CN116716203A - 一株具有高抗氧化性的植物乳杆菌sc75-2-2及其应用 - Google Patents
一株具有高抗氧化性的植物乳杆菌sc75-2-2及其应用 Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A—HUMAN NECESSITIES
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- C12R2001/25—Lactobacillus plantarum
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Abstract
本发明公开一株具有高抗氧化性的植物乳杆菌(Lactobacillus plantarum)SC75‑2‑2,该植物乳杆菌植物乳杆菌保藏于中国典型培养物保藏中心,保藏编号为CCTCC M 20221516,所述植物乳杆菌具有高耐酸耐胆盐、高抗氧化性、在肠道中定植的特性,该植物乳杆菌酸处理3 h其存活率为85.5%,耐胆盐处理3h其存活率为73%,DPPH的清除率为73.25%、羟基自由基的清除率为6.65%、超氧阴离子的清除率为35%,具有较强的抗氧化效果,本发明的植物乳杆菌粉在制备抗氧化和调节肠道菌群食品或保健品中有广泛的应用前景。
Description
技术领域
本发明涉及微生物领域,尤其涉及一株具有高抗氧化性的植物乳杆菌(Lactobacillus plantarum)SC75-2-2及其应用。
背景技术
正常情况下,人体拥有完整的氧化-抗氧化平衡机制,如果出现机体病变或者受外界环境毒害影响导致这种平衡发生改变,机体产生的活性氧及其他自由基不能被清除,就会对机体产生有害影响。而服用外援性抗氧化物质会引发其他各种疾病,如关节炎、癌症、高血压等,寻找天然与高性价比的食品抗氧化剂以保护细胞免受ROS的影响极其重要。
益生菌具有长期的安全性及潜在的健康益处,被认为是有效抗氧化剂的一个新兴来源,在食品中加入益生菌在体内赋予微生物平衡,特别是在胃肠道。近年来,从生物资源中寻找更安全的天然抗氧化剂取代合成抗氧化剂受到了广泛关注,而植物源性食品中含有较多抗氧化物质,茶叶是重要的抗氧化物质来源之一。但国内外对酸茶来源益生菌的抑菌特性研究较少,目前也有通过其它途径筛选出的具有抗氧化性质的菌株,但这些菌株的耐胃肠特性不佳,定植受限。
发明内容
针对上述现有技术中存在的不足,本发明提供一株具有高抗氧化活性、耐酸耐胆盐、可有效在肠道中定植的植物乳杆菌及其应用。
本发明采取的技术方案如下:
一株具有高抗氧化性的植物乳杆菌(Lactobacillus plantarum)SC75-2-2,所述植物乳杆菌已于2022年9月27日保藏于中国典型培养物保藏中心(CCTCC),保藏编号为:CCTCC M20221516,保藏地址为:湖北省武汉市武昌区八一路珞珈山。
进一步地,所述植物乳杆菌具有高耐酸耐胆盐、高抗氧化性、在肠道中定植的特性。
一株具有高抗氧化性的植物乳杆菌粉在制备抗氧化和调节肠道菌群食品或保健品中的应用本发明的有益效果如下:
本发明的植物乳杆菌SC75-2-2,是从云南省德昂族传统厌氧发酵酸茶样品中分离得到的50株乳酸菌中筛选得到的一株具有高抗氧化活性的植物乳杆菌,该植物乳杆菌SC75-2-2具有较强的耐酸耐胆盐特性、能够在肠道中定植的特性,实验结果表明,植物乳杆菌酸处理3h其存活率为85.5%,耐胆盐处理3h其存活率为73%;DPPH的清除率为73.25%、羟基自由基的清除率为6.65%、超氧阴离子的清除率为35%,具有较强的抗氧化效果,本发明的植物乳杆菌粉在制备抗氧化和调节肠道菌群食品或保健品中的应用有广泛的应用前景。
附图说明
图1为本发明的植物乳杆菌SC75-2-2在显微下的形态图;
图2为本发明的植物乳杆菌SC75-2-2***进化树;
图3为本发明实施例3植物乳杆菌DPPH自由基清除率图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
植物乳杆菌株SC75-2-2的分离纯化、鉴定及保藏
