CN105779346A - 一种产细菌素的屎肠球菌及其应用 - Google Patents
一种产细菌素的屎肠球菌及其应用 Download PDFInfo
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- CN105779346A CN105779346A CN201610187192.0A CN201610187192A CN105779346A CN 105779346 A CN105779346 A CN 105779346A CN 201610187192 A CN201610187192 A CN 201610187192A CN 105779346 A CN105779346 A CN 105779346A
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- enterococcus faecalis
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- bacteriocin
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- C—CHEMISTRY; METALLURGY
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Abstract
本发明涉及一种产细菌素的屎肠球菌菌株,该菌株的保藏名称为:屎肠球菌(Enterococcus faecium)R1,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心,保藏日期:2016年1月20日,保藏号:CGMCC No.12085。该菌株来源为农家传统发酵豆酱,在MRS琼脂培养基中呈小于1mm的圆形或椭圆形的白色光滑不透明菌落;在MRS液体培养基中呈均匀浑浊生长;电镜下菌体为球型,成对或链状排列,革兰氏染色呈阳性。本发明的菌株对抗生素敏感,不含毒力基因,属于安全菌株。其产生的细菌素为肠球菌素P,在酸性条件下稳定,具有很好的热稳定性,对常见的肠道致病菌具有良好的抑菌效果。
Description
技术领域
本发明属于食品生物技术领域,特别是涉及一种产细菌素的屎肠球菌及其应用。
背景技术
近年来食品安全问题频繁爆发,其主要原因就是致病菌和腐败微生物的生长繁殖。食品源病原微生物可引起人类疾病,WHO估测全世界每年有成千上万例食品源疾病,危害人们的健康。为抑制病原微生物的生长繁殖,化学防腐剂的添加成为解决问题的方式之一。然而,化学防腐剂的滥用,使其在饲料及是食品中大量残留,对禽畜类及人类身体健康产生严重威胁。此时生物防腐剂应运而生,乳酸链球菌素Nisin是从乳酸菌中提取的天然食品防腐剂,其安全无毒的特点很大程度上避免了化学防腐剂所产生的危害,已经广泛应用在乳制品、肉制品和饮料等食品保藏中。但最近研究发现,Nisin分子中的“羊毛硫氨酸环”特征结构能够抑制体内的益生菌,人们开始对此产生怀疑和担忧。因此,寻找广谱、高效、稳定和更为安全的天然食品防腐剂是保障食品安全的关键和必然趋势。
传统发酵豆酱具有丰富的营养价值和乳酸菌资源,其中存在着大量的屎肠球菌(Enterococcusfaecium)资源。屎肠球菌作为乳酸菌的一种,具有良好的生物学特性。屎肠球菌是目前农业部令(2013年2045号)动物微生物饲料添加剂栏所允许使用的益生菌种类,研究表明该菌所产生的类细菌素可作为生物防腐剂抑制腐败菌和致病菌的生长繁殖,屎肠球菌产生的类细菌素具有分子质量小、热稳定性强等特点,具有良好的应用前景。
发明内容
针对上述存在的技术问题,本发明提供一种产细菌素的屎肠球菌(Enterococcusfaecium)R1及其应用,该菌株对模拟人工消化液有很好的耐受性,该菌株对抗生素敏感,不含毒力基因,属于安全菌株。
