CN114381390A - 长双歧杆菌me-875及其应用 - Google Patents
长双歧杆菌me-875及其应用 Download PDFInfo
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Abstract
本发明提供长双歧杆菌ME‑875及其应用。该菌株可有效促进细胞生长,并可将所述长双歧杆菌应用于与细胞生长障碍有关的疾病,以此改善和调节肠道微生物菌群,进而用于预防或治疗相关病症。
Description
技术领域
本发明涉及微生物技术领域,更具体地,涉及一种长双歧杆菌ME-875及其应用。
背景技术
双歧杆菌属(Bifidobacterium)广泛存在于人和动物的消化道、阴道和口腔等生境中,双歧杆菌属的细菌是人和动物肠道菌群的重要组成成员之一。人体肠道中定植着大量的微生物,肠道微生物与人体健康与疾病之间存在着十分密切的关系,其中双歧杆菌是一种重要的肠道有益微生物,对人体健康具有生物屏障、营养作用、抗肿瘤作用、免疫增强作用、改善胃肠道功能、抗衰老等多种重要的生理功能。
长双歧杆菌(Bifidobacterium longum,BL)是益生菌的一种,属于双歧杆菌,其具有调节人体肠道健康的功效。长双歧杆菌是一种获取自健康人体肠道,经筛选出的适合人体食用的双歧杆菌。
现有技术表明,肠道微生物菌群与多种疾病相关,肠道微生物群主要是通过免疫系统介导,产生活化的免疫因子,进而调控各种疾病的发生和进程。然而,长双歧杆菌作为一种肠道益生菌,其在细胞生长调控方面还没有相关研究。
发明内容
针对现有技术所存在的技术问题,本发明要解决的技术问题是提供一种可有效促进细胞生长的长双歧杆菌,并将所述长双歧杆菌应用于与细胞生长障碍有关的疾病,以此改善和调节肠道微生物菌群,进而用于预防或治疗相关病症。
针对此,本发明首先提供一种长双歧杆菌ME-875,该菌株已于2021年11月15日在中国微生物菌种保藏管理委员会普通微生物中心保藏,其保藏编号为CGMCC No.23903,分类命名为长双歧杆菌Bifidobacterium longum,保藏地址为:中国,北京市朝阳区,邮政编码:100101;所述菌株的检测结果为存活。
本发明提供的长双歧杆菌ME-875能够用于有效地促进细胞的生长率,可应用于与细胞生长障碍有关的疾病。
进一步地,本发明的另一个目的在于提供长双歧杆菌ME-875在制备提高细胞生长率、预防或治疗与细胞生长障碍有关的疾病的产品中应用。
优选地,所述产品包括固体或液体发酵剂、益生菌固体饮料、食品、药品、功能性保健食品等。
优选地,在上述应用中,所述长双歧杆菌ME-875的活菌数量≥1×106CFU/g。
本申请的另一目的在于提供一种用于提高细胞生长率、预防或治疗与细胞生长障碍有关的疾病的产品,所述产品含有所述的长双歧杆菌ME-875,所述产品包括固体或液体发酵剂、益生菌固体饮料、食品、药品、功能性保健食品。
优选地,所述产品中所述长双歧杆菌ME-875的活菌数量≥1×106CFU/g。
优选地,所述产品,还包括药物载体和/或药用辅料。
进一步地,所述药用辅料可选自抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、润湿剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏合剂、整合剂、渗透促进剂、pH值调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂与反絮凝剂、助滤剂以及释放阻滞剂中至少一种。
优选地,所述产品的剂型可选自颗粒剂、胶囊剂、片剂、丸剂或口服液。
本发明的有益效果是:本申请提供一种长双歧杆菌ME-875可有效促进细胞生长,并可将所述长双歧杆菌应用于与细胞生长障碍有关的疾病,以此改善和调节肠道微生物菌群,进而用于预防或治疗相关病症。
具体实施方式
下面结合具体实施例对本发明作进一步的详细说明,以使本领域的技术人员更加清楚地理解本发明。
以下各实施例,仅用于说明本发明,并不用来限制本发明的范围。基于本发明中的具体实施例,本领域普通技术人员在没有做出创造性劳动的情况下,所获得的其他所有实施例,都属于本发明的保护范围。
在本发明实施例中,若无特殊说明,所有原料组分均为本领域技术人员熟知的市售产品;在本发明实施例中,若未具体指明,所用的技术手段均为本领域技术人员所熟知的常规手段。
实施例1长双歧杆菌ME-875的获取
1.1长双歧杆菌ME-875的分离和鉴定
16S rDNA鉴定:取5g取自健康人体的粪便样品,加入到50mL无菌水中,震荡混匀后梯度稀释,在MRS细菌培养基(由de Man,Rogosa and Sharpe三位发明人名字首字母命名)+莫匹罗星锂盐的选择性培养基平板涂布,置于37℃,厌氧过夜培养,挑取单菌落纯化,并提取基因组DNA,采用如下通用引物:
27:5'-AGAGTTTGATCCTGGCTCAG-3(SEQ ID NO:2)
1492R:5'-TACGGCTACCTTGTTACGACTT-3'(SEQ ID NO:3);
进行16S rDNA片段的PCR扩增,扩增片段提交测序公司进行测序,测序结果如SEQID NO:1所示,并将测序结果进行NCBI Blast比对分析。
经比对分析鉴定,获得本发明的长双歧杆菌ME-875,该菌株已于2021年11月15日在中国微生物菌种保藏管理委员会普通微生物中心保藏,其保藏编号为CGMCC No.23903,分类命名为长双歧杆菌Bifidobacteriumlongum,保藏地址为:中国,北京市朝阳区,邮政编码:100101;所述菌株的检测结果为存活。
优选地,所述健康人体选自25岁左右健康成人;优选地,所述选择性培养基中添加有终浓度为2.5mg/L的莫匹罗星锂盐。
1.2长双歧杆菌ME-875的培养和保存
