于预防和治疗林麝腹泻。
本发明提供的具体技术方案如下:
本发明第一方面,提供一种林麝源艾克曼菌株,其保藏于中国典型培养物保藏中心,保藏编号为CCTCC M 2024488。
本发明第二方面,提供一种所述艾克曼菌株的发酵液,具体是按照以下步骤进行制备:
将所述艾克曼菌株在BHI培养基中厌氧培养,得到活化菌液;
将所述活化菌液在含有III型猪胃粘蛋白的BHI培养基中厌氧培养,得到所述发酵液。
其中,活化菌液制备时是在厌氧培养基中培养24~72小时,推荐为48小时,至复苏状态;培养温度:30~40℃,推荐为37℃;保护气:N2/CO2(80:20,v/v)或N2/CO2/H2(80:10:10,v/v/v)。随后,在相同的厌氧条件及温度下以1%~5%的接种量将已活化菌悬液接种至装有BHI液体培养基(在BHI液体培养基中添加III型猪胃粘蛋白至其质量浓度为2.5~4%)的血清瓶中培养48~72小时,收集得到发酵液。进一步优选地,活化菌悬液的接种量为4%。可将得到的发酵液进一步在转速4000r/min离心得到菌体。作为一种优选实施方式,III型猪胃粘蛋白在BHI液体培养基中的质量浓度为4%,活化菌悬液的培养时间为48h。
本发明第三方面,提供一种所述艾克曼菌株或所述发酵液在制备改善腹泻的药物中的用途。
优选地,所述腹泻是由肠道产毒素大肠杆菌引起的。
优选地,所述艾克曼菌株或所述发酵液用于制备改善腹泻导致的炎症反应的药物。
优选地,所述艾克曼菌株或所述发酵液用于制备改善腹泻导致的肠道黏膜屏障紊乱的药物。
优选地,所述艾克曼菌株或所述发酵液用于制备维持腹泻导致的肠道菌群稳态的药物。
优选地,所述艾克曼菌株或所述发酵液用于增加Muribaculaceae、Muribaculum、Lachnospiraceae在肠道中的占比。
优选地,所述艾克曼菌株或所述发酵液用于制备改善林麝腹泻的药物。
本发明第四方面,提供一种预防或治疗腹泻的微生物制剂,其包括所述艾克曼菌株或所述发酵液,以及可接受的辅料。其中可接受的辅料包含一种或多种渗透剂、乳化剂、黏贴剂、稳定剂、表面活性剂、分散剂、缓冲剂或粘合剂。所述的微生物制剂是悬液、药浴液、浓缩物、粉剂、颗粒剂、浆液、可注射液或糊剂的形式。也可以是控释或缓释制剂的形式。
对比现有技术,本发明的有益效果为:
本发明首次报道了从林麝粪便中分离到的艾克曼菌(命名为:Akkermanniamuciniphila FMD01)。通过一系列的体外实验来评价FMD01的益生菌作用,结果表明FMD01具有与人源标准菌株ATCC BAA-835(命名为:AKK-H)相似的益生菌功能。此外,FMD01显著改善了肠毒素大肠杆菌(ETEC)诱导的小鼠腹泻模型。FMD01还能改善器官损伤,抑制炎症反应,并改善肠道屏障通透性,促进肠道菌群的重建和维持稳态。因此,FMD01菌株可能作为一种潜在的饲料添加剂用于林麝养殖。
生物材料保藏信息:
艾克曼菌株FMD01保藏于中国典型培养物保藏中心,保藏编号为CCTCC M2024488,分类命名为Akkermannia muciniphila FMD01,拉丁文名称为Akkermanniamuciniphila,保藏时间为2024年3月14日;保藏地址为中国·武汉·武汉大学。
附图说明
图1是林麝源Akk菌株FMD01的外观形态和分子鉴定。(A)菌落形态;(B)革兰氏染色;(C)在扫描电子显微镜下以10,000倍的放大倍数观察Akk菌株的形态;(D)对疑似Akk菌株进行了PCR扩增后的凝胶电泳图。泳道M:DL2000 DNA Marker,其他所有泳道均来自林麝粪便样品的PCR产物;(E)疑似Akk菌株经PCR特异性引物扩增片段的DNA测序结果。