本发明的目的是通过以下技术方案来实现的:一种产细菌素的屎肠球菌菌株,该菌株的保藏名称为:屎肠球菌(Enterococcusfaecium)R1,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心,保藏地址:北京市朝阳区北辰西路1号院,中国科学院微生物研究所,邮政编码:100101,保藏日期:2016年1月20日,保藏号:CGMCCNo.12085。
本发明中,所述产细菌素的屎肠球菌菌株,所述屎肠球菌菌株来源于农家传统发酵豆酱中,在MRS琼脂培养基中呈小于1mm的圆形或椭圆形的白色光滑不透明菌落;在MRS液体培养基中均匀浑浊生长;电镜下菌体为球型,成对或链状排列,革兰氏染色阳性。
本发明中,所述MRS琼脂培养基为:在MRS液体培养基中加入2%的琼脂粉;所述MRS液体培养基为:蛋白胨10g,牛肉浸膏8g,酵母粉4g,葡萄糖20g,无水乙酸钠5g,吐温1g,磷酸氢二钾2g,柠檬酸铵2g,七水硫酸镁0.58g,一水硫酸锰0.25g,蒸馏水1000mL,pH值6.5。
本发明中,所述产细菌素的屎肠球菌的选育方法,使用MRS琼脂培养基从农家传统发酵豆酱中筛选分离出乳酸菌菌株,通过平板划线分离纯化出乳酸菌菌株;以常见致病菌为指示菌,应用牛津杯法,筛选出产细菌素的乳酸菌,经16SrDNA测序分析,得到屎肠球菌。
所述产细菌素的屎肠球菌的选育方法,所述从农家传统发酵豆酱中筛选分离出乳酸菌菌株是采用平板稀释法,将稀释后豆酱样品涂布在MRS固体培养基上,37℃培养24h;挑取疑似乳酸菌形态单菌落于MRS液体培养基中,37℃培养24h。
所述产细菌素的屎肠球菌的选育方法,对纯化后的菌株进行革兰氏染色和过氧化氢酶试验;选取革兰氏染色为阳性的球菌、过氧化氢酶试验为阴性的菌株为球状乳酸菌。所述牛津杯法是指以常见致病菌为指示菌,用屎肠球菌R1发酵上清液进行抑菌试验。
一种细菌素,其活性成分产生自屎肠球菌(Enterococcusfaecium)R1产生,保藏号:CGMCCNo.12085。
所述的细菌素,其细菌素种类为肠球菌素中的肠球菌素P,酸性条件下稳定。
所述的细菌素对金黄色葡萄球菌、大肠杆菌或单核细胞增生李斯特菌的抑菌的应用。
该菌株采用BLAST分析法,将本发明屎肠球菌R1的16SrDNA全序列与NCBI注册的屎肠球菌BAB-7371的16SrDNA的基因序列比较,同源性为100%。
本发明的有益效果为:
本发明的菌株对模拟人工消化液有很好的耐受性,该菌株对抗生素敏感,不含毒力基因,属于安全菌株。其产生的细菌素为肠球菌素P,在酸性条件下稳定,并具有很好的热稳定性,对常见的肠道致病菌如金黄色葡萄球菌、大肠杆菌、单核细胞增生李斯特菌等具有良好的抑菌效果。
附图说明
序列表SEQIDNO:1是本发明分离株屎肠球菌株R1的16SrDNA基因全序列。
图1为本发明屎肠球菌R1在MRS琼脂培养基的菌落照片。
图2为本发明屎肠球菌R1的革兰氏染色照片。
图3为本发明屎肠球菌R1对金黄色葡萄球菌的体外抑菌性试验初筛结果。
图4为本发明屎肠球菌R1对金黄色葡萄球菌的体外抑菌性试验复筛结果。
图4A为原菌液的体外抑菌性试验结果。
图4B为排除过氧化氢后的体外抑菌性试验结果。
图4C为排除酸后的体外抑菌性试验结果。
图4D为胰蛋白酶处理后的体外抑菌试验结果
图5为本发明屎肠球菌R1的16SrDNA基因PCR扩增检测结果。
图6为本发明屎肠球菌R1构建系统发育树。
图7为屎肠球菌R1细菌素基因种类测定电泳图。
图8为屎肠球菌R1药敏试验结果。
具体实施方式
下面结合附图和实施例对本发明进行详细描述。
下述实施例中所用的培养基配方如下:
1.MRS液体培养基:蛋白胨10g,牛肉浸膏8g,酵母粉4g,葡萄糖20g,无水乙酸钠5g,吐温1g,磷酸氢二钾2g,柠檬酸铵2g,七水硫酸镁0.58g,一水硫酸锰0.25g,蒸馏水1000mL,pH值6.5,121℃灭菌20min。
2.MRS固体培养基:在MRS液体培养基中加入2%的琼脂粉。
3.LB液体培养基:胰蛋白胨10g/L、酵母粉5g/L、氯化钠10g/L,调节pH至7.0左右,121℃灭菌15min。