1、从云南省德昂族传统厌氧发酵酸茶样品中分离所述菌株。
MRS(de Man,Rogsa Sharpe)培养基:将10g蛋白陈、8g牛肉粉、4g酵母粉、20g葡萄糖、1mL吐温80、2g磷酸氢二钾、5g乙酸钠、2g柠檬酸三铵、0.2g硫酸镁、0.05g硫酸锰、mL1000mL蒸馏水混合,pH调整为6.2±0.2。
方法:采集云南省德昂族传统厌氧发酵酸茶样品:每个样品采集100g,用灭菌样品袋将采集的样品包装后,迅速移至低温盒中保存,并在两天内对样品进行分离纯化。
酸茶样品中乳酸菌的分离纯化与保存:在无菌条件下精确称取酸茶样品1.0g,置于5mL灭菌生理盐水中,然后对样品进行研磨,充分洗脱附着微生物,将20μL的洗脱液接种至5mL MRS肉汤培养基中,置35℃进行静止厌氧培养24h;随后用0.9%灭菌生理盐水进行梯度稀释,将20μL适宜稀释度的稀释液涂布于MRS改良琼脂培养基,置于35℃进行厌氧培养48~72h,反复涂布于MRS琼脂平板进行菌种分离纯化并保存。
2、形态学鉴定:将上述菌株进行过氧化氢酶试验、革兰染色与镜检鉴定,结果如图1所示,该菌SC75-2-2为革兰染色呈阳性菌的短杆菌;过氧化氢酶试验呈阴性;在MRS琼脂培养基平板上菌落呈乳白色、表面光滑、凸起明显、边缘整齐、不透明、有光泽。
3、分子生物学鉴定:采用改良CTAB法提取其总DNA,用细菌16S rRNA基因通用引物27F(5′-AGAGTTTGATC ATGGCTCAG-3′)和1492R(5′-TACG-GTTACCTTGTTACGACTT-3′)对所分离得到乳酸菌的16S rRNA基因进行扩增。PCR反应体系(50μL):上下游引物各1μL(浓度为10μmol/L),模板DNA2μL,5UTaq酶混合液(TaKaRa)25.0μL,超纯水21.0μL。PCR扩增程序:95℃预变性5min;95℃1min,60℃1min,72℃2min,循环35次;72℃延伸10min,4℃保温。PCR产物应用UNIQ-10柱式DNA胶回收试剂盒进行纯化,由生物工程公司进行核苷酸序列测定,测序结果见序列表;所得到的核苷酸序列通过BLAST在GenBank+PDB基因库中进行同源性比较,找到同源性最接近的已知种属的序列,用软件MEGA7.0进行序列分析,并采用Neighbor-Joining法进行***发育分析,构建进化树见图2,研究分离菌株与模式株之间的亲缘关系进行菌种鉴定。
序列分析:将该菌提取基因组DNA后进行16S rRNA扩增后送与生工生物工程(上海)股份有限公司进行测序,进行16S rRNA扩增后测序,使用MEGA 6.0软件结合测序结果,应用邻接法(Neighbor-Joining Method)构建***进化树,分析该菌株与模式株之间的亲缘关系如图2所示,本发明分离得到的乳酸菌SC75-2-2与植物乳杆菌的亲缘关系最近,其同源性达到100%,同属植物乳杆菌属,因此,鉴定该菌株为植物乳杆菌,命名为植物乳杆菌(Lactobacillus plantarum)SC75-2-2。
该菌株已于2022年9月27日保藏于中国典型培养物保藏中心(CCTCC,地址:湖北省武汉市武昌区八一路珞珈山17),分类命名为植物乳杆菌(Lactobacillus plantarum)SC75-2-2,保藏编号为:CCTCC M 20221516。
实施例2
植物乳杆菌耐酸、耐胆盐能力分析
1、耐酸试验
将1mL菌株培养液置于1.5mL的离心管中,8 000×g离心5min,将收集到的菌体用灭菌的培养基冲洗两次,然后将菌体重新悬浮于pH为3.0的MRS肉汤培养基中,最后置于35℃的CO2培养箱中厌氧培养,并于0h、3h分别取菌液1mL,用0.9%生理盐水进行10倍梯度稀释(10-1-10-7),然后取适当浓度涂布于MRS琼脂平板,35℃厌氧培养48-72h,培养结束后计数测定活菌数,计算其存活率。
菌株存活率计算公式
N 1――菌株处理后活菌数;
N 0――菌株初始活菌数。
将上述植物乳杆菌酸处理3h后统计的菌落数为1.3×109cfu,其存活率为85.5%。
2、耐胆盐试验
