CN115491323B - 一株沙福芽孢杆菌sc-11及其应用 - Google Patents
一株沙福芽孢杆菌sc-11及其应用 Download PDFInfo
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Abstract
本发明公开了一株沙福芽孢杆菌SC‑11，所述沙福芽孢杆菌(Bacillussafensis)SC‑11保藏号为CCTCC M 2022543，所述沙福芽孢杆菌SC‑11在合成绿原酸中的应用。本发明从华南忍冬叶片中分离筛选得到一株能够产生绿原酸的沙福芽孢杆菌SC‑11，产物通过超高效液相色谱筛选，质谱定性，确定为绿原酸，其分泌绿原酸的能力可达45.73mg/L，在农业、养殖业都具有良好的应用前景。
Description
技术领域
本发明属于微生物技术领域，具体涉及的是一株沙福芽孢杆菌SC-11及其应用。
背景技术
绿原酸(Chlorogenic acids,CGA)，又称3-O-咖啡酰奎尼酸(3-O-caffeoylquinicacid)。是由咖啡酸和奎宁酸缩合而成的缩酚酸，是经过苯丙氨酸反应途径从苯丙氨酸生物合成而来的一种苯丙素类物质。绿原酸广泛存在于药用植物如杜仲、金银花、忍冬等。咖啡豆也是较丰富的绿原酸资源。绿原酸具有抗菌、抗炎、抗氧化等生物活性，对高血压、高血糖、糖尿病等也有一定的预防和治疗作用。
目前对于绿原酸的提取主要来源于药用植物，但是由于绿原酸本身的不稳定性以及植物的生长周期过长、提取过程的繁杂以及开发其产品的漫长过程都需要较高的成本，为绿原酸的大规模工业应用带来了阻碍。而植物内生菌作为一种新型的微生物资源，具有与植物宿主合成相同或者相似活性成份的能力，这也为次生代谢产物的新来源提供了崭新的方向。目前，从植物中分离得到的菌株合成绿原酸的能力较低，因此利用微生物发酵方法、改良发酵工艺大规模发酵合成绿原酸有重要的意义。
在已报道和公开的文献和专利中，沙福芽孢杆菌多被发现具有解磷、固氮等植物促生能力，还可以通过去除有机物和抑制病原体生长改善水质，多被应用于微生物制剂及水产养殖中。但是未见报道能够分泌合成绿原酸。
发明内容
本发明提供一株能够合成绿原酸的菌株SC-11，通过优化发酵条件进一步提高合成绿原酸的产量。
为实现上述目的，本发明提供的技术方案如下：
一株沙福芽孢杆菌(Bacillus safensis)SC-11，于2022年05月03日保藏于中国典型培养物保藏中心，保藏地址：湖北省武汉市武昌区八一路299号中国典型培养物保藏中心，保藏号为CCTCC M 2022543。
如上所述沙福芽孢杆菌SC-11在合成绿原酸中的应用。
如上所述沙福芽孢杆菌SC-11在合成绿原酸中的应用，操作方法为：将所述沙福芽孢杆菌SC-11置于基础培养基中培养至对数期，以体积百分比1％的接种量置于优化发酵培养基中，装液量30mL，32℃、200rpm、pH为7的条件下培养5天。
优选的，所述优化发酵培养基组分为果糖55.12g/L、玉米浆25.31g/L、KH2PO45.05g/L、FeSO4 0.1g/L、MgSO4 0.05g/L和L-酪氨酸2g/L。
优选的，所述基础培养基组分为葡萄糖10g/L，酵母浸粉5g/L，胰蛋白胨10g/L，NaCl 10g/L。
与现有技术相比，本发明具有如下有益的技术效果：
本发明从华南忍冬叶片中分离筛选得到一株能够产生绿原酸的沙福芽孢杆菌SC-11，产物通过超高效液相色谱筛选，质谱定性，确定为绿原酸，其分泌绿原酸的能力可达45.73mg/L，在农业、养殖业都具有良好的应用前景。
保藏信息说明
沙福芽孢杆菌(Bacillus safensis)SC-11于2022年05月03日保藏于中国典型培养物保藏中心(简称CCTCC)，保藏号为CCTCC M 2022543。
附图说明
图1为绿原酸与沙福芽孢杆菌(Bacillus safensis)SC-11产物的液相色谱图。
图2为绿原酸与沙福芽孢杆菌(Bacillus safensis)SC-11产物的二级特征谱图。
图3为沙福芽孢杆菌(Bacillus safensis)SC-11在牛肉膏蛋白胨固体培养基上的菌落形态。
图4为本发明沙福芽孢杆菌(Bacillus safensis)SC-11的显微镜观察。
图5为本发明沙福芽孢杆菌(Bacillus safensis)SC-11的扫描电镜图。
图6为本发明沙福芽孢杆菌(Bacillus safensis)SC-11的电泳图。
具体实施方式
下面结合附图具体实施方式进行详细描述，但应当理解本发明的保护范围并不受具体实施方式的限制。实施例中采用的原料、试剂若无特殊说明，皆为市售所得。下述实施例中所用的实验方法如无特殊说明，均为常规方法。下述实施例中的定量实验，均设置三次重复实验，结果取平均值。
实施例中采用的培养基组分如下：
牛肉膏蛋白胨液体培养基(BE)：牛肉浸粉5g，蛋白胨10g，NaCl 5g，去离子水1L，pH调节至7.2-7.4；
牛肉膏蛋白胨固体培养基(BA)：牛肉浸粉5g，蛋白胨10g，NaCl 5g，琼脂20g，去离子水1L，pH调节至7.2-7.4。
实施例1
沙福芽孢杆菌SC-11的分离与合成绿原酸的鉴定
1、沙福芽孢杆菌SC-11的分离