实施例1:乳酸菌的分离与纯化:
采用常规平板稀释法,称取1g豆酱样品于10mL灭菌生理盐水中充分震荡,进行10倍稀释,分别取10-5g/mL、10-6g/mL、10-7g/mL稀释度的样品100μL,均匀涂布在MRS固体培养基上,37℃培养24h。挑取疑似乳酸菌形态单菌落于MRS液体培养基中,37℃培养24h。采用平板划线分离法进行纯化后,进行革兰氏染色和过氧化氢酶试验。选取革兰氏染色为阳性的球菌、过氧化氢酶试验为阴性的菌株暂定为球状乳酸菌,进行穿刺保藏。
该菌命名为屎肠球菌(Enterococcusfaecium)R1,于2016年1月20日保藏于中国微生物菌种保藏管理委员会普通微生物中心,地址:北京市朝阳区北辰西路1号院3号,保藏单位:中国科学院微生物研究所,邮编:100101,菌种保藏号为No.12085。本发明的菌株在MRS琼脂培养基的菌落照片见图1,其革兰氏染色照片见图2。
实施例2:产细菌素乳酸菌的筛选:
㈠抑菌性乳酸菌的筛选
采用牛津杯琼脂扩散法进行抑菌性乳酸菌的筛选。
⑴细菌发酵液的制备:将乳酸菌菌株接种于MRS液体培养基中,37℃培养24h。然后按2﹪的接种量接到MRS液体培养基中进行一代菌的活化,37℃培养24h。同样方法将乳酸菌活化至三代。培养液于10000r/m离心10min,取发酵上清液备用,4℃保存。
⑵指示菌菌悬液制备和处理:将金黄色葡萄球菌接种到LB液体培养基中,37℃培养36h-48h。然后按2﹪的接种量接到LB液体培养基中进行一代菌的活化,37℃培养36h-48h。同样方法将金黄色葡萄球菌活化至三代。培养好的指示菌菌悬液用无菌生理盐水稀释成107CFU/mL,备用。
⑶制备LB平板:每个平板中倒入20-25mLMRS琼脂培养基,涂布指示菌前,保证平皿干燥。
⑷取100μL指示菌菌悬液均匀涂布在LB琼脂培养基上,4℃静置1h。用灭菌的镊子夹取无菌牛津杯,在琼脂培养基平板上等距放置3个牛津杯,每个牛津杯中加入100μL无菌发酵上清液,于37℃培养18h-24h,用游标卡尺测量抑菌圈直径,重复3次取均值,确定抑菌效果。本发明菌株R1在抑菌性乳酸菌筛选的过程中具体试验结果见表1和图3。
表1屎肠球菌R1发酵上清液对金黄色葡萄球菌的体外抑菌性试验初筛结果
㈡产细菌素乳酸菌的确定
⑴有机酸的排除
将乳酸菌发酵上清液用1mol/L的NaOH溶液或HCL溶液调节pH至5.0,采用牛津杯琼脂扩散法测定发酵液的抑菌活性。同时测定pH为5.0的乙酸和乳酸的抑菌活性。重复3次取均值。
⑵过氧化氢的排除
将乳酸菌发酵上清液于80℃水浴加热10min,采用牛津杯琼脂扩散法测定发酵液的抑菌活性,重复3次取均值。
⑶蛋白酶类抑菌物质的确定
将胰蛋白酶、胃蛋白酶分别溶解到50mmol/L的磷酸缓冲液(pH7.6、2.0)中配成母液,加入到试验菌株发酵上清液中,使其终浓度为1mg/mL,调节pH至各酶的最适作用范围,37℃水浴1h后,将pH调回到原发酵上清液初始pH,并用经缓冲液稀释相同倍数后的发酵上清液作为对照,检测各种蛋白酶对乳酸菌发酵上清液抑菌活性的影响。
试验结果表明,本发明菌株R1属于细菌素的产生菌株且具有良好的抑菌活性,具体结果见图4和表2。
表2屎肠球菌R1发酵上清液对金黄色葡萄球菌的体外抑菌性试验复筛结果
实施例3:产细菌素菌株的鉴定
㈠乳酸菌生理生化鉴定
对筛选出的产细菌素的菌株R1进行生理生化试验,对其种属进行初步鉴定。根据《伯杰细菌鉴定手册(第八版)》和《常见细菌系统鉴定手册》里面对屎肠球菌的描述内容,初步确定该菌株R1为屎肠球菌。具体结果见表3。
表3屎肠球菌R1生理生化鉴定结果
㈡16SrDNA序列同源性分析鉴定
⑴总DNA的提取及纯度的检测
采用优化后的CTAB法提取供试菌株基因组DNA。具体操作步骤如下:
①取1.5mL对数生长末期的菌体培养物,4℃,4500rpm离心5min收集菌体,去尽培养液。用1mL灭菌水冲洗,4℃,4500rpm离心5min,将水倒掉,在沉淀中加入567μL的TE(10*TE)缓冲液,用吸管反复吹打,使之重悬。
