CN117511826A - 尿路粘液乳杆菌及其应用 - Google Patents
尿路粘液乳杆菌及其应用 Download PDFInfo
- Publication number
- CN117511826A CN117511826A CN202410000615.8A CN202410000615A CN117511826A CN 117511826 A CN117511826 A CN 117511826A CN 202410000615 A CN202410000615 A CN 202410000615A CN 117511826 A CN117511826 A CN 117511826A
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- lactobacillus
- strain
- lvagi
- food
- pharmaceutical composition
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A61K35/66—Microorganisms or materials therefrom
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P15/02—Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P31/04—Antibacterial agents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
本发明涉及微生物领域,具体涉及尿路粘液乳杆菌及其应用。该尿路粘液乳杆菌无毒力因子、无耐药基因、不溶血,具有良好的安全性。该菌产乳酸能力强,能够降低女性阴道pH,抑制致病菌的繁殖。此外,该菌产生的抑菌物质,如过氧化氢、乳酸和细菌素等,对阴道加德纳菌表现出较好的抑菌能力。
Description
技术领域
本发明涉及微生物领域,具体涉及尿路粘液乳杆菌及其应用。
背景技术
女性的生殖道是开放的腔道,寄居着大量不同种类的微生物,是人体微生物的主要分布区之一,与女性生殖健康关系密切。女性生殖道中约有300多种微生物共生,包括细菌、病毒和真菌等,其中细菌是主要的微生物。它们彼此制约、相互制衡,形成动态平衡,多种阴道炎的发生与阴道微生态环境失衡有关。
生殖道不同部位的微生物组成不同,在健康的育龄妇女中,大多数细菌存在于下生殖道(阴道和子宫颈),上生殖道的细菌至今尚未得到很好的表征。阴道微生物群的微生物多样性较低,以乳杆菌为主。总体上可将阴道菌群分为五种社区群落(community statetypes,CST)类型。CST-I以卷曲乳杆菌为主,CST-II以格氏乳杆菌为主,CST-III以惰性乳杆菌为主,CST-V以詹氏乳杆菌为主,而CST-IV无优势乳杆菌群,并可进一步细分为CSTⅣ-A和CSTⅣ-B亚型,其中CSTⅣ-B与细菌性阴道病(Bacterial Vaginosis,以下简称“BV”)联系紧密,以厌氧菌为主, 如奇异杆菌、芬戈尔德菌和加德纳菌。
在疾病状态下,微生物组成和生物量负荷变化很大,多种病原体过度生长,阴道微生态环境进入一个脆弱的状态,不容易抵御致病菌的侵染、繁殖,出现各种阴道炎症。BV是一种常见的妇科疾病,感染率在15%-52%,是主要因阴道加德纳菌和其他厌氧菌过度繁殖取代乳杆菌,造成阴道内菌群失调而出现临床症候群的一种阴道感染性疾病。据资料报道,BV是导致组织性绒毛膜炎、羊水感染、剖腹产后子宫内膜炎及其他妊娠不良和妊娠并发症的危险因素。