1.1、取样：在广西壮族自治区广西药用植物园采集植物样本华南忍冬叶片，置于样品袋中，与4℃冰箱保存；
1.2、筛选：采用涂布平板法，取采集的植物叶片，浸泡在75％酒精中60s，用无菌水冲洗干净，浸泡在5％次氯酸钠溶液中表面消毒15s，消毒处理的样品用无菌剪刀剪切成小块置于研钵中，研磨后，吸取100μL，涂布至牛肉膏蛋白胨固体培养基平板上，于37℃恒温培养箱培养2d；挑取生长较好的多株菌株分别接种于牛肉膏蛋白胨液体培养基(BE)，发酵5d，75％酒精和发酵上清液1:1混合，浸提5h，浓缩至1mL，加2mL甲醇复溶，过0.22μm滤膜，制备样品溶液；在325nm下用超高效液相色谱UPLC检测；UPLC的参数为：色谱柱：Thermo FisherScientific Hypersil GOLD aQ 100×2.1mm 3μm，流动相A：乙腈，B：0.1％甲酸，条件为：0-2min，5％A，95％B；2-8min，95％A，5％B；8-10min，95％A，5％B；10-15min，5％A，95％B，进样量，1μL；
1.3、定性：在UPLC中与绿原酸标准品有相同出峰时间的样品，利用Thermo FisherQ-Exactive检测，利用电喷雾电离源(ESI)的负离子[MH]-检测模式，绿原酸标准品在m/z353.0867中被检测，去质子化物质在m/z191.0552处碎裂，与绿原酸标品比对，发现一株命名为SC-11的菌株具有产绿原酸的能力(图1-2)；
1.4、定量：上述样品溶液利用高效液相色谱HPLC定量，HPLC的参数为：Waters色谱系统，色谱柱(JADE-PAK ODS-AQ C18柱；250mm×4.6mm，5μm；Echway corporation，Guangzhou，China)，流动相0.5％乙酸水(A)和乙腈(B)；梯度洗脱，条件为：0min，95％A和5％B；5min，92％A和8％B；15min，50％A和50％B；20min，10％A和90％B；21-25min，95％A和5％B；柱温35℃；流速，1mL/min；进样量，10μL；检测波长327nm。
2、菌株SC-11的鉴定
2.1、将得到的菌株SC-11进行一系列生理生化鉴定，并提取DNA进行16SrDNA的扩增和测序，所得结果如下：
该菌株在牛肉膏蛋白胨固体培养基上形成边缘褶皱的乳白色菌落(图3)；
显微镜镜检显示短杆状、革兰氏阳性细菌(图4)；
扫描电镜观测菌体成短圆杆状，无鞭毛，菌体大小为(1.8-2.0)μm，(0.8-0.9)μm(图5)；
其他检测项目如表1所示：
表1菌株SC-11的生理生化特征
注：“+”表示阳性反应，存在或有，“-”表示阴性反应，不存在或无。
2.2、利用引物27F和1492R进行16SrDNA扩增，引物序列如下：
27F:5′-AGAGTTTGATCATGGCTCAG-3′
1492R：5′-TAGGGTTACCTTGTTACGACTT-3′
PCR扩增条件为95℃2min，95℃30s，55℃30s，72℃1min，35cycles，72℃2min，4℃保存；
对PCR产物进行1％的琼脂糖凝胶电泳验证(电泳图如图6所示)然后送公司测序，测序结果如SEQ.ID.NO.1所示，菌株SC-11的16S rDNA的序列长度为1438bp，经同源比对，其与Bacillus safensis strain MXR1709B01(GeneBank accession no MN17650.1)具有99％的高度相似性，结合菌株SC-11的菌株形态学和生理生化特征，鉴定该菌株为Bacillussafensis菌属的一个菌株，命名为沙福芽孢杆菌(Bacillus safensis)SC-11，将其保藏于中国典型培养物保藏中心(CCTCC)，保藏号为CCTCC M 2022543。
实施例2
沙福芽孢杆菌SC-11在合成绿原酸中的应用
绿原酸产量的检测方法与实施例1中1.4的方法一致。
将沙福芽孢杆菌SC-11置于基础培养基(基础培养基组分为葡萄糖10g/L，酵母浸粉5g/L，胰蛋白胨10g/L，NaCl 10g/L)中培养至对数期，以体积百分比1％的接种量置于优化发酵培养基(优化发酵培养基组分为果糖55.12g/L、玉米浆25.31g/L、KH2PO4 5.05g/L、FeSO4 0.1g/L、MgSO4 0.05g/L和L-酪氨酸2g/L)中，装瓶量30mL，32℃、200rpm、pH为7的条件下培养5天，测定绿原酸产量，菌株分泌绿原酸的能力为45.73mg/L。
前述对本发明的具体示例性实施方案的描述是为了说明和例证的目的。这些描述并非想将本发明限定为所公开的精确形式，并且很显然，根据上述教导，可以进行很多改变和变化。对示例性实施例进行选择和描述的目的在于解释本发明的特定原理及其实际应用，从而使得本领域的技术人员能够实现并利用本发明的各种不同的示例性实施方案以及各种不同的选择和改变。本发明的范围意在由权利要求书及其等同形式所限定。
序列表
<110> 广西大学
<120> 一株沙福芽孢杆菌SC-11及其应用
<130> sbyx
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1438
<212> DNA