图2是FMD01的分离、表征及其益生潜力评估。(A)在酸性条件下24小时内FMD01的存活率。(B)在0.5%和1%胆汁盐条件下FMD01的存活率。(C)FMD01的生长曲线。(D)正常状态下Caco-2细胞的显微形态(10倍)。(E)FMD01对Caco-2细胞的粘附能力。(F)FMD01的自聚集性。(G)溶血性葡萄球菌、FMD01和AKK-H的溶血活性(从左到右)。所有数据均以平均值±SD表示。**P<0.01;***P<0.001,与指示组相比。
图3是FMD01缓解ETEC引起的腹泻小鼠症状。(A)实验过程示意图。最后一天对小鼠进行安乐死,以观察体重波动和病原体通过粪便排出模式。(B)用于确定肠道水分平衡的腹泻指数。(C)DAI评分。(D)结肠组织的宏观观察。(E)结肠长度。(F)脾脏的宏观观察。(G)脾脏指数[脾脏重量/体重]。(H)肝脏的宏观观察。(I)肝脏指数[脾脏重量/体重]。(J)确定了各种器官和组织中的细菌载量。所有数据均表示为平均值±SD。*P<0.05;**P<0.01;***P<0.001,与指示组相比。
图4是FMD01抑制由ETEC引起的腹泻小鼠的炎症反应。(A~C)通过qRT-PCR确定血清中促炎细胞因子TNF-α(A)、IL-1β(B)和IL-6(C)的水平。(D~G)通过qRT-PCR确定结肠组织中促炎细胞因子TNF-α(D)、IL-1β(E)和IL-6(F)以及抗炎细胞因子IL-4(G)的水平。数据表示为平均值±SD。统计分析采用单因素方差分析,然后进行Dunnett的多重比较检验。*p<0.05,**p<0.01,和***p<0.001与指示组相比。
图5是FMD01缓解了ETEC引起的腹泻小鼠结肠组织损伤和肠道屏障通透性。(A)结肠组织的苏木精和伊红染色及组织学评分。(B)绒毛高度/隐窝深度比率。(C)杯状细胞密度。(D)通过免疫荧光染色检测紧密连接相关蛋白,Bar=100μm。(E)Occludin、(F)ZO-1和(G)Mucin2的相对蛋白水平。(H)ZO-1的mRNA水平。(I)Claudin2的mRNA水平。
图6是FMD01调控了小鼠由ETEC引起的腹泻中的肠道微生物群落组成。(A)Observed_OTUs。(B)Chao1指数。(C)Shannon指数。(D)Simpson指数。(E)Venn图。Venn图展示了对照、ETEC和ETEC+Akk个体中共有的OTU(97%序列一致性)数量。(F)MDS分析,以及(G~L)每组小鼠中Lachnospiraceae_unclassified、Lachnospiraceae_NK4A136、Muribaculum、Parab acteroides、Dubosiella和Escherichia-Shigella属的差异显著性分析(n=10)。(M~O)Proteobacteria门、Bacteroidetes门和Proteobacteria门的相对丰度。
具体实施方式
下面结合具体实施方式对本发明所述的技术方案做进一步的说明,但本发明不仅限于此。
实施例1
1、艾克曼菌株的分离纯化
2019年10月从陕西凤县林麝养殖场采集20份新鲜林麝粪便标本,密封保存于无菌离心管中,储存于4℃冰箱中,在到达实验室后立即进行分析。用于分离菌株的样品首先在灭菌的含有L型半胱氨酸0.05%(w/v)的磷酸盐缓冲液中均质(样品在均质体系中的含量为10%w/v),并10倍梯度稀释至肉眼不可见浑浊,再分别稀释至10-1、10-2、10-3后分别加入富集培养液中,在37℃厌氧条件下富集培养。富集培养液(1L)由以下原料配制而成:0.3gNH4Cl、0.3g NaCl、0.11g CaCl2、0.1g MgCl2、1mL碱性微量元素溶液(该1mL碱性微量元素溶液中含有7.5mM FeCl2、1mM H3BO4、0.5mM ZnCl2、0.1mM CuCl2、0.5mM MnCl2、0.5mM CoCl2、0.1mM NiCl2、50mM HCl)、1mL酸性微量元素溶液(该1mL酸性微量元素溶液中含有0.1mMNa2SeO3、0.1mM Na2WO4、0.1mM Na2MoO4、10mM NaOH)、0.5mg刃天青、0.5g L型半胱氨酸、4gNaHCO3和2.5g猪III型粘蛋白。