CN114891678A - 多粘芽孢杆菌cpl258及其筛选和应用 - Google Patents
多粘芽孢杆菌cpl258及其筛选和应用 Download PDFInfo
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- CN114891678A CN114891678A CN202210536972.7A CN202210536972A CN114891678A CN 114891678 A CN114891678 A CN 114891678A CN 202210536972 A CN202210536972 A CN 202210536972A CN 114891678 A CN114891678 A CN 114891678A
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- cpl258
- bacillus polymyxa
- polymyxa
- fermentation
- bacillus
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
本发明公开了一株多粘芽孢杆菌CPL258及其筛选和应用,该菌株CPL258经鉴定为多粘芽孢杆菌(Paenibacillus polymyxa),已保藏于中国典型微生物保藏中心,保藏时间为2021年09月13日,保藏编号为CCTCC NO.M20211157。本发明多粘芽孢杆菌CPL258是一种产芽孢的革兰氏阳性短杆状细菌,可产生具有抑菌作用的抗菌肽类、核苷类物质、抗生素及抗菌蛋白等,且其具有防治动物性埃希氏大肠杆菌导致的猪腹泻,从而增强仔猪免疫力,降低死亡率的作用。综上,本发明多粘芽孢杆菌CPL258在仔猪饲料应用方面具有广阔的前景。
Description
技术领域
本发明属于生物技术应用领域,具体涉及一株多粘芽孢杆菌CPL258及其筛选和应用。
背景技术
多粘芽孢杆菌于2002年被美国环境保护署列为商业应用微生物之一,也是我国农业部规定免做安全鉴定的一级菌种之一。该种属微生物能在不利条件下形成芽孢,即使在苛刻的胃酸环境中也能保持较高的活性,目前在畜禽生产养殖中具有较为广泛的应用。芽孢杆菌能改善畜禽肠道微生态环境,治疗和预防细菌性肠道疾病,提高动物抗病能力,促进动物生长。以猪腹泻疾病为例,已经有饲料添加多种芽孢杆菌(枯草芽孢杆菌、解淀粉芽孢杆菌、地衣芽孢杆菌等)提高仔猪的抵抗力,维持其肠道平衡的研究报道。
多粘芽孢杆菌代谢产物之一为Colistin,一种多组分碱性多肽类抗生素。本项目发现ColistinA是Colistin的成分之一,它可以用来可用来对抗一些由革兰氏阴性菌引起的感染。Colistin A的分子结构如图1所示,它的结构主要由三部分组成,环状7肽包括:4个L-Dab,1个L-Leu,1个L-Thr,1个D-Leu;线性3肽包括:2个L-Dab,1个L-Thr;疏水酰基尾包括:甲基辛酸,异辛酸。分子量约为1169.5g/mol。相对于传统的抗生素,Colistin A具有更高的稳定性,但是Colistin A的产量很低,如何高效生产Colistin A是继续解决的问题。
Colistin A如下所示:
发明内容
发明目的:针对现有技术存在的问题,本发明提供一株产抗细菌代谢产物的菌株多粘芽孢杆菌(Paenibacillus polymyxa)CPL258,本发明筛选到的多粘芽孢杆菌CPL258可以高产Colistin A,对于猪腹泻埃希氏大肠杆菌疾病具有非常好的效果。
本发明还提供所述多粘芽孢杆菌(Paenibacillus polymyxa)CPL258的筛选方法和应用。
技术方案:为了实现上述目的,本发明所述一株多粘芽孢杆菌(Paenibacilluspolymyxa)CPL258,已保藏于中国典型微生物保藏中心,保藏地址:中国.武汉.武汉大学,邮编:430072,保藏时间为2021年09月13日,保藏编号为CCTCC NO.M20211157。该菌株来源于淮安市水产养殖所附近土壤中,在LB培养基上菌落形态为圆形、表面光滑、稍凸起、边缘不整齐、乳白色、不透明。
本发明所述多粘芽孢杆菌(Paenibacillus polymyxa)CPL258是一种产芽孢的革兰氏阳性细菌,可产生具有抑菌作用的肽类、核苷类物质、抗生素及抗菌蛋白等;对金黄色葡萄球菌、埃希氏大肠杆菌、鰤鱼诺卡氏菌具有抑制作用。
本发明所述的多粘芽孢杆菌CPL258的筛选方法,包括如下步骤:
(1)取土壤样品与生理盐水混匀,热处理后加入NaCl,水浴后取土壤悬液用无菌水稀释;
(2)取稀释后的稀释液滴加在LB固体培养基上,涂布均匀,培养得到初筛菌株,以分离筛选得到的初筛菌株为供试菌;
(3)以金黄色葡萄球菌为指示菌,采用牛津杯法筛选出具有金黄色葡萄球菌拮抗效果多粘芽孢杆菌CPL258。
作为优选,本发明所述的多粘芽孢杆菌CPL258的筛选,包括如下步骤:
将在江苏省淮安市水产养殖所(33.28′24,54″Net 119.9′48,79″E)附近选取的10~20cm深处的土壤约10g,加入100ml生理盐水混匀。在80℃条件下水浴1h,再加入4g NaCl,37℃水浴2h。取1mL土壤悬液用无菌水10倍梯度稀释,取100μL稀释后的菌液滴加在LB固体培养基上,涂抹均匀,倒置37℃过夜培养。观察培养基上单个菌落的形态,包括菌落的颜色、大小、菌落边缘以及表面粗糙情况,最终获得86株芽孢杆菌初筛菌株。以分离筛选获得的初筛菌株为供试菌,以金黄色葡萄球菌为指示菌,采用牛津杯法筛选出金黄色葡萄球菌拮抗效果最好的多粘芽孢杆菌CPL258。
本发明所述的多粘芽孢杆菌CPL258在生产具有抑菌作用的肽类、核苷类物质、抗菌蛋白、抗生素中的应用。
其中,所述抗生素为Colistin A。
进一步地,所述多粘芽孢杆菌CPL258接种于发酵培养基中进行发酵,从发酵液中分离获得所述的Colistin A。
其中,所述发酵培养基的配方为每1L中包括:蛋白胨4.5-5g,玉米浆0.02-0.05L,淀粉8.0-11.0g,(NH4)2SO4 2.8-3.3g,CaCO3 10.0-12.0g,K2HPO4 6.0-8.0g,VB1、VB3、VB5、VB12、VH各1-3mg,,pH 7.0-7.2。
其中,所述发酵条件为通气比5.8-6.2vvm,转速130-180r/min,接种量1.8-2.0%,发酵时间为48小时。
作为优选,所述菌株多粘芽孢杆菌CPL258接种于培养基中进行发酵的条件为37℃,180rpm恒温培养,发酵时间为48h。
本发明所述的多粘芽孢杆菌CPL258在制备防治猪腹泻病害的生防制剂或饲料中的应用。
进一步地,所述多粘芽孢杆菌CPL258在制备防治猪腹泻埃希氏大肠杆菌疾病生防制剂或饲料中的应用。
其中,所述生物制剂或者饲料中含有所述的多粘芽孢杆菌CPL258的菌体、芽孢、发酵液或者由多粘芽孢杆菌CPL258发酵液制备的Colistin A。
本发明的所述多粘芽孢杆菌CPL258具有广谱抑菌性,具有拮抗革兰氏阳性菌、革兰氏阴性菌以及真菌的活性。尤其多粘芽孢杆菌CPL258对埃希氏大肠杆菌、鰤鱼诺卡氏菌和金黄色葡萄球菌有明显的抑制作用。本发明提供了所述的多粘芽孢杆菌CPL258在针对防治仔猪猪腹泻的猪饲料中的应用,尤其是猪腹泻埃希氏大肠杆菌疾病。本发明通过饲喂含有多粘芽孢杆菌CPL258的饲料,感染猪腹泻埃希氏大肠杆菌的仔猪的存活率远高于普通饲料喂养的仔猪。
本发明分离获得一株多粘芽孢杆菌(Paenibacillus polymyxa),该菌是一种产芽孢的革兰氏阳性细菌,细胞形态呈短杆状、单生,能产生肽类、核苷类抗菌物质、抗菌蛋白等多种具有抑菌作用的物质。
本发明从土壤中分离获得一株多粘芽孢杆菌(Paenibacillus polymyxa),经鉴定为厚壁菌门,芽孢杆菌科,类芽孢杆菌属(Paenibacillus)。该菌是一种产芽孢的革兰氏阳性细菌,细胞形态呈短杆状、单生,能产生抗菌肽类、核苷类抗菌物质、抗菌蛋白等多种具有抑菌作用的物质。本发明该菌株可以高产Colistin A,并且通过优化的培养基可以进一步提高其产量,本发明的菌株或者含Colistin A的发酵液,可以用于猪腹泻埃希氏大肠杆菌的防治,效果非常好。
有益效果:与现有技术相比,本发明具有如下优点:
1、本发明提供一种全新的多粘芽孢杆菌CPL258,具体来说是高产抗细菌代谢产物Colistin A且对猪腹泻埃希氏大肠杆菌具有防治作用的菌株。
2、本发明将注射猪腹泻埃希氏大肠杆菌的仔猪分别喂食基础饲料和添加了多粘芽孢杆菌CPL258的基础饲料(每1kg基础饲料添加200g多粘芽孢杆菌CPL258发酵液),结果显示喂食添加多粘芽孢杆菌CPL258发酵液的饲料,感染埃希氏大肠杆菌性猪腹泻的猪存活率远远高于喂食普通饲料,证明多粘芽孢杆菌CPL258对猪腹泻埃希氏大肠杆菌病有很好的防治作用,可以有效应用在制备防治猪腹泻病害的生防制剂或饲料中。
3、本发明制备了全新组成的发酵培养基可以进一步有效提高其次级代谢产物Colistin A产量。
综上,本发明多粘芽孢杆菌CPL258对仔猪猪腹泻具有良好的防治效果,可用于制成相关的生物制剂和饲料。
附图说明
图1为多粘芽孢杆菌CPL258在LB培养基上的菌落形态示意图;
图2为用牛津杯法测定多粘芽孢杆菌CPL258分别对鰤鱼诺卡氏菌,埃希氏大肠杆菌和金黄色葡萄球菌的抑菌活性图;
图3为多粘芽孢杆菌CPL258革兰氏染色结果图;
图4为多粘芽孢杆菌CPL258次级代谢产物预测图;
图5为多粘芽孢杆菌CPL258次级代谢产物合成基因簇所在位点分析图;
图6为多粘芽孢杆菌CPL258基于16srDNA片段构建的进化树;
图7为多粘芽孢杆菌CPL258次级代谢产物ColistinA的高效液相色谱分析;
图8为多粘芽孢杆菌CPL258不同方案中ColistinA产量检测对比。
具体实施方式
以下结合附图和实施例对本发明作进一步说明。
本发明实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂家建议的条件。其中使用的埃希氏大肠杆菌、金黄色葡萄球菌、鰤鱼诺卡氏菌由淮阴工学院益生制剂与功能化合物实验室提供,也可以使用其他野生型的猪腹泻埃希氏大肠杆菌。
实施例1
土壤中多粘芽孢杆菌CPL258的分离筛选和抑菌活性的测定:
多粘芽孢杆菌CPL258从江苏省淮安市水产养殖所(33.28′24,54″Net 119.9′48,79″E)附近土壤中分离获得,分离过程如下:选取10~20cm深处的土壤约10g,加入100ml生理盐水混匀。在80℃条件下水浴1h,再加入4g NaCl,37℃水浴2h。取1mL土壤悬液用无菌水10倍稀释,取100μL稀释后的试液滴加在LB固体培养基上,涂抹均匀,倒置37℃过夜培养。观察培养基上单个菌落的形态,包括菌落的颜色、大小、菌落边缘以及表面粗糙情况,获得367株菌株。将从土壤中分离出的多粘芽孢杆菌属菌株进行平板培养,次日平板上挑取单菌落,将单菌落进行梯度稀释,涂布于含kan(5mg/mL)的LB固体培养基的平板上50℃培养。并在37℃条件下,用LB液体培养基活化金黄色葡萄球菌与埃希氏大肠杆菌。取单菌落的多粘芽孢杆菌划线备用。第二天,分别向融化后的固体培养基中添加金黄色葡萄球菌和埃希氏大肠杆菌,然后倒平板。