CN109666601B - 一株具有抑菌特性的植物乳杆菌及其在腹泻预防中的应用 - Google Patents
一株具有抑菌特性的植物乳杆菌及其在腹泻预防中的应用 Download PDFInfo
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- CN109666601B CN109666601B CN201811469469.4A CN201811469469A CN109666601B CN 109666601 B CN109666601 B CN 109666601B CN 201811469469 A CN201811469469 A CN 201811469469A CN 109666601 B CN109666601 B CN 109666601B
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Abstract
本发明提供一株具有腹泻预防与肠道护卫功能的植物乳杆菌新菌株,该菌株对常见的肠道致病菌的生长与肠道定植有抑制作用,且所述菌株对胃酸、胆盐具有良好的耐特受性,可以顺利到达肠道并与肠道上皮细胞发生黏附,发挥其肠道保护功能。本发明菌株除对肠道致病菌的生产具有抑制作用外,本发明菌株可通过上述特性更好地发挥肠道守卫和预防腹泻的功能。
Description
技术领域
本发明涉及生物技术领域,具体涉及一株植物乳杆菌新菌株,涉及该菌株在腹泻预防与肠道护卫功能中的应用。
背景技术
腹泻是人们日常生活中的一种常见病症,呈现季节性、地理性差异。夏秋时节是腹泻的高发季节,除疲劳受凉等因素外,细菌感染是夏秋腹泻发生的重要原因。在夏秋较为湿热的环境中,致病微生物繁殖较快,食物容易变质,如不注意饮食卫生,食用了大肠埃希氏菌、志贺氏菌、沙门氏菌、弯曲杆菌、弧菌等污染的食物可引发不同程度的腹泻症状。另一方面,随着人们生活水平的提高,出门旅行甚至出境旅行逐渐成为人们生活中的一部分,然而在旅行中易发的胃肠疾病如旅行者腹泻(TD)常给人们的旅行生活带来困扰。大约80-90%的旅行者腹泻也是由细菌感染引起,轻者可能仅有轻微的痛性痉挛,伴有急性稀薄便,重者则有严重腹痛、发热、呕吐,伴有血性腹泻。以上腹泻症状对于老人、小孩等肠胃较弱且免疫力较弱的人群则更为严重。
腹泻在日常生活中如此普遍但所受重视却不足。相比于腹泻发生后服用治疗药物,食用对腹泻有预防作用的食品防范于未然,未来将更受到消费者的青睐。近年来,国内益生菌市场发展迅速,但产品形式仍以酸奶和乳酸菌饮料等发酵乳制品为主,功能宣称仍以调节胃肠道功能、平衡肠道菌群为主,缺乏进一步的功能细分产品。同时,国内益生菌市场所用菌种基本依赖于杜邦、科汉森等国外企业垄断性供应,菌株同质化严重。本发明提供了一种新的益生菌选择。除具备基本的乳酸菌特性外,也应具备活着到达肠道从而发挥益生功能的能力,同时对腹泻的预防作用也应具有机理层面的解释说明。基于此本发明筛选和开发出一种具备抗腹泻功效的益生菌株,可应用于相应益生菌产品的开发,满足国内功能细分市场的需求。
发明内容
本发明的目的在于提供一株具有腹泻预防与肠道护卫功能的植物乳杆菌新菌株,该菌株对常见的肠道致病菌的生长与肠道定植有抑制作用,且所述菌株对胃酸、胆盐具有良好的耐特受性,可以顺利到达肠道并与肠道上皮细胞发生黏附,发挥其肠道保护功能。
本发明提供的菌株分离自四川泡菜样品,经鉴定其属于植物乳杆菌(Lactobacillus plantarum),命名为589菌株,于2018年5月25日在中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏地址为北京市朝阳区北辰西路1号院3号,微生物保藏编号为CGMCC No.15811
该菌株通过如下方法分离筛选得到:
(1)菌株分离:从四川随机收集泡菜样品溶入无菌稀释液,经一定稀释度稀释后采用选择性培养平板,进行分离培养。挑选具有典型乳酸菌菌落形态的单菌落进行进一步的划线纯化,完成镜检、鉴定等工作后,接种于液体培养基中获得纯培养物并制备保藏管。
(2)菌株筛选:采用双层平板扩散法对菌株的抑菌特性进行筛选。共进行577株菌株的初筛,获得25株优选菌株又进行进一步的复筛实验。同时结合菌株的耐受性、黏附性等基本特性,获得1株优选菌株,即为本发明中涉及的植物乳杆菌589菌株。
一种植物乳杆菌(Lactobacillus plantarum)589在肠道守卫与腹泻预防中的应用。
一种植物乳杆菌(Lactobacillus plantarum)589作为发酵剂或食品添加剂在食品、饮料、保健食品、药品或饲料中的应用。
一种含有植物乳杆菌(Lactobacillus plantarum)589的组合物。
一种含有植物乳杆菌(Lactobacillus plantarum)589的组合物在肠道守卫与腹泻预防中的应用。
一种含有植物乳杆菌(Lactobacillus plantarum)589的组合物作为发酵剂或食品添加剂在食品、饮料、保健食品、药品或饲料中的应用。
本发明菌株的生物学性质如下:
(1)形态学特征:在MRS琼脂培养基生长形态为乳白色菌落、不透明、圆形、表面光滑湿润、边缘整齐、中央凸起。革兰氏染色呈典型阳性,显微镜下观察细胞呈短杆状,有圆形末端,呈单个、成对、或短链状出现,无鞭毛,不产芽孢,不运动(图1所示)。
(2)培养特征:最适生长温度为37℃,兼性厌氧,生长于MRS培养基中。培养基成分:蛋白胨10g,牛肉膏10g,酵母膏5g,葡萄糖20g,乙酸钠5g,K2HPO4 2g,MgSO4·7H2O 0.2g,MnSO4·4H2O 0.2g,
(3)生物学鉴定:鉴定采用16s rDNA PCR扩增。扩增序列测序后所得结果于NCBI的GenBank 数据库中进行同源性比对分析,结果显示该菌株为植物乳杆菌(Lactobacillusplantarum)。基因序列如下所示:
本发明的优势:
(1)本发明的植物乳杆菌(Lactobacillus plantarum)589菌株分泌的代谢产物具有抑制藤黄微球菌、大肠杆菌、伤寒沙门氏菌、金黄色葡萄球菌、志贺氏菌生长的作用。其中,大肠杆菌、伤寒沙门氏菌、金黄色葡萄球菌、志贺氏菌均为常见的腹泻感染菌,与夏秋腹泻、旅行性腹泻发生相关,本发明菌株可通过抑制上述致病菌的生长对常见腹泻起到预防作用。且其抑菌作用优于目前市面所售菌株,作用效果应不弱于商业菌株。
(2)本发明的植物乳杆菌(Lactobacillus plantarum)589菌株具有良好的耐酸耐胆盐特性。在pH=2.5酸环境作用下3h后,活菌数仅降低0.20个数量级,在0.3%胆盐作用8h后,活菌数仅降低0.42个数量级;同时该菌株还具有良好的黏附特性。因此,所述植物乳杆菌589 菌株能够顺利到达肠道,并与肠道上皮细胞发生黏附,发挥其生理作用。
(3)本发明的植物乳杆菌(Lactobacillus plantarum)589菌株的另一项优势是在肠道内可发挥竞争性黏附作用。提前服用本发明菌株或当使用有致病菌风险的食物同时服用了本发明菌株,可以显著降低致病菌的黏附率。从体外实验结果看,对典型的肠道致病菌如沙门氏菌的黏附抑制率达53.8%。因此,本发明菌株除对肠道致病菌的生产具有抑制作用外,本发明菌株可通过上述特性更好地发挥肠道守卫和预防腹泻的功能。
(4)本发明使用的植物乳杆菌(Lactobacillus plantarum)589菌株是被囊括于卫生部 2010年颁布的《可用于食品的菌种名单》的食品级微生物;
附图说明
图1为本发明分离得到的植物乳杆菌新菌株的菌株外观特性。其中,菌株菌落特征如图1A所示,革兰氏染色显微镜观察特征如图1B所示。
图2为本发明植物乳杆菌对多种致病菌的抑菌结果图。其中,图2A为对照,图2B-图2F分别为植物乳杆菌589对藤黄微球菌、大肠杆菌、金黄色葡萄球菌、沙门氏菌、志贺氏菌的抑菌圈结果图。
图3为本发明植物乳杆菌与商业菌株的抑菌效果对比。其中,图3A为本发明菌株的抑菌结果图,图3B-图3D分别为对照菌1-对照菌3的抑菌结果图。
图4为本发明植物乳杆菌的黏附实验镜检结果图。其中,图4A为对照菌的黏附实验镜检结果图,图4B为本发明植物乳杆菌的黏附实验镜检结果图。
具体实施方式
以下结果具体实施例,对本发明进行详细说明,但本发明并不受这些实例的限制。
实施例1
植物乳杆菌589的抑菌特性
