CN116445340B - 提高抗病性和抑制致病菌生长的民猪肠源暹罗芽孢杆菌 - Google Patents
提高抗病性和抑制致病菌生长的民猪肠源暹罗芽孢杆菌 Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Abstract
本发明公开一种提高抗病性和抑制致病菌生长的民猪肠源暹罗芽孢杆菌(Bacilluus siamensis)MZ16,属于农业畜牧应用领域。该菌保藏在中国典型培养微生物保藏中心,保藏日期为2022年3月21日,保藏编号为CCTCC M2022297,分类命名为Bacilluus siamensis MZ16。该菌有助于减轻致病菌微生物对动物的危害,提高动物的抗病性和免疫力,调节动物肠道,提高动物生产性能,是一株具有较高应用价值的猪源益生菌。
Description
技术领域
本发明属于农业畜牧应用领域,具体涉及一种提高抗病性和抑制致病菌生长的民猪肠源暹罗芽孢杆菌(Bacilluus siamensis)MZ16。
背景技术
随着饲用抗生素作为生长促进剂在动物饲料中的大量使用,越来越多耐药性菌株的出现引起了全世界的广泛关注,普遍存在细菌感染一直是困扰畜牧养殖业的一大问题。尤其是对于新生仔猪和断奶仔猪,其发育尚不完善,使得它们极易遭受细菌感染而发生严重腹泻甚至死亡,造成巨大的经济损失。并且,感染畜禽的致病菌如沙门氏菌和致病性大肠杆菌等容易随畜禽产品进入人类口中而造成食物中毒,严重危害人类健康。因此寻找具有高效广谱抗菌活性、毒性低、不易诱导耐药菌株及其产生的抗菌物质来替代抗生素是解决这一问题的有效途径。随着农业部194号文件的实施,全面禁抗时代的来临,研制新型安全抗菌制剂,已经迫在眉睫。
我国是养猪大国,健康的猪是生产优质猪肉的基本要求。东北民猪是中国特有的地方猪种,分布在气候寒冷的东北地区,具有抗病能力强、肉品质优良、抗逆强、耐粗饲、瘦肉率低等优良性状,近年来多组学整合分析将这些优良性状与其肠道微生物区系联系起来,发现与其体内的微生物密切相关,而其消化道内丰富的微生物也为抑制致病菌物质的分离和筛选提供了丰富的来源。结肠作为流动速度慢,大量物质被发酵,微生物区系最复杂,微生物种类和数量较多,因此选用结肠作为抑菌筛选的主要部位,通过对民猪结肠内容物中的微生物进行筛选,得到了很多可以抑制有害菌的芽孢杆菌,其中暹罗芽孢杆菌(Bacilluus siamensis)就是其中一种。
发明内容
基于以上畜牧养殖生产过程中实际问题及需求,本发明目的在于提供一种提高抗病性和抑制致病菌生长的民猪肠源暹罗芽孢杆菌(Bacilluus siamensis)MZ16,有助于提高动物生产性能,调节动物肠道,提高动物的抗病能力。
本发明的目的通过以下技术方案实现:一种提高抗病性和抑制致病菌生长的民猪肠源暹罗芽孢杆菌,保藏于中国典型培养物保藏中心,地址:湖北省武汉市洪山区八一路武汉大学中国典型培养物保藏中心,邮政编码:430072,保藏日期为2022年3月21日,保藏编号为CCTCC M 2022297,分类命名为Bacilluus siamensis MZ16。
进一步的,所述的民猪肠源暹罗芽孢杆菌对抗生素具有敏感性,所述的抗生素包括对链霉素、卡那霉素、万古霉素、庆大霉素、克林霉素、红霉素、四环素和氯霉素。
本发明还提供如上所述的贝莱斯芽孢杆菌的培养方法,如下:取OD600=1.0种子液按体积比1%的接种量接种至培养基中,温度30℃-45℃,pH=4-8,所述的培养基采用LB液体培养基。
进一步,在温度37℃,pH=6,220r/min条件下摇床振荡培养。
本发明还提供如上所述的民猪肠源暹罗芽孢杆菌在制备动物肠道有害菌抑制剂的应用。
如上所述的民猪肠源暹罗芽孢杆菌,对大肠杆菌ATCC 25922、大肠杆菌K88、大肠杆菌K99、鸡白痢沙门氏菌C7913、粪链球菌29212、肺炎链球菌31001、表皮葡萄球菌12228、鼠伤寒沙门氏菌C7731、铜绿假单胞菌PA01和/或粪肠球菌29212、
