CN105647836A - 一株枯草芽孢杆菌及其应用 - Google Patents
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Abstract
本发明公开了一株枯草芽孢杆菌及其应用,属于发酵技术领域。所述枯草芽孢杆菌已于2016年1月21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,保藏编号为CGMCC?No.12090,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。该菌株具有较好的抑菌性能,能有效地控制豆瓣酱发酵过程中蜡状芽孢杆菌的生长,而蜡状芽孢杆菌是一种潜在病原菌。
Description
技术领域
本发明涉及一株枯草芽孢杆菌及其应用,属于发酵技术领域。
背景技术
芽孢杆菌的菌体呈杆状,形态为长直或接近直,大小为0.3~2.2×1.2~7.0微米。多数运动,鞭毛典型侧生。菌体可形成耐热内生孢子,在一个孢子囊细胞中,孢子不多于1个。菌体暴露在空气中并不妨碍孢子的形成,革兰氏反应阳性,仅在生长早起可能出现阴性。有机化能营养,可利用多种底物进行严格呼吸代谢,严格发酵代谢或者呼吸和发酵二者兼有的代谢。在呼吸代谢中,最终的电子受体是分子氧,在一些种中可以用硝酸盐代替氧,大多数的种可以产生氢氧化物酶。严格好氧或兼性厌氧。
枯草芽孢杆菌是芽孢杆菌属中的一个种,呈杆状,很少成链,染色均匀,鞭毛侧生。内生孢子的大小为0.8×1.5~1.8微米,游离孢子的表面着色较弱,萌发时芽孢壳赤道裂,营养细胞露出后,孢子壳消解缓慢。菌落形态为圆形或不规则形,菌落直径2~4mm,边缘粗糙。菌落表面粗糙褶皱,白色不透明。最适生长温度为28~30℃,最适生长pH在5.5到8.5之间。该种可产生多肽抗菌素,一个菌株可产生一种或多种抗菌素,释放对菌体有溶菌作用的酶。
蚕豆酱是以蚕豆等为原料发酵酿制而成的保持蚕豆酱原形的半流动粘稠体或半固态调味品。蚕豆酱在我国有着悠久的历史,营养丰富,味道鲜美,有助消化,增进食欲,因此深受广大消费者的欢迎。然而,在蚕豆酱的发酵制备过程中,常因酱醅中有害微生物的繁殖,而破坏发酵过程,影响酱体的风味,甚至产生不利于身体健康的有害物质。
发明内容
本发明提供了一株枯草芽孢杆菌(Bacillussubtilis)。
所述枯草芽孢杆菌(Bacillussubtilis),已于2016年1月21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,保藏编号为CGMCCNo.12090,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。
本发明所述的枯草芽孢杆菌分离自安徽某知名酱品传统方法酿制的酱醅,该菌株具有典型特征的M13-PCR指纹,即菌株专一性的特征指纹,如图1所示;测序分析,其16srDNA序列基因型如SEQIDNO.1所示,NCBI登录号为KT982215。
所述枯草芽孢杆菌对蜡状芽孢杆菌以及酵母菌具有较明显的抑制作用。其基因组内存在编码细菌素Subtilosin以及制磷脂菌素Plipastatin的基因。
所述枯草芽孢杆菌为从风味口感较佳的传统方法酿制的豆瓣酱酱醅中筛选得到的,其保藏条件如下:从生长良好的LB培养基平板中取单菌落移入新鲜的液体LB培养基中,37℃摇床培养24h,取1.3mL菌液移入盛有0.5mL无菌甘油(40%,v/v)的甘油管中,放-80℃冰箱保存。
所述LB培养基为:蛋白胨1%;酵母粉0.5%;NaCl1%;pH自然。
所述枯草芽孢杆菌的培养条件为:将甘油管保藏的枯草芽孢杆菌接入LB液体培养基,在37℃、180rpm摇瓶培养24h即可到达对数期中后期。
本发明还提供了一种所述枯草芽孢杆菌的应用,用于抑制发酵过程中酱醅中蜡状芽孢杆菌等潜在病原菌的生长。
本发明提供的枯草芽孢杆菌是蚕豆酱发酵过程中的无害菌,对酱醅中有害微生物的抑制有一定的作用,对酱醅中微生物组成以及病原菌的抑制有着深刻的影响,可用于抑制豆瓣酱生产过程中的有害菌。
生物材料保藏
枯草芽孢杆菌(Bacillussubtilis),已于2016年1月21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,保藏编号为CGMCCNo.12090,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。
附图说明
图1Rep-PCR(M13-PCR)扩增指纹图谱,M:DL10000marker,1.12:样品。
具体实施方式
实施例1枯草芽孢杆菌的筛选
从传统发酵豆瓣酱60天左右的酱醅中取1g样品加入到9mL无菌水中,轻微振荡制成悬浊液,将悬浊液梯度稀释,涂布于LB平板,37℃好氧培养24至48h。挑取白色扁平有褶皱的单菌落反复进行平板划线分离,重复三次得到纯化的单菌落,供下一步鉴定用。
