CN116496929A - 一种猪源芽孢杆菌及其应用 - Google Patents
一种猪源芽孢杆菌及其应用 Download PDFInfo
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Abstract
本发明公开了一种猪源芽孢杆菌及其应用,猪源芽孢杆菌的分类命名为Bacillus tequilensis YB‑2,已于2022年09月29日在中国典型培养物保藏中心登记保藏,保藏编号为CCTCC No:M 20221531。所述猪源芽孢杆菌来源于新鲜猪粪便。本发明的猪源芽孢杆菌Bacillus tequilensis YB‑2可抑制大肠杆菌、沙门氏菌和金色葡萄球菌,具有优异的抗菌效果,可应用在制备抑制大肠杆菌、沙门氏菌和金色葡萄球菌的产品中;同时该猪源芽孢杆菌具有良好的耐酸、耐胆盐的特性,可应用在制备耐酸、耐胆盐的产品中。
Description
技术领域
本发明涉及微生物技术领域,具体涉及一种猪源芽孢杆菌及其应用。
背景技术
随着养猪业集约化和早期断奶行为的推广,仔猪呼吸道疾病、腹泻、链球菌等成为了当前最流行的仔猪疾病,抗生素作为添加剂的饲料可以有效预防和治疗常见猪患病,还能够增加仔猪的生长品质,因此人们为了追求养猪生产效益的最大化,开发了多种带有抗生素的饲料。但是,抗生素的耐药性和抗生素残留等负面问题逐渐突出,抗生素还会打破猪群体内原有的微生态平衡,目前各国都严格限制抗生素在猪饲料的使用。养殖业中对于安全和高效养殖的需求,促进了对抗生素替代添加剂的开发。微生态制剂(益生菌)成为一种重要的时代选择。益生菌从分类上可以分为细菌和非细菌微生物,前者包括乳酸菌、双岐杆菌、芽孢杆菌和肠球菌等,非细菌包括米曲霉、假丝酵母、布拉酵母和酿酒酵母等;益生菌在畜禽养殖中具有重要的作用,包括可以改变肠道菌群,提高有益菌群、降低大肠杆菌含量;增加营养物的消化和吸收;产生抗菌物;改变病原微生物的基因表达;免疫调节等。益生菌的菌种选择上,需要为非病原菌、耐受复杂的肠胃环境生活(低pH,高胆汁酸)和加工运输储藏等环节,综合考虑以上要求以及降低生产成本方面综合考虑,芽孢杆菌成为应用在畜禽养殖中的首选菌类;从动物自身分离得到的原籍菌能更好的适应同种类动物的胃肠道环境,从而能更好地发挥其益生特性,取得最大化的治疗和保健效果;研究表明,芽孢杆菌能够改善肠道微生态环境,从而改善仔猪健康状况,但现有技术中猪源芽孢杆菌的报道较少,并且报道的猪源芽孢杆菌在抗菌、耐酸、耐胆盐等方面的性能存在一定劣势。
发明内容
本发明的一个目的是解决至少上述问题和/或缺陷,并提供至少后面将说明的优点。
为了实现根据本发明的这些目的和其它优点,提供了一种猪源芽孢杆菌,其分类命名为Bacillus tequilensis YB-2,已于2022年09月29日在中国典型培养物保藏中心登记保藏,保藏编号为CCTCC No:M 20221531。
优选的是,所述猪源芽孢杆菌来源于新鲜猪粪便。
本发明还提供一种如上所述的猪源芽孢杆菌在制备抑制大肠杆菌产品中的应用。
本发明还提供一种如上所述的猪源芽孢杆菌在制备抑制沙门氏菌产品中的应用。
本发明还提供一种如上所述的猪源芽孢杆菌在制备抑制金色葡萄球菌产品中的应用。
本发明还提供一种如上所述的猪源芽孢杆菌在制备耐酸产品中的应用。
优选的是,将所述猪源芽孢杆菌置于pH=2的环境下2小时,成活率为96.42%;将所述猪源芽孢杆菌置于pH=3的环境下2小时,成活率为88.23%。
本发明还提供一种如上所述的猪源芽孢杆菌在制备耐胆盐产品中的应用。
优选的是,将所述猪源芽孢杆菌置于0.15%胆盐的环境下2小时,成活率为100%;将所述猪源芽孢杆菌置于0.3%胆盐的环境下2小时,成活率为92.59%。
优选的是,所述猪源芽孢杆菌的16S rDNA的序列如SEQ ID NO.1所示。。
本发明至少包括以下有益效果:本发明的猪源芽孢杆菌Bacillus tequilensisYB-2可抑制大肠杆菌、沙门氏菌和金色葡萄球菌,具有优异的抗菌效果,同时该猪源芽孢杆菌具有优良的耐酸、耐胆盐的特性。
本发明的其它优点、目标和特征将部分通过下面的说明体现,部分还将通过对本发明的研究和实践而为本领域的技术人员所理解。
