CN111961625A - 一株戊糖片球菌smm914及其筛选方法和应用 - Google Patents
一株戊糖片球菌smm914及其筛选方法和应用 Download PDFInfo
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Abstract
本发明公开了一株戊糖片球菌SMM914及其筛选方法和应用,所述戊糖片球菌(Pediococcus Pentosaceus)SMM914保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC:20160。本发明提供的筛选方法工艺过程操作简单、筛选效率高,所得菌株具有较好的抗菌能力和抗氧化作用,可提高仔猪肠道中有益细菌群类的比例,对预防断奶仔猪炎性肠病(IBD)具有良好的应用前景。
Description
技术领域
本发明涉及一株戊糖片球菌,具体涉及一株戊糖片球菌SMM914及其筛选方法和应用。
背景技术
母乳被认为是一种珍贵的天然食品,哺乳动物的后代在新生儿的初期发育过程中完全依赖母乳。近年来,一些研究通过分离细菌并进行菌株水平研究,表明动物母乳和新生儿粪便中存在相同的细菌菌株,证实在母乳喂养过程中母乳微生物群可以通过垂直转移从母体传递给新生儿。同时,断奶又是哺乳动物不可避免的一个生命阶段,在这个阶段断奶动物经历了一系列的应激源,包括与母亲分离、运输、混合窝仔、饮食转换和频繁接触潜在的病原体,特别是从液体奶到固体饲料的转变会改变肠道菌群的组成,肠道菌群的紊乱易发生消化道疾病,如断奶仔猪腹泻、传染性胃肠炎、沙门氏菌病、慢性代谢性疾病如炎性肠病(IBD)等,严重影响生产性能,甚至造成死亡。母猪生产性能与断奶仔猪肠道健康之间的平衡问题有待解决。
益生菌治疗是一种理想的替代抗生素治疗各种疾病,包括胃肠道疾病的方法。根据联合国粮食及农业组织和世界卫生组织的定义,益生菌被定义为“当摄入足够浓度时,能给宿主带来健康益处的活微生物”。早期对益生菌的定义强调了益生菌在改善肠道微生物生态系统中的作用,表明益生菌对肠道和免疫系统具有普遍的益处。乳酸菌是肠道菌群的一员,被广泛用作益生菌。乳酸菌是正常肠道细菌的一员,也是最常用的益生菌,具有“公认安全”(generally recognized as safe)的地位。乳酸菌被广泛用于发酵食品的加工,并越来越多地添加在食品中,如奶酪、酸奶、蔬菜汁、水果和谷类食品。从乳酸菌可以形成微生物群落和生物屏障,减缓腹泻,食物过敏和炎症性肠病。乳酸菌可以产生被称为细菌素的特异性抑制蛋白,通过竞争效应抑制病原体的生长,刺激宿主的免疫功能。乳酸乳球菌是世界上最古老的“驯化”细菌之一,被用来制造营养健康的食品。目前,它已被修饰为多种抗生素的抗菌肽和抗菌蛋白的表达宿主,并可被修饰成口服疫苗的载体。
在作为益生菌使用的过程中,应探索每个候选乳酸菌,以确保菌株的安全性和可取性。动物胃肠道是一个非常复杂的微生物生态系统,影响着营养物质的吸收和代谢以及宿主的营养和保护功能。同时,作为健康肠道菌群的正常居民,乳酸菌菌株通过改善肠道内微生物群落的平衡来预防和/或治疗肠道疾病,这表明了乳酸菌能够在肠道内生存并定植和粘附宿主组织。此外,由于猪的肠道生理病理学与人类有着惊人的相似性,猪不仅被认为是世界上最重要的家畜物种之一,而且也是模拟人类的理想模型。虽然从人和牛乳中分离得到了丰富的益生菌,但目前对母猪乳中自然菌的保存、修复和研究策略还很有限。本发明旨在建立起母猪乳源菌种资源库并从中筛选分离具有益生菌潜能的菌株,为新型益生菌的市场化开发和产业化应用奠定了基础。