将富集瓶中获得的样品在添加0.05%(w/v)L型半胱氨酸和0.5% III型猪胃粘蛋白的BHI琼脂平板上涂布。将平板置于厌氧工作站(ELECTROTEK,OR,USA)中,在37℃条件下孵育48小时。最后,仔细选择具有明显形态特征的菌落,将其划线至BHI平板上,以获得纯分离样本。
2、结果
Akk是人类肠道黏膜的特异性共生菌,是与肥胖、糖尿病等疾病相关的下一代益生菌。然而,其体外培养的低丰度和难度使其难以分离到新的菌株。开发保护林麝的益生菌,最安全的策略应该是利用自体肠道细菌。此外,还可以比较不同来源的Akk菌的优缺点。因此,在本研究中,我们使用富集培养液结合特异性引物(AM1:5’-CAGCACGTGAAGGTGGGGAC-3’,如SEQ ID NO:2所示;AM2:5’-CCTTGCGGTTGGCTTCAGAT-3’,如SEQ ID NO:3所示)扩增筛选,从20份林麝粪便样本中分离出Akk(图1A)。最终从3份阳性粪便样本中分离纯化1株疑似Akk菌株(图1D)。经过革兰氏染色初步鉴定(图1B)和扫描电镜形态学观察(图1C),通过16SrDNA测序和Akk特异性引物PCR扩增进一步确认候选菌株,最终鉴定1株为林麝源Akk(图1E),命名为Akkermannia muciniphila FMD01。
菌株16S rDNA测序显示其序列如SEQ ID NO.1所示:
GGTAGTGGCAAGTCGAACGAGAGAATTGCTAGCTTGCTAATAATTCTCTAGTGGCGCACGGGTGAGTAACACGTGAGTAACCTGCCCCCGAGAGCGGGATAGCCCTGGGAAACTGGGATTAATACCGCATAGTATCGAAAGATTAAAGCAGCAATGCGCTTGGGGATGGGCTCGCGGCCTATTAGTTAGTTGGTGAGGTAACGGCTCACCAAGGCGATGACGGGTAGCCGGTCTGAGAGGATGTCCGGCCACACTGGAACTGAGACACGGTCCAGACACCTACGGGTGGCAGCAGTCGAGAATCATTCACAATGGGGGAAACCCTGATGGTGCGACGCCGCGTGGGGGAATGAAGGTCTTCGGATTGTAAACCCCTGTCATGTGGGAGCAAATTAAAAAGATAGTACCACAAGAGGAAGAGACGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGTCTCAAGCGTTGTTCGGAATCACTGGGCGTAAAGCGTGCGTAGGCTGTTTCGTAAGTCGTGTGTGAAAGGCGCGGGCTCAACCCGCGGACGGCACATGATACTGCGAGACTAGAGTAATGGAGGGGGAACCGGAATTCTCGGTGTAGCAGTGAAATGCGTAGATATCGAGAGGAACACTCGTGGCGAAGGCGGGTTCCTGGACATTAACTGACGCTGAGGCACGAAGGCCAGGGGAGCGAAAGGGATTAGATACCCCTGTAGTCCTGGCAGTAAACGGTGCACGCTTGGTGTGCGGGGAATCGACCCCCTGCGTGCCGGAGCTAACGCGTTAAGCGTGCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGAAATTGACGGGGACCCGCACAAGCGGTGGAGTATGTGGCTTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTAATGAACAACATGTGAAAGCATGCGACTCTTCGGAGGCGTTACACAGGTGCTGCATGGCCGTCGTCAGCTCGTGTCGTGAGATGTTTGGTTAAGTCCAGCAACGAGCGCAACCCCTGTTGCCAGTTACCAGCACGTGAAGGTGGGGACTCTGGCGAGACTGCCCAGATCAACTGGGAGGAAGGTGGGGACGACGTCAGGTCAGTATGGCCCTTATGCCCAGGGCTGCACACGTACTACAATGCCCAGTACAGAGGGGGCCGAAGCCGCGAGGCGGAGGAAATCCTAAAAACTGGGCCCAGTTCGGACTGTAGGCTGCAACCCGCCTACACGAAGCCGGAATCGCTAGTAATGGCGCATCAGCTACGGCGCCGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACATCATGGAAGCCGGTCGCACCCGAAGTATCTGAAGCCAACCGCAAGGAGGCAGGTCGTAAGGAGACTC
实施例2
林麝源Akk菌FMD01益生功能的研究
为了表征FMD01的益生菌功能,我们进行了一系列体外试验,评估了FMD01和人源标准菌株AKK-H的生长能力、模拟胃肠道液体耐受性、酸和胆盐耐受性、自聚集能力、疏水性、抗生素敏感性、溶血、有害代谢物产生、生物膜形成能力以及对胃肠道粘膜的粘附能力。
1、菌悬液制备及生长曲线测定
将FMD01与AKK-H于装有BHI液体培养基的试管中在厌氧培养基中培养24~72小时,推荐为48小时,至复苏状态;培养温度:30~40℃,推荐为37℃;保护气:N2/CO2(80:20,v/v)或N2/CO2/H2(80:10:10,v/v/v)。随后,在该厌氧条件及温度下以1%~5%的接种量(推荐为4%)将已活化菌悬液接种至装有BHI液体培养基(在该BHI液体培养基中添加III型猪胃粘蛋白至其质量浓度为2.5~4%,推荐添加量为4%)的血清瓶中培养48~72小时(推荐时间为48h)。收集该菌体(离心转速4000r/min),于PBS溶液中重悬至菌浓度为每0.2mL中达到2×1010CFU。
生长曲线测定:接种0小时开始,每5小时测定600nm波长处的光密度(OD)值,直至培养100小时。
2、菌株耐酸性、耐胆盐性评估
耐酸性测定:将FMD01、AKK-H菌株分别活化后,以1%接种量分别接种于pH 2.0、pH3.0、pH 4.0和pH 6.5(作为对照)的BHI液体培养基中37℃孵育24h,分别于培养后0h和24h两个时间点测定600nm波长处的OD值。
耐胆盐性测定:在BHI液体培养基中加入牛胆盐,使其浓度分别为0.5%(g/100mL)和1%(g/100mL),同时以普通BHI培养基作为对照(0%),以1%的接种量将培养至对数生长期的FMD01、AKK-H菌株分别接种到上述含胆盐培养基中,37℃孵育4h,分别于培养后0h和4h两个时间点测定600nm波长处的OD值。将不同OD600nm样品稀释涂布后测定活菌细胞总数(CFU),绘制OD600nm与CFU标准曲线。根据标准曲线,得到OD600nm相对应的CFU。
菌株存活率(%)=孵育后的活菌数(CFU/mL)/初始活菌数(CFU/mL)×100%。
3、菌株疏水性检测
生理盐水洗涤培养至对数生长期的FMD01及AKK-H洗涤两次,用生理盐水调整菌液至OD600=0.5±0.05(菌液浓度为107~108CFU/mL)。取2mL调好浓度的菌液与2mL碳氢化合物(二甲苯或甲苯)混合,漩涡震荡10min,室温分别静置15min、30min、60min。取水相测定600nm处吸光值。表面疏水性(%)=(混合前OD600值-混合后OD600值)/混合前OD600值×100%。