所述菌株的培养条件、检测存活条件(培养基成分、pH、温度等)为:均于MRS细菌培养基中,pH 6.2-6.6,在37℃恒温培养箱静置过夜培养。且所述培养物不会危及人类健康或动、植物病原或污染环境。
优选地,所述菌株的建议的长期保藏方法:超低温冻结保存。
1.3长双歧杆菌ME-875的的毒力实验及安全性检测
将本发明的长双歧杆菌ME-875接种于MRS细菌培养基中,37℃恒温培养箱静置过夜培养,pH 6.2-6.6,计数培养液中长双歧杆菌ME-875活菌数为3.7×108cfu/mL,将培养物的原液和5倍浓缩液,经口以20.0mL/kg BW给受试小鼠连续灌胃3天,观察7天。试验设培养基原液和5倍浓缩液对照组。试验结果表明:长双歧杆菌ME-875的MRS培养物原液和5倍浓缩液组与各自对照组相比,对小鼠体重增长的影响无统计学意义(p>0.05),同时未观察到受试小鼠有毒性反应或死亡。
采用SN/T 1944-2007《动物及其制品中细菌耐药性的测定》方法,评估长双歧杆菌ME-875的抗生素敏感性能。评价结果显示,长双歧杆菌ME-875对氨苄西林Ampicillin、青霉素G PenicillinG、红霉素Erythromycin、氯霉素Chloramphenicol、克林霉素Clindamycin、万古霉素Vancomycin和四环素Tetracycline等敏感。符合欧洲食品安全委员会(EuropeanFood Safety Authority)对食用细菌耐药性评价规范中的要求。试验结果表明:长双歧杆菌ME-875不含外源抗生素耐药基因,食用安全。
实施例2长双歧杆菌ME-875的性能测试
2.1对骨细胞C3H10T1/2增殖的影响
菌株上清液的制备:
实验组:将本发明的长双歧杆菌ME-875接种于MRS细菌培养基中,37℃恒温培养箱静置过夜培养,pH 6.2-6.6,计数培养液中长双歧杆菌ME-875活菌数为3.7×108cfu/mL。
对照组:采用实施例1方法分离的其他长双歧杆菌(cb9Y4、cb10Y5、cb24Y5、cb26Y1);
骨细胞C3H10T1/2的培养:采用含有10%胎牛血清、100U/mL青霉素和100mg/L链霉素的DMEM培养基培养骨细胞C3H10T1/2,置于37℃、5%CO2条件下过夜培养。
灭活菌株上清液刺激:分设CK组、空白组、实验组和对照组,并取上述骨细胞C3H10T1/2接种于96孔板,每孔5×103个;实验组和对照组是通过收集上述长双歧杆菌活菌,并于2.0%甲醛处理进行灭活,离心收集灭活菌株上清液;分取100μL灭活菌株上清液逐次加入96孔板中进行刺激处理,处理时间为1h;CK组是不经任何刺激;空白组是采用与实验组相同体积的2.0%甲醛进行刺激。
MTT法检测细胞生长:刺激结束后,每孔加入20μL 5mg/mL MTT溶液,4h后弃培养基,每孔加入DMSO 100μL,置摇床上低速振荡10min,使结晶物充分溶解。在酶联免疫检测仪490nm处测值,并以此计算细胞的生长率。
结果如下表1所示:
表1对骨细胞C3H10T1/2增殖的影响
结果分析:相比于其他分离的长双歧杆菌而言,本发明的长双歧杆菌ME-875可显著性提高骨细胞C3H10T1/2的生长率。
2.2对胚胎干细胞CE3增殖的影响
具体方法步骤参见2.1节所述,其中所述胚胎干细胞CE3是在mES完全培养液中,置于37℃、5%CO2条件下过夜培养。所述对照组为:cb10Y2、cb20Y2、cb39Y4、cb40Y1。
结果如下表2所示:
表2.对胚胎干细胞CE3的影响
结果分析:相比于其他分离的长双歧杆菌而言,本发明的长双歧杆菌ME-875可显著性提高胚胎干细胞CE3的生长率。
在此有必要指出的是,以上实施例仅限于对本发明的技术方案做进一步的阐述和说明,并不是对本发明的技术方案的进一步的限制,本发明的方法仅为较佳的实施方案,并非用于限定本发明的保护范围。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 美益添生物医药(武汉)有限公司
<120> 长双歧杆菌ME-875及其应用
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<212> DNA
<213> 人工序列(artificial sequence)
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gttgctgatt cgcgattact agcgactccg ccttcacgca gtcgagttgc agactgcgat 180
ccgaactgag accggttttc agggatccgc tccgcgtcgc cgcgtcgcat cccgttgtac 240
cggccattgt aggatgcgtg aagccctgga cgtaaggggc atgatgatct gacgtcatcc 300
ccaccttcct ccgagttaac cccggcggtc ccccgtgagt tcccggataa tccgctggca 360
acacggggcg agggttgcgc tcgttgcggg acttaaccca acatctcacg acacgagctg 420
acgacgacca tgcaccacct gtgaacccgc cccgaaggga agccgtatct ctacgaccgt 480
cgggaacatg tcaagcccag gtaaggttct tcgcgttgca tcgaattaat ccgcatgctc 540
cgccgcttgt gcggcccccg tcaatttctt tgagttttag ccttgcggcc gtactcccca 600
ggcggatgct taacgcgtta gctccgacac ggaacccgtg gaacgggccc cacatccagