CN116333922A - 一株来源于深海大鱿鱼的鼠李糖乳杆菌菌株及其应用 - Google Patents
一株来源于深海大鱿鱼的鼠李糖乳杆菌菌株及其应用 Download PDFInfo
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Abstract
本发明提供了一株来源深海大鱿鱼的鼠李糖乳杆菌(Lacticaseibacillus rhamnosus),所述鼠李糖乳杆菌菌株于2022年11月28日保藏于广东省微生物菌种保藏中心(GDMCC),保藏地址为广州,保藏编号为GDMCC No:63001。本发明还提供了一株鼠李糖乳杆菌在表达耐酸性和耐胆盐能力中的应用,本发明中的鼠李糖乳杆菌易培养,鼠李糖乳杆菌耐酸耐胆盐能力较强。
Description
技术领域
本发明涉及微生物技术领域,具体而言,涉及一株鼠李糖乳杆菌菌株及其应用。
背景技术
鼠李糖乳杆菌(Lacticaseibacillus rhamnosus),属异型发酵的无芽孢短杆菌,乳杆菌属,是产L-乳酸,不产芽孢,厌氧耐酸的革兰氏阳性共生菌。在显微镜下观察到的鼠李糖乳杆菌常以单链状或成链状存在,具有调节机体免疫、改善脂质代谢等功能。
益生发酵酸乳由于具备调节人体肠道菌群、缓解乳糖不耐症、降胆固醇以及延缓衰老等功效而深受消费者青睐。一般情况下,嗜热链球菌和保加利亚乳杆菌的混合菌株是酸奶发酵常用的主要菌种。发酵的过程中也可以添加双歧杆菌、乳酸链球菌、鼠李糖乳杆菌等菌种。由于对鼠李糖乳杆菌的研究不断加强,人们也发现了其对人体健康的有益功效。比如对人体的免疫调节,在食品行业形成了重要地位。由于鼠李糖乳杆菌具有耐酸、耐胆盐能力强的特点,也常常单独用于食品发酵中。本发明筛选的鼠李糖乳杆菌,并评价其生物学活性,以期为益生菌的开发和利用提供候选菌株。
发明内容
本发明要解决的技术问题之一是提供一株鼠李糖乳杆菌(Lacticaseibacillusrhamnosus),所述鼠李糖乳杆菌菌株于2022年11月28日保藏于广东省微生物菌种保藏中心(GDMCC),保藏地址为广州,保藏编号为GDMCC No:63001。
可选地,所述鼠李糖乳杆菌DNA序列为SEQ ID NO.1。
可选地,所述鼠李糖乳杆菌是从深海大鱿鱼的消化系统中分离得到的。
本发明要解决的技术问题之二是提供一株鼠李糖乳杆菌在表达中的应用。
与现有技术相比,本发明具有如下的有益效果:
1、本发明分离和鉴定了一株鼠李糖乳杆菌(Lacticaseibacillus rhamnosus),通过基因组序列分析和构建系统发育树,确定该菌株为鼠李糖乳杆菌。
2、与现有技术相比,本发明中的鼠李糖乳杆菌易培养,鼠李糖乳杆菌耐酸耐胆盐能力较高。
附图说明
图1为本发明实施例2中菌株S400M01的菌落图;
图2为本发明实施例2中菌株S400M01的镜检图;
图3为本发明实施例2中菌株S400M01的PCR扩增产物的琼脂糖凝胶电泳图;
图4为本发明实施例2中菌株S400M01的系统发育树。
图5为鼠李糖乳杆菌在pH2.5、pH3.5环境中的耐受性;
图6为鼠李糖乳杆菌在1.5g/L胆盐环境中的耐受性;
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明,但不以任何形式限制本发明。应当指出的是,对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。
实施例1鼠李糖乳杆菌(Lacticaseibacillus rhamnosus)菌株的获得。
1、培养基的配制
培养基(g/L):MRS培养基
2、菌株的筛选
将从深海捕捞到的大鱿鱼样本进行解剖分离,将分离的消化系统进行研磨,稀释后涂布于MRS平板培养基中,对应的平板分别编号,将平板置于37℃恒温培养箱厌氧培养24h,挑选长势较好的单菌落,将挑出的单菌落分别接到试管上中再次培养,进行保菌。挑选的菌落轮廓明显,颜色呈乳白色(如1.2)。
实施例2鼠李糖乳杆菌(Lacticaseibacillus rhamnosus)菌株的基因序列分析。
1.真菌DNA基因组提取
取实施例1得到的菌株培养液,离心后取沉淀,用CTAB法进行16SrDNA提取。
2.对该菌株进行16S rDNA的PCR扩增:
PCR扩增:引物515F和806R进行PCR扩增,515F序列为5′-GTGYCAGCMGCCGCGGTAA-3′,806R序列为5′-GGACTACNVGGGTWTCTAAT-3′。PCR反应体系为:TAQ酶25μL,515F引物2μL,806R引物2μL,DNA模板5μL,无菌水16μL。PCR扩增程序为:94℃预变性5min;94℃变性60s,48℃退火60s,72℃延伸60s,28个循环;72℃延伸7min,4℃终止反应。凝胶电泳及PCR产物胶回收:PCR产物经1.5%琼脂糖凝胶电泳鉴定。利用快捷型琼脂糖凝胶DNA回收试剂盒(北京百泰克生物技术有限公司)对目的PCR产物进行回收纯化,进行16SrDNA测序。PCR产物经1%琼脂糖凝胶电泳鉴定(如图3)。观察是否扩增出目的条带。
3.16S rDNA测序与同源性分析
利用快捷型琼脂糖凝胶DNA回收试剂盒(北京百泰克生物技术有限公司)对目的PCR产物进行回收纯化,将纯化后的PCR产物送至上海生工生物工程技术服务有限公司进行测序,基因序列如SEQ ID NO:1所示。将测得的序列提交美国NCBI的GENBANK获取相似序列,通过BLAST工具和DNAMAN软件进行对比分析并以Neighbor-Joining方法构建系统发育树,如图4所示。
通过系统发育树发现该菌株与Lacticaseibacillus rhamno序列相似高达99.69%,因此可确定为一株深海大鱿鱼来源的鼠李糖乳杆菌,我们将其命名为Lacticaseibacillus rhamnosus S400M01。
由上述筛选得到的鼠李糖乳杆菌菌株,命名为鼠李糖乳杆菌菌株(Lacticaseibacillus rhamnosus),菌株编号S400M01,其保藏日期为2022年11月28日,保藏于广东省微生物菌种保藏中心(GDMCC),保藏地址为广州,保藏编号为GDMCCNo:63001。
实施例3鼠李糖乳杆菌(Lacticaseibacillus rhamnosus)菌株表达的活性能力。
将发酵液于4 000r/min、4℃条件下离心3min,弃上清液,PBS洗涤菌体2次,以1%的量接种于pH值分别为2.5、3.5的MRS培养液中,37℃厌氧培养,分别于0、3、6h取样,用平板计数法进行活菌计数。如图5所示0h活菌数对数值均在6.5以上,3h后活菌数有所下降,但其活菌数对数值均在6.3以上,表明了短乳杆菌S400M01具有较好的耐酸性。
同样将鼠李糖乳杆菌接种于牛胆盐质量浓度为1.5g/L的MRS培养液中,分别于0、3、6h取样后进行活菌计数。如图6所示,筛选的菌株具有良好的耐胆盐能力,菌株在1.5g/L胆盐质量浓度条件下孵育6h仍维持活菌数对数值在6.0以上,为其在肠道进一步发挥益生作用提供了保证。
虽然本公开披露如上,但本公开的保护范围并非仅限于此。本领域技术人员,在不脱离本公开的精神和范围的前提下,可进行各种变更与修改,这些变更与修改均将落入本发明的保护范围。
序列
SEQ ID NO:1
TCGACGAGTTCTGATTATTGAAAGGGTGCTTGCATCTTGATTTAATTTTGGACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAAATCCAAAAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGGTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCGAAG
Claims (4)
1.一株鼠李糖乳杆菌(Lacticaseibacillus rhamnosus),其特征在于:所述鼠李糖乳杆菌菌株于2022年11月28日保藏于广东省微生物菌种保藏中心(GDMCC),保藏地址为广州,保藏编号为GDMCC No:63001。
2.根据权利要求1所述的一株鼠李糖乳杆菌,其特征在于:所述鼠李糖乳杆菌DNA序列为SEQ ID NO.1。
3.根据权利要求1所述的一株鼠李糖乳杆菌,其特征在于:所述鼠李糖乳杆菌是从深海大鱿鱼的消化系统中分离得到的。
4.如权利要求1-3任一所述的一株鼠李糖乳杆菌耐酸耐胆盐的能力。
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