本发明还提供一种益生菌制剂,含有如上所述的民猪肠源暹罗芽孢杆菌。
本发明的优点及有益效果:本发明所提供的民猪肠源暹罗芽孢杆菌菌株对大肠杆菌ATCC 25922、大肠杆菌K88、大肠杆菌K99、鸡白痢沙门氏菌C7913、粪链球菌29212、肺炎链球菌31001、表皮葡萄球菌12228、鼠伤寒沙门氏菌C7731、铜绿假单胞菌PA01和粪肠球菌29212均具有抑菌活性,对链霉素(Streptomycin)、卡那霉素(Kanamycin)、万古霉素(Vancomycin)、庆大霉素(Gentamicin)、克林霉素(Clindamycin)、红霉素(Erythromycin)、四环素(Tetracycline)和氯霉素(Chloramphenicol)均具有敏感性。民猪肠源暹罗芽孢杆菌MZ16通过维持TJ蛋白和分泌性抗体IgA、SIgA的含量,维持肠道黏膜机械屏障和免疫屏障稳态,从而抵抗肠炎;能够显著的提高白羽肉鸡回肠黏膜炎症因子中抗炎因子IL-4和IL-10mRNA的表达量,显著降低促炎因子IL-2、IL-和IL-1βmRNA的表达量。综上所述,民猪肠源暹罗芽孢杆菌MZ16是一株具有较高应用价值的猪源益生菌,其对畜牧业和饲料发酵工业的发展具有重要意义。
附图说明
图1为本发明的民猪肠源暹罗芽孢杆菌MZ16菌体形态图;
图2为本发明的民猪肠源暹罗芽孢杆菌MZ16的系统发育树图;
图3为不同温度条件下、本发明的民猪肠源暹罗芽孢杆菌MZ16生长能力实验结果图;
图4为不同pH条件下、本发明的民猪肠源暹罗芽孢杆菌MZ16生长能力实验结果图;
图5为不同NaCl条件下、本发明的民猪肠源暹罗芽孢杆菌MZ16生长能力实验结果图;
图6为不同胆盐浓度条件下、本发明的民猪肠源暹罗芽孢杆菌MZ16生长能力实验结果图;
图7为本发明的民猪肠源暹罗芽孢杆菌MZ16的抗菌药物敏感性实验结果图;
图8为本发明的民猪肠源暹罗芽孢杆菌MZ16对小鼠生长性能影响的实验结果图,其中,CON:对照组;MZ16:民猪肠源暹罗芽孢杆菌MZ16处理组;
图9为本发明的民猪肠源暹罗芽孢杆菌MZ16对小鼠腹泻率影响的实验结果图,其中,(a)体重,(b)平均日采食,CON:对照组;MZ16:民猪肠源暹罗芽孢杆菌MZ16处理组;
图10为本发明的民猪肠源暹罗芽孢杆菌MZ16对炎症造模小鼠结肠组织中免疫因子的影响图,其中,CON:对照组;DSS:DSS处理组;MZ16:民猪肠源暹罗芽孢杆菌MZ16处理组,以P<0.05作为显著差异标准;
图11为本发明的民猪肠源暹罗芽孢杆菌MZ16对白羽肉鸡回肠炎症因子表达量的影响图,(a)IL-4,(b)IL-10,(c)IL-2,(d)IL-8,CON:对照组;MZ16:民猪肠源暹罗芽孢杆菌MZ16处理组。
具体实施方式
通过以下实施例对本发明内容进一步说明,但这并不构成对本发明任何意义的限定。
供试材料:大肠杆菌25922、大肠杆菌K88、大肠杆菌K99、鸡白痢沙门氏菌C7913、粪链球菌29212、肺炎链球菌31001、表皮葡萄球菌12228、鼠伤寒沙门氏菌C7731、铜绿假单胞菌PA01和粪肠球菌29212由东北农业大学动物营养研究所提供;民猪粪便采集自东北农业大学阿城实验基地。
实施例1
菌株筛选与鉴定具体按以下步骤进行:
1、培养基的配置:本实验选择LB液体培养基作为用于抑菌活性测定的筛选培养基。营养琼脂培养基:琼脂15g,酵母膏3g,胰蛋白胨5g,氯化钠5g,水1000mL,pH值调至7.4,121℃高温高压灭菌20min;LB液体培养基:酵母膏3g,胰蛋白胨5g,氯化钠5g,水1000mL,121℃高温高压灭菌20min;LB固体培养基:琼脂15g,胰蛋白胨5g,酵母膏3g,氯化钠5g,水1000mL,121℃高温高压灭菌20min。