指示菌选取藤黄微球菌(Micrococcus luteus),以及大肠杆菌(Escherchia coliCICC 23689)、金黄色葡萄球菌(Staphylococcus aureus CICC 10301)、伤寒沙门氏菌(Salmonella typhimurium CICC 10437)、志贺氏菌(Shigella flexneri CICC 21534)四种常见的肠道感染致病菌。采用 NB培养基,于37℃培养条件下,连续活化三代后用于抑菌实验。采用双层平板打孔扩散法进行本发明菌株抑菌特性的测定。将已灭菌的1.5%琼脂倾倒在无菌培养皿中,待凝固后,将已分装灭菌的固体培养基(含1.1%琼脂)降温至一定温度后,与指示菌混合并立即倾注于已摆放牛津杯的琼脂层上。指示菌需根据生长情况调整至合理浓度。待上层凝固后,取出牛津杯,并在孔中分别加入100μL受试菌上清液与对照试剂。其中上清液制备方法如下:取菌株发酵液以4000g离心10min后,取上清,经pH调整后,采用0.22μm滤膜进行过滤,获得的无菌上清液用于抑菌实验。随后,将平板经扩散后放入37℃培养过夜后,观察指示菌的生长状况及抑菌圈的大小。
结果如下表及图2所示,本发明菌株植物乳杆菌589的菌培养上清液对藤黄微球菌
及四种致病菌均可观察到明显的抑菌圈,其中对大肠杆菌的抑菌效果最为明显,抑菌圈直
径达 24.86±2.55mm。表明植物乳杆菌589菌株在培养过程中分泌产生的菌代谢物对致病
菌具有抑菌效果,且其抑菌活性具有一定的广谱性。
指示菌名称 | 抑菌圈直径(mm) |
藤黄微球菌 | 17.74±1.52 |
大肠杆菌 | 24.86±2.55 |
金黄色葡萄球菌 | 19.99±0.21 |
伤寒沙门氏菌 | 21.77±1.74 |
志贺氏菌 | 22.57±0.35 |
对照 | - |
对比例1
植物乳杆菌589的抑菌特性比较
将本发明的植物乳杆菌589与商业菌株进行比较。对照菌1为目前在益生菌市场上广泛使用的具有调节肠道功能的鼠李糖乳杆菌,对照菌2与对照菌3为从加拿大市场收集的对腹泻有良好预防功能的鼠李糖乳杆和干酪乳杆菌。分别对本发明菌株与对照菌株进行培养,培养至对数生长末期后,取菌株发酵液以4000g离心10min后,取上清,经pH调整后,采用0.22μm 滤膜进行过滤,获得的无菌上清液用于抑菌实验。
指示菌选取藤黄微球菌,连续活化三代后用于抑菌实验。采用琼脂打孔扩散法进行本发明菌株与商业菌株抑菌特性的比较测定。采用1.5%的琼脂倾倒在无菌培养皿中制备琼脂层,随后将上层的固体培养基与指示菌混合并倾注于已摆放牛津杯的琼脂层上。待上层凝固后,取出牛津杯,并在孔中分别加入100μL本发明菌株的菌上清液与对照菌的菌上清液将平板经扩散后放入37℃培养过夜,观察指示菌的生长状况及抑菌圈的大小。
所得结果如下表及图3所示,本发明菌株与对照菌株的菌分泌物对藤黄微球菌均
具有抑菌作用。其中,植物乳杆菌589的菌培养上清液对藤黄微球菌的抑菌圈直径达 17.74
±1.52mm,结果均优于3款对照菌,说明本专利菌株拥有不弱于商业菌株的抑菌特性。
菌种 | 抑菌圈(mm) |
植物乳杆菌589 | 17.74±1.52 |
对照1(鼠李糖乳杆菌) | 11.85±2.21 |
对照2(鼠李糖乳杆菌) | 10.86±2.69 |
对照3(干酪乳杆菌) | 11.71±3.25 |
实施例2
植物乳杆菌589的耐受特性
将本发明菌株与市售商业菌株均培养至对数生长末期后,获取菌悬液,将菌悬液分成2组,分别进行耐酸特性与耐胆盐特性的测定,操作如下。(1)取一定量菌液,4000g/10min离心后弃上清。加入相同体积pH=2.5的MRS溶液,吹打混匀后,在37℃环境下孵育,用稀释涂布计数法测定0点及孵育1h、2h、3h后菌数的变化。(2)取一定量菌液,4000g,10min离心后弃上清。加入相同体积的含0.3%胆盐的MRS溶液,吹打混匀后,在37℃环境下孵育,用稀释涂布计数法测定0点及孵育4h、8h后菌数的变化。菌株存活率计算公式:菌株存活率(%)=N1/N0*100%。N1为菌株处理后活菌数,N0为菌株初始活菌数。
结果如下表所示,植物乳杆菌589表现出良好的耐酸、耐胆盐特性。在酸环境下孵育 3h后,菌数降低0.20个log值。在胆盐环境下孵育8h后,菌数降低0.42个log值。其中,酸环境下的耐受性优于对照的商业菌株,对照菌株在酸环境下孵育3h后,菌数降低0.74个log值。
实施例3
植物乳杆菌589的肠道黏附能力
本发明菌株与对照商业菌株培养至对数末期后收集菌液。4000g,10min离心后,用
含10%胎牛血清的DMEM完全培养基重悬,并调整菌液至一定浓度。HT29细胞培养后,采用胰