附图说明:
图1为本发明猪源芽孢杆菌YB-2的革兰氏染色图(×1000);
图2为本发明猪源芽孢杆菌YB-2的芽孢染色图(×1000);
图3为本发明猪源芽孢杆菌YB-2的抑菌圈效果图,其中:A大肠杆菌;B沙门氏菌;C金色葡萄球菌;1:YB-1;2:YB-2;5:YB-5;
图4为本发明猪源芽孢杆菌YB-2的遗传进化树;
图5为本发明猪源芽孢杆菌YB-2的生长曲线。
具体实施方式:
下面结合附图对本发明做进一步的详细说明,以令本领域技术人员参照说明书文字能够据以实施。
应当理解,本文所使用的诸如“具有”、“包含”以及“包括”术语并不配出一个或多个其它元件或其组合的存在或添加。
实施例1:
一种猪源芽孢杆菌,其分类命名为Bacillus tequilensis YB-2,已于2022年09月29日在中国典型培养物保藏中心登记保藏,保藏编号为CCTCC No:M 20221531;
所述猪源芽孢杆菌来源于新鲜猪粪便;
猪源芽孢杆菌筛选分离:
以四川绵阳本地猪的新鲜猪粪为样本,无菌称取适量粪便样本,放入PBS缓冲液中,振摇使其充分混合,90℃水浴放置10min,杀灭其他非芽孢菌,然后用无菌水梯度稀释,取适量稀释液涂布于大豆酪蛋白琼脂培养基(TSA),置于37℃培养箱,倒置培养48h,观察平板菌落生长状况,挑单个菌落划线接种于新的平板,纯化培养,初步筛选分离到10株革兰阳性的芽孢杆菌,编号分别为YB-1、YB-2、YB-3、YB-4、YB-5、YB-7、YB-8、YB-9、YB-10、YB-11。
猪源芽孢杆菌的形态学鉴定:
在无菌操作台中,将纯化后的芽孢杆菌分离株分别于LB平板上划线,37℃分别培养24h,观察并记录单菌落的大小、形状、光泽度、颜色、透明程度等特征。对所有分离株进行革兰氏染色(图1)和芽孢染色(图2),染色步骤参照染色试剂盒,显微镜下观察并记录菌体形态;各菌株菌落形态如表1;
表1
猪源芽孢杆菌拮抗病原菌特性筛选:
LB肉汤固体培养基灭菌后冷却至约50℃时,倒入无菌培养皿中,待平板自然晾干后,分别移取0.1mL指示菌菌液(108CFU/mL)均匀涂布于平板上,自然晾干后,平板上打孔,孔中加入0.1mL待测菌液,静置,待菌液渗透之平板中后,37℃恒温培养24h,测量抑菌圈大小;抑制病原菌试验,初步筛选得到能够抑制大肠杆菌、金黄色葡萄球菌和沙门菌的芽孢杆菌2株,分别为YB-1、YB-2,且YB-2的抑菌效果强于YB-1,抑菌结果见表2,抑菌圈效果图如图3(注:A大肠杆菌;B沙门氏菌;C金色葡萄球菌;1:YB-1;2:YB-2;5:YB-5)。将通过抑菌试验所的优势菌株保存,进行生理生化特性鉴定和分子生物学鉴定。
表2
通过上述的猪源芽孢杆菌拮抗病原菌特性筛选可知,所述的猪源芽孢杆菌Bacillus tequilensis YB-2可以应用在制备抑制大肠杆菌的产品中,同样可以应用在制备抑制沙门氏菌的产品和抑制金色葡萄球菌的产品中。
实施例2:
猪源芽孢杆菌耐酸耐胆盐筛选:
耐胆盐实验:
分别将YB-1、YB-2两株菌接种至LB液体培养基中,培养12-24h后,取菌液按5%的比例接种于不同胆盐水平(0.15%和0.3%)的LB液体培养基中,37℃放置2h;分别在0和2h取100μL菌液10倍梯度稀释,取10-5梯度的稀释液涂布LB平板,37℃培养24h,菌落计数。每个样品3个重复。
存活率(%)=“2h”菌落数/“0h”菌落数×100%
耐酸实验:
分别将YB-1、YB-2两株菌接种至LB液体培养基中,培养12-24h后,取菌液按5%比例接种到pH为2和3的LB液体培养基中,37℃放置2h;分别在0和2h取100μL菌液10倍梯度稀释,取10-5梯度的稀释液涂布LB平板,37℃培养24h,菌落计数。每个样品3个重复。
存活率(%)=“2h”菌落数/“0h”菌落数×100%
为进一步对候选菌株耐受消化道环境的复筛,进行了耐酸耐胆盐的试验,试验结果如表3。结果表明,YB-1、YB-2两株菌均表现为较强的耐酸耐胆盐特性。综合抑菌试验的结果,最终确定YB-2为最终候选菌株。
表3
项目 | YB-1 | YB-2 |