针对BV,临床治疗方法是采用抗生素甲硝唑或克林霉素,甲硝唑是一种前体药,在无氧环境中,细菌胞内酶促还原将甲硝唑的硝基还原成氨基,从而将抗生素转化为活性形式,然后通过与病原体DNA共价结合破坏其螺旋结构并导致单链和双链断裂,进而使病原体DNA降解并死亡;克林霉素能够与细菌核糖体上的50S核糖体亚基结合,阻止肽链的延长,从而抑制细菌细胞的蛋白质合成,导致细菌死亡。采用抗生素治疗虽然见效快,但也存在很大缺陷,有以下两方面:(1)对阴道微环境中所有抗生素敏感微生物均有抑制作用,因此,治疗后阴道微生态没有恢复到一个健康的、能抵御致病菌侵袭的平衡状态,被抑制或杀灭的病原微生物或者外来的病原微生物还会再度繁殖甚至致病而出现复发或新的阴道炎症;(2)微生物产生耐药性、且抗生素无法使得阴道微生态环境平衡,导致了难治性BV。因而,抗生素治疗虽然见效快,但复发率高,3月内复发率高达30%。
阴道微生态失衡的治疗包括杀菌、黏膜修复、恢复阴道微生态平衡三步。杀菌是治疗阴道炎症的第一步,抑制或灭杀病原微生物,包括过度增殖的需氧菌和厌氧菌、芽生孢子或者真菌、滴虫等。病原微生物被抑制或灭杀后,阴道黏膜的免疫修复和优势乳杆菌的恢复才是治疗阴道炎症的最终目标。在这期间,如果阴道黏膜的修复、乳杆菌的恢复过程被影响、阴道内理化环境未恢复至正常,被抑制的病原微生物或者外来的病原微生物还会再度繁殖甚至致病而出现复发或新的阴道炎症。而益生菌在阴道内可以迅速占据阴道上皮的受体,产生对阴道的保护作用,从而促使阴道恢复至正常微环境,减少阴道炎症的复发。因此,采用益生菌微生态制剂治疗BV是以现在的技术手段看来,最为优选的一种方式。
目前国内上市的阴道微生态药物仅有2种,一种以肠链球菌(Streptococcus faecalis)为活性成分,另一种则以德氏乳杆菌(Lactobacillus delbrueckii)为活性成分。此外,还有一些药物管线处于临床开发阶段,如欧赛微科含卷曲乳杆菌Lc262-1的活菌胶囊最近刚完成3期临床试验,四川厌氧生物含4种优势乳杆菌的KAL-001活菌胶囊正处于2期研究中。除了药品外,本领域还出现一些口服益生菌,比如科汉森开发的UREX®和ASTARTE®,已经被广泛应用在各种针对女性生殖道健康问题的口服产品中,据报道,其原理是益生菌经口腔-肠道-肛门的途径传播至阴道。
中国专利文件CN102851248 A公开了一种用于防治细菌性阴道病的詹氏乳杆菌。中国专利文件CN 107794236 A公开了一种卷曲乳杆菌及其应用。中国专利文件CN108004187 A公开了一种格氏乳杆菌及其用于制备阴道抑制菌药物中的应用。
由于人体阴道内乳杆菌多样性丰富,不同乳杆菌表现出不同的益生能力,在阴道微环境协同发挥作用,且存在个体差异,不同女性阴道内优势菌株略有区别,因此在选择对应的阴道用乳杆菌益生菌时,需要综合考虑乳杆菌的种类及不同菌种的益生能力。
2021年首次报道了分离自健康女性尿液的尿路粘液乳杆菌,暂未报道其对人体助益及相关功效。本发明提供的尿路粘液乳杆菌菌株分离自健康女性阴道分泌物,具有良好的安全性,同时在产酸、过氧化氢及抑菌等方面有良好益生功能,有助于解决女性生殖道健康问题。
发明内容
本发明的第一目的在于提供一种尿路粘液乳杆菌(Limosilactobacillus urinaemulieris)菌株,所述菌株选自保藏号为CCTCC NO: M 20231491的尿路粘液乳杆菌Lvagi-34。
在一些具体实施方式中,所述菌株的16S rDNA序列如SEQ ID NO.1所示。