2、指示菌液的制备:取冻存的致病菌作为指示菌划线至营养琼脂培养基中,37℃恒温培养,活化菌株;挑取单菌落接种于LB液体培养基中,220rpm、37℃过夜培养,然后再将过夜菌传代于新的液体培养基中培养2-8h,直至指示菌处于生长对数期,离心收集细菌菌体,用无菌PBS冲洗,再将所得细菌的浓度调整为105CFU/ml;
3、微生物代谢物的收集:称取1.0g民猪结肠内容物,溶解于盛有50mL灭菌去离子水的三角瓶中,于摇床中室温震荡30min,制成均一的浓度为10 -2的稀释液,取适量此稀释液,进行十倍梯度连续稀释,得到10 -3-10 -5稀释液。将30μL各浓度的稀释液均匀涂布于营养琼脂培养板,置于37℃恒温箱培养24h,挑取单菌落。将单菌落接入LB液体培养基内,37℃、220rpm震荡培养24h后,12000rpm离心5min。吸取上清液,经0.22微米水系过滤器过滤备用;
4、抑制致病菌微生物菌株生长的筛选:初筛选用琼脂扩散法。挑取各保存菌种的单个菌落种于LB液体培养基中,37℃摇瓶培养24h,12000rpm离心5分钟后,取50μL上清液滴加至含有指示菌的初筛培养板的牛津杯(1×10 5CFU,8mm深×10mm外径)孔中,37℃恒温培养48h后,用游标卡尺记录每种保存菌种的抑菌圈的有无和直径(d),作为评判菌种拮抗指示菌能力的标准。挑选抑菌圈直径较大的菌种进行摇瓶和划线培养,如此反复分离纯化培养4-5代后,置于50%甘油溶液中,-80℃保存备用。复筛:将初筛中筛选出的拮抗菌-80℃保存一周后,重新取出进行活化培养,按照琼脂扩散法,继续观察这几种菌的抑菌活性,比较菌种保存过程中拮抗菌抑菌活性的变化,以鉴别其拮抗性能是否稳定。
5、筛选得到的菌株生物学特性如下:如图1所示,菌落为浅黄色,不透明,表面褶皱。将菌液态培养24h后,取菌液送至吉林省库美生物科技有限公司进行16s rDNA扩增回收以及序列测定。所得测序结果在Ezbiocloud数据库(https://www.ezbiocloud.net/identify)16S-based ID比对,采用邻接法(Neighobr-joining)来构建系统发育树,分析菌种。
6、经形态观察,生理生化反应和16S rDNA分子鉴定,将具有抑制致病菌生长能力的菌株确定为暹罗芽孢杆菌(Bacilluus siamensis)MZ16。
实施例2
菌株对致病菌抑菌能力的测定
1、菌株活化:取冻存菌株划线至营养琼脂培养基上,37℃恒温培养活化菌株,挑取单菌落接种至LB液体培养基,220rpm、37℃培养24h;
2、指示菌液的制备:取大肠杆菌25922、大肠杆菌K88、大肠杆菌K99、鸡白痢沙门氏菌C7913、粪链球菌29212、肺炎链球菌31001、表皮葡萄球菌12228、鼠伤寒沙门氏菌C7731、铜绿假单胞菌PA01、粪肠球菌29212分别作为指示菌,制备具体方法参考实施例1的指示菌液制备方法;
3、微生物代谢物的收集:具体方法参考实施例1;
4、抑菌活性测定:具体方法参考实施例1;
5、菌株抑菌活性结果:菌株抑菌效果如表1所示,菌株MZ16对多种致病菌均具有抑制作用。
表1Bacillus siamensis MZ16对致病微生物的抑菌率
实施例3
菌株在不同温度下生长能力测定
1、菌株活化:取待复苏冻存菌株原液以1%的接种量(50μl+5ml LB)接种至LB液体培养基,220rpm、37℃过夜培养12h;
2、不同温度下菌株的生长测定:调整OD600=1时,以体积比1%的接种量(50μl+5mlLB)传代至LB液体培养基,于20℃、30℃、37℃、45℃、50℃、55℃培养9h,测其OD600值。并接稀释液100μl到营养琼脂培养基。通过吸光度值和活菌计数分析所选菌株在不同pH环境条件下的生长情况,并绘制温度生长曲线;
3、如图2所示,菌株生长能力实验结果图。
4、菌株生长能力实验结果显示:37℃时最适合菌株生长。37℃前,随着温度升高菌株的生长能力逐渐升高;超过37℃后,随着温度的升高菌株的生长能力逐渐降低。30℃-45℃为较适合菌株生长的温度范围。
实施例4