4、菌株自聚集能力检测
将培养至对数生长期的两株菌的OD600值调整为0.5(±0.05),菌悬液于37℃静置5h和24h后,测定OD600值。
5、生物膜形成检测
菌株在37℃的胰蛋白酶大豆肉汤(TSB,Oxoid)中培养过夜。收集细菌8000rpm离心10min,PBS洗涤2次,重悬于不含葡萄糖的TSB和添加0.25%、1%和2.5%葡萄糖的TSB。每个细菌悬液混匀后,取200μL装入96孔聚苯乙烯微量滴定板中。阴性对照是未接种菌的空白培养基。微量滴定板在37℃下孵育24h,每孔去除培养基,清洗三次,其余附着的细胞用每孔200μL的99%甲醇(Sigma-Aldrich)固定。15min后晾干,然后每孔用200μL的2%结晶紫染色5min。用无菌生理盐水洗涤三次,去除多余的污渍。将平板风干后,将贴壁细胞在160μL的33%(v/v)冰醋酸(Sigma-Aldrich)中重悬。使用酶标仪在600nm处测量每个孔的OD值。临界值(ODC)定义为阴性对照的平均OD值。根据OD值,将菌株分为非生物膜产生菌株(OD≤ODC)、弱菌株(ODC<OD≤2×ODC)、中度菌株(2×ODC<OD≤4×ODC)和强生物膜产生菌株(4×ODC<OD)。
6、菌株对结肠癌Caco-2细胞系粘附性能检测
Caco-2细胞的培养:将含有10%胎牛血清的RPMI 1640培养基作为细胞培养、传代培养基,Caco-2细胞于37℃、5%CO2恒温培养箱中培养,生长1d细胞换液,3d后传代(传代时用胰酶消化,1:5传代)。
调整细胞浓度为5×105cell/mL,每孔1mL细胞液平铺于24孔细胞培养板中继续培养。观察24孔板中细胞培养至单层(大约培养24h)后弃去细胞培养液,用无菌PBS清洗3次,将l mL菌液与l mL RPMI-1640细胞培养液混合后加入到6孔培养板的各孔中,置于37℃CO2培养箱中培养1~1.5h,取出盖玻片,用PBS清洗5次,甲醇固定,革兰氏染色及HE染色,显微镜下观察粘附情况。细菌粘附指数计算:于显微镜下随机选取20个视野,计数细胞上粘附的细菌个数,粘附指数=细菌数/100个细胞(即平均每100个细胞粘附的细菌个数)。黏附率(%)=黏附的细菌总数/每孔中添加的细菌数×100%。
7、菌株溶血性检测
将两株菌及金黄色葡萄球菌(阳性对照)分别涂在含有5%羊血(w/v)的哥伦比亚固体平板上,37℃孵育48h。孵育后,根据菌落周围培养基中红细胞的裂解情况评估其溶血活性并进行分类。菌落周围出现草绿色半透明溶血环表示α-溶血;菌落周围出现完全透明、界限分明的溶血环表示β-溶血;菌落周围无溶血现象表示为γ-溶血。
8、抗生素敏感性检测
利用纸琼脂扩散法(Kirby-Bauer test,K-B法)进行药敏实验,将培养至对数生长期的两种菌液调整菌浓为2×106CFU/mL,用涂布器将菌液分别均匀涂布于BHI固体平板表面,待自然晾干15~20min后,把药敏纸片放于平板表面中,分别在37℃厌氧培养24h和48h,用游标卡尺测量药敏片的抑菌圈,以大肠埃希菌作为质控菌株。药敏结果参照美国临床实验室标准化研究所(clinical and laboratorystandards institute,CLSI)标准判定结果为:敏感(S)、中度敏感(I)和耐药(R)。试验所用的抗生素共8类12种,包括β-内酰胺类氨苄西林(Ampicillin,10ug/片)、阿莫西林(Amoxicillin,20ug/片)、头孢噻肟(CefotaximeSodium,30ug/片);氨基糖苷类卡那霉素(Kanamycin,30ug/片)、链霉素(Streptomycin,10ug/片)、庆大霉素(Gentamicin,10ug/片);喹诺酮类诺氟沙星(Norfloxacin,10ug/片);大环内酯类红霉素(Erythromycin,15ug/片);四环素类四环素(Tetracycline,30ug/片);糖肽类万古霉素(Vancomycin,30ug/片);氯霉素类氯霉素(Chloramphenicol,30ug/片);林可胺类克林霉素(Clindamycin,2ug/片)。
9、有害代谢物生成检测
氨基酸脱羧酶检测试验:将连续传代三次的两株菌分别接入到不含氨基酸和三组氨基酸(酪氨酸、鸟氨酸、组氨酸)培养基中,37℃静置培养24h到48h,滴加1.6%溴甲酚紫-乙醇溶液1mL,观察培养基颜色变化。与未添加前体氨基酸的培养基对比,颜色变为黄色为阴性,颜色为紫色为阳性。酪氨酸、鸟氨酸、组氨酸分别用于检测酪氨、腐胺和组胺。
硝酸盐还原酶检测试验:将连续传代三次的两株菌分别接入到硝酸盐培养基中,37℃静置培养48小时后滴加等量的5% KI溶液和5%淀粉溶液,混匀后观察培养基颜色变化,以未接种菌的空白培养基作为对照,变蓝则说明产生硝酸盐还原酶。
10、结果
结果表明,FMD01与AKK-H具有相似的抗酸、抗胆盐能力,两株菌均表现出良好的抗酸、抗胆盐能力(图2A、B)。在繁殖能力方面,FMD01的增殖能力较AKK-H强(图2C)。考虑到益生菌需要经过消化道才能发挥作用,我们测量了AKK-H与FMD01对胃酸和肠液的抵抗力,如图2D所示。
AKK-H与FMD01的疏水性、生物膜形成能力、对不同抗生素的敏感性及有害代谢物的产生情况具体见表1~4。
表1疏水性测定结果
表2FMD01和AKK-H菌株生物膜形成能力
SA,强粘附;MA,中度粘附;WA,弱粘附。
表3FMD01和AKK-H菌株对不同抗生素的敏感性
H代表高度敏感;I代表中度敏感;L代表低度敏感。
表4有害代谢物实验结果
由表1~4及图2可知,FMD01和AKK-H菌株的耐酸及耐胆盐性质均较好。FMD01对胃肠道粘膜上皮细胞系Caco-2的粘附能力是由细菌计数决定的,其对Caco-2的粘附指数为17.0±3.9,这意味着每个Caco-2细胞可以平均粘附17个FMD01活菌(图2D,E)。事实上,益生菌对肠道上皮细胞的粘附能力通常决定了它们的定殖能力,即对宿主肠道菌群稳态的维持或重组能力。因此,FMD01显示出更强的益生菌潜力。FMD01也比AKK-H具有更快、更高的自聚集和疏水能力(图2F和表1)。两种Akk菌株都不会在血琼脂板上引起溶血(γ-溶血),表明它们对红细胞没有副作用(图2G)。在所有检测病例中,AKK-H均被归为弱粘附(WA),但在2.5%葡萄糖浓度下,FMD01在生物膜形成方面具有相对较高的倾向(表2)。在抗生素敏感性方面,AKK-H菌株对大多数抗生素具有高敏感性,但对青霉素、诺氟沙星和卡那霉素不敏感。而FMD01对大多数抗生素高度敏感(表3)。两株Akk均不产生有害代谢物(表4)。
综上所述,从林麝粪便中分离到的FMD01菌株可以作为潜在的益生菌菌株进行进一步的系统研究。
实施例3
为了评估FMD01在体内的益生菌作用,我们建立了小鼠腹泻模型,并评估了FMD01对其的保护作用(图3A)。
1、小鼠腹泻模型建立和样品收集
从陕西师范大学(西安,中国)购买6-8周龄雄性无特定病原体(SPF)C57BL/6小鼠,并将小鼠置于控制环境(23±2℃,40~60%湿度,以及12小时的光/暗周期)中饲养。经过1周的适应期后,小鼠被随机分为三组:对照组、模型组(标记为ETEC组)以及模型+Akk组(标记为ETEC+AKK),各组均喂食基础饲料,模型+Akk组另外口服灌胃2×1010CFU/mL FMD01菌悬液0.2mL/天,持续14天。在第15天,对照组小鼠通过腹腔注射0.1mL PBS,模型组及模型+AKK组小鼠腹腔注射ETEC溶液(2.75×108CFU/mL)/10g体重。
为了评估FMD01的腹泻模型和抗腹泻效果,我们采用了腹泻指数进行评估,包括相对器官重量/胴体重量比、粪便含水量、形态学分析以及血清细胞因子(IL-6、IL-10和TNF-α)。最后一次给药后三天,对所有小鼠实施安乐死,称重,并分离指示器官,利用以下公式计算相对重量:相对器官重量=器官重量(g)/活体重×100%。提取血样,离心(3500g×10min)以分离血清。用手术剪刀采集远端结肠、脾脏和肝脏组织,并用冰冷的生理盐水短暂冲洗。收集新鲜粪便并测定细菌组成。
2、结果
2.1、FMD01改善小鼠肠毒素大肠杆菌诱导的腹泻
我们发现,在模型小鼠中可以观察到眼皮分泌物、肛门未形成的粪便、粪便含水量(图3B)和DAI(图3C)显著增加,而连续两周的FMD01活菌(2×1010CFU/mL)预给药后,所有这些指标都明显改善(p<0.001)。
腹泻病以结肠组织的表型和病理改变为主要特征。我们发现,ETEC诱导的腹泻小鼠的结肠长度明显缩短(图3D,3E),但FMD01使结肠长度大大恢复。同时,FMD01显著缓解ETEC诱导的脾肿胀和肝肿大(图3F~I)。我们进一步测量了各种组织中的ETEC负荷,数据显示肝脏、脾脏、肾脏、盲肠内容物、结肠和结肠组织内容物中的细菌负荷显著增加(p<0.01或p<0.001),而FMD01处理有效地抑制了细菌负荷,维持在正常水平(图3J)。此外,ETEC小鼠血清和结肠组织中经典炎症因子IL-1β、IL-6和TNF-α上调,而抗炎细胞因子IL-4mRNA和蛋白水平均下调(图4A~G)。相反,所有这些细胞因子都被FMD01反向调节,这表明FMD01可以保护小鼠免受腹泻时的炎症反应。
2.2、FMD01改善腹泻小鼠肠道黏膜屏障紊乱
肠道黏膜屏障是保护宿主免受病原体感染的第一线屏障,在腹泻中起着关键作用。H&E染色时,与正常对照组相比,模型小鼠远端结肠杯状细胞数量减少,隐窝萎缩,淋巴细胞浸润(p<0.001),提示肠黏液层受损,屏障功能减弱。然而,FMD01显著减轻了ETEC诱导腹泻小鼠结肠组织的病理损伤(图5A~C)。考虑到紧密连接(TJs)是肠屏障最重要的组成部分之一,我们检测了结肠组织中TJs相关蛋白的水平。如图5D~H所示,与正常小鼠相比,ETEC小鼠结肠中Claudin2和ZO-1的表达均显著降低,而FMD01则显著上调,甚至更高。综上所述,这些结果共同表明,FMD01干预可以有效降低ETEC诱导小鼠腹泻的严重程度。
2.3、FMD01维持大肠杆菌诱导腹泻小鼠肠道菌群稳态
越来越多的证据表明,肠道微生物菌群失调是肠外源性大肠杆菌引起腹泻的关键因素。因此,我们采用16S rRNA测序来研究肠道微生物群在FMD01缓解腹泻中的作用。我们从30个小鼠结肠内容物样本中获得1485145个细菌的16S rRNA序列,平均每个样本82509个序列。数据分析发现,ETEC组α-多样性指数(Observed_OTUs、Chao1、Shannon和Simpson)显著降低,而FMD01干预显著增加了肠道微生物群α-多样性(p<0.001,图6A~D)。维恩图表示共有ASV和唯一ASV的数量(图6E)。此外,多维尺度(MDS)分类学差异(β-多样性)分析显示,在操作分类学单位(OTU)水平上,三组样品的肠道菌群有一定距离,但是FMD01处理组的分布更接近对照组,并与对照组部分重叠(图6F),说明FMD01预防了ETEC感染引起的肠道菌群失衡。此外,三组样品有6个科的微生物组成存在显著差异,包括Muribaculum、Paraacteroides、Lachnospiraceae_NK4A136、Lachnospiraceae_Unclassified、Dubosella和Escherichia Shigella(图6G~L)。在丰度前50的属中,与对照组相比,ETEC组有5个属的微生物丰度显著上调,14个下调(p<0.05)。然而,当在门和属水平上分析细菌组成时,结果显示,与ETEC组相比,FMD01干预组变形菌门的丰度显著降低(图6M),但拟杆菌门(图6N)和厚壁菌门(图6O)的丰度显著增加。益生菌相对丰度的增加表明FMD01可以增加其他益生菌,如Muribaculaceae、Muribaculum、Lachnospiraceae_Unclassified的丰度,从而增强益生菌作用,改善机体肠道损伤状况。