发明内容
针对上述问题,本发明的目的在于提供一株戊糖片球菌SMM914及其筛选方法和应用,本发明提供的筛选方法工艺过程操作简单、筛选效率高,所得菌株具有较好的抗菌能力和抗氧化作用,尤其是可提高仔猪肠道中有益细菌群类的比例,对预防仔猪断奶仔猪炎性肠病具有良好的应用前景。
为实现上述目的,本发明采用如下技术方案。
一株戊糖片球菌SMM914,其特征在于,所述戊糖片球菌(PediococcusPentosaceus)SMM914保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,保藏编号为CGMCC:20160,保藏日期为2020年06月30日。
作为具体技术方案,所述戊糖片球菌对鼠伤寒沙门氏菌、肠道出血性大肠杆菌、产肠毒素大肠杆菌、克雷伯氏肺炎菌、点状气单胞菌、金黄色葡萄球菌、李斯特菌和产气荚膜梭菌均具有抑菌活性。
作为具体技术方案,所述戊糖片球菌对金黄色葡萄球菌的抗菌活性大于其对鼠伤寒沙门氏菌、肠道出血性大肠杆菌、产肠毒素大肠杆菌、克雷伯氏肺炎菌、点状气单胞菌、李斯特菌和产气荚膜梭菌的抗菌活性。
作为具体技术方案,所述戊糖片球菌具有抗氧化作用。
作为具体技术方案,所述戊糖片球菌可提高仔猪肠道中有益细菌群类的比例。
作为具体技术方案,所述有益细菌群类包括瘤胃菌科、毛螺菌科、乳酸杆菌科。
一种戊糖片球菌SMM914的筛选方法,其特征在于包括以下步骤:
(1)建立母猪乳益生菌菌种资源库;
(2)根据抗菌能力对菌株进行初筛;
(3)根据抗氧化能力对菌株进行复筛;
(4)仔猪体内验证试验。
作为具体技术方案,所述步骤(1)包括以下步骤:
a、菌种分离:从泌乳期母猪采集新鲜猪奶,以猪乳为接种剂,采用MRS、M17、TPY和GYP四种液体培养基,在厌氧培养箱中,在37℃下连续培养30天,每隔1天取样铺平板分离纯化;
b、DNA测序:从分离纯化的培养物中提取DNA,以5′-gctcaggaacgcygg-3′和5′-cacctacacatgradtc-3′为引物,扩增出16SrDNA基因的V1-V5区域,经桑格测序鉴定;
c、乳酸菌的定性与分类:利用BLASTN软件在NCBI核苷酸采集数据库中搜索部分16SrDNA序列,为每个序列选择最佳匹配,并进行分类,最终从猪乳中分离出1240个细菌分离株;
d、构建似然树:采用CD-HIT算法对序列同源性阈值低于99%的16SrDNA基因序列进行非冗余选择;之后通过对16SrDNA基因序列进行非冗余校正,利用Fasttree构建最大似然树,确定各分离株之间的系统发育关系;
e、序列存档:利用BLASTN对1240个细菌分离株进行16SrDNA序列比对,比较了Silva132版16srRNA数据库、NCBI核苷酸采集数据库(nr/nt)和DAIRYdb参考数据库,其阈值为1e-05e,覆盖率为99%,最低相似性阈值为99%,之后将序列读取数据存入美国国家生物技术信息中心的序列读取存档。
作为具体技术方案,所述步骤(3)具体为:
采用CO2麻醉法收集6日龄交配雌果蝇w1118,饥饿2小时分成若干组,每组由3个小瓶组成,每个小瓶装有20只雌性果蝇;之后用戊糖片球菌菌株的纯培养物喂食3d,以不含戊糖片球菌的蒸馏水作为阴性对照组;然后将处理后的果蝇转移到装有200μL 5%w/v蔗糖和12mmol/ml百草枯的2片惠特曼纸的小瓶中并不断更新小瓶,最后统计果蝇存活率,筛选出抗氧化作用最强的菌株。
