CN111254087B - 一株具有增强免疫力功能的高黏附性能瑞士乳杆菌及其应用 - Google Patents
一株具有增强免疫力功能的高黏附性能瑞士乳杆菌及其应用 Download PDFInfo
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- CN111254087B CN111254087B CN201911382679.4A CN201911382679A CN111254087B CN 111254087 B CN111254087 B CN 111254087B CN 201911382679 A CN201911382679 A CN 201911382679A CN 111254087 B CN111254087 B CN 111254087B
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Abstract
本发明属于微生物技术领域。本发明公开了一株具有增强免疫力功能的高黏附性能瑞士乳杆菌,其命名为WHH2580,于2019年10月23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,其微生物保藏编号为:CGMCC No.18730。微生物分类命名为瑞士乳杆菌Lactobacillus helveticus。本发明还公开了一种具有增强免疫力功能的高黏附性能瑞士乳杆菌的应用。本发明中菌株活菌形式和灭活形式均具备优良的黏附性能,且通过提高调节机体的脏器指数、非特异性免疫及特异性免疫反应来提高机体免疫力;本发明的乳杆菌具有免疫调节作用,如摄取相应组合物,则可通过赋活免疫功能来改善免疫功能下降。
Description
技术领域
本发明涉及微生物技术领域,尤其是涉及一株具有增强免疫力功能的高黏附性能瑞士乳杆菌及其应用。
背景技术
益生菌是能够在人体肠道内发挥生理功能而有助于健康的有益微生物,它们被宿主摄入后黏附于肠道粘膜上皮细胞,定殖形成稳定菌群。经大量研究证明,一些乳酸菌种属可对宿主产生有益作用,不仅有助于维持肠道微生态的平衡,营养物质的吸收,还可以调节宿主免疫功能,促进肠道粘膜屏障功能完善以及增强抗感染的能力。
黏附不仅是益生菌在肠道中定殖的前提,还能引起宿主细胞发挥一定的生物学效应,参与正常菌群生物屏障形成的机制,具有重要的生态学意义。在益生菌制品的开发过程中,外源性的益生菌能否在肠道中黏附和定殖时评价其效果的重要指标之一。
随着人们生活和工作节奏的加快,很大一部分人群由于饮食营养不均衡、工作压力大、缺乏运动等,不同程度上出现体质虚弱、营养不良、精神萎靡、疲乏无力、食欲降低、睡眠障碍等免疫力低下的现象。预防疾病最重要的手段是增强机体免疫力。
对于免疫力低下的人群来说,尤其是儿童,由于其生理结构和功能发育的不完善,更容易受到外来病原菌的威胁,再加上一些抗生素的滥用,则更需要较为安全的微生态食品(益生菌产品)为其增强免疫力。
目前对于免疫力的调节手段主要是通过补充各种各样的维生素、氨基酸等,服用期间是有效果的,但停用很短一段时间后,免疫力又会下降,没有达到从根本上改善人体脏器机能,使人体自身具备免疫力的功效。
近年,大量的研究表明,肠道微生物对于调节人体健康有着重要的作用。肠道是机体最大的免疫器官,机体50%以上的淋巴组织分布于肠黏膜,并且,益生菌可以通过抑制病原微生物的生长来平衡局部微生物群,或益生菌通过分泌各种酶和酸刺激肠道黏膜和肠上皮细胞代谢,进而增强局部和全身免疫反应。因此,或许可从肠道微生物入手,尝试找到调节免疫力的新方法,以克服现有治疗药物以及治疗方法副作用的缺陷。
益生菌可以冻干菌粉、乳制品发酵剂等形式应用于食品、保健品、膳食补充剂和药品中,并以多种产品形式呈现,满足消费者的营养健康需求。目前,市场上多数声称可提高免疫力的益生菌产品中所用的菌株多为国外益生菌。
许多研究显示乳酸菌具有免疫调节功效。然而益生菌功效具有菌株特异性,且自然界尚有许多乳酸菌未被发现或予以充分的研究。