本发明的第二目的在于提供前述尿路粘液乳杆菌菌株的培养方法,所述方法包括将所述尿路粘液乳杆菌菌株接种至培养基,进行增殖培养,得到增殖的尿路粘液乳杆菌菌株。
在一些具体实施方式中,所述培养基为MRS培养基。
本发明的第三目的在于提供一种食品、保健品或药物组合物,其活性成分含有前述的尿路粘液乳杆菌菌株或前述培养方法得到的尿路粘液乳杆菌菌株。
在一些具体实施方式中,所述组合物的活性成分还包括选自卷曲乳杆菌、詹氏乳杆菌、约氏乳杆菌、德氏乳杆菌和格氏乳杆菌中的一种或多种。
在一些具体实施方式中,所述尿路粘液乳杆菌菌株作为唯一活性成分。
在一些具体实施方式中,所述组合物的单个制剂中含有106~1015 CFU的尿路粘液乳杆菌(Limosilactobacillus urinaemulieris)菌株。
本发明的第四目的在于提供前述尿路粘液乳杆菌菌株在制备改善女性生殖道健康的产品中的应用。
在一些具体实施方式中,所述改善女性生殖道健康的产品作为抑菌剂或杀菌剂。
在一些具体实施方式中,所述改善女性生殖道健康的产品用于抑制或杀灭阴道加德纳菌。
在一些具体实施方式中,本发明所述的尿路粘液乳杆菌Lvagi-34具有如下形态特征:
(1)显微镜检为革兰氏阳性短杆菌;
(2)在MRS固体培养基中菌落形态呈浅白色半透明状圆形菌落,中间凸起、表面光滑湿润。
本发明的尿路粘液乳杆菌Lvagi-34无毒力因子、无耐药基因、不溶血,具有良好的安全性。产乳酸能力强,能够降低女性阴道pH,并且可产抑菌物质过氧化氢,可有效抑制致病菌繁殖。
本发明提供的菌株保藏信息如下:
菌株名称:尿路粘液乳杆菌(Limosilactobacillus urinaemulieris)Lvagi-34
保藏日期:2023年08月17日
保藏单位:中国典型培养物保藏中心(China Center for Type CultureCollection,CCTCC),地址:湖北省武汉市武汉大学,邮编:430072,电话:027-68754052
保藏编号:CCTCC NO: M 20231491。
附图说明
图1为实施例1中尿路粘液乳杆菌Lvagi-34菌落形态正面照片。
图2为实施例1中尿路粘液乳杆菌Lvagi-34革兰氏染色照片。
具体实施方式
定义与说明
针对本发明所请求保护的特定保藏编号的尿路粘液乳杆菌菌株,其含义包括但不限于:
存放于保藏中心的微生物保藏号为CCTCC NO: M 20231491的尿路粘液乳杆菌(Limosilactobacillus urinaemulieris)Lvagi-34株;
与Lvagi-34株具有相同基因组的尿路粘液乳杆菌菌株;
基于前述1或2的没有突变的传代菌株;
基于前述1、2或3的在传代中积累微小突变的,但毒性、免疫原性与生物活性没有实质变化的传代菌株;
基于前述1-4任一所述菌株的活菌或灭活形式,其可以是完整的菌体或裂解物或发酵产物等衍生物。
如本领域所知,菌株经传代应用不可避免引入微小的突变,当突变发生在非编码序列区或者编码区的同义突变或者不影响菌株毒性、免疫原性与生物活性的突变(比如,可能是两个结构域之间的连接氨基酸残基,或者位于蛋白质高级结构内部因不与免疫细胞接触而不影响毒性、免疫原性与生物活性的微小突变的残基),可以合理预期,当这些微小变化没有明显影响后代毒株的毒性、免疫原性与生物活性的情况下,仍然能实现本发明的目的,且其源于本发明贡献的菌株,因此仍在本发明的实质技术贡献范围内。这些微小的突变仍属于非实质性突变,应当视为毒性、免疫原性与生物活性没有变化的突变菌株。