一种上述戊糖片球菌SMM914在预防断奶仔猪炎性肠病的应用。
本发明有益效果:
本发明提供了一株戊糖片球菌SMM914,该菌株对鼠伤寒沙门氏菌、肠道出血性大肠杆菌、产肠毒素大肠杆菌、克雷伯氏肺炎菌、点状气单胞菌、金黄色葡萄球菌、李斯特菌和产气荚膜梭菌均具有抑菌活性,特别具有强大的抑制金黄色葡萄球菌增殖的能力,同时还具备较好的抗氧化作用。本发明提供的筛选方法工艺过程操作简单、筛选效率高,所得菌株可提高仔猪肠道中有益细菌群类的比例,对预防断奶仔猪炎性肠病具有良好的应用前景。同时本发明还建立了相应的猪乳源益生菌菌种库,便于通过基于功能的定向检测体系筛选菌株,为益生菌的筛选提供资源。
附图说明
图1为基于最大似然估计法建立的系统发育树;图1中,灰色SMM号代表疑似新种,灰色圆圈代表乳制品数据库中已知菌种。
图2为不同菌株的抑菌活性检测结果图。
图3为不同菌株的体外抗氧化活性检测结果图。
图4为仔猪口服戊糖片球菌SMM914后激活氧化应激有关的Nrf2信号通路测定结果图;图4中,A-E分别为Western blot检测肝脏中抗氧化蛋白Keap1、NQO-1、HO-1、SOD1、CAT的表达量结果,不同组蛋白表达量均与PCNA表达量进行均一化;F-H分别为GSH-Px,SOD andCAT的抗氧化酶活测定结果;I为膜脂过氧化指标丙二醛MDA的含量测定结果。
图5为细菌群落主成分分析散点图;图5中,A为对照组与低剂量组的分离分布,B为对照组与高剂量组的分离分布。
图6为结肠菌群中益生菌和有害菌的变化图;图6中,A为乳杆菌(Lcatobacillus),B为毛螺菌FCS200(Lachnospiraceae FCS200),C为不能培养的毛螺菌(Lcanhospireaeca_uncultured),D为克里斯滕森菌科R7(Christensenellaceae R7),E为瘤胃球菌科UCG-005(Ruminococcaceae UCG-005),F为瘤胃球菌科UCG-014(Ruminococcaceae UCG-014),G为拟杆菌(Bcateroides),H为普雷沃氏菌科(Prevotellaceae),I为普雷沃氏菌2(Prevotellaceae 2)。
具体实施方式
下面结合具体实施方式对本发明做进一步的说明,需要指出的是以下实施方式仅是以例举的形式对本发明所做的解释性说明,但本发明的保护范围并不仅限于此,所有本领域的技术人员以本发明的精神对本发明所做的等效的替换均落入本发明的保护范围。
实施例
本发明菌株戊糖片球菌SMM914的筛选方法,包括如下步骤:
1)建立母猪乳益生菌菌种资源库
a、菌种分离
选用长沙市某种猪场饲养的具有相似繁殖日期的二胎健康母猪进行试验。母猪在母乳取样前4周内未接受抗生素治疗。依次用酒精棉条和温盐水润滑的无菌棉签255擦拭乳头周围的乳晕皮肤。采用无菌试管法,从6头母猪泌乳期采集新鲜猪奶,以猪乳为接种剂,采用MRS、M17、TPY和GYP四种液体培养基,在厌氧培养箱中(N2=90%,CO2=5%,H2=5%),在37℃下连续培养30天,每隔1天取样铺平板分离纯化;
b、DNA测序:从分离纯化的培养物中提取DNA,以5′-gctcaggaacgcygg-3′和5′-cacctacacatgradtc-3′为引物,扩增出16SrDNA基因的V1-V5区域,经桑格测序鉴定。
c、乳酸菌的定性与分类:利用BLASTN软件在NCBI核苷酸采集数据库中搜索部分16SrDNA序列,为每个序列选择最佳匹配,并进行分类,最终从猪乳中分离出1240个细菌分离株。