②加30μL10%SDS(w/v)和3μL20mg/mL蛋白酶K,混匀,于37℃水浴保温1h。
③加入100μL10mol/LCTAB溶液(4.1gNaCl溶于80mL水中缓慢加入CTAB10g)及100μL浓度为0.7mol/LNaCl溶液,混匀,65℃水浴保温10min,得到粗提取液。
④在此粗提取液中加入700μL的酚/氯仿/异戊醇(25∶24∶1,v/v),颠倒混匀,静置一分钟,12000rpm离心5min。
⑤吸上清至另一组1.5mlEP管中,并加入700μL氯仿/异戊醇(24∶1,v/v),颠倒混匀,静置一分钟,12000rpm离心5min,弃下层,重复2次。
⑥在第二次所得到的上清中,加入500μL冰的异丙醇,轻轻混合,-20℃放置,30min,12000rpm离心,5min。
⑦弃去上清,得到DNA沉淀,用1mL70%的乙醇(v/v)洗涤DNA沉淀,4℃,10000rmp,10min,弃去上清液,并在空气中自然干燥。
⑧最后用30-60μL灭菌去离子水溶解DNA,4℃放置过夜后,置于-20℃保存。
⑵PCR扩增16SrDNA序列
16SrDNA扩增引物采用通用引物:
正向引物为27f:5,-AGAGTTTGATCCTGGCTCAG-3,;
反向引物为1495r:5,-CTACGGCTACCTTGTTACGA-3,。
PCR扩增反应体系见表4。PCR扩增反应程序为:95℃预变性5min;95℃变性30s,55℃退火30s,72℃延伸1min30s,循环24次:72℃末端延伸10min,10℃保温。
表4菌株R116SrDNAPCR扩增反应体系
⑶检测16SrDNA扩增片段及测序
预先制备1.0%的琼脂糖凝胶(含溴化乙锭),取5μL的PCR产物与1μL的Loadingbuffer混合,加入1%的琼脂糖凝胶点样孔中,在电压为5V/cm,电泳液为1×TAE中电泳。电泳结束后,将胶板置于凝胶成像系统中观察。R1PCR产物检测后,片段长度约为1500bp,直接送上海美吉生物医药科技有限公司进行序列测定。该菌株R1的16SrDNA基因PCR扩增检测结果见图5。该菌株R1的16SrDNA基因全序列见序列表SEQIDNO:1。
⑷乳酸菌同源性分析和系统发育数的构建
将所得序列在在NCBI上使用BLAST进行同源性比对,同时从GenBank数据库中获得公认标准序列数据,并用MEGA软件包以NeighborJoin法制作系统发育树。具体结果见图6。
实施例4:屎肠球菌R1所产细菌素特性分析
㈠抑菌谱测定
参照实施例2中的方法,将指示菌分别为金黄色葡萄球菌、大肠杆菌、单核细胞增生李斯特菌、志贺氏菌、鼠伤寒沙门氏菌、地衣芽孢杆菌和植物乳杆菌,检测屎肠球菌R1发酵上清液对其他常见致病菌的抑菌效果。具体结果见表5。
表5屎肠球菌R1发酵上清液对常见致病菌的体外抑菌性试验结果
注:“----”无抑菌性;“+”抑菌圈直径<10mm;“++”抑菌圈直径10~15mm;“+++”抑菌圈直径>15mm。
㈡细菌素酸碱稳定性试验
以单核细胞增生李斯特菌为指示菌,用1mol/L的NaOH溶液或HCL溶液调节乳酸菌发酵上清液pH至2-10,37℃保温2h后,采用牛津杯琼脂扩散法测定发酵液的抑菌活性,以相应pH的无菌MRS培养基作为空白对照,检测菌株R1所产细菌素的酸碱稳定性。结果表明屎肠球菌R1所产细菌素在pH2-6均具有抑菌活性,该细菌素酸性条件下稳定。具体结果见表6。
表6屎肠球菌R1细菌素的酸碱稳定性
㈢细菌素热稳定性试验
以单核细胞增生李斯特菌为指示菌,用1mol/L的NaOH溶液将乳酸菌发酵上清液pH调至5.0,分别在60℃、80℃和100℃条件下处理30min,121℃条件下处理15min,以相同pH值的未经加热处理的发酵上清液作为空白对照,检测菌株R1所产细菌素的热稳定性。结果表明,121℃条件下处理15min后,抑菌活性仍然保留78%,屎肠球菌R1所产细菌素具有一定的热稳定性。具体结果见表7。