我国地大物博,发酵传统食品资源丰富,从中进行可提高免疫力益生菌的挖掘和筛选,开发自主知识产权菌株,从而制造适合国人体质和提高免疫力需要的益生菌产品显得意义重大。
发明内容
为解决上述问题,本发明提供了一株具有增强免疫力功能的高黏附性能瑞士乳杆菌。该菌株具有优良的黏附性能,可提高体外脾淋巴细胞增殖和脾淋巴细胞分泌白细胞介素IL-12,促进人结肠癌上皮细胞HT-29分泌白细胞介素IL-8;此外,在免疫低下动物模型中,该菌株还可显著提高小鼠脏器指数、促进脾淋巴细胞增殖和提高NK细胞活性。
本发明还提供了具有增强免疫力功能的高黏附性能瑞士乳杆菌的应用。
为实现上述目的,本发明采用的技术方案如下:
一株具有增强免疫力功能的高黏附性能瑞士乳杆菌,其命名为WHH2580,于2019年10月23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,其微生物保藏编号为:CGMCC No.18730。保藏单位地址为北京市朝阳区北辰西路1号院3号,微生物分类命名为瑞士乳杆菌Lactobacillus helveticus。其具有如SEQ ID NO.1所示的16s rRNA基因序列。
该菌株是发明人在前期工作中从新疆那拉提红山沟采集的酸奶中筛选得到。
同时,该具有增强免疫力功能的高黏附性能瑞士乳杆菌具备黏附特性,具备耐酸耐胆盐特性。
本发明所提供的瑞士乳杆菌WHH2580菌株(活体和非活体)具备优良的黏附特性。在人结肠癌上皮细胞HT-29细胞模型试验中表现出优良的黏附能力,单细胞黏附活体菌数达15.69±5.94个,单细胞黏附非活体菌数达11.59±4.02个,显著优于阳性对照菌株鼠李糖乳杆菌LGG和干酪乳杆菌代田株LcS的黏附性能。该菌株可与肠道上皮细胞发生黏附,发挥生理功效。
本发明所提供的瑞士乳杆菌WHH2580菌株具备良好的耐酸耐胆盐特性。PBSpH3.0、PBS+0.3%pepsin pH2.0体系中3h降低一个数量级。0.1%Trypsin体系中3h降低0.8个数量级,0.3%BS体系中3h降低2.8个数量级,0.5%BS体系中3h降低3.6个数量级。
一种具有增强免疫力功能的高黏附性能瑞士乳杆菌的应用,将具有增强免疫力功能和高黏附性能瑞士乳杆菌的活菌形式、灭活形式或菌株衍生物用于制备药品、食品、保健品或膳食补充剂。
作为优选,药品为胶囊、粉剂或片剂。
作为优选,食品为发酵乳、乳酪、含乳饮料、乳粉或发酵果蔬。
本发明同时提供了具有增强免疫力功能的高黏附性能瑞士乳杆菌的应用,其是将瑞士乳杆菌WHH2580菌株的活菌形式、灭活形式或菌株衍生物与生理上可接受的赋形剂或稀释剂相结合,可以是食品或药品。其中的食品可以是发酵乳、乳酪、含乳饮料、乳粉、发酵果蔬或其他含有该菌株或该菌株衍生物的食品的任何一种,药品可以是胶囊、粉剂、片剂或其他药学上可以接受的赋形剂或稀释剂的任何一种。
上述由瑞士乳杆菌WHH2580菌株的活菌形式、灭活形式或菌株衍生物与生理上可接受的赋形剂或稀释剂相结合制得的食品、药品、保健品或膳食补充剂用于提高机体的免疫脏器指数、非特异性免疫及特异性免疫反应来提高机体免疫力。
因此,本发明具有以下有益效果:
(1)本发明所提供的瑞士乳杆菌WHH2580菌株(活体和非活体)具备优良的黏附特性。
在人结肠癌上皮细胞HT-29细胞模型试验中表现出优良的黏附能力,单细胞黏附活体菌数达15.69±5.94个,单细胞黏附非活体菌数达11.59±4.02个,显著优于阳性对照菌株鼠李糖乳杆菌LGG和代田株LcS的黏附性能。该菌株可与肠道上皮细胞发生黏附,发挥生理功效。
(2)本发明所提供的瑞士乳杆菌WHH2580菌株在体外细胞试验中,可促进体外脾淋巴细胞增殖,不加诱导剂的增值倍数为1.912±0.087,加诱导剂的增值倍数为1.533±0.109。
本发明所提供的瑞士乳杆菌WHH2580菌株在体外细胞试验中,可促进体外脾淋巴细胞分泌白细胞介素IL-12。瑞士乳杆菌WHH2580 IL-12/IL-10比值与鼠李糖乳杆菌LGG无显差。