综上所述,这些结果共同表明肠道微生物群的重建和维持可能在FMD01预防腹泻中发挥关键作用。
肠道菌群失调引起的肠道疾病被认为是限制林麝养殖和种群数量的关键因素,因此肠道菌群对林麝健康的影响越来越受到关注。目前对林麝肠道微生物群落的研究主要集中在不同种群间肠道微生物多样性的比较分析。北京林业大学胡德福团队对林麝在严重腹泻、中度腹泻和健康状况等不同生理阶段的肠道微生物进行了研究。研究结果表明,健康组和两组腹泻组的肠道微生物多样性存在显著差异。具体而言,健康林麝肠道微生物的多样性水平明显高于两个腹泻组。LEfSe分析进一步表明,肠道功能障碍程度与主要病因之间存在显著关联。在轻度腹泻病例中,大肠杆菌-志贺氏菌是主要致病菌,而在重度腹泻病例中,梭杆菌和铜绿假单胞菌被确定为关键致病菌。研究结果表明,Akk与其抵御疾病的能力之间存在显著相关性,突出了肠道微生物群的显著影响。
考虑到Akk通过调节共生菌群对肠道免疫稳态的安全性和较强的调节功能,它可能是一种适合饲养林麝的益生菌。本研究从林麝中分离出Akk,评价其益生菌功能,并验证其对肠道产毒素大肠杆菌(ETEC)引起的小鼠腹泻模型的保护作用。本研究提示FMD01可用于预防和治疗林麝腹泻。
基于以上说明,本发明所属技术领域的技术人员可以理解在没有变更本发明的技术思想或者必要的特征的情况下本发明还可以以其他具体方式实施。对此,应当理解,以上说明的实施例在所有的方面均为示例性的而非限制性的。本发明的范围应包括后述的权利要求书的意思以及范围和从其等同概念导出的所有的变更或者变形的方式,而不仅仅包括上述的详细描述。
Claims (10)
1.一种林麝源艾克曼菌株,其特征在于,其保藏于中国典型培养物保藏中心,保藏编号为CCTCC M 2024488。
2.一种林麝源艾克曼菌株发酵液,其特征在于,具体是按照以下步骤制备得到:
将权利要求1所述艾克曼菌株在BHI培养基中厌氧培养,得到活化菌液;
将所述活化菌液在含有III型猪胃粘蛋白的BHI培养基中厌氧培养,得到所述发酵液。
3.根据权利要求2所述的发酵液,其特征在于,
活化菌液的培养条件为:在30~40℃培养24~72小时;
发酵液的培养条件为:30~40℃下培养48~72小时;
所述III型猪胃粘蛋白在BHI培养基中的质量浓度为2.5~4%。
4.一种权利要求1所述艾克曼菌株或权利要求2所述发酵液在制备改善腹泻的药物中的用途。
5.根据权利要求4所述的用途,其特征在于,所述腹泻是由肠道产毒素大肠杆菌引起的。
6.根据权利要求4所述的用途,其特征在于,所述艾克曼菌株或所述发酵液用于制备改善腹泻导致的炎症反应的药物。
7.根据权利要求4所述的用途,其特征在于,所述艾克曼菌株或所述发酵液用于制备改善腹泻导致的肠道黏膜屏障紊乱的药物。
8.根据权利要求4所述的用途,其特征在于,所述艾克曼菌株或所述发酵液用于制备维持腹泻导致的肠道菌群稳态的药物。
9.根据权利要求4所述的用途,其特征在于,所述艾克曼菌株或所述发酵液用于制备改善林麝腹泻的药物。
10.一种预防或治疗腹泻的微生物制剂,其特征在于,其包括权利要求1所述艾克曼菌株或权利要求2所述发酵液,以及可接受的辅料。
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