表7屎肠球菌R1细菌素的热稳定性
㈣细菌素种类的确定
⑴肠球菌素基因PCR检测
参照实施例3中的方法,提取菌株基因组DNA。根据Genbank中9种常见肠球菌素基因设计引物,引物由天根生化科技有限公司合成,引物序列见表8。
表8菌株R1细菌素相关基因PCR扩增引物
以基因组DNA为模板扩增细菌素基因序列,25μL反应体系见表9。PCR反应条件为:扩增EntQ的反应程序为94℃预变性5min;94℃变性40s,60℃退火40s,72℃延伸40s,30个循环;最后72℃延伸5min。扩增其他引物反应程序为94℃预变性5min;94℃变性30s,56℃退火30s(EntA、Ent31和Cyl为58℃),72℃延伸30s,30个循环;最后72℃延伸5min。所得PCR产物按照实施例3的方法用1%琼脂糖凝胶电泳在标准条件下进行检测。只有以EntP-F/R为引物的PCR产物大小与预期片段长度相同,结果见图7。
表9PCR扩增体系
⑵细菌素基因片段的克隆
①目的基因与载体连接
采用胶回收试剂盒(SangonBiotechSK8132)对PCR产物进行纯化回收,具体步骤参照试剂盒的说明书。按照载体说明,将纯化后的PCR产物与pEASY-T1载体相连接,连接产物于-20℃保存。
②重组质粒的转化
向50μL大肠杆菌感受态细胞中加入5μL连接产物,混匀,冰浴静置30min。于42℃水浴中放置60-90s,迅速放于冰上5min。加入500μLLB液体培养基,混匀,于37℃,150r/m摇床培养1h,使菌体复苏。取150μL菌体均匀涂布于含有氨苄的LB固体平板,37℃培养12-16h。
③重组质粒的验证
挑取平板上的白斑,接种于10μLRNase-FreeWater中,混匀,以该混合物为模板进行PCR检测,对于有条带的样品,将其转移到10mL含有氨苄的LB液体培养基中,37℃,150r/m摇床培养12-16h。将培养好的新鲜菌液送至上海桑尼生物科技有限公司进行测序。将测序结果在NCBI数据库进行Blast比对,发现此序列与肠球菌素P(EEI61669.1)基因序列同源性达99%。屎肠球菌R1所产细菌素种类为肠球菌素P。
实施例5:屎肠球菌R1的益生性
㈠模拟胃液耐受性
模拟胃液的配制:取质量浓度为0.1kg/L的HCL溶液16.4mL,加蒸馏水稀释使pH值分别达到2.0、2.5和3.0,按照1g/100mL的量加入胃蛋白酶,待充分溶解后,用0.2μm的微孔滤膜过滤除菌,备用。
取活化好的菌株于4℃,6000rpm离心2min,向得到的菌体中加入与培养基等量的模拟胃液,37℃培养3h,采用倾注培养法对0h和3h的培养液进行活菌计数。
存活率=[N1/N0]×100%
式中,N0—0h活菌数;N1—经模拟胃液消化3h后的活菌数。
结果显示,经3h培养,菌株R1在pH2.0的模拟胃液中可以存活,在pH2.5的模拟胃液中存活率达到44.10%,在pH3.0的模拟胃液中,存活率达到86.35%,可见,该菌株对模拟胃液环境具有很好的耐受性。见表10。
表10屎肠球菌R1对模拟胃液的耐受性
㈡模拟肠液耐受性
模拟肠液的配制:取6.8gKH2PO4,加500mL蒸馏水充分溶解,用质量浓度为4g/L的NaOH溶液调pH值至6.8,再加水稀释至1000mL,按照1g/100mL的量加入胰蛋白酶,充分溶解后,用0.2μm的微孔滤膜过滤除菌,备用。
取活化好的乳酸菌菌株于4℃,6000rpm离心2min,向得到的菌体中加入与培养基等量的模拟肠液,37℃培养6h,采用倾注培养法对0h、2h、4h和6h的培养液进行活菌计数,计算存活率。
试验结果显示,屎肠球菌R1在模拟肠液中培养6h后,存活率为87.72%,菌株R1对模拟肠液有一定的耐受性,具体结果见表11。
㈢胆盐耐受性
将试验菌株发酵液以3%接种量接种到含有0.3%胆盐浓度的MRS液体培养基中,37℃培养6h后,采用倾注培养法对0h、2h、4h和6h的培养液进行活菌计数,计算存活率。
结果显示菌株在含0.3%胆盐的培养基中培养6h后,存活率为32.21%,其对胆盐有一定的耐受性。