本发明所提供的瑞士乳杆菌WHH2580菌株在体外细胞试验中,可促进人结肠癌上皮细胞HT-29分泌白细胞介素IL-8。瑞士乳杆菌WHH2580刺激分泌的IL-8显著高于商业菌株LGG。
本发明所提供的瑞士乳杆菌WHH2580菌株在体内免疫低下动物试验中,活性和非活性菌体均可显著提高小鼠脾淋巴细胞增殖和NK细胞活性。
(3)本发明所提供的瑞士乳杆菌WHH2580菌株相关的产品可以是其活体形式,也可以是其非活体形式。目前对于非活体益生菌的研究相对较少。与活菌制剂相比,非活菌制剂有一些优势,如货架期更长,安全性更高,且在储存和运输过程中不需要冷藏,成本更低。
附图说明
图1为瑞士乳杆菌WHH2580在显微镜下观察到的形态;
图2为瑞士乳杆菌WHH2580活体菌、非活体菌、商业菌株LGG和Lcs与HT-29细胞黏附效果图;
图3为实施例动物试验中各组BALB/c小鼠的体重变化图;
图4为实施例动物试验中各组BALB/c小鼠的脏器比值图;
图5为实施例动物试验中各组BALB/c小鼠的脾淋巴细胞转化数值图;
图6为实施例中各组BALB/c小鼠的NK细胞活性图。
具体实施方式
下面结合具体实施方式对本发明的技术方案作进一步的说明。
显然,所描述的实施例仅是本发明的一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例,都属于本发明保护的范围。
在本发明中,若非特指,所有的设备和原料均可从市场上购得或是本行业常用的,下述实施例中的方法,如无特别说明,均为本领域常规方法。
实施例
1.菌株的分离与培养
一株具有增强免疫力功能的瑞士乳杆菌,该菌株是发明人在前期工作中从新疆那拉提红山沟采集的酸奶中筛选得到。经鉴定属于瑞士乳杆菌(Lactobacillushelveticus),命名为WHH2580,于2019年10月23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,其微生物保藏编号为:CGMCC No.18730。该菌株的生物学性质如下:
形态学特征:在MRS琼脂培养基中生长状态为:在MRS琼脂培养基中生长形态为浅乳白色菌落,半透明、圆形、表面粗糙、边缘不整齐、平展。革兰氏染色呈典型阳性,显微镜下观察细胞呈长杆状,无鞭毛,不产芽孢,不运动。
培养特征:最适生长温度为37度,兼性厌氧,生长于MRS培养基中。
2.菌株的鉴定
2.1生物学鉴定:
针对16s rRNA基因序列测序,所得结果与NCBI GenBank数据库中进行同源性比对分析,结果显示该菌株为瑞士乳杆菌(Lactobacillus helveticus)。
基因序列如SEQ ID NO.1所示:
CCTTCCCGAAGGTTAGGCCACCGGCTTTGGGCATTGCAGACTTCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTTCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTGCAGACTGCAGTCCGAACTGAGAACAGCTTTCAGAGATTCGCTTGCCTTCGCAGGCTCGCTTCTCGTTGTACTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGCTGGCAACTAATAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTTAGCGTCCCCGAAGGGAACTCCTAATCTCTTAGGATGGCACTAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGAAAAACAGTTTCCGATGCAGTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTATTCTTCCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTGATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCCTTCTTCACCAACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATTGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATGCACCGCGGGGCCATCCCATAGCGACAGCTTACGCCGCCTTTTATAAGCTGATCATGCGATCTGCTTTCTTATCCGGTATTAGCACCTGTTTCCAAGTGGTATCCCAGACTATGGGGCAGGTTCCCCACGTGTTACTCACCCATCCGCCGCTCGCGTCCCCAGCGTCATTACCGAAGTAAATCTGCTGGTTCTGCTCGCTCGACTTGC
2.2WHH2580菌株的API50CHL系统鉴定:
WHH2580发酵菌液1mL离心,菌泥用生理盐水重悬。调整OD600至5mL重悬菌液的OD600为0.5左右。离心去上清,用API50培养基5mL重悬。加入API50板中,每孔100uL。37度孵育24h和48h。
WHH2580菌株可利用6种碳源:D-葡萄糖、D-果糖、D-甘露糖、N-乙酰葡萄糖胺、D-乳糖和D-海藻糖。
3.菌株耐受性
瑞士乳杆菌WHH2580菌株的耐酸耐胆盐性能:
3.1耐酸性能:WHH2580菌株二代活化后的菌液20mL/管离心,pH7.2 PBS洗两次。分别将菌泥置于20mL无菌PBS pH3.0、PBS+0.3%pepsin pH3.0和PBS pH7.2中,每一个体系进行两个重复。置于37℃,分别于0h、1h、2h、3h取样,进行活菌计数。
耐酸性实验结果显示:pH3.0在2h对该菌的存活性没有显著的影响,3h活菌数降低0.9个数量级。胃蛋白酶pH3.0在2h对该菌的存活性没有显著的影响,3h活菌数降低0.7个数量级。上述结果表明该菌有优良的耐酸性。
3.2耐胆盐性能:WHH2580菌株二代活化后的菌液20mL/管离心,pH7.2PBS洗两次。分别将菌泥置于20mL无菌PBS+0.1%Trypsin pH8.0、PBS+0.3%BS pH8.0、PBS+0.5%BSpH8.0和PBS pH7.2中,每一个体系进行两个重复。置于37℃,分别于0h、1h、2h、3h取样,进行活菌计数。
耐胆盐性实验结果显示:0.1%Trypsin体系中3h降低0.8个数量级,0.3%BS体系中3h降低2.8个数量级,0.5%BS体系中3h降低3.6个数量级。上述结果表明该菌有良好的耐胆盐性。
4.菌株黏附性瑞士乳杆菌WHH2580菌株的黏附性:
益生菌通过黏附作用定殖于宿主肠上皮细胞,逐步形成生物膜并产生多种代谢产物,从而构成一层天然的保护屏障,对宿主产生多种生理功效。因此,益生菌黏附并定殖于宿主肠上皮细胞上是发挥其益生作用的前提。
采用HT-29细胞作为黏附性试验的细胞株,具体实验方法如下:
HT-29细胞传至第三代,采用0.25%的胰酶消化后1000rpm离心5min,加入5mL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,直至细胞呈单细胞悬液。取适量细胞用血球细胞计数板计数,用含10%胎牛血清的DMEM培养基(含青霉素100U/mL,链霉素100mg/mL)将细胞稀释至细胞悬液浓度为到1×106cells/mL,取1mL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2天。
WHH2580菌株(鼠李糖乳杆菌LGG和代田株LcS为阳性对照菌株)二代活化后,取10mL菌液室温下4200g离心10min收集菌体,用10mL含10%胎牛血清的DMEM完全培养基中培养(不加双抗)重悬,调整菌浓度至2×108CFU/mL。取5mL菌株调整液95℃灭活5min。菌株调整液和灭活菌株调整液各取1mL接种于已放置细胞爬片的12孔细胞培养板中,各3个平行,于CO2培养箱中37℃孵育2h。