毒性、免疫原性与生物活性没有实质变化,包括担不限于,在检测灵敏度、检测限等检测技术的局限性和可接受或不可避免误差的范围内视为毒性、免疫原性与生物活性是相同的。用细胞、动物等测定Lvagi-34株后代的毒性、免疫原性与生物活性,由于细胞品系、动物品种、年龄、性别、健康状况、培养条件等体现的差别以及可预期或不可避免的系统误差属于没有实质性变化。
本发明所述的组合物含有活性成分Lvagi-34株,以及其他成分,比如不具有生理功效的辅料成分,或者其他功效性成分。功效性成分包括但不限于其他功效菌株,或益生元、后生元成分等。
作为一种较优的方式,本发明的组合物含有其他活性乳杆菌,如选自卷曲乳杆菌、詹氏乳杆菌、约氏乳杆菌、德氏乳杆菌和格氏乳杆菌中的一种或多种。
辅料成分包括添加剂、药物载体和赋型剂。药物载体是指不对受试者引起显著刺激且不消除所施用的益生菌的生物活性及特性的药学载体。药学上可接受的载体可增强或稳定组合物,或可用于促进组合物的制备。药学上可接受的载体可包括溶剂、分散介质、涂层、表面活性剂、抗氧化剂、等渗剂、吸收延迟剂、盐、药物稳定剂、结合剂、赋形剂、崩解剂、润滑剂、甜味剂、调味剂、染料等及其组合,正如本领域技术人员已知的(参见例如Remington's Pharmaceutical Sciences,第18版MackPrinting Company,1990,第1289-1329页)。除非常规载体与活性成分不相容,否则考虑将其用于治疗性或药物组合物中。载体可经选择以使受试者的不利副作用降至最低和/或使活性成分的失活降至最低。
赋型剂是指添加至药物组合物中以使药物具有一定形状或一定浓度的物质。例如无菌水、生理盐水、聚亚烷基二醇(诸如聚乙二醇)、植物油或氢化萘、碳酸氢钙、磷酸钙、多种糖、各种类型淀粉、纤维素衍生物、明胶等。
本发明所述的组合物可以制备成任一种便于使用的形式,例如临床或食品中常见的粉剂、片剂、颗粒剂、凝胶剂、胶囊或液体剂。
本发明所述的组合物以能发挥功效的含量(治疗有效量)和频率给予使用对象,推荐单次使用剂量中含有106~1015 CFU、107~1013 CFU或107~1012 CFU的尿路粘液乳杆菌(Limosilactobacillus urinaemulieris)。
本发明中的特定温度参数,如无特殊说明,应理解为恒温处理,并允许在一定温度区间内存在变动。如在±5℃、±4℃、±3℃、±2℃、±1℃的范围内波动。
下面将结合附图对本发明实施例中的技术方案进行清楚、完整地描述。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例,基于本发明中的实施例,本领域技术人员在没有做出创造性劳动的前提下所获得的所有其它实施例,均应属于本发明保护的范围。
以下实施例中所用到的材料的配制方法或购买渠道如下:
MRS肉汤制备:称量MRS成品培养基(Thermo Scientific™,CM0359B)粉末52.0 g,溶解至1 L蒸馏水中;加热煮沸,凉至室温加入0.55 g半胱氨酸盐酸盐,搅拌溶解后调节pH值至6.5;装上定量分液器并通N2,加热至沸腾,在微沸状态下煮20 min,冷却后分装至10mL厌氧管中,118 ℃高温湿热灭菌20 min,阴凉、避光存放、备用。
MRS固体培养基制备:称量MRS成品培养基(Thermo Scientific™,CM0359B)粉末52.0 g,琼脂粉15.0 g,溶解至1 L蒸馏水中,加热煮沸,沸腾后加入0.55 g半胱氨酸盐酸盐,调节pH值至6.5,118 ℃高温湿热灭菌20 min,阴凉、避光存放、备用。
过氧化氢半定量培养基制备:称量MRS成品培养基(Thermo Scientific™,CM0359B)粉末52.0 g,琼脂粉15.0 g,溶解至1 L蒸馏水中,调节pH值至6.5,118 ℃高温湿热灭菌20 min,灭菌结束后放入50 ℃水浴锅保温30 min,加入3,3',5,5'-四甲基联苯胺(TMB)(终浓度为0.25 mg/mL)和辣根过氧化物酶(HRP)(终浓度为0.01 mg/mL)混匀;冷却凝固后,放于4 ℃冰箱待用。
无氧PBS的配制:称量磷酸二氢钾0.27 g,磷酸氢二钠1.42 g,氯化钠8 g,氯化钾0.2 g,溶解至1 L的蒸馏水中,加热煮沸,凉至室温加入0.55 g半胱氨酸盐酸盐,搅拌溶解后调节pH值至6.5,装上定量分液器并通N2,加热至沸腾,在微沸状态下煮30 min,冷却后分装至10 mL厌氧管中,121 ℃高温湿热灭菌30 min,阴凉、避光存放、备用。
无氧BHI液体培养基配制:称量BHI成品培养基(Thermo Scientific™,CM1135B)粉末37.0 g,溶解至1 L蒸馏水中,加热煮沸,凉至室温加入0.55 g半胱氨酸盐酸盐,搅拌溶解后调节pH值至6.5,装上定量分液器并通N2,加热至沸腾,在微沸状态下煮20 min,冷却和分装过程中通入N2和CO2(1:1比例),分装至10 mL厌氧管中,118 ℃高温湿热灭菌20 min,阴凉、避光存放、备用。
无氧BHI半固体培养基配制:称量BHI成品培养基(Thermo Scientific™,CM1135B)粉末37.0 g,溶解至1 L蒸馏水中;加热煮沸,凉至室温加入6 g琼脂粉,0.55 g半胱氨酸盐酸盐,搅拌溶解后调节pH值至6.5,装上定量分液器并通N2,加热至沸腾,在微沸状态下煮20 min,稍稍冷却,冷却和分装过程中通入N2和CO2(1:1比例),及时分装至10 mL厌氧管中,118 ℃高温湿热灭菌20 min,阴凉、避光存放。
实施例1 菌株的分离与鉴定
用阴道棉拭子采集20-40岁中国健康女性的阴道分泌物样品,取2 mL无菌无氧的PBS缓冲液于装有上述棉拭子的厌氧管中,充分震荡混匀,并以其为原液连续十倍梯度稀释;取100 μL稀释10000倍的液体涂布于MRS固体培养基,置于厌氧培养箱中37℃培养,培养48 h后,挑取若干单菌落分别在MRS肉汤培养基中培养24 h,并将培养后得到菌液的一部分转接继续培养,菌液的另一部分进行细菌DNA提取;通过细菌16S rRNA基因扩增及测序,并将测序结果进行BLAST对比,最终确定3株尿路粘液乳杆菌(Limosilactobacillus urinaemulieris),分别命名为Lvagi-34、Lvagi-35、Lvagi-42。
取尿路粘液乳杆菌Lvagi-34菌株PCR验证后菌液划线至MRS固体培养基,37℃厌氧静置培养24-48 h,其菌落形态呈浅白色半透明状圆形菌落,中间凸起、表面光滑湿润,正面照片见图1。
取1环尿路粘液乳杆菌Lvagi-34落于载玻片,滴加适量无菌ddH2O涂布为稀薄菌液层,载玻片置于酒精灯上加热至水分蒸发,并在火焰快速通过2-3次以固定菌体。随后按照革兰氏染色试剂盒说明书(广东环凯微生物科技有限公司,029010)操作染色。显微镜检观察拍照,菌体染色镜检为革兰氏阳性短杆菌,见图2。
实施例2 菌株全基因组新颖性分析与保藏
将尿路粘液乳杆菌Lvagi-34接种至5 ml MRS肉汤培养基,培养至对数生长期后,提取菌株全基因组DNA,利用Illumina高通量测序平台NovaSeq 6000进行全基因组测序。按常规方法组装及注释后,将蛋白序列输入VFDB(Virulence Factor Databases)及CARD(TheComprehensive Antibiotic Resistance Database)数据库分别进行毒力因子及耐药基因分析。结果显示,该菌不具有毒力因子和耐药基因。