表11屎肠球菌R1对模拟肠液和胆盐的耐受性
实施例6:屎肠球菌R1的安全性
㈠抗生素敏感性试验
选取青霉素、氨苄西林、庆大霉素、红霉素、诺氟沙星、环丙沙星和氯霉素等7种药敏纸片进行药敏试验,参照麦氏比浊管,将活化好的试验菌株发酵液用无菌生理盐水稀释成107CFU/mL,取100μL均匀涂布在MRS固体培养基上,4℃静置1h。采用药敏纸片法,在平皿中等距放置药敏纸片,空白纸片作为对照,37℃培养16-18h后,测量抑菌圈直径。
试验菌株敏感性参照CLSI的最新版本标准进行判定。
试验结果表明,屎肠球菌R1对青霉素类(青霉素、氨苄西林)、氨基糖苷类(庆大霉素)药物、环丙沙星和氯霉素敏感,对大环内酯类(红霉素)和诺氟沙星呈中介。具体结果见表12和图8。
表12屎肠球菌R1药敏试验结果
㈡毒力因子的检测
参照实施例3的方法,提取菌株基因组DNA。毒力因子主要包括gelE、cylA、ccf、esp、ace和asal。由天根生化科技有限公司合成六种常见的肠球菌相关毒力基因的引物,引物序列见表13。以基因组DNA为模板,对目的毒力因子基因进行PCR扩增,反应体系同表9。PCR反应条件为:95℃预变性4min;94℃变性1min,53℃退火45s(其中cylA为60℃,ace为55℃),72℃延伸1min,35个循环;最后72℃延伸10min。所得PCR产物用1%琼脂糖凝胶电泳在标准条件下进行检测,结果显示并未出现和预期片段大小相同的条带。
上述药敏试验和毒力因子检测试验结果表明,菌株屎肠球菌R1是安全的。
表13毒力因子基因PCR扩增引物
Claims (9)
1.一种产细菌素的屎肠球菌菌株,其特征在于:该菌株的保藏名称为:屎肠球菌(Enterococcusfaecium)R1,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心,保藏日期:2016年1月20日,保藏号:CGMCCNo.12085。
2.根据权利要求1所述产细菌素的屎肠球菌菌株,其特征在于:所述屎肠球菌菌株来源于农家传统发酵豆酱中,在MRS琼脂培养基中呈小于1mm的圆形或椭圆形的白色光滑不透明菌落;在MRS液体培养基中均匀浑浊生长;电镜下菌体为球型,成对或链状排列,革兰氏染色阳性。
3.根据权利要求2所述产细菌素的屎肠球菌菌株,其特征在于:所述MRS琼脂培养基是在MRS液体培养基中加入2%的琼脂粉,所述MRS液体培养基为:蛋白胨10g,牛肉浸膏8g,酵母粉4g,葡萄糖20g,无水乙酸钠5g,吐温1g,磷酸氢二钾2g,柠檬酸铵2g,七水硫酸镁0.58g,一水硫酸锰0.25g,蒸馏水1000mL,pH值6.5。
4.如权利要求1所述产细菌素的屎肠球菌的选育方法,其特征在于:使用MRS琼脂培养基从农家传统发酵豆酱中筛选分离出乳酸菌菌株,通过平板划线分离纯化出乳酸菌菌株;以常见致病菌为指示菌,应用牛津杯法,筛选出产细菌素的乳酸菌,经16SrDNA测序分析,得到屎肠球菌。
5.如权利要求4所述产细菌素的屎肠球菌的选育方法,其特征在于:所述从农家传统发酵豆酱中筛选分离出乳酸菌菌株是采用平板稀释法,将稀释后豆酱样品涂布在MRS固体培养基上,37℃培养24h;挑取疑似乳酸菌形态单菌落于MRS液体培养基中,37℃培养24h。
6.如权利要求4所述产细菌素的屎肠球菌的选育方法,其特征在于:对纯化后的菌株进行革兰氏染色和过氧化氢酶试验;选取革兰氏染色为阳性的球菌、过氧化氢酶试验为阴性的菌株为球状乳酸菌。
7.一种细菌素,其活性成分产生自屎肠球菌(Enterococcusfaecium)R1,保藏号:CGMCCNo.12085。
8.如权利要求7所述的细菌素,其细菌素种类为肠球菌素中的肠球菌素P,酸性条件下稳定。
9.一种如权利要求7所述的细菌素对金黄色葡萄球菌、大肠杆菌或单核细胞增生李斯特菌的抑菌的应用。
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