<213> Bacillus safensis(沙福芽孢杆菌)
<400> 1
ggcgggtgct atacatgcag tcgagcggac agaagggagc ttgctcccgg atgttagcgg 60
cggacgggtg agtaacacgt gggtaacctg cctgtaagac tgggataact ccgggaaacc 120
ggagctaata ccggatagtt ccttgaaccg catggttcaa ggatgaaaga cggtttcggc 180
tgtcacttac agatggaccc gcggcgcatt agctagttgg tggggtaatg gctcaccaag 240
gcgacgatgc gtagccgacc tgagagggtg atcggccaca ctgggactga gacacggccc 300
agactcctac gggaggcagc agtagggaat cttccgcaat ggacgaaagt ctgacggagc 360
aacgccgcgt gagtgatgaa ggttttcgga tcgtaaagct ctgttgttag ggaagaacaa 420
gtgcgagagt aactgctcgc accttgacgg tacctaacca gaaagccacg gctaactacg 480
tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggaattatt gggcgtaaag 540
ggctcgcagg cggtttctta agtctgatgt gaaagccccc ggctcaaccg gggagggtca 600
ttggaaactg ggaaacttga gtgcagaaga ggagagtgga attccacgtg tagcggtgaa 660
atgcgtagag atgtggagga acaccagtgg cgaaggcgac tctctggtct gtaactgacg 720
ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc cacgccgtaa 780
acgatgagtg ctaagtgtta gggggtttcc gccccttagt gctgcagcta acgcattaag 840
cactccgcct ggggagtacg gtcgcaagac tgaaactcaa aggaattgac gggggcccgc 900
acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac caggtcttga 960
catcctctga caaccctaga gatagggctt tcccttcggg gacagagtga caggtggtgc 1020
atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc 1080
ttgatcttag ttgccagcat tcagttgggc actctaaggt gactgccggt gacaaaccgg 1140
aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct 1200
acaatggaca gaacaaaggg ctgcaagacc gcaaggttta gccaatccca taaatctgtt 1260
ctcagttcgg atcgcagtct gcaactcgac tgcgtgaagc tggaatcgct agtaatcgcg 1320
gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccacg 1380
agagtttgca acacccgaag tcggtgaggt aacctttatg gagccagccg ccgaaggt 1438
Claims (5)
1.一株沙福芽孢杆菌(Bacillus safensis)SC-11，其特征在于：所述沙福芽孢杆菌SC-11保藏号为CCTCC M 2022543。
2.一株如权利要求1所述沙福芽孢杆菌SC-11在合成绿原酸中的应用。
3.根据权利要求2所述沙福芽孢杆菌SC-11在合成绿原酸中的应用，其特征在于，所述应用操作方法为：将所述沙福芽孢杆菌SC-11置于基础培养基中培养至对数期，以体积百分比1％的接种量置于优化发酵培养基中，装液量30mL，32℃、200rpm、pH为7的条件下培养5天。
4.根据权利要求3所述所述沙福芽孢杆菌SC-11在合成绿原酸中的应用，其特征在于：优化发酵培养基组分为果糖55.12g/L、玉米浆25.31g/L、KH2PO4 5.05g/L、FeSO4 0.1g/L、MgSO4 0.05g/L和L-酪氨酸2g/L。
5.根据权利要求3所述所述沙福芽孢杆菌SC-11在合成绿原酸中的应用，其特征在于：所述基础培养基组分为葡萄糖10g/L，酵母浸粉5g/L，胰蛋白胨10g/L，NaCl 10g/L。
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