菌株在不同pH环境下生长能力测定
1、菌株活化:实验操作方法同实施例3;
2、菌株相应pH条件下的生长测定:用1mol/L的HCl和1mol/L的NaOH将LB液体培养基的pH调整到2.0、3.0、4.0、5.0、6.0、7.0、8.0、9.0。将已经活化的菌液以1%的接种量(50μl+5ml LB)转接至调好pH值的LB培养基中,相同条件培养9h。培养结束后,检测培养物的OD600值,并接种稀释液100μL至固体培养基上,37℃培养12h,以确定所选菌株的生长情况;
3、如图3所示,菌株生长能力实验结果。
4、菌株生长能力实验结果显示:pH=6时最适合菌株生长,pH=4-8是菌株可以存活的。
实施例5
菌株在不同NaCl环境下生长能力的测定
1、菌株活化:实验操作方法同实施例3;
2、菌株相应NaCl条件下的生长测定:活化后的菌液以体积比1%的接种量(50μl+5ml LB)接种于含1%、2%、3%、4%、5%、6%、7%、8%、9% NaCl的LB液体培养基中,同时以不接种菌液的相同液体培养基作为对照,37℃、220rpm条件下培养9h。培养结束后,检测培养物的OD 600值,并接种稀释液100μL至固体培养基上,37℃培养12h,以确定所选菌株的生长情况;
3、如图4所示,菌株生长能力实验结果。
4、菌株生长能力实验结果显示:菌株在不同NaCl浓度条件下均可存活,但随着NaCl浓度升高,菌株生长能力降低。
实施例6
菌株不同胆盐浓度条件下生长能力的测定
1、菌株活化:实验操作方法同实施例3;
2、菌株相应胆盐条件下的生长测定:活化后的菌液以体积比1%的接种量(50μl+5ml LB)接种于含0%、0.1%、0.2%、0.3%、0.5%、0.8%、1%、3%胆盐的LB液体培养基中,同时以不接种菌液的相同液体培养基作为对照,37℃、220rpm条件下培养9h。培养结束后,检测培养物的OD 600值,并接种稀释液100μL至固体培养基上,37℃培养12h,以确定所选菌株的生长情况;
3、如图5所示,菌株生长能力实验结果。
4、菌株生长能力实验结果显示:菌株在无胆盐添加的情况下,生长情况较好;在添加胆盐0.3%后,菌株几乎无法生长。
实施例7
菌株的抗菌药物敏感性
1、菌株活化富集
取待复苏冻存菌株原按体积比为1%的接种量接种于选择药物敏感性试验专用培养基,摇菌管在220rpm、37℃条件下过夜培养18h后,再以1%的接种量传3代。
2、活菌数调整
菌株活化传代后,4℃、6000rpm离心收集菌体,并用PBS洗涤后将活菌细胞与药物敏感性试验专用液体培养基混匀,将浓度调整至约105cfu/mL。
3、菌株抗生素敏感性测定与分析
试验根据美国临床实验室标准化研究所(CLSI)推荐的测定最小抑菌浓度(MIC)的方法,评价菌株对链霉素(Streptomycin)、卡那霉素(Kanamycin)、万古霉素(Vancomycin)、庆大霉素(Gentamicin)、克林霉素(Clindamycin)、红霉素(Erythromycin)、四环素(Tetracycline)和氯霉素(Chloramphenicol)的敏感性。将初始浓度为32mg/L的梯度抗生素溶液加入到第1号孔内,之后的孔梯度稀释,随后与细菌溶液混合,37℃培养20±2h。通过将测定的MIC值与《直接饲喂微生物和发酵制品生产菌株鉴定及其安全性评价指南》给定的各抗菌药物的临界值进行比较,以区分耐药菌株和敏感菌株。如低于该值,说明菌株对该抗生素未超过界定范围较敏感,否则即为耐药。
4、如图6所示,菌株的抗菌药物敏感性实验结果:菌株的抗菌药物敏感性实验结果显示:与《直接饲喂微生物和发酵制品生产菌株鉴定及其安全性评价指南》的临界值相比,MZ-16的MIC值较低,表明菌株对所有试验抗生素均敏感。
实施例8
菌株的动物致病性试验
1、菌株活化富集
取待复苏冻存菌株原液以体积比1%的接种量接种于LB液体培养基,摇菌管在220rpm、37℃条件下过夜培养18h后,再以体积比1%的接种量传3代。