d、构建似然树:采用CD-HIT软件对序列同源性阈值低于99%的16SrDNA基因序列进行非冗余选择,进而将1240个菌株分为271个细菌类群,而并确定为三大类:原核生物种类、疑似新种类和乳制品中已知种类。通过对16SrDNA基因序列进行非冗余校正,利用Fasttree构建最大似然树,基于最大似然估计法确定各分离株之间的系统发育关系,建立系统发育树,见图1。结果显示,1240株菌中有922株为乳酸菌目,乳杆菌目以乳酸球菌属为主;葡萄球菌属和链球菌属分别占菌株总数的5.81%和4.03%。这些菌种资源使深入的研究它们与哺乳动物健康和疾病有关的功能成为可能。
e、序列存档:利用BLASTN对1240个细菌分离株进行16SrDNA序列比对,比较Silva132版16srRNA数据库、NCBI核苷酸采集数据库(nr/nt)和DAIRYdb参考数据库,其阈值为1e-05e,覆盖率为99%,相似性阈值为99%。序列读取数据存入美国国家生物技术信息中心的序列读取存档。
2)根据抗菌能力对菌株进行初筛
传统益生菌(乳酸杆菌、乳酸球菌和双歧杆菌)的研究非常多,戊糖片球菌也是益生菌,但针对它的研究要少很多。本专利以1240个细菌分离株中得到的80株戊糖片球菌为研究对象,建立一套益生菌筛选方法,为后续大规模鉴定其它细菌的益生性奠定基础。首先采用采用琼脂平板扩散法测定80株戊糖片球菌对病原菌的拮抗性。待测病原菌为鼠伤寒沙门氏菌ATCC14028、肠道出血性大肠杆菌ATCC43894、产肠毒素大肠杆菌O149:K88、克雷伯氏肺炎菌ATCC13883、点状气单胞菌subsp.15468、金黄色葡萄球菌ATCC25923、李斯特菌ATCC19115和产气荚膜梭菌ATCC13124。
具体方法如下:病原体在LB培养基中在37℃培养8小时,然后以20μL/4mlLB的体积比稀释混合,稀释液均匀涂布在LB琼脂平板上。然后,用深度为6毫米、直径为5毫米的无菌移液管在每个琼脂平板上打孔。戊糖片球菌在37℃的MRS培养基中培养18小时,然后将戊糖片球菌发酵液的上清液以30μL的体积精确地加入到孔中。以30μLMRS空白培养基作为每个平板中心的阴性对照。在37℃培养48h后,测定抑菌圈的直径,每个样本重复三次,根据抑菌圈大小数值构建热图,结果见图2。
由图2可知,不同菌株的抑菌活性存在明显差异,而近三分之一的戊糖片球菌菌株表现出对病原菌没有拮抗能力,最后初步筛选出对鼠伤寒沙门氏菌、肠道出血性大肠杆菌、产肠毒素大肠杆菌、克雷伯氏肺炎菌、点状气单胞菌、金黄色葡萄球菌、李斯特菌和产气荚膜梭菌均具有抑菌活性的戊糖片球菌SMM914、戊糖片球菌SMM847、戊糖片球菌SMM853、戊糖片球菌SMM906、戊糖片球菌SMM907、戊糖片球菌SMM908、戊糖片球菌SMM881、戊糖片球菌SMM867、戊糖片球菌SMM918、戊糖片球菌SMM862,一共10株戊糖片球菌,其中戊糖片球菌SMM914表现出更好的抑制金黄色葡萄球菌增殖的能力。
3)根据抗氧化能力对菌株进行复筛
采用CO2麻醉法收集6日龄交配雌果蝇w1118,饥饿2小时分成若干组,每组由3个小瓶组成,每个小瓶装有20只雌性果蝇。用步骤(2)筛选出的10株戊糖片球菌菌株的纯培养物(1×1010CFU)喂食3天,以不含戊糖片球菌的蒸馏水作为阴性对照组。然后将这些处理后的果蝇转移到装有200μL 5%(w/v)蔗糖和12mmol/ml百草枯的2片惠特曼纸的小瓶中并不断更新小瓶,最后统计果蝇存活率,结果见图3。