鉴定主要通过:1)菌落特征观察:用肉眼直接观察,挑选菌落直径在2~4mm之间,圆形或不规则,表面粗糙有褶皱,白色不透明等符合枯草芽孢杆菌菌落特征的菌株。2)个体形态观察:用光学显微镜革兰氏染色观察。挑选杆状、革兰氏阳性的菌株。将满足鉴定条件的菌株挑出甘油管保藏。
以1~2%接种量取-80℃甘油贮存液接种于LB培养基中,37℃振荡静置培养24h。取适量培养液离心2min(4000rpm,4℃),弃尽上清液,得到适量菌体,用CTAB法提取基因组DNA。用细菌通用引物对上游引物27F扩增16srDNA,反应条件为:94℃预变性3min后进入以下循环:94℃变性40s,56℃退火35s,72℃延伸80s,30个循环;72℃延伸10min。经1%琼脂糖凝胶电泳鉴定结果良好。将扩增产物寄送上海生工基因测序,如SEQIDNO.1所示,在NCBI网站上进行BLAST序列比对,确定为枯草芽孢杆菌。
实施例2枯草芽孢杆菌CGMCCNo.12090对酱醅中蜡状芽孢杆菌的抑制(滤纸片法)
该实验所用的培养基为LB培养基:蛋白胨1%;酵母粉0.5%;NaCl1%;pH自然。三角瓶分装,装液量40mL,121℃灭菌,20min。固体LB琼脂含量为2%。
以1%接种量取-80℃甘油贮存液接种于新鲜LB液体培养基,37℃摇床培养24小时,转速180rpm。接着以2%接种量传代培养,培养24h左右可用作实验菌。实验所用蜡状芽孢杆菌活化及传代过程同上。传代后的实验菌(枯草芽孢杆菌)和指示菌(蜡状芽孢杆菌)的菌浓均在108CFU/mL左右。将指示菌稀释至106级别,均匀涂布于LB平板,用直径为1.5cm的滤纸片蘸取实验菌菌液,轻轻放置于涂有指示菌的LB平板上,所有操作均在无菌环境中进行。将LB平板放置于37℃培养箱中培养24h即可观察实验结果。实验结果见附表1。
表1枯草芽孢杆菌CGMCCNo.12090抑菌谱
注:指示菌株均为酱醅中分离所得;
-:没有抑菌圈;
+:抑菌圈直径2~3cm;
++:抑菌圈直径大于3cm;
实施例3应用于传统发酵蚕豆酱
CGMCCNo.12090BacillussubtilisHYM05菌株在LB液体培养基中培养1天,作为种子液。将种子液接种至酱醅,发酵过程中蜡状芽孢杆菌的数量变化有明显区别,对照为未接种种子液的酱醅,未接种所述芽孢杆菌的酱体中检测到较大数量(约2.55×104CFU/g)的蜡状芽孢杆菌,而接种的酱体中蜡状芽孢杆菌数量维持在一个较低的水平(约1.32×102CFU/g)。
可以理解的是,对本领域普通技术人员来说,可以根据本发明的技术方案及其发明构思加以等同替换或改变,而所有这些改变或替换都应属于本发明所附的权利要求的保护范围。
Claims (7)
1.一株枯草芽孢杆菌(Bacillussubtilis),于2016年1月21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,保藏编号为CGMCCNo.12090,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。
2.权利要求1所述的枯草芽孢杆菌在抑制豆瓣酱发酵过程中蜡状芽孢杆菌的生长中的应用。
3.权利要求1所述的枯草芽孢杆菌在抑制豆瓣酱发酵过程中酵母菌的生长中的应用。
4.权利要求1所述枯草芽孢杆菌的培养方法,其特征在于,是将甘油管保藏的枯草芽孢杆菌接入LB液体培养基中,在37℃、转速180rpm的摇床中振荡培养24h至对数中后期。
5.应用权利要求1所述枯草芽孢杆菌发酵豆瓣酱的方法,其特征在于,是将枯草芽孢杆菌种子液在豆瓣酱发酵过程的中前期添加入到酱醅中。
6.权利要求1所述的枯草芽孢杆菌在抑制蜡状芽孢杆菌生长中的应用。
7.权利要求1所述的枯草芽孢杆菌在抑制酵母生长中的应用。
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CN111248409A (zh) * | 2020-02-27 | 2020-06-09 | 江南大学 | 一种低盐豆瓣酱发酵方法 |
CN116063111A (zh) * | 2022-11-23 | 2023-05-05 | 河北冀微绿色农业科技有限公司 | 枯草芽孢杆菌m-15在制备微生物肥料中的应用 |
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CN111172079A (zh) * | 2020-02-18 | 2020-05-19 | 北京工商大学 | 任清芽孢杆菌新菌种及其应用 |
CN111248409A (zh) * | 2020-02-27 | 2020-06-09 | 江南大学 | 一种低盐豆瓣酱发酵方法 |
CN116063111A (zh) * | 2022-11-23 | 2023-05-05 | 河北冀微绿色农业科技有限公司 | 枯草芽孢杆菌m-15在制备微生物肥料中的应用 |
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