酶进行消化,用含10%胎牛血清的DMEM培养基(含青霉素100U/mL,链霉素100mg/mL)进行重
悬。经细胞计数后,将细胞悬液浓度调整至1×106cells/mL,接种于已放置细胞爬片的 12
孔细胞培养板中,于37℃,5%CO2培养24h。加入受试菌菌悬液1mL,37℃条件下于CO2培养箱
中孵育2h。孵育结束后,经PBS洗涤3次、甲醇固定后,取出细胞爬片。经革兰氏染色后,用中
性树脂封片,于光学显微镜下进行观察。结果如下表所示,植物乳杆菌589的黏附率略优于
对照商业菌。
菌株 | 黏附率(菌数/细胞数) |
植物乳杆菌589 | 1.96±0.15 |
对照菌 | 1.75±0.30 |
实施例4
植物乳杆菌589抑制致病菌黏附肠上皮细胞
本发明菌株与对照商业菌株培养至对数末期后收集菌液。4000g,10min离心后,经PBS洗涤,用含10%胎牛血清的DMEM完全培养基重悬,并调整菌液浓度为1×108cfu/mL。致病菌选用大肠杆菌H10407(Escherchia coli ATCC 35401)与伤寒沙门氏菌(Salmonellatyphimurium ATCC 14028),培养后收集菌液,于4000g,10min条件下离心后,经PBS洗涤,重悬于DMEM 完全培养基,并调整菌液浓度为1×108cfu/mL。HT29细胞采用胰酶进行消化,DMEM完全培养基重悬后,调整细胞悬液浓度至1×106cells/mL,接种于已放置细胞爬片的12孔细胞培养板中,于37℃,5%CO2培养箱内培养24h。
竞争性黏附实验:将受试菌与致病菌的菌悬液各取0.5mL加入到已含单层HT-29细胞的12孔培养板中,对照孔加入1mL致病菌菌悬液。将细胞培养板于细胞培养箱中孵育2h后,用PBS缓冲液清洗除去未黏附的菌体。加入1mL 0.5%的Triton X-100溶液,孵育5min,使细胞溶解。采用选择性固体平板分别计数两种致病菌(大肠杆菌与沙门氏菌)在每孔细胞内的黏附数。
排除性黏附实验:将本发明菌株与对照商业菌株的菌悬液1mL加入已含有单层HT-29 细胞的12孔培养板中。将其在细胞培养箱中孵育1h后,用PBS缓冲液清洗去除未黏附的菌株。将1mL致病菌菌悬液加入到12孔培养板中,继续孵育1h。对照孔不加入受试菌而直接加入致病菌菌悬液,孵育1h。用PBS缓冲液清洗后,加入1mL 0.5%的Triton X-100溶液,孵育5min使细胞溶解。采用选择性固体平板分别计数两种致病菌(大肠杆菌与沙门氏菌) 在每孔细胞内的黏附数。
如上表所示,本发明菌株植物乳杆菌589在肠道内可发挥竞争性黏附与排除性黏附作用。当大肠杆菌、沙门氏菌等致病菌到达肠道与肠上皮细胞发生黏附时,如同时也服用本发明菌株可以显著降低致病菌的黏附率,从本实验结果看,对大肠杆菌的黏附抑制率达21.1%,对沙门氏菌的黏附抑制率达53.8%。同时,如提前服用本发明菌株对肠道也会起到一定的保护作用,减少致病菌进入肠道后在肠上皮细胞上的黏附率。除对肠道致病菌的抑菌作用外,本发明菌株可通过上述特性更好地发挥肠道守卫和预防腹泻的功能。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
序列表
<110> 杭州娃哈哈科技有限公司
杭州娃哈哈集团有限公司
<120> 一株具有抑菌特性的植物乳杆菌及其在腹泻预防中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1442
<212> DNA
<213> 植物乳杆菌(Lactobacillus plantarum)
<400> 1
tacatgcagt cgaacgaact ctggtattga ttggtgcttg catcatgatt tacatttgag 60
tgagtggcga actggtgagt aacacgtggg aaacctgccc agaagcgggg gataacacct 120
ggaaacagat gctaataccg cataacaact tggaccgcat ggtccgagtt tgaaagatgg 180
cttcggctat cacttttgga tggtcccgcg gcgtattagc tagatggtgg ggtaacggct 240
caccatggca atgatacgta gccgacctga gagggtaatc ggccacattg ggactgagac 300
