CN111849799A - 益生菌株、其组合物及其用于制备泌尿生殖道保健组合物的用途 - Google Patents
益生菌株、其组合物及其用于制备泌尿生殖道保健组合物的用途 Download PDFInfo
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Abstract
本发明涉及微生物领域,尤其涉及益生菌株、其组合物及其用于制备泌尿生殖道保健组合物的用途。本发明提供一种泌尿生殖道保健的益生菌株,是寄存编号为DSM33288的约氏乳杆菌。本发明提供一种约氏乳杆菌或其代谢产物用于制备泌尿生殖道保健组合物的用途。本发明还提供一种泌尿生殖道保健组合物。该约氏乳杆菌及其代谢产物能抑制常见泌尿生殖道感染的致病细菌及真菌的生长,因此减少泌尿生殖道感染的风险或减轻其病症。
Description
相关申请的交叉引用
本申请主张在2019年04月26日在中国台湾提交的中国台湾专利申请号108114789的优先权,但关于约氏乳杆菌(Lactobacillus johnsonii)菌株的内容不主张该优先权。
技术领域
本发明涉及微生物领域,尤其涉及益生菌株、其组合物及其用于制备泌尿生殖道保健组合物的用途。
背景技术
在解剖学上,女性的直肠、阴道、及膀胱三者相邻,因此有研究指出肠道中的微生物可经由细菌黏附而进入泌尿生殖道。此过程被认为是一种自然现象,其为阴道及尿道外部提供维持健康所需的微生物菌群。在正常情况下,阴道主要寄宿有多种乳酸杆菌,例如卷曲乳杆菌(Lactobacillus crispatus)、詹氏乳杆菌(Lactobacillus jenesenii),其群体会排除病原体寄宿阴道、产生乳酸与维持阴道pH值为3.8至4.5以抑制病原体繁殖、以及产生抗菌物质与过氧化氢以杀死其他微生物。
当阴道及尿道外部因为各种原因,例如雌激素缺乏、抗生素治疗、在性交期间使用杀精子剂等,而丧失正常微生物菌群,泌尿生殖系统感染率就会上升。此外,由于女性较男性的尿道短且尿道与肛门及阴道口距离较近,相比男性有更高机率因为病菌由阴道口或肛门侵入尿道而罹患泌尿系统感染。常见的泌尿生殖道感染包括细菌性阴道感染、真菌性阴道感染、及泌尿道感染。细菌性阴道感染的主要病原体是厌氧革兰氏阴性杆菌,如阴道加德纳菌(Gardnerella vaginalis);真菌性阴道感染主要起因于白色念珠菌(Candidaalbicans);泌尿道感染的病原体通常是泌尿道致病型大肠杆菌(Escherichia coli)。该些感染虽可以藉由抗生素治疗,但是病原体抗药性的增加及感染的高复发率已成为有待解决的问题。
目前预防泌尿生殖系统感染的方法包括保持私密处清洁与通风、避免使用刺激性清洁用品、足量饮水、及生活规律与适度运动以维持免疫力等。然而,对于学业繁重或工作忙碌的现代人而言,恪守健康生活习惯有执行上的困难。因此,开发一种使用方便且有助于保持泌尿生殖道健康的新颖组合物,实有其必要。
发明内容
缘此,本发明的一目的在提供一种泌尿生殖道保健的益生菌株,其是可供食用的约氏乳杆菌(Lactobacillus johnsonii)的新颖菌株。该菌株是寄存于DSMZ-德国微生物保藏中心,其寄存编号为DSM33288。
本发明的另一目的在提供一种前述约氏乳杆菌或其代谢产物用于制备泌尿生殖道保健组合物的用途。
本发明的又一目的在提供一种泌尿生殖道保健组合物,包含一有效量的前述约氏乳杆菌或其代谢产物。
在本发明的一实施例中,该约氏乳杆菌或其代谢产物抑制一泌尿道感染病原菌的生长。该泌尿道感染病原菌包括大肠杆菌、葡萄球菌等革兰氏阴性菌。
在本发明的一实施例中,该约氏乳杆菌或其代谢产物抑制一细菌性阴道感染病原菌的生长。该细菌性阴道感染病原菌包括阴道加德纳菌、链球菌、及葡萄球菌。
在本发明的一实施例中,该约氏乳杆菌或其代谢产物抑制一真菌性阴道感染病原菌的生长。该真菌性阴道感染病原菌包括念珠菌,例如白色念珠菌。
本发明揭示的约氏乳杆菌及其代谢产物能抑制多种造成泌尿生殖道感染的细菌及真菌的生长,因此可用于制备泌尿生殖道保健组合物,以便减少泌尿生殖道感染的风险或减轻其病症。该组合物可具有粉末、颗粒、溶液、或胶体的剂型,且可制成食品、饮品、营养补充剂、或医药品,藉由口服或局部施用方式给予一个体。
鉴于先前研究显示人类肠道中的微生物可经由细菌黏附而进入泌尿生殖道,本发明所属技术领域熟习此技艺者当可理解,适量补充约氏乳杆菌DSM33288可增加该益生菌于肠道中的群体大小,进而提高该益生菌进入泌尿生殖道的机会,最终达到泌尿生殖道保健的目的。
以下将配合图式进一步说明本发明的实施方式,下述所列举的实施例是用以阐明本发明的发明特点及应用,而非以限定本发明的范围,任何熟习此技艺者,在不脱离本发明的精神和范围内,当可做些许更动与润饰,因此本发明的保护范围当视后附的申请专利范围所界定者为准。
附图说明
图1A是大肠杆菌未经处理(空白对照组)的生长情形的照片;
图1B是大肠杆菌经过本发明的一实施例的约氏乳杆菌培养物上清液处理后的生长情形的照片;
图2A是阴道加德纳菌未经处理(空白对照组)的生长情形的照片;
图2B是阴道加德纳菌经过本发明的一实施例的约氏乳杆菌培养物上清液处理后的生长情形的照片;
图3A是念珠菌未经处理(空白对照组)的生长情形的照片;
图3B是念珠菌经过本发明的一实施例的约氏乳杆菌培养物上清液处理后的生长情形的照片;
图4是本发明的约氏乳杆菌在人工胃液模拟试验中的菌数变化图;
图5是本发明的约氏乳杆菌在人工肠液模拟试验中的菌数变化图。
具体实施方式
本发明提供一种具有泌尿生殖道保健功效的约氏乳杆菌,其是分离自人类母乳,经培养及菌种鉴定后确认为一新颖菌株,寄存于DSMZ-德国微生物保藏中心(DeutscheSammlung von Mikroorganismen und Zellkulturen,DSMZ);寄存地址:德国布伦瑞克市38124因霍芬大街7B;保藏日期2019年9月19日;保藏编号DSM 33288。以下实施例显示该菌株及其代谢产物能显著抑制多种造成泌尿生殖道感染的细菌及真菌的生长,且该菌株具备耐胃酸与耐胆盐的特性。
定义
本文中所使用数值为近似值,所有实验数据皆表示在20%的范围内,较佳为在10%的范围内,最佳为在5%的范围内。
本文中所谓「有效量」是指一活性物质在一个体引发特定功效所需要的量。如本技术领域熟习技艺者所认知,有效剂量将依给予途径、赋形剂的使用、以及与其它物质共享的可能而变化。
本文中有关细菌培养的操作程序与参数条件等是落在熟习此项技术者的专业素养与例行技术范畴内。
本文中所谓「代谢产物」是指培养细菌时,经该细菌因代谢而分泌至细菌培养液中的物质。
材料与方法
细菌及真菌
以下实施例使用的细菌或真菌包括分离自母乳且经16S rRNA基因序列比对而确定的约氏乳杆菌DSM33288,其16S rRNA基因具有SEQ ID NO:1的核苷酸序列;购自食品工业发展研究所生物资源保存及研究中心的阴道加德纳菌(BCRC 17040);以及分离自人类粪便的大肠杆菌及分离自芥菜菜心的念珠菌。
约氏乳杆菌的培养
约氏乳杆菌DSM33288依1%的接种量在37℃静置培养于乳杆菌属MRS培养基(BDDifco Lactobacilli MRS Broth;Thermo Fischer Scientific)约16小时。所得细菌培养物(菌量约为5×109CFUs/mL)经过5000rpm离心20分钟后,收集上清液以供进一步分析。该上清液包含细菌代谢产物。
