CN116179404B - 一株霍氏肠杆菌及其应用 - Google Patents
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Abstract
本发明涉及一株霍氏肠杆菌,所述霍氏肠杆菌命名为Enterobacter hormaechei,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.1.19421,保藏地址为北京市朝阳区北辰西路1号院3号,保藏日期为2021年12月28日。所述霍氏肠杆菌RH3基因序列如SEQ ID NO.1所示,并且,所述霍氏肠杆菌RH3缺失hdc基因。该菌能利用组氨酸产生组胺,但缺失形成组胺的关键基因—hdc基因,可以为hdc基因缺失型细菌合成组胺的机制提供研究素材。
Description
技术领域
本发明属于微生物应用领域,尤其是涉及一株霍氏肠杆菌及其应用。
背景技术
组氨酸脱羧酶(hdc,EC 4.1.1.22)是氨基酸脱羧酶的一种,可以催化组氨酸脱羧生成组胺,是微生物形成组胺的关键控制酶,其编码基因为hdc基因。现有的可以产生组胺的微生物都是通过组氨酸脱羧酶将氨基酸脱羧形成组胺。本申请人发现了一株缺失hdc基因的霍氏肠杆菌菌株,同样可以产组胺。
发明内容
本发明的目的是提供一株霍氏肠杆菌RH3及其应用。
为了实现上述目的,本发明采用的技术方案如下:
第一个方面,本发明提供一株霍氏肠杆菌RH3,所述霍氏肠杆菌RH3(Enterobacter hormaechei RH3)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.1.19421,保藏地址为北京市朝阳区北辰西路1号院3号,保藏日期为2021年12月21日。
进一步地,所述霍氏肠杆菌RH3,其基因序列如SEQ ID NO.1所示,并且,所述霍氏肠杆菌RH3缺失hdc基因。
第二个方面,本发明提供一种菌剂,其包含上述第一个方面所述的霍氏肠杆菌RH3。
进一步地,所述的一种菌剂,还包含或者包含上述第一个方面所述的霍氏肠杆菌RH3的代谢产物。
第三个方面,本发明提供上述第二个方面所述的菌剂在产组胺上的应用。
第四个方面,本发明提供上述第一个方面所述的霍氏肠杆菌RH3在产组胺上的应用。
进一步地,所述霍氏肠杆菌RH3利用组氨酸产生组胺。
进一步地,所述霍氏肠杆菌RH3利用组氨酸产生组胺,包括以下步骤:
将所述霍氏肠杆菌RH3接种至含有组氨酸的LB液体培养基中,在pH 7.0、37℃,静置条件下培养28 h,开始产生组胺。
进一步地,所述LB液体培养基中组氨酸含量为1%(w/v)。
本发明所具有的优点和有益效果是:
本发明提供一株霍氏肠杆菌RH3(Enterobacter hormaechei RH3),该菌缺失形成组胺的关键基因—hdc基因,但依然能利用组氨酸产生组胺,可以为hdc基因缺失型细菌合成组胺的机制提供研究素材。
生物材料保藏:
一株霍氏肠杆菌RH3(Enterobacter hormaechei RH3),该菌于2021年12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,保藏地址:北京市朝阳区北辰西路1号院3号,保藏编号CGMCC NO.1.19421。
附图说明
图1为组胺浓度标准曲线;
图2为霍氏肠杆菌RH3在LB固体平板上形成的菌落形态;
图3为霍氏肠杆菌RH3中hdc基因特异性扩增;
图4为霍氏肠杆菌RH3中hdc的表达量;
图5为霍氏肠杆菌RH3全基因组中与组氨酸相关的KEGG代谢通路图;
图6为霍氏肠杆菌RH3发酵曲线和产组胺曲线。
具体实施方式
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
下述实施例中的实验方法,如无特殊说明,均为常规方法,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中涉及的培养基如下:
LB液体培养基:酵母膏5 g/L,蛋白胨10 g/L,氯化钠10 g/L,pH 7.0。
TSB肉汤:胰蛋白胨 15 g/L,大豆蛋白胨 5 g/L,氯化钠 5 g/L,pH 7.2。
LB固体培养基:酵母膏5 g/L,蛋白胨10 g/L,氯化钠10 g/L,琼脂20 g/L, pH7.0。
组氨酸脱羧酶液体培养基:氨基酸10 g/L,蛋白胨5 g/L,酵母浸膏 3 g/L,葡萄糖1 g/L,16%溴甲酚紫乙醇溶液1mL/L,pH 6.8。
活化方法:霍氏肠杆菌RH3以OD600=0.8±0.27,1%的接种量接种于LB液体培养基培养至16-18 h。
实施例1
霍氏肠杆菌RH3的筛选及鉴定:
(1)霍氏肠杆菌RH3的筛选:
本发明的霍氏肠杆菌RH3,是从四川传统发酵香肠中经过筛选培养基分离得到。
发酵香肠制作于四川农业大学食品科学学院肉制品加工实验室。将猪瘦肉75.27%、猪肥膘18.82%、盐2.40%、白砂糖0.80%、辣椒粉0.94%、花椒粉0.38%、十三香0.1%、味精0.14%、葡萄糖0.09%、大蒜粉0.05%、黑胡椒0.04%、白酒0.94%、NaNO20.03%(均购于雅安市雨城区吉选超市)充分混合,并填充到猪肠衣中,然后进行为期28天的自然发酵。
霍氏肠杆菌RH3的初筛:用无菌剪刀剪取15 g发酵香肠于含有1%组氨酸的TSB肉汤中富集,37℃培养24 h。将富集液进行10倍梯度稀释,分别取1 mL培养液和各稀释梯度溶液,涂布于LB固体培养基平板上,37℃条件下培养24-72 h。经溴甲酚紫显色后,根据菌落形态特征挑取蓝色/紫色菌落/有紫色晕环的菌落,此为疑似产组胺菌,于LB固体培养基上划线传代培养,直至分离出单菌落,取单菌落于LB固体培养基斜面保种。
取保种后的霍氏肠杆菌连续活化两次后,以OD值为0.8±0.27 nm,1%的接种量接种于组氨酸脱羧酶液体培养基中,37℃恒温静置培养48±2 h,初步筛选疑似产组胺的肠细菌。
霍氏肠杆菌RH3的复筛:采用柱前丹磺酰氯衍生高效液相色谱法检测发酵液中组胺的含量,具体方法如下:
挑取分离纯化后的组胺产生菌株接种于LB液体培养基活化2次,以OD600 =0.8±0.27,1%的接种量接种于含有1%组氨酸的LB液体培养基中,静置培养48 h,进行衍生,衍生方法如下:
标准溶液配制与柱前衍生:准确称取组胺标品0.02 g,用0.4 mol/L的高氯酸定容至10 mL,分别吸取0.025、0.05、0.10、0.20、0.50、1.0、5.0 mL用0.4 mol/L的高氯酸定容至10 mL。各取1 mL依次加入200 μL 2mol/L的NaOH、300 μL饱和NaHCO3、2 mL10 mg/mL的丹磺酰氯丙酮溶液混匀,40℃暗反应45 min之后加入100 μL氨水,反应30 min后加入乙腈定容至5 mL,并用0.22 μm有机滤膜过滤,绘制组胺浓度标准曲线如图1所示。
采用高效液相色谱(Ultimate 3000,Thermo Fisher Scientific Inc.,MA,USA)测定组胺含量,色谱柱为C18柱(4.6 mm×250 mm,5 μm),流速为0.8 mL/min,紫外检测波长为254 nm,进样量10 μL,柱温30℃,流动相A为超纯水,流动相B为乙腈。洗脱程序为:0-5min:流动相A 35%,流动相B 65%;5-20 min:流动相A 30%,流动相B 70%;20-25 min:流动相A 0%,流动相B 100%;25-30 min:流动相A 35%,流动相B 65%。
(2)霍氏肠杆菌RH3的形态与生理生化鉴定:
将霍氏肠杆菌RH3按OD600=0.8±0.27,1%的接种量接种到10 mL LB液体培养基中37℃培养16-18 h后,活化两次后,挑取一环菌液接种至LB固体平板上,37℃培养20-24 h。观察RH3在LB固体平板上形成的菌落形态特征为圆型,边缘齐整,表面光滑湿润,菌落呈透明白色,形成的菌落大小为0.5-1.1mm,如图2所示。经格兰仕染色后发现该菌为革兰氏阴性菌,呈杆状,无芽孢,无荚膜,无鞭毛,兼性厌氧。