由图3可知,百草枯处理45小时后,对照组果蝇的存活率只有16.67%,而喂食戊糖片球菌SMM914的果蝇存活率达到了53.33%,并且跟其他戊糖片球菌菌株相比,喂食SMM914后果蝇的存活率最高,因此戊糖片球菌SMM914具有最强的抗氧化能力。
4)仔猪体内验证试验
a、从9头二胎母猪中选取54头长白仔猪,随机分为3个处理组(n=18),其中对照组(生理盐水,每次2.0mL,对照组)、戊糖片球菌SMM914低剂量组(108cfu/mL,每次2.0mL,LD组)和戊糖片球菌SMM914高剂量组(109cfu/mL,每次2.0mL,HD组)。用无针头的注射器将生理盐水或细菌细胞溶液注入每头猪的口中。从仔猪10-18日龄开始,每隔一天口服一次,每隔一天口服一次,21日龄断奶。
b、每组7窝仔猪分别于28日龄处死,解剖,提取肝脏样本在液氮下粉末化。在放射免疫沉淀试验缓冲液中用蛋白酶抑制剂苯甲基磺酰氟(PMSF,Beyotime Biotechnology)溶解。上清液在12 000g下离心,在4℃下离心10分钟。
c、变性蛋白质用10%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离,然后在200mA电流下转移到聚偏二氟乙烯膜上1小时。用5%脱脂牛奶和0.5%吐温-20(TBST)混合盐溶液在室温下封闭细胞膜2h,然后与kelch样ECH相关蛋白1(Keap1)抗体、核因子-e2相关因子2(Nrf2)、NADPH醌氧化还原酶-1(NQO-1)、血红素氧合酶-1(HO-1)、过氧化氢酶(CAT)、铜锌超氧化物歧化酶(SOD1)、增殖细胞核抗原(60097-1-Ig,Proteintech)、肌动蛋白(SC-47778,Proteintech)共孵育。
d、在TBST中冲洗膜3次,然后与第二抗体共同孵育。最后,用TBST溶液清洗上述膜,并用化学发光仪器进行可视化显示,测定结果见图4。
由图4可知,无论是高剂量还是低剂量的SMM914都显著抑制了仔猪的Keap1蛋白水平。我们还发现,SMM914不仅明显增加了Nrf2核内表达的蛋白水平,而且以浓度依赖的方式提高了NQO-1、HO-122 1、CAT和SOD1的蛋白水平。在酶活性测定方面,HD组大鼠肝组织中谷胱甘肽过氧化物酶(GSH-Px)活性、过氧化氢酶(CAT)活性和超氧化物歧化酶(SOD)活性均显著升高(p<0.05)。同时,HD组肝组织MDA含量明显低于对照组。总之SMM914作为一种有前途的益生菌,通过激活nrf2/keap1抗氧化信号通路,提高断奶仔猪的抗氧化能力。
本发明菌株戊糖片球菌SMM914在预防断奶仔猪炎性肠病的应用效果:
上述仔猪宰后取肠道食糜标本提取细菌DNA,PCR扩增细菌16SrDNA V3-V4区,在95℃ 2min,95℃ 30s,55℃ 30s,72℃ 30s,72℃ 5min的程序下进行三重扩增。对PCR产物进行纯化、定量和测序。使用UPARSE系统在基于97%的相似性截止条件下进行高质量的读取并集群到操作单元(otu)中。使用UCHIME对嵌合序列进行鉴定和删除,根据Silva132版的细菌数据库,用RDP分类器分析了每个16SrDNA序列的分类(http://RDP.cme.msu.edu/)。结果见图5、图6。
为了更直观地测量整体微生物菌群结构相似性的程度,由图5可知,基于距离的PCA结果显示高剂量组和对照组之间的样本是分开的聚类,表明高剂量的SMM914明显重塑了结肠的微生物群结构。