2、活菌数调整
将活化好的菌株5000r/min离心10min,生理盐水洗涤,重复4次后,重悬于生理盐水中。用细菌浊度仪确定细菌浓度为1MCF。
3、致病性测定分析
将活菌作为受试物,通过经口灌胃途径给予BALB/C小鼠(5周龄,19.43±0.3g),每只鼠灌胃0.3mL,并设置对照组灌胃等量生理盐水,试验期为8天,试验期间,每天记录小鼠体重。通过对比评价该冻存菌株对小鼠的致病性。动物致病性试验按照国家、行业标准方法开展。试验显示受试物组动物在试验期间出现中毒症状或死亡,或试验期间体重等指标与对照组相比有显著性差异时,则判定菌株具有致病性。
4、如图7和图8所示,菌株致病性实验结果。
5、菌株致病性实验结果显示:与正常组相比,灌服民猪肠源暹罗芽孢杆菌MZ16对小鼠的体重、平均日采食量均无显著影响、腹泻率也无显著变化(P>0.05)。对其行为状态进行观察,试验期灌菌小鼠毛色细密,饮水正常,行为活跃,反应敏捷,与对照组无差异,说明该菌株没有致病性。
实施例9
1、具体实施方法见实施例9,研究民猪肠源暹罗芽孢杆菌MZ16对炎症造模小鼠结肠组织中免疫因子的影响。
2、如图10所示,民猪肠源暹罗芽孢杆菌MZ16对炎症造模小鼠结肠组织中免疫因子的影响。
3、实验结果显示:DSS处理显著降低SIgA浓度(P<0.05),而同对照相比,预先灌服民猪肠源暹罗芽孢杆菌MZ16在进行DSS处理则不会降低SIgA浓度(P>0.05),且同DSS组相比,SIgA浓度显著升高(P<0.05)。这表明民猪肠源暹罗芽孢杆菌MZ16可增强黏膜免疫功能,以抵抗DSS损伤。同样,结肠组织中的IgA浓度也呈现与SIgA相同的趋势。说明民猪肠源暹罗芽孢杆菌MZ16通过维持TJ蛋白和分泌性抗体IgA、SIgA的含量,维持肠道黏膜机械屏障和免疫屏障稳态,从而抵抗肠炎。
实施例10
(1)组织总RNA的提取
1)研磨裂解:将-80℃保存的回肠黏膜组织样品置于研钵中,在液氮环境中迅速研磨,研磨的过程中适量加入液氮。待样品研磨至粉末状,取100mg左右粉末置于含1mLTrizol试剂的1.5mL无RNA酶的离心管中,利用漩涡振荡器充分震荡后,在室温下静置5min使组织充分裂解。
2)分相:将裂解完成的组织样品置于4℃离心机中,12000rpm离心5min,弃沉淀。将上清液转移至新的1.5mL离心管中,每个离心管中加入200μL氯仿并充分震荡摇匀,冰面静置5min后,在4℃条件下12000rpm离心15min。
3)RNA沉淀:离心后吸取上层水相转移至新的1.5mL离心管中,并加入等体积预冷的异丙醇充分混匀后,冰面静置5min,然后在4℃条件下12000rpm离心10min,可见白色RNA沉淀。
4)洗涤RNA:离心后弃上清留沉淀,然后加入1mL DEPC配制的75%乙醇,充分震荡摇匀,冰面静置5min后,在4℃条件下7500rpm离心5min。
5)RNA溶解:离心后弃上清,将离心管轻轻倒扣在滤纸上空干,加入50uL DEPC水充分混合溶解,置于-80℃冰箱保存。
(2)RNA浓度和完整性检测
RNA纯度与浓度检测:取2uL总RNA置于P330超微量分光光度计测量孔上,测定RNA浓度和A260/A280的吸光度值,当A260/A280比值处于1.8-2.0之间,则认为RNA纯度符合实验标准,否则重新提取。
RNA完整性检测:取2μL总RNA与1μL Loading Buffer充分混匀,在1.2%琼脂糖变性凝胶上120V电泳30min,将凝胶置于凝胶成像分析系统上,在电脑上观察RNA条带情况,出现28S、18S和5S RNA三条亮带,表明RNA完整性较好,符合实验标准。
RNA浓度定量:在纯度和完整性符合试验条件的情况下,根据RNA检测的浓度,根据测定浓度将RNA稀释至200ng/μL,以便后续实验。
(3)RNA反转录反应