用牙签挑取一个多粘芽孢杆菌单菌落,点于平板中央;再用牙签挑取具有一定抗性的多粘芽孢杆菌属菌株单菌落点于平板四周(注意每挑一株菌株均需要更换牙签,避免污染),放37℃下培养12h。次日观察实验结果,得到具有优异抑菌效果的86株初筛菌株。将培养的初筛菌株为供试菌,以金黄色葡萄球菌为指示菌,采用牛津杯法筛选出对金黄色葡萄球菌具有良好拮抗效果的菌株,将该菌株命名为CPL258。菌株CPL258在LB培养基上菌落形态为圆形、表面光滑、稍凸起、边缘不整齐、乳白色、不透明,如图1所示。
采用牛津杯法进行抑菌活性的测定,具体步骤包括:取埃希氏大肠杆菌、金黄色葡萄球菌或鰤鱼诺卡氏菌单菌落接种于LB液体培养基中,37℃,180r/min条件下培养至OD值为0.5-0.6,与冷却至60℃左右的LB固体培养基等体积混合后倒平板,待培养基凝固后,无菌操作夹取牛津杯垂直放置在平板上。菌株CPL258于LB液体培养基中,37℃,180r/min条件培养48h,发酵液离心后取50μl上清打入到牛津杯中,37℃条件下培养2h,观察牛津杯周围抑菌圈直径。抑菌圈直径/mm=抑菌圈总直径/mm-牛津杯直径/mm,结果如图2所示,CPL258对鰤鱼诺卡氏菌,埃希氏大肠杆菌和金黄色葡萄球菌均具有良好的抑制效果,抑菌圈直径分别为为2.5±0.3mm,4.1±0.3mm,0.9±0.3mm。CPL258革兰氏染色结果显示其为革兰氏阳性短杆状细菌,如图3所示。本发明的菌株的发酵液可以在非常短的时间(2h内)抑制埃希氏大肠杆菌、金黄色葡萄球菌或鰤鱼诺卡氏菌。
实施例2
关于多粘芽孢杆菌CPL258的全基因组测序和种属鉴定。
将筛选出的菌株CPL258培养至对数生长期,提取其基因组DNA,利用PacBio RS II和Illumina HiSeq 4000平台进行测序。测序结果显示,多粘芽孢杆菌CPL258,序列全长为5982321bp,GC含量为45.44%,含有5833个基因。antiSMASH次级代谢产物预测结果(如图4所示)显示,多粘芽孢杆菌CPL258含有13个次级代谢产物编码基因簇,其中Region1与BGC0001089来源的bacillaene合成基因簇相似性为78%;Region2与BGC0001192来源的colistin A/colistin B(粘菌素)合成基因簇相似性为100%;Region4与BGC0001152来源的fusaricidin B(杀镰孢菌素)合成基因簇相似性为100%;Region6与BGC0000402来源的paenilarvins合成基因簇相似性为75%;Region8与BGC0001356来源的paeninodin合成基因簇相似性为100%;Region12与BGC0000449来源的tridecaptin(十三肽菌素)合成基因簇相似性为100%;Region13与BGC0001090来源的bacillomycin D(芽胞菌霉素D)合成基因簇相似性为100%;而Region3与菌株BGC0000406来源的基因簇相似性仅为6%,说明该基因簇可能合成新的抑菌物质。上述8个基因簇在基因组上所在位点分布如图5所示,其他未标注基因簇因未发现与其同缘关系比较近的序列,功能未知。
将CPL258的16srDNA(SEQ ID NO.1)片段于NCBI网站在线BLAST,并将比对结果构建进化树,结果如图6所示,其与Paenibacillus polymyxa strain YC0573同缘关系最近,相似性达到99.1%,结合其生理生化特性,即CPL258鉴定为多粘芽孢杆菌,目前已保藏于中国典型微生物保藏中心,保藏时间为2021年09月13日,保藏编号为CCTCC NO.M20211158。
实施例3
利用工业发酵培养基进行多粘芽孢杆菌CPL258发酵培养。
(1)种子培养:取在斜面保存的多粘芽孢杆菌CPL258一环,划线接种在LB固体培养基(胰蛋白胨1%、酵母粉0.5%、NaCl 1%,琼脂1.5%,余量为水,pH 7.0)中,12h后取单菌落接种于LB试管培养基中,37℃,180r/min,培养24h后,制成种子液。
(2)发酵培养:将种子液按体积比2%的量接种至发酵培养基中,培养条件为37℃,通气比6.0vvm,转速180r/min,发酵时间为48h得到发酵液。
培养方案一:采用LB培养基,其配方为胰蛋白胨1%、酵母粉0.5%、NaCl1%,余量为水,pH 7.0,121℃灭菌15分钟。
培养方案二:发酵培养基,其配方组成为每L含有:蛋白胨3g,玉米浆0.03L,淀粉10.0g,(NH4)2SO4 3g,CaCO3 10.0g,K2HPO4 7g,VB1、VB3、VB5、VB12、VH各2mg,pH 7.0,121℃灭菌15分钟。
培养方案三:发酵培养基,其配方组成为每L含有:蛋白胨5g,玉米浆0.03L,淀粉10.0g,(NH4)2SO4 3g,CaCO3 10.0g,K2HPO4 7g,VB1、VB3、VB5、VB12、VH各2mg,pH 7.0,121℃灭菌15分钟。
培养方案四:发酵培养基,其配方组成为每L含有:蛋白胨8g,玉米浆0.03L,淀粉10.0g,(NH4)2SO4 3g,CaCO3 10.0g,,K2HPO4 7g,VB1、VB3、VB5、VB12、VH各2mg,pH7.0,121℃灭菌15分钟。
(3)高效液相色谱:检测条件:缓冲液配定:900mL超纯水中加入4.46g无水硫酸钠,用磷酸调节到pH为2.3并用超纯水定容到1000mL。将缓冲液用水相过滤膜过滤两次;色谱柱:Agilent zorbax sb-Aq C18(规格:4.6×250mm,粒径:5μm);柱温为25℃;进样量为5μL;流动相:乙腈:水(20:80,v/v);流速为1ml/min;检测波长为215nm。
如图7所示,其中A为ColistinA标准品检测结果,B为多粘芽孢杆菌CPL258发酵液的高效液相色谱分析(方案三),并结合图4证明本发明发酵液中主要产物为ColistinA。(4)进一步通过液相分析系统分析得到发酵产物中,Colistin A产量在四次不同培养方案中分别为2.5g/L,3.5g/L,6.0g/L,4.5g/L。如图8所示在不同培养方案中Colistin A产量检测对比。
实施例4
多粘芽孢杆菌CPL258在最优发酵培养方案下获得的发酵液进行粗提制备
粗提物制备:先将实施例3制备的发酵液降温至20℃;再用4mol/L的NaOH溶液调节pH至11.0后保持温度20℃搅拌30分钟;将碱化的发酵液在3000转速下离心15分钟后,收集离心物,用生理盐水洗涤三次后,然后用1.0mol/L的H2SO4溶液调节上清液pH 3.0-4.0使沉淀溶解;抽滤溶解液,得到浓缩液加0.1%高锰酸钾搅拌30分钟,抽滤所得脱色浓缩液,纳滤处理得到除盐的浓缩液,喷干进风温度110℃,出风温75℃,得浓缩产物。
实施例5
猪饲料添加剂对断乳仔猪的生长和健康的影响研究
选取240头(28±2)日龄健康的断奶仔猪,将所选仔猪按品种、胎次相同,体重相近、公母各半的原则随机分为6个组,每个组进行2个重复,每个重复10头。各组基础日粮相同,试验从28日龄断奶仔猪开始,为期30天。
实验组A:饲喂喷洒本发明的猪饲料(每1kg基础饲料添加200g多粘芽孢杆菌CPL258发酵液,实施例3培养方案一)。
实验组B:饲喂喷洒本发明的猪饲料(每1kg基础饲料添加100g多粘芽孢杆菌CPL258发酵液浓缩产物,实施例4方法采用方案三)。
实验组C:饲喂喷洒本发明的猪饲料(每1kg基础饲料添加200g多粘芽孢杆菌CPL258发酵液,实施例3方法采用方案三)。