根据糖发酵实验显示RH3能够菌株能水解淀粉、葡萄糖、乳糖,不产色素,不产生H2S,不能利用乳糖、尿素、蛋白胨水,不产酸,能利用甘露醇、蔗糖、柠檬酸盐和丙二酸盐,组氨酸脱羧酶、赖氨酸脱羧酶、鸟氨酸脱羧酶、精氨酸脱羧酶阳性,VP阳性,MR阴性。
3、霍氏肠杆菌RH3的分子鉴定:
DNA的提取和引物序列:使用天根生化科技(北京)有限公司的细菌基因组DNA提取试剂盒(DP302)提取DNA,以提取的DNA为模板,使用引物27F-AGAGTTTGATCCTGGCTCAG(SEQID NO.2)和1492R-TACGGCTACCTTGTTACGACTT(SEQ ID NO.3)扩增该菌的16S rDNA基因。
扩增体系:12.5μL 2×Taq Master MIX、引物各1μL、1μL模板DNA和9.5 μL ddH2O。
扩增条件:95℃预变性5 min后,95℃变性60 s,56℃复性90 s,72℃延伸120 s,共29个循环,最后72℃延伸10 min,4℃保存。PCR扩增产物进行电泳分析,将符合预期的PCR产物送至上海生工生物工程股份有限公司测序,测序结果如下所示。将所得序列在NCBI数据库中进行分析,与Enterobacter hormaechei相似度为100%。因此鉴定RH3为霍氏肠杆菌(Enterobacter hormaechei)。并于2021年12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,保藏地址:北京市朝阳区北辰西路1号院3号,保藏编号CGMCCNO.1.19421。
16S rDNA基因测序结果如下:
>Enterobacter hormaecheiRH3序列 如SEQ ID NO.1所示。
4、RH3中hdc基因的特异性扩增:
Primer Premier 5.0软件设计了6对扩增hdc基因的引物。
扩增引物为引物1、引物2、引物3、引物4、引物5和引物6。其中:
引物1:hdc F1-TGGCTCACCGATTCCT(SEQ ID NO.4)/hdc R1-TCTGATGGGCAAGGGA(SEQ ID NO.5),
引物2:hdc F2-GGCTGCTATCTGGGTC(SEQ ID NO.6)/ hdc R2-GCAAGGAATCGGTGAG(SEQ ID NO.7),
引物3:hdc F3-TTGGCTGCTATCTGGG(SEQ ID NO.8)/hdc R3-GCAAGGAATCGGTGAG(SEQ ID NO.9),
引物4:hdc F4-ATTGCCATCATTCAGC(SEQ ID NO.10)/hdc R4-ATTGCCTCCCACATCA(SEQ ID NO.11),
引物5:hdc F5-AATCCACAGCCGTTTA(SEQ ID NO.12)/hdc R5-ATTGCCTCCCACATCA(SEQ ID NO.13),
引物6:hdc F6-GCGACAATGAAAAGCACCCC(SEQ ID NO.14)/hdc R6-TGACCGTTACGCGAACCTGA(SEQ ID NO.15)。
扩增体系为:10 μL rTaq DNA聚合酶,上下游引物各0.5μL,0.5μL模板DNA,8.5μLddH2O。
扩增程序为:95℃预变性5 min,94℃变性30 s,56℃复性30 s,72℃延伸2.5 min,72℃终延伸5 min,30个循环;4℃保存1 h。在1%琼脂糖凝胶电泳检测PCR产物,用凝胶成像仪成像。
扩增结果:如图3所示,M:Maker,A: hdc F1/R1,B: hdc F2/R2,C: hdc F3/R3,D:hdc F4/R4,E: hdc F5/R5,F: hdc F6/R6。
5、RH3中hdc表达量的检测:
样品的制备:在含有菌体的PBS缓冲液中加入RIPA裂解液后冰上裂解30 min,冰浴超声3 min,4℃ 12000 g离心10 min,取上清,调蛋白质浓度1 mg/mL,并加入等体积的1×SDS loading bufer,100℃煮沸10 min,-20℃保存。
Western Blot:分别配置10%的分离胶、5%的浓缩胶和含有0.3% Tris,1.44%甘氨酸和0.1%SDS的电极缓冲液(pH 8.3)。凝胶厚度为1.0 mm,上样体积为10 μL,上样浓度为5μg。将电压调节到80 V,开始电泳,30 min后当样品进入分离凝胶后调节到120 V,当样品距离分离凝胶下边缘1 mm时,停止电泳。将电泳结束后的凝胶取下,在转膜缓冲液中平衡10min,将PVDF膜在甲醇中饱和1 min,ddH2O处理2 min,然后在转膜缓冲液中浸泡10 min。在阴极板依次放上海绵、两层滤纸、凝胶、PVDF膜、滤纸以及海绵,阳极板覆盖在海绵上,固定好后置于电转槽中,200 mA电转1.5 h后,将电转好的PVDF膜取出用TBST清洗,用5% milk/TBST室温摇床封闭60 min后,用TBST漂洗2 min,添加一抗(1:500),4℃孵育过夜后,加入辣根过氧化物酶标记的二抗(1:5000),室温孵育60 min,用PBST洗涤3次,滴加显影液,显影图像采用Image J软件计算灰度值。RH3中不存在hdc条带,其灰度值为0.034,见图4。
6、RH3全基因组的检测:
将天根DNA提取试剂盒提取RH3的基因组,送至北京擎科生物科技有限公司检测菌株的全基因组序列。如图5所示,根据全基因组注释信息,在KEGG pathway数据库中得到RH3全基因组中与组氨酸相关的KEGG代谢通路图,RH3中hdc基因在其全基因组中并未被标注,表明RH3菌株中不存在hdc基因。
实施例2
本实施例提供一种菌剂,所述菌剂包括霍氏肠杆菌RH3以及所述霍氏肠杆菌RH3的代谢产物。
实施例3
本实施例提供霍氏肠杆菌RH3在产生组胺上的应用。霍氏肠杆菌RH3利用组氨酸产生组胺。
实施例4
本实施例提供霍氏肠杆菌RH3利用组氨酸产生组胺的方法,包括以下步骤:
将活化以后的霍氏肠杆菌RH3,以OD600=0.8±0.27,1%的接种量,接种至组氨酸含量为1%(w/v)的LB液体培养基中,在pH 7.0、37℃,静置条件下培养28 h,开始产生组胺。如图6所示,采用柱前丹磺酰氯衍生高效液相色谱法检测发酵液中组胺的含量,每隔2 h取一次样,分别测定菌株发酵过程中的OD600值以及产组胺的浓度,RH3在发酵28 h后产生0.0008 mg/mL组胺,在发酵64 h之后,产组胺浓度稳定在2.25 mg/mL。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (9)
1.一株霍氏肠杆菌,其特征在于:所述霍氏肠杆菌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.1.19421,保藏地址为北京市朝阳区北辰西路1号院3号,保藏日期为2021年12月21日。
2.根据权利要求1所述的霍氏肠杆菌,其特征在于,其16S rDNA基因序列如SEQ IDNO.1所示,并且,所述霍氏肠杆菌缺失hdc基因。
3.一种菌剂,其特征在于:包含权利要求1或2所述的霍氏肠杆菌。
4.根据权利要求3所述的一种菌剂,其特征在于:还包含所述霍氏肠杆菌的代谢产物。
5.权利要求3或4所述的菌剂在产组胺上的应用。
6.权利要求1或2所述的霍氏肠杆菌在产组胺上的应用。
7.根据权利要求6所述的霍氏肠杆菌在产组胺上的应用,其特征在于:所述霍氏肠杆菌利用组氨酸产生组胺。
8.根据权利要求7所述的霍氏肠杆菌在产组胺上的应用,其特征在于:所述霍氏肠杆菌利用组氨酸产生组胺,包括以下步骤:
将所述霍氏肠杆菌接种至含有组氨酸的LB液体培养基中,在pH 7.0、37℃条件下,静置培养28 h,开始产生组胺。
9.根据权利要求8所述的霍氏肠杆菌在产组胺上的应用,其特征在于:所述LB液体培养基中组氨酸含量为1%(w/v)。
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