实施例1
约氏乳杆菌抑制泌尿道感染病原菌的生长
本实施例以分离自粪便的大肠杆菌为例示病原菌,评估本发明的约氏乳杆菌对泌尿道感染病原菌的抑制作用。简言之,预先将该大肠杆菌依1%的接种量在37℃、150rpm震荡培养于LB培养基(BD Difco LB Broth,Miller;Thermo Fischer Scientific)约16小时,再将100μL所得菌液(菌量约为5×108CFU/mL)涂布于具有一凹陷处的LB琼脂培养基(含1.5%琼脂的LB培养基)。其后,将50μL本发明的约氏乳杆菌的培养物上清液加入已涂有大肠杆菌的LB琼脂培养基的凹陷处(实验组)。作为对比,另设置一不予约氏乳杆菌培养物上清液处理的空白对照组。前述二组LB琼脂培养基在37℃静置培养24小时以观察是否形成抑菌圈。
图1A及1B分别显示空白对照组及实验组的LB琼脂培养基在24小时培养后的照片。依据图1A-1B,空白对照组培养基的凹陷处周围生长着大量大肠杆菌,但实验组培养基的凹陷处周围可观察到一直径约为1.3公分的抑菌圈(内径约0.6公分),此结果说明约氏乳杆菌DSM33288及其代谢产物可有效抑制泌尿道感染病原菌的生长。
实施例2
约氏乳杆菌抑制细菌性阴道感染病原菌的生长
本实施例以阴道加德纳菌BCRC 17040为例示病原菌,评估本发明的约氏乳杆菌对细菌性阴道感染病原菌的抑制作用。简言之,预先将该阴道加德纳菌依1%的接种量在37℃静置培养于添加5%绵羊血的LB培养基约16小时,再将100μL所得菌液(菌量约为1×108CFU/mL)涂布于具有一凹陷处的血液琼脂培养基(含5%绵羊血的胰化蛋白大豆琼脂培养基(BD BBL TrypticaseTM Soy Agar;Thermo Fischer Scientific))。其后,将50μL本发明的约氏乳杆菌的培养物上清液加入已涂有阴道加德纳菌的血液琼脂培养基的凹陷处(实验组)。作为对比,另设置一不予约氏乳杆菌培养物上清液处理的空白对照组。前述二组血液琼脂培养基在37℃静置培养24小时以观察是否形成抑菌圈。
图2A及2B分别显示空白对照组与实验组的血液琼脂培养基在24小时培养后的照片。依据图2A-2B,空白对照组培养基的凹陷处周围有阴道加德纳菌分布,但实验组培养基上的凹陷处周围可观察到一直径约为1.6公分的抑菌圈(内径约0.6公分),此结果说明约氏乳杆菌DSM33288及其代谢产物可有效抑制细菌性阴道感染病原菌的生长。
实施例3
约氏乳杆菌抑制真菌性阴道感染病原菌的生长
本实施例以分离自芥菜菜心的念珠菌为例示病原菌,评估本发明的约氏乳杆菌对真菌性阴道感染病原菌的抑制作用。简言之,预先将该白色念珠菌依1%的接种量在30℃、150rpm震荡培养于YPD培养基(BD Difco YPD Broth;Thermo Fischer Scientific)约16小时,再将100μL所得菌液(菌量约为2×107CFU/mL)涂布于具有一凹陷处的YPD琼脂培养基(含1.5%琼脂的YPD培养基)。其后,将50μL本发明的约氏乳杆菌的培养物上清液加入已涂有念珠菌的YPD琼脂培养基的凹陷处(实验组)。作为对比,另设置一不予约氏乳杆菌培养物上清液处理的空白对照组。前述二组YPD琼脂培养基在37℃静置培养24小时以观察是否形成抑菌圈。
图3A及3B分别显示空白对照组与实验组的YPD琼脂培养基在24小时培养后的照片。依据图3A-3B,空白对照组培养基的凹陷处周围为念珠菌所覆盖,但实验组培养基的凹陷处周围可观察到一直径约为2.3公分的抑菌圈(内径约0.6公分),此结果说明约氏乳杆菌DSM33288及其代谢产物可有效抑制真菌性阴道感染病原菌的生长。