孵育结束后,缓慢吸去培养液,用PBS洗涤3次,用100%甲醇固定8min。取出细胞爬片静置20min,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。
黏附实验结果如下表。
瑞士乳杆菌WHH2580活体菌、非活体菌、商业菌株LGG和Lcs与HT-29细胞黏附效果附图2所示。
瑞士乳杆菌WHH2580活体菌和非活体菌的黏附能力显著高于阳性对照菌株鼠李糖乳杆菌LGG和代田株LcS。瑞士乳杆菌WHH2580非活体菌的黏附率稍低于其活体菌的黏附率,但非活体菌的黏附效果比活体菌好,HT-29细胞间的菌体较少,绝大部分非活体菌紧紧黏附在细胞上。
5.菌株耐药性瑞士乳杆菌WHH2580菌株抗生素的敏感性:
益生菌对抗生素的敏感性是检测菌株安全性的重要指标。为了检测WHH2580菌株的安全性,选取下列9种抗生素进行测试,检测WHH2580菌株对9种抗生素的敏感性,具体实验设计如下。
抗生素配制浓度是0.5ug/mL、1ug/mL、2ug/mL、4ug/mL、8ug/mL、16ug/mL、32ug/mL、64ug/mL、128ug/mL、256ug/mL,实际作用浓度是0.125ug/mL、0.25ug/mL、0.5ug/mL、1ug/mL、2ug/mL、4ug/mL、8ug/mL、16ug/mL、32ug/mL、64ug/mL。
无菌的96孔聚苯乙烯板中每个孔加入100μL培养基,2-11列按下表浓度标示每孔加50μL抗生素稀释液(ug/mL),1-11列每孔加入菌悬液50μL(菌液终浓度在105-106CFU/mL),密封后置37℃培养箱中24-48h,肉眼观察和测OD625。其中鼠李糖乳杆菌LGG和代田株LcS为阳性对照菌株。
实验结果如下表,MIC值ug/mL。
根据CLSI M4菌株对抗生素的敏感性的判断标准,得到下列结论。WHH2580菌株对达托霉素Daptomycin和克林霉素Clindamycin有抗性,对红霉素Erythromycin中性,对Penicillin、Ampicillin、Imipenem、Meropenem、Vancomycin和Linezolid敏感。LGG菌株对Imipenem、Meropenem、Vancomycin、Daptomycin、Clindamycin有抗性,对红霉素Erythromycin中性,对Penicillin、Ampicillin和Linezolid敏感。LcS菌株对Vancomycin和Daptomycin有抗性,对Imipenem、Meropenem和Erythromycin中性,对Penicillin、Ampicillin、Clindamycin和Linezolid敏感。
6.菌株抑制病原菌瑞士乳杆菌WHH2580菌株抑菌实验:
事先分装灭菌的1.5%素琼脂于无菌培养皿。摆放好牛津杯。灭菌1%琼脂的MRS培养基或NB培养基,50℃以下接种106-107CFU/mL浓度的五种指示菌(大肠埃希氏菌、金黄色葡萄球菌、福氏志贺氏菌、乙型副伤寒沙门氏菌、藤黄微球菌),振荡混匀后倾倒在之前倒好的有牛津杯的素琼脂培养基上。待以上培养基冷却凝固后,拔去牛津杯。在不同的小孔中分别加入100uL的相应菌液(WHH2580)和阳性对照(10g/mL多粘菌素B或1%Nisin),放入4℃冰箱中扩散7h。放入37℃培养16h后,观察并记录抑菌圈的大小。
实验结果如下表,表格中的数值为抑菌圈的大小cm。
实验结果显示WHH2580菌株对5种致病菌均有抑制效果,其对乙型副伤寒沙门氏菌、大肠埃希氏菌和福氏志贺氏菌的抑制作用和10g/mL多粘菌素B无明显差异,对金黄色葡萄球菌的抑制作用和1%Nisin无明显差异,稍低于其他对照菌株。
7.菌株增强免疫力功能
增强免疫功能的体外和体内实验:
7.1体外脾淋巴细胞增殖:
处死小鼠,无菌取脾脏,用眼科剪将脾脏剪碎后,用无菌的玻璃注射器芯将其碾碎,经200目金属筛网过滤后,加入含10%胎牛血清的无菌Hank’s液,1000rpm,4℃离心5min,收获细胞。加入5倍于细胞体积的细胞裂解液,裂解5min,加入含10%胎牛血清的无菌Hank’s液终止裂解,1000rpm,4℃离心5min去上清。用含10%胎牛血清的RPMI-1640培养基洗涤,1000rpm,4℃离心5min。洗涤两次,沉淀重悬于5mL含10%胎牛血清的RPMI-1640培养基。细胞计数,调整细胞浓度为5×106cells/mL。
乳酸菌活化菌液4000rpm室温下离心10min去上清,PBS洗涤两次,加入适量含10%胎牛血清的RPMI-1640培养基,调整其OD600至0.50±0.10。将细胞悬液加入96孔细胞培养板,每个样品3个平行。设置调零组(细胞培养基),空白对照组(细胞悬液+细胞培养基,诱导剂组(细胞悬液+10μg/mL Con A),乳酸菌组(细胞悬液+10μg/mL Con A+菌悬液),置二氧化碳培养箱37℃培养72h。每孔加入20μL MTT溶液(2.5mg/ml),37℃显色4h后,1500rpm,4℃离心10min,吸出上清,再加入100μL DMSO,用酶标仪于490nm下测定吸光值。
数据分析:在Con A诱导和不诱导的情况下,均能显著促进脾淋巴细胞增殖的益生菌作为优选菌株。
结果如下表所示,在Con A诱导和不诱导的情况下,瑞士乳杆菌WHH2580脾淋巴细胞增殖倍数高于两株商业菌株。
7.2体外脾淋巴细胞细胞因子IL-10和IL-12测定
细胞取材、红细胞裂解、洗涤、细胞计数、益生菌培养同上述体外脾淋巴细胞增殖步骤。将细胞悬液加入96孔细胞培养板,每个处理5个重复,分为调零组(细胞培养基100μL),空白对照组(1×107cells/mL的细胞悬液100μl,细胞培养基100μl),益生菌组(1×107cells/mL的细胞悬液100μL,1×108cfu/mL菌悬液100μL),37℃5%CO2培养箱中培养24h。1500rpm离心10min,吸取上清,0.22μm滤膜过滤,采用BD试剂盒测定IL-10和IL-12含量。
IL-12/IL-10比值高的菌株可作为免疫调节潜力菌株。
结果如下表所示,瑞士乳杆菌WHH2580 IL-12/IL-10比值与鼠李糖乳杆菌LGG接近,没有显差。
菌株编号 | IL-12浓度(pg/mL) | IL-10浓度(pg/mL) | IL-12/IL-10比值 |
鼠李糖乳杆菌LGG | 245.595 | 210.889 | 1.165 |
瑞士乳杆菌WHH2580 | 711.881 | 640.918 | 1.111 |
7.3体外人结肠癌细胞HT-29细胞因子IL-8测定
将生长融合至70%-80%的人结肠癌细胞HT-29进行消化,调整细胞浓度,1*106cell/孔接入24孔板,37℃培养,24h后换液。加入20ng/mL LPS处理6小时,再加入8*108CFU/mL菌液处理18小时后,3000r/min,5min离心得上清,用相应ELISA试剂盒检测白细胞介素IL-8的含量。
分泌IL-8高的菌株可作为免疫调节潜力菌株。
结果如下表所示。
菌株编号 | IL-8浓度(ng/mL) |
鼠李糖乳杆菌LGG | 2981±95 |
瑞士乳杆菌WHH2580 | 4021±145 |
瑞士乳杆菌WHH2580 IL-8高于商业菌株LGG。
7.4动物试验:
采取随机分组设计,将近交系BALB/C雄性小鼠(6-8周龄,16-20g),192只,适应期5天,随机分为8组,每组12只。动物饲养保持环境温度为21±2℃,湿度为30-70%,12h光照交替,自由饮水,自由摄入饲料。每三天换一次垫料,及时添水和饲料。分组如下:
对照组1:空白对照组,灌胃水;
对照组2:模型对照组,灌胃水+腹腔注射50mg/kg环磷酰胺造模;
对照组3:阳性药物组,灌胃40mg/kg盐酸左旋咪唑+腹腔注射50mg/kg环磷酰胺造模;
实验组1:灌胃本发明菌株菌悬液,灌胃剂量为2*108CFU/d;
实验组2:灌胃本发明菌株灭活菌悬液,灌胃剂量为2*108CFU/d;
其中灭活菌是通过80℃10min处理的。经平板培养,证实无活菌存在。