对照组:饲喂基础猪饲料(购买自淮安市养殖市场,膨化饲料,粗蛋白质均为42.2%,粗脂肪11.9%,粒径为2.5mm,即实验组中不加发酵液的饲料)。
饲料采用粉料拌湿的方式饲喂,饲料加水拌湿即可。仔猪定时饲喂,饲喂时间分别在每天的8、12、16和20点。仔猪在试验期间自由饮水,猪舍管理按照试验猪场常规进行。
每天早晨7:00到晚上21:00连续观测仔猪的排粪情况,对每头仔猪每次排出的粪便进行感官评分,统计每天记录仔猪粪便情况(按照0~3四级记录,0级干、1级-软、2级-稀、3级-水样,其中0和1级为正常,2级和3级为腹泻),计算腹泻率(腹泻率=[腹泻头次数/(仔猪头数×试验天数)]×100%),并计算最后的统计结果见表1。
表1仔猪食用不同饲料在30天内的腹泻率
由表1此可得:本发明多粘芽孢杆菌CPL258发酵液浓或者缩产物作为预防仔猪腹泻的饲料添加剂具有显著的降低仔猪腹泻率的效果。
序列表
<110> 淮阴工学院
<120> 多粘芽孢杆菌CPL258及其筛选和应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1140
<212> DNA
<213> 多粘芽孢杆菌 CPL258(Paenibacillus polymyxa CPL258)
<400> 1
attctcttcc gcgctatttc acccgacgca ggcgcgcgat ataaaatccg tcgctgtgga 60
aatgctgcgg taacagctgt accgagcctc cctgtatact atccttttcg cggctcacgt 120
ccgggaagga atgaccctct gccaattcat actctggatg ttcgcttaaa aattgagtca 180
attgtccttc attttcgtcc ggttcaatcg tgcatgtgct gtataccaga ataccacctg 240
gcttcaacag tcccgccaca ctatccagca gctcatgctg aagctgtgta atgtcgcgaa 300
catcctgcgc agttttgctc caacgcaggt ccggtttacg acgaatcact ccaaagccag 360
agcatggcgc atccagcaaa atacggtcaa aagaagctgg agcatatcgt tccttcagtt 420
ctagcgcatc accagtgact gtttccacgg catccagacc aagtcgatta gcctgttcac 480
gaattaattg atgcttatgg gcatgcagat cattcgcaac tatccgtccg tgatccttca 540
tcagctcagc catatgagct gttttgccac ctggagcggc acagcaatcc agtaccagca 600
tgccaggctc aggagcgact gcttcggcaa cgagcatgga gctttcatcc tgtacggaca 660
gcaagccgtc cgtataccac gaggtaagcg ccatatttcc accgctacga acaacaatcc 720
catagggact aactgccgaa ggaacggcat caagcccttt tgagttcatc tcgtccagca 780
gctgatcacg gctgatcatt gtcgtattca cccgtacgct gaccgcaggg ggctcattat 840
tagcctgaca gatagcctca gcagtatctg caccgtactg cttaatccag cgcttaacca 900