实施例4
约氏乳杆菌的耐酸与耐胆盐特性
为验证本发明的约氏乳杆菌能否抵抗胃部的酸性环境与肠道中的胆盐,将该菌株的隔夜培养菌液(菌量约为5x109CFU/mL)用于人工胃液或肠液模拟试验。在人工胃液模拟试验中,该菌液依1%的添加量添加至人工胃液(0.2%氯化钠水溶液,pH 1.2、pH 2、或pH3),并于37℃、50rpm震荡培养3小时。在人工肠液模拟试验中,该菌液依1%的添加量添加至人工肠液(含0.68%磷酸二氢钾及0.1%、0.2%、或0.3%胆盐(oxgall)的水溶液,pH 6.8),并于37℃、50rpm震荡培养3小时。作为对比,另设置对照组,是将本发明的约氏乳杆菌的菌液培养于pH 7且不含胆盐的0.2%氯化钠或0.68%磷酸二氢钾水溶液。前述试验后所得菌液取100μL涂布于乳杆菌属MRS琼脂培养基,于37℃培养隔夜以计算菌数。
依据图4,本发明的约氏乳杆菌经过人工胃液(pH 3)内培养3小时的菌数与在pH 7下培养的菌数相当,显示其具有耐胃酸性。依据图5,本发明的约氏乳杆菌经过人工肠液(0.3%胆盐)内培养3小时的菌数与在无胆盐环境下培养的菌数相当,显示其对胆盐的耐受性。该些结果说明约氏乳杆菌DSM33288经由口服进入人体后得以在消化道中生存,因此能发挥抑制泌尿生殖道内病原菌生长的效用。
综上所述,本发明揭示的约氏乳杆菌能抑制常见泌尿生殖道感染的致病细菌及真菌的生长,因此可用于制备泌尿生殖道保健组合物。该组合物可具有粉末、颗粒、溶液、或胶体的剂型,且可制成食品、饮品、营养补充剂、或医药品,藉由口服或局部施用方式给予一个体。
【生物材料寄存】
约氏乳杆菌(Lactobacillus johnsonii),寄存于DSMZ-德国微生物保藏中心(Deutsche Sammlung von Mikroorganismen und Zellkulturen,DSMZ);地址:德国布伦瑞克市38124因霍芬大街7B;保藏日期2019年9月19日;保藏编号DSM 33288。
Claims (10)
1.一种泌尿生殖道保健的益生菌株,是寄存编号为DSM33288的约氏乳杆菌。
2.一种约氏乳杆菌或其代谢产物用于制备泌尿生殖道保健组合物的用途,其中所述约氏乳杆菌的寄存编号为DSM33288。
3.根据权利要求2所述的用途,其特征在于,所述约氏乳杆菌或其代谢产物抑制一泌尿道感染病原菌的生长。
4.根据权利要求3所述的用途,其特征在于,所述泌尿道感染病原菌是大肠杆菌。
5.根据权利要求2所述的用途,其特征在于,所述约氏乳杆菌或其代谢产物抑制细菌性阴道感染病原菌的生长。
6.根据权利要求5所述的用途,其特征在于,所述细菌性阴道感染病原菌是阴道加德纳菌。
7.根据权利要求2所述的用途,其特征在于,所述约氏乳杆菌或其代谢产物抑制真菌性阴道感染病原菌的生长。
8.根据权利要求7所述的用途,其特征在于,所述真菌性阴道感染病原菌是念珠菌。
9.一种泌尿生殖道保健组合物,包含一有效量的约氏乳杆菌或其代谢产物,其特征在于,所述约氏乳杆菌的寄存编号为DSM33288。
10.根据权利要求9所述的组合物,其特征在于,所述组合物是具有粉末、颗粒、溶液、或胶体的剂型。
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CN115181712A (zh) * | 2022-09-14 | 2022-10-14 | 中国农业科学院北京畜牧兽医研究所 | 约氏乳杆菌ljm20及其菌剂和应用 |
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