采用免疫功能低下模型动物进行试验。连续给予受试物38天,在第32天后开始给予免疫抑制剂。抑制剂前每周称一次体重,抑制剂后每天称一次体重。腹腔注射免疫抑制剂第5天开始检测,检测指标有脏器比,脾淋巴细胞增殖,NK细胞活性。
实验结果如下:
体重结果见图3。除空白对照外的各实验组的体重变化趋势类似,在注射完抑制剂的第一天体重下降,后面逐渐恢复。
脏器比结果见图4。模型组的胸腺/体重比和脾脏/体重比显著低于对照组。低剂量组和阳性药物组的胸腺/体重比显著高于模型组(P<0.05),高剂量组和死菌组的胸腺/体重比极显著高于模型组(P<0.001)。低剂量组、中剂量组、死菌组和阳性药物组的脾脏/体重比显著高于模型组(P<0.05)。
小鼠脾淋巴细胞增殖结果见图5。超低剂量组、低、中、高剂量组OD值显著高于模型组灭活菌组极显著高于模型组。表明WHH2580超低剂量、低、中、高剂量和灭活菌均能显著促进免疫低下小鼠脾淋巴细胞增殖。
小鼠NK细胞活性的测定结果见图6。中剂量组的NK细胞活性极显著高于模型组,灭活菌组的NK细胞活性显著高于模型组。表明WHH2580中剂量和灭活菌均能显著提高免疫低下小鼠NK细胞活性。
根据保健品功能评价方法,在免疫功能低下模型成立条件下,血液白细胞总数、细胞免疫功能(小鼠脾淋巴细胞转化实验,迟发型变态反应实验)、体液免疫功能(抗体生成细胞检测,血清溶血素测定)、单核-巨噬细胞功能(小鼠碳廓清实验,小鼠腹腔巨噬细胞吞噬荧光微球实验)、NK细胞活性,任两个方面试验结果为阳性,判定该受试样品对免疫功能低下者具有增强免疫力作用。WHH2580菌株在细胞免疫功能和NK细胞活性两个方面试验结果为阳性,可判定WHH2580菌株对免疫功能低下者具有增强免疫力作用。
WHH2580灭活菌株同样可以通过提高调节机体的脏器指数、非特异性免疫及特异性免疫反应来提高机体免疫力。
本发明所提供的瑞士乳杆菌WHH2580菌株相关的产品可以是其活体形式,也可以是其非活体形式。目前对于非活体益生菌的研究相对较少。与活菌制剂相比,非活菌制剂有一些优势,如货架期更长,安全性更高,且在储存和运输过程中不需要冷藏,成本更低。
本发明所提供的瑞士乳杆菌WHH2580菌株高黏附性能是其发挥免疫功效的前提。
应当理解的是,对于本领域普通技术人员来说,可以根据上述说明加以改进或变换,而所有这些改进和变换都应属于本发明所附权利要求的保护范围。
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<110> 杭州娃哈哈科技有限公司
杭州娃哈哈集团有限公司
<120> 一株具有增强免疫力功能的高黏附性能瑞士乳杆菌及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1425
<212> DNA
<213> 瑞士乳杆菌(Lactobacillus helveticus)
<400> 1
ccttcccgaa ggttaggcca ccggctttgg gcattgcaga cttccatggt gtgacgggcg 60
gtgtgtacaa ggcccgggaa cgtattcacc gcggcgttct gatccgcgat tactagcgat 120
tccagcttcg tgcagtcgag ttgcagactg cagtccgaac tgagaacagc tttcagagat 180
tcgcttgcct tcgcaggctc gcttctcgtt gtactgtcca ttgtagcacg tgtgtagccc 240
aggtcataag gggcatgatg acttgacgtc atccccacct tcctccggtt tatcaccggc 300
agtctcatta gagtgcccaa cttaatgctg gcaactaata acaagggttg cgctcgttgc 360
gggacttaac ccaacatctc acgacacgag ctgacgacag ccatgcacca cctgtcttag 420