由图6可知,在科水平上,与对照组相比,低剂量组和高剂量组的乳酸杆菌科(Lactobacillaceae),毛螺菌科(Lachnospiraceae),克里斯滕森菌科(Christensenellaceae)和瘤胃球菌科(Ruminococcaceae)的相对丰富度分别增加了11.61%,10.08%,29.31%,80.77%和123.22%,11.34%,54.21%,503.96%。在高剂量组中观察到乳杆菌属(Lactobacillus)有增加趋势(与对照组相比,p=0.068)。在该剂量组组中,SMM914还促进了毛螺菌科AC2044(Lachnospiraceae AC2044)(p<0.001)和毛螺菌科FCS020(Lachnospiraceae FCS020)(p<0.05)的生长,具体参见图6中A-F。
已有研究表明,在哺乳期间,乳杆菌通过上调谷胱甘肽还原酶和谷胱甘肽S-转移酶的表达发挥抗氧化损伤的保护作用。众所周知,毛螺菌科(Lachnospiraceae)细菌参与碳水化合物的分解,并可能有助于抗氧化性能。例如,蛋氨酸可减轻大鼠的氧化应激,它是通过增加乳酸杆菌(Lactobacillus)和毛螺菌科(Lachnospiraceae)细菌的丰度实现的。
球菌科与疾病的严重程度呈负相关,克里斯滕森菌科R7(Christensenellaceae_R_7)因其在肠道环境和免疫调节中的积极作用而被认为是潜在的有益细菌(肥胖会改变肠道微生物生态),在本专利中,克里斯滕森氏菌科R7属(Christensenellaceae R7)(p<0.05),瘤胃球菌科UCG-005(Ruminococcaceae UCG-005)属(p<0.01)和瘤胃球菌科UCG-014(Ruminococcaceae UCG-014)属(p<0.05)在高剂量组中富集,具体参见图6中D-F。
相反,在科水平上,低剂量组和高剂量组的拟杆菌科(Bacteroidaceae)和普雷沃氏菌科(Prevotellaceae)的相对丰富度分别下降了34.24%,5.19%和80.92%,62.92%,具体参见图6中G-I。具体而言,在高剂量组中观察到了拟杆菌科(Bacteroidaceae)的下降趋势(与对照组相比,p=0.056)。SMM914还抑制了普雷沃氏菌科(Prevotellaceae)细菌的相对丰度降低(p<0.05),这些减少的细菌与氧化应激有关。同时,IBD患者的病原微生物群主要表现为普雷沃氏菌科(Prevotellaceae)增加,瘤胃球菌科(Ruminococcaceae)和毛螺菌科(Lachnospiraceae)细菌减少。因此,通过增加有利的细菌而同时淘汰那些有害的细菌,SMM914的喂食选择性地促进了更适应氧化应激的微生物群落的转变。
个体水平上,同样选取54头长白仔猪,随机分为3个处理组(n=18),其中对照组(生理盐水,每次2.0mL,对照组)、戊糖片球菌SMM914低剂量组(108cfu/mL,每次2.0mL,LD组)和戊糖片球菌SMM914高剂量组(109cfu/mL,每次2.0mL,HD组)。通过对6日内各组仔猪腹泻情况进行统计结果如下表:
分组 | 对照组 | 低浓度组 | 高浓度组 |
断奶后腹泻率/% | 5.56 | 2.78 | 0 |
结果表明,高剂量戊糖片球菌SMM914处理能够显著降低断奶仔猪腹泻率,是预防断奶仔猪炎性肠病IBD的有效方法。
Claims (10)