acggcccaaa ctcctacggg aggcagcagt agggaatctt ccacaatgga cgaaagtctg 360
atggagcaac gccgcgtgag tgaagaaggg tttcggctcg taaaactctg ttgttaaaga 420
agaacatatc tgagagtaac tgttcaggta ttgacggtat ttaaccagaa agccacggct 480
aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg atttattggg 540
cgtaaagcga gcgcaggcgg ttttttaagt ctgatgtgaa agccttcggc tcaaccgaag 600
aagtgcatcg gaaactggga aacttgagtg cagaagagga cagtggaact ccatgtgtag 660
cggtgaaatg cgtagatata tggaagaaca ccagtggcga aggcggctgt ctggtctgta 720
actgacgctg aggctcgaaa gtatgggtag caaacaggat tagataccct ggtagtccat 780
accgtaaacg atgaatgcta agtgttggag ggtttccgcc cttcagtgct gcagctaacg 840
cattaagcat tccgcctggg gagtacggcc gcaaggctga aactcaaagg aattgacggg 900
ggcccgcaca agcggtggag catgtggttt aattcgaagc tacgcgaaga accttaccag 960
gtcttgacat actatgcaaa tctaagagat tagacgttcc cttcggggac atggatacag 1020
gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc 1080
gcaaccctta ttatcagttg ccagcattaa gttgggcact ctggtgagac tgccggtgac 1140
aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac 1200
acgtgctaca atggatggta caacgagttg cgaactcgcg agagtaagct aatctcttaa 1260
agccattctc agttcggatt gtaggctgca actcgcctac atgaagtcgg aatcgctagt 1320
aatcgcggat cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1380
caccatgaga gtttgtaaca cccaaagtcg gtggggtaac cttttaggaa ccagccgcct 1440
aa 1442
Claims (6)
1.一株具有抑菌特性的植物乳杆菌(Lactobacillus plantarum)589,其特征在于,于2018年5月25日在中国微生物菌种保藏管理委员会普通微生物菌种保藏中心保藏,微生物保藏编号为CGMCC No.15811。
2.一种如权利要求1所述的植物乳杆菌(Lactobacillus plantarum)589在制备肠道守卫与腹泻预防的药物中的应用。
3.一种如权利要求1所述的植物乳杆菌(Lactobacillus plantarum)589作为发酵剂或食品添加剂在制备食品、药品或饲料中的应用。
4.一种含有如权利要求1所述的植物乳杆菌(Lactobacillus plantarum)589的组合物。
5.一种如权利要求4所述的含有植物乳杆菌(Lactobacillus plantarum)589的组合物在制备肠道守卫与腹泻预防的药物中的应用。
6.一种如权利要求4所述的含有植物乳杆菌(Lactobacillus plantarum)589的组合物作为发酵剂或食品添加剂在制备食品、药品或饲料中的应用。
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