RNA反转录反应使用TAKARA公司的可以除去基因组DNA进行Real Time RT-PCR反应的反转录试剂(PrimeScriptTM RT reagent Kit with gDNA Eraser,RR047A)。将按照实际和说明处理进行操作。
1)去DNA反应
按表3中的成分配制反应混合液,此步骤需要在冰上进行,将混合液按反应数分装至反应管中,最后加入RNA样品,放入PCR仪中反应,42℃反应2min,取出4℃放置,等待下一步操作。
表3去除基因组DNA反应体系
2)反转录反应
从4℃冰箱取出步骤1)的反应液,按试剂盒说明操作加入10μL反应液2)混合均匀后放入PCR仪进行反转录反应,37℃反应15min,85℃反应5s,最后4℃反应30min。反应液成分见表4。
表4反转录反应体系的组成
(4)引物的设计与合成
根据Genbank(https://www.ncbi.nlm.nih.gov/genbank/)数据库相关基因序列信息,设计引物并由生工生物工程有限公司(上海)合成,按照引物使用说明,对引物用1×TE进行稀释后使用,。
(5)实时荧光定量PCR
按照Premix Ex TaqTM(Tli RNaseH Plus)试剂盒说明书步骤操作,反应体系为10.0μL。按照表5配制PCR反应液,此步骤在冰上进行。将点好反应液的96孔板放入ABI7500型PCR仪中进行两步法PCR扩增反应,扩增标准程序为:第一步,预变性:95℃ 30s,循环1次;第二步,PCR反应:95℃ 5s,60℃ 34s,循环40次。同时,根据上述反转录产生的cDNA在相同进程设定下进行目标基因和内标基因(β-actin)进行实时荧光定量PCR反应,以便通过基因引物的Tm值优化反应条件。待反应结束后确认PCR的扩增曲线和融解曲线。
表5实时荧光定量PCR的反应体系组成
(6)目的基因相对表达量的计算
样品经过实时荧光定量PCR反应后,可获得一个反应目的基因和内参基因含量的Ct值,此Ct值为反应管中荧光信号达到设定阈值时所经历的循环数。通过计算每个样品目的基因与内参基因的差值获得ΔCt值,并计算试验组和对照组ΔCt值之间的差值获得ΔΔCt值,再通过建立扩增曲线数学模型,最终计算2-ΔΔCt值,即为目的基因的mRNA相对表达量。图11为民猪肠源暹罗芽孢杆菌MZ16对白羽肉鸡回肠炎症因子表达量的影响。如图11所示,饲喂民猪肠源暹罗芽孢杆菌MZ16能够显著的提高白羽肉鸡回肠黏膜炎症因子中抗炎因子IL-4和IL-10mRNA的表达量,显著降低促炎因子IL-2、IL-8mRNA的表达量。
Claims (5)
1.一种提高抗病性和抑制致病菌生长的民猪肠源暹罗芽孢杆菌(Bacilluus siamensis),保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 2022297,分类命名为Bacilluus siamensis MZ16。
2.根据权利要求1所述的一种提高抗病性和抑制致病菌生长的民猪肠源暹罗芽孢杆菌的培养方法,其特征在于,取OD600=1.0种子液按体积比1%的接种量接种至培养基中,温度30℃-45℃,pH=4-8,所述的培养基采用LB液体培养基。
3.根据权利要求2所述的一种提高抗病性和抑制致病菌生长的民猪肠源暹罗芽孢杆菌的培养方法,在温度37℃,pH=6,220r/min条件下摇床振荡培养。
4.根据权利要求1所述的一种提高抗病性和抑制致病菌生长的民猪肠源暹罗芽孢杆菌在制备动物肠道有害菌抑制剂的应用,所述的动物肠道有害菌为大肠杆菌ATCC 25922、大肠杆菌K88、大肠杆菌K99、鸡白痢沙门氏菌C7913、粪链球菌29212、肺炎链球菌31001、表皮葡萄球菌12228、鼠伤寒沙门氏菌C7731、铜绿假单胞菌PA01和/或粪肠球菌29212。
5.一种益生菌制剂,其特征在于:含有如权利要求1所述的一种提高抗病性和抑制致病菌生长的民猪肠源暹罗芽孢杆菌。
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