1.一株戊糖片球菌SMM914,其特征在于,所述戊糖片球菌(Pediococcus Pentosaceus)SMM914保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC:20160。
2.根据权利要求1所述的戊糖片球菌SMM914,其特征在于,所述戊糖片球菌对鼠伤寒沙门氏菌、肠道出血性大肠杆菌、产肠毒素大肠杆菌、克雷伯氏肺炎菌、点状气单胞菌、金黄色葡萄球菌、李斯特菌和产气荚膜梭菌均具有抑菌活性。
3.根据权利要求2所述的戊糖片球菌SMM914,其特征在于,所述戊糖片球菌对金黄色葡萄球菌的抗菌活性大于其对鼠伤寒沙门氏菌、肠道出血性大肠杆菌、产肠毒素大肠杆菌、克雷伯氏肺炎菌、点状气单胞菌、李斯特菌和产气荚膜梭菌的抗菌活性。
4.根据权利要求1所述的戊糖片球菌SMM914,其特征在于,所述戊糖片球菌具有抗氧化作用。
5.根据权利要求1所述的戊糖片球菌SMM914,其特征在于,所述戊糖片球菌可提高仔猪肠道中有益细菌群类的比例。
6.根据权利要求5所述的戊糖片球菌SMM914,其特征在于,所述有益细菌群类包括瘤胃菌科、毛螺菌科、乳酸杆菌科。
7.一种如权利要求1所述的戊糖片球菌SMM914的筛选方法,其特征在于包括以下步骤:
(1)建立母猪乳益生菌菌种资源库;
(2)根据抗菌能力对菌株进行初筛;
(3)根据抗氧化能力对菌株进行复筛;
(4)仔猪体内验证试验。
8.根据权利要求7所述的筛选方法,其特征在于,所述步骤(1)包括以下步骤:
a、菌种分离:从泌乳期母猪采集新鲜普通牛奶在厌氧培养箱中,在37℃下连续发酵30d,收集4h;以猪乳为接种剂,采用MRS、M17、TPY和GYP四种液体培养基共培养,富集并分离牛乳中的乳酸菌,之后分离纯化;
b、DNA测序:从分离纯化的培养物中提取DNA,以5′-gctcaggaacgcygg-3′和5′-cacctacacatgradtc-3′为引物,扩增出16SrDNA基因的V1-V5区域,经桑格测序鉴定;
c、乳酸菌的定性与分类:利用BLASTN软件在NCBI核苷酸采集数据库中搜索部分16SrDNA序列,为每个序列选择最佳匹配,并进行分类,最终从猪乳中分离出1240个细菌分离株;
d、构建似然树:采用CD-HIT软件对序列同源性阈值低于99%的16SrDNA基因序列进行非冗余选择;之后通过对16SrDNA基因序列进行非冗余校正,利用Fasttree构建最大似然树,确定各分离株之间的系统发育关系;
e、序列存档:利用BLASTN对1240个细菌分离株进行16SrDNA序列比对,比较Silva132版16srRNA数据库、NCBI核苷酸采集数据库nr/nt和DAIRYdb参考数据库,其阈值为1e-05e,覆盖率为99%,截止率为99%,之后将序列读取数据存入美国国家生物技术信息中心的序列读取存档。
9.根据权利要求7所述的筛选方法,其特征在于,所述步骤(3)具体为:
采用CO2麻醉法收集6日龄交配雌果蝇w1118,饥饿2小时分成若干组,每组由3个小瓶组成,每个小瓶装有20只雌性果蝇;之后用戊糖片球菌菌株的纯培养物定殖3d,以不含戊糖片球菌的蒸馏水作为阴性对照组;然后将处理后的果蝇转移到装有200μL 5%w/v蔗糖和12mmol/ml百草枯的2片惠特曼纸的小瓶中并不断更新小瓶,最后统计果蝇存活率,筛选出抗氧化作用最强的菌株。
10.一种如权利要求1至6任一所述的戊糖片球菌SMM914在预防断奶仔猪炎性肠病的应用。
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