利用平均核苷酸相似度(Average Nucleotide Identity,ANI)进行菌株的新颖性分析。通过在Genbank中进行搜索,找到了5个已公开的Limosilactobacillus urinaemulieris全基因组,通过fastANI(v1.33)比较发现,其中2个菌株与Lvagi-34全基因组最相近并低于99.9%,分别为GCA_014838745.1(98.8398%)和GCA_932750685.1(97.5919%)。故可认为Lvagi-34为新菌株,其16S rDNA序列如SEQ ID NO.1所示。
实施例3 低pH生长耐受实验
将保藏的尿路粘液乳杆菌Lvagi-34在pH值6.5的MRS肉汤活化,37 ℃过夜培养。将活化菌液按照10%比例转接至pH值4-5的MRS肉汤培养基中,每2-3 h测量一次OD600值。
结果显示,尿路粘液乳杆菌Lvagi-34能在低pH环境(pH值4-5)中生长,具有耐酸特性。
实施例4 产乳酸试验
分别活化尿路粘液乳杆菌Lvagi-34、Lvagi-35、Lvagi-42,转接至MRS肉汤培养基中,每株菌设置2个平行,37 ℃条件下培养48 h,用pH 0.5-5.0试纸测定并记录培养48 h后的乳杆菌菌液的pH值,按以下两个条件选择菌株进行液相色谱。将上清液稀释5倍,加入浓硫酸进行前处理,上样前用 0.22 μm 针头滤器过滤。液相色谱相关参数如下:
仪器型号:Agilent,分析型液相色谱1200
色谱柱型号:伯乐,Aminex HPX-87H
流动相:0.005 M H2SO4,速度0.6 mL/min
检测器及检测波长:DAD,207 nm;RID,示差折光信号
进样量:20 μL。
结果如表1所示,尿路粘液乳杆菌Lvagi-34能产生乳酸降低pH值。相同实验条件下,Lvagi-34产乳酸能力强于同种属的对照菌株Lvagi-35和Lvagi-42。
表1 3珠尿路粘液乳杆菌产乳酸能力及培养48 h pH值
微生物 | 乳酸含量平均值(mg/L) | pH平均值(48 h) |
尿路粘液乳杆菌Lvagi-34 | 13369.9780 | 3 |
尿路粘液乳杆菌Lvagi-35 | 11797.2532 | 3.5 |
尿路粘液乳杆菌Lvagi-42 | 10413.3975 | 3.5 |
实施例5 产过氧化氢能力
尿路粘液乳杆菌Lvagi-34、Lvagi-35、Lvagi-42经活化后,用移液器吸取2 μL菌液点种于含0.25 mg/mL TMB及0.01 mg/mL HRP的MRS固体培养基中,将平皿置于相同的厌氧密封罐中,加入厌氧产气袋,37 ℃条件下培养,在48 h观察时间点,取出相应平皿,暴露于空气中,30 min后观察显色反应并拍照记录:以德氏乳杆菌为阳性对照,比德氏乳杆菌产生的蓝色深计为4分,与德氏乳杆菌产生的蓝色相当计为3分,比德氏乳杆菌产生的蓝色浅计为2分,显蓝色非常弱计为1分(轻微显色反应),不变色的计0分。
结果如表2所示,尿路粘液乳杆菌Lvagi-34在培养48 h时可产生一定量过氧化氢。
表2 3珠尿路粘液乳杆菌产过氧化氢能力
微生物 | 产过氧化氢评分-48 h |
尿路粘液乳杆菌Lvagi-34 | 1 |
尿路粘液乳杆菌Lvagi-35 | 1 |
尿路粘液乳杆菌Lvagi-42 | 1 |
实施例6 抑制阴道加德纳菌试验