将1mL菌株培养液直接接种于含0.3%胆盐的MRS新鲜培养基中,置于35℃培养,并于培养0h,3h时分别取样,稀释后涂布于MRS琼脂平板,将涂布平板置于35℃的CO2培养箱中厌氧培养48-72h。待培养结束后计算MRS琼脂平板上的活菌数,计算其存活率。
菌株存活率计算公式
N 1――菌株处理后活菌数;
N 0――菌株初始活菌数。
将上述植物乳杆菌酸耐胆盐处理3h后,MRS琼脂平板上菌落数统计为3.5×108cfu,其存活率为73%。
实验结果表明,植物乳杆菌SC75-2-2具有较强的耐酸及耐胆盐特性。
实施例3
植物乳杆菌的抗氧化性能力分析
1、无细胞提取上清液的制备
当该菌株培养浓度达到109cfu/mL和1010cfu/mL时,在5000g下离心5min去除培养基。收集菌体,用生理盐水重悬沉淀;在冰浴中用细胞破碎仪在20kHz频率下破碎30min,再在10000g下离心5min得到无细胞提取上清液,即为待测样品;采用2,2-dipHenyl-1-picrylhydrazyl(DPPH)、超氧阴离子和羟基自由基清除率确定菌株的抗氧化能力。
2、清除DPPH自由基实验
将无细胞提取液与2mL DPPH甲醇溶液(0.5mM)混合,在室温下避光放置30min,DPPH与生理盐水作为对照组,样品与乙醇作为空白组。测量517nm处的吸光度值。
DPPH自由基清除率(%)=1-[A517(样品组)-A517(空白组)/A517(对照组)]×100%
样品组:样品+2mL DPPH溶液;空白组:样品+2mL乙醇溶液;对照组:2mL DPPH+生理盐水
测定结果如图3所示,酸茶中分离出的50株植物乳杆菌中菌株SC75-2-2(1010CFU/mL)的DPPH清除率为73.25%。清除率与Vc(0.8mg/mL)的清除率72.49%相近,由此可知植物乳杆菌SC75-2-2具有很强的清除DPPH自由基的能力,表现出其较强的抗氧化活性;对109CFU/mL和1010CFU/mL植物乳杆菌进行DPPH清除测试,菌株的清除率随着浓度的升高而升高。
3、清除羟基自由基实验
无细胞提取液与1mL水杨酸、1mL硫酸亚铁FeSO4和1mL过氧化氢混合在37℃放置15min.在510nm吸光度下检测:
羟基自由基清除率(%)=1-[(A1-A2)/A0]×100%
其中:A1为样品溶液(FeSO4+水杨酸+样品+过氧化氢),A2为样品溶液的空白对照(FesoSO4+水杨酸+样品+RO水),A0为对照溶液(FeSO4+水杨酸+生理盐水+过氧化氢)
结果为:羟基自由基的清除率为6.65%。
4、清除超氧阴离子自由基实验
无细胞提取上清液与pH为8.2的Tris(0.1M)和二乙酰胺五乙酸(2mM)混合液以及邻苯三酚(6mM)混匀。在室温放置10min。测量325nm处的吸光度值。
超氧阴离子自由基清除率(%)=1-[(A11-A10)/(A01-A00)]×100%
其中:A11为样品溶液(样品+Tris+二乙酰胺五乙酸+邻苯三酚),A10为样品溶液的空白对照(样品+Tris+二乙酰胺五乙酸+RO),A01为对照溶液(生理盐水+Tris+二乙酰胺五乙酸+邻苯三酚),A00为对照溶液的空白对照(生理盐水+Tris+二乙酰胺五乙酸+RO),结果为超氧阴离子的清除率为35%;
上述清除DPPH自由基实验、清除羟基自由基实验、清除超氧阴离子自由基实验结果表明:该植物乳杆菌具有耐酸耐胆盐和强抗氧化活性,可用于制备食品或保健品中具有抗衰老、抗氧化、以及调节肠道菌群的作用。
Claims (3)
1.一株具有高抗氧化性的植物乳杆菌(Lactobacillus plantarum)SC75-2-2,其特征在于,所述植物乳杆菌SC75-2-2保藏于中国典型培养物保藏中心,保藏编号为CCTCC M20221516。
2.如权利要求1所述的一株具有高抗氧化性的植物乳杆菌,其特征在于,所述植物乳杆菌具有高耐酸耐胆盐、高抗氧化性、在肠道中定植的特性。
3.如权利要求1或2所述的一株具有高抗氧化性的植物乳杆菌粉在制备抗氧化和调节肠道菌群食品或保健品中的应用。
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