cgtccccgaa gggaactcct aatctcttag gatggcacta gatgtcaaga cctggtaagg 480
ttcttcgcgt tgcttcgaat taaaccacat gctccaccgc ttgtgcgggc ccccgtcaat 540
tcctttgagt ttcaaccttg cggtcgtact ccccaggcgg agtgcttaat gcgttagctg 600
cagcactgag aggcggaaac ctcccaacac ttagcactca tcgtttacgg catggactac 660
cagggtatct aatcctgttc gctacccatg ctttcgagcc tcagcgtcag ttgcagacca 720
gagagtcgcc ttcgccactg gtgttcttcc atatatctac gcattccacc gctacacatg 780
gagttccact ctcctcttct gcactcaaga aaaacagttt ccgatgcagt tcctcggtta 840
agccgagggc tttcacatca gacttattct tccgcctgcg ctcgctttac gcccaataaa 900
tccggacaac gcttgccacc tacgtattac cgcggctgct ggcacgtagt tagccgtgac 960
tttctggttg attaccgtca aataaaggcc agttactacc tctatccttc ttcaccaaca 1020
acagagcttt acgatccgaa aaccttcttc actcacgcgg cgttgctcca tcagacttgc 1080
gtccattgtg gaagattccc tactgctgcc tcccgtagga gtttgggccg tgtctcagtc 1140
ccaatgtggc cgatcagtct ctcaactcgg ctatgcatca ttgccttggt aagccgttac 1200
cttaccaact agctaatgca ccgcggggcc atcccatagc gacagcttac gccgcctttt 1260
ataagctgat catgcgatct gctttcttat ccggtattag cacctgtttc caagtggtat 1320
cccagactat ggggcaggtt ccccacgtgt tactcaccca tccgccgctc gcgtccccag 1380
cgtcattacc gaagtaaatc tgctggttct gctcgctcga cttgc 1425
Claims (5)
1.一株具有增强免疫力功能的高黏附性能瑞士乳杆菌,其特征在于:
其命名为WHH2580,于2019年10月23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,其微生物保藏编号为:CGMCC No.18730,保藏单位地址为北京市朝阳区北辰西路1号院3号,微生物分类命名为瑞士乳杆菌(Lactobacillus helveticus)。
2.一种根据权利要求1所述具有增强免疫力功能的高黏附性能瑞士乳杆菌的应用,其特征在于:将具有增强免疫力功能和高黏附性能瑞士乳杆菌的活菌形式或灭活形式用于制备药品、食品或保健品。
3.根据权利要求2所述的具有增强免疫力功能的高黏附性能瑞士乳杆菌的应用,其特征在于:所述的药品为胶囊、粉剂或片剂。
4.根据权利要求2所述的具有增强免疫力功能的高黏附性能瑞士乳杆菌的应用,其特征在于:所述食品为发酵乳。
5.根据权利要求2所述的具有增强免疫力功能的高黏附性能瑞士乳杆菌的应用,其特征在于:所述食品为乳酪、含乳饮料、乳粉或发酵果蔬。
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