尿路粘液乳杆菌经活化后,取0.1 mL菌液与融化的MRS固体培养基混匀,倾注入6cm平皿内,完全凝固后37 ℃培养48 h,取出平皿,用内径6 mm的打孔器在琼脂培养基上打孔,获得菌饼;阴道加德纳菌(购买自北京北纳创联生物技术研究院,BNCC337545)经活化转接后,用无氧无菌PBS缓冲液以10倍梯度,取10-1稀释液0.5 mL与5.25 mL含5%马血清和融化的BHI固体培养基混匀,倾注入9 cm平皿内,完全凝固后,将尿路粘液乳杆菌菌饼轻放在BHI固体培养基表面,每皿对称放4个菌饼,每株菌设置2个平行,放入厌氧的密封罐中,加厌氧产气袋,平皿正置培养48 h,用游标卡尺测量抑菌圈大小计算平均值。
结果如表3所示,尿路粘液乳杆菌Lvagi-34能有效抑制加德纳菌生长。相同实验条件下,Lvagi-34抑菌能力强于同种属的对照菌株Lvagi-35和Lvagi-42。
表3 抑制阴道加德纳菌能力
微生物 | 抑菌圈直径(mm)-平均值 |
尿路粘液乳杆菌Lvagi-34 | 9.18 |
尿路粘液乳杆菌Lvagi-35 | 8.59 |
尿路粘液乳杆菌Lvagi-42 | 8.66 |
实施例7 溶血实验
按10%接种量将保藏的尿路粘液乳杆菌Lvagi-34接种至5 mL MRS肉汤培养基中,以粪肠球菌(β溶血,CICC23658,购自中国工业微生物菌种保藏管理中心)作为阳性对照,以空白培养基作为阴性对照。所有菌株均在MRS肉汤培养基体培养基中37℃厌氧培养12 h,得到活化菌株。各取2.5 μL活化菌株接种至哥伦比亚血平板(上海科玛嘉微生物技术有限公司)上,每组设置3个平行,于37℃厌氧培养48 h后进行观察。
结果显示:阳性对照菌株菌落周围形成界限明显、完全透明的溶血环,为β溶血;尿路粘液乳杆菌Lvagi-34菌落周围的培养基没有变化,为γ溶血,即不溶血。
综上所述,尿路粘液乳杆菌Lvagi-34能产乳酸并耐受酸性环境,可适应并维持女性生殖道的酸性环境;该菌还可产一定量的过氧化氢,过氧化氢是细菌间发生拮抗作用的自净物质,可以抑制生殖道的有害菌并促进有益菌的生长;该菌在对阴道加德纳菌的抑制上也表现出良好的效果,具备预防和/或治疗与阴道加德纳菌相关的疾病。
Claims (9)
1.一种尿路粘液乳杆菌(Limosilactobacillus urinaemulieris)菌株,所述菌株选自保藏号为CCTCC NO: M 20231491的尿路粘液乳杆菌Lvagi-34。
2.权利要求1所述的尿路粘液乳杆菌(Limosilactobacillus urinaemulieris)菌株的培养方法,包括将尿路粘液乳杆菌接种至培养基,进行增殖培养,得到增殖的尿路粘液乳杆菌菌株。
3.根据权利要求2所述的培养方法,其特征在于,所述培养基为MRS培养基。
4.一种食品、保健品或药物组合物,其活性成分含有权利要求1所述的尿路粘液乳杆菌(Limosilactobacillus urinaemulieris)菌株或权利要求2所述的培养方法得到的尿路粘液乳杆菌菌株。
5.根据权利要求4所述的食品、保健品或药物组合物,其特征在于,所述组合物的活性成分还包括选自卷曲乳杆菌、詹氏乳杆菌、约氏乳杆菌、德氏乳杆菌和格氏乳杆菌中的一种或多种。
6.根据权利要求4所述的食品、保健品或药物组合物,其特征在于,所述尿路粘液乳杆菌为唯一活性成分。
7.根据权利要求4所述的食品、保健品或药物组合物,其特征在于,所述组合物的单个制剂中含有106~1015 CFU的尿路粘液乳杆菌。
8.权利要求4所述的食品、保健品或药物组合物在制备改善女性生殖道健康的产品中的应用。
9.根据权利要求8所述的应用,其特征在于,所述改善女性生殖道健康的产品用于抑制或杀灭阴道加德纳菌。
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