pH=2 | 93.33% | 96.42% |
pH=3 | 84.00% | 88.23% |
0.15%胆盐成活率 | 100.00% | 100.00% |
0.3%胆盐成活率 | 89.29% | 92.59% |
通过上述的猪源芽孢杆菌耐酸耐胆盐筛选可知,所述的猪源芽孢杆菌Bacillustequilensis YB-2可以应用在制备耐酸的产品中,同样可以应用在制备耐胆盐的产品中。
实施例3:
猪源芽孢杆菌Bacillus tequilensis YB-2的鉴定:
按生化检测试剂盒中的步骤对YB-2菌株的生理生化特征进行检测;YB-2菌株的生理生化特征见表4。参照《常见细菌系统鉴定手册》和《伯杰氏细菌鉴定手册》进行对照分析,符合芽孢杆菌的生理生化特性。
表4
项目 | 结果 |
过氧化氢酶 | + |
VP反应 | - |
甲基红反应 | - |
淀粉水解 | - |
明胶 | - |
注:+表示阳性;-表示阴性
分子生物学鉴定:
采用细菌鉴定16S rDNA通用引物27F及1492R进行PCR扩增,引物序列如表5所示,引物由上海生工生物科技有限责任公司合成;
表5
PCR扩增模板为活化后的新鲜菌液,体系为50μL,详见表6。
表6
PCR扩增程序如下:95℃5min;95℃45s;55℃45s;72℃1min;重复35个循环;72℃8min。PCR扩增产物经1%琼脂糖凝胶电泳鉴定后于上海生工生物有限公司测序,在GenBank进行BLAST比对分析;在GenBank进行BLAST比对,绘制遗传进化树,结果显示,YB-2与参考菌株Bacillus tequilensis(KCTC 13622)在同一分枝上,亲缘性极高(图4),因此可判定YB-2为特基拉芽胞杆菌;且测序得到所述猪源芽孢杆菌Bacillus tequilensis YB-2的16SrDNA的序列如SEQ ID NO.1所示。
实施例4:
猪源芽孢杆菌YB-2的生物学特性分析:
将菌种活化一代后按照2%的接种量接入100mL LB液体培养基,于37℃条件下震荡培养,每4h取5mL菌液待测,利用紫外分光光度计在600nm波长下测定菌液浓度(OD值),共记录24h。通过对猪源特基拉芽胞杆菌YB-2生长曲线的测定(图5),可以发现YB-2在2h后进入对数生长期,10h后进入稳定期。
尽管本发明的实施方案已公开如上,但其并不仅仅限于说明书和实施方式中所列运用,它完全可以被适用于各种适合本发明的领域,对于熟悉本领域的人员而言,可容易地实现另外的修改,因此在不背离权利要求及等同范围所限定的一般概念下,本发明并不限于特定的细节和这里示出与描述的图例。
Claims (10)
1.一种猪源芽孢杆菌,其特征在于,其分类命名为Bacillus tequilensis YB-2,已于2022年09月29日在中国典型培养物保藏中心登记保藏,保藏编号为CCTCC No:M 20221531。
2.如权利要求1所述的猪源芽孢杆菌,其特征在于,所述猪源芽孢杆菌来源于新鲜猪粪便。
3.一种如权利要求1所述的猪源芽孢杆菌在制备抑制大肠杆菌产品中的应用。
4.一种如权利要求1所述的猪源芽孢杆菌在制备抑制沙门氏菌产品中的应用。
5.一种如权利要求1所述的猪源芽孢杆菌在制备抑制金色葡萄球菌产品中的应用。
6.一种如权利要求1所述的猪源芽孢杆菌在制备耐酸产品中的应用。
7.如权利要求6所述的猪源芽孢杆菌在制备耐酸产品中的应用,其特征在于,将所述猪源芽孢杆菌置于pH=2的环境下2小时,成活率为96.42%;将所述猪源芽孢杆菌置于pH=3的环境下2小时,成活率为88.23%。
8.一种如权利要求1所述的猪源芽孢杆菌在制备耐胆盐产品中的应用。
9.如权利要求8所述的猪源芽孢杆菌在制备耐胆盐产品中的应用,其特征在于,将所述猪源芽孢杆菌置于0.15%胆盐的环境下2小时,成活率为100%;将所述猪源芽孢杆菌置于0.3%胆盐的环境下2小时,成活率为92.59%。
10.如权利要求1所述的猪源芽孢杆菌,其特征在于,所述猪源芽孢杆菌的16S rDNA的序列如SEQ ID NO.1所示。
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