gccactgtgg atgggaatgt tctaatgaaa tccgttcctc cggtgacaaa ttgtcaggaa 960
tacgcagctt gtccggctca cgcagcatgc tgcggagcac gccgttgacc ataccggaaa 1020
taccctgatg cccacgccgc ttggcaatgg tcaccgcttc actaacgact gcatgatccg 1080
gaactcgatc cagatacacc acctgataca cactcatccg cagcaacgaa cgtacccaag 1140
Claims (10)
1.一株多粘芽孢杆菌(Paenibacillus polymyxa)CPL258,已保藏于中国典型微生物保藏中心,保藏时间为2021年09月13日,保藏编号为CCTCC NO.M 20211157。
2.一种权利要求1所述的多粘芽孢杆菌CPL258的筛选方法,其特征在于,包括如下步骤:
(1)取土壤样品与生理盐水混匀,热处理后加入NaCl,水浴后取土壤悬液用无菌水稀释;
(2)取稀释后的稀释液滴加在LB固体培养基上,涂布均匀,培养得到初筛菌株,以分离筛选得到的初筛菌株为供试菌;
(3)以金黄色葡萄球菌为指示菌,采用牛津杯法筛选出具有金黄色葡萄球菌拮抗效果多粘芽孢杆菌CPL258。
3.一种权利要求1所述的多粘芽孢杆菌CPL258在生产具有抑菌作用的肽类、核苷类物质、抗菌蛋白、抗生素中的应用。
4.根据权利要求3所述的应用,其特征在于,所述抗生素优选为Colistin A。
5.根据权利要求4所述的应用,其特征在于,所述多粘芽孢杆菌CPL258接种于发酵培养基中进行发酵,从发酵液中分离获得所述的Colistin A。
6.根据权利要求5所述的应用,其特征在于,所述发酵培养基的配方为每1L中包括:蛋白胨4.5-5g,玉米浆0.02-0.05L,淀粉8.0-11.0g,(NH4)2SO42.8-3.3g,CaCO3 10.0-12.0g,K2HPO4 6.0-8.0g,VB1、VB3、VB5、VB12、VH各1-3mg,pH 7.0-7.2。
7.根据权利要求5所述的应用,其特征在于,所述发酵过程为通气比5.8-6.2vvm,转速130-180r/min,接种量1.8-2.0%。
8.一种权利要求1所述的多粘芽孢杆菌CPL258在制备防治猪腹泻病害的生防制剂或饲料中的应用。
9.根据权利要求8所述的应用,其特征在于,所述多粘芽孢杆菌CPL258在制备防治猪腹泻埃希氏大肠杆菌疾病生防制剂或饲料中的应用。
10.根据权利要求8或者9所述的应用,其特征在于,所述生物制剂或者饲料中含有权利要求1所述的多粘芽孢杆菌CPL258的菌体、芽孢、发酵液或者由多粘芽孢杆菌CPL258发酵液制备的Colistin A。
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Application publication date: 20220812 Assignee: Nanjing spomu Biotechnology Co.,Ltd. Assignor: HUAIYIN INSTITUTE OF TECHNOLOGY Contract record no.: X2023980047354 Denomination of invention: Bacillus polymyxin CPL258 and its screening and application Granted publication date: 20230822 License type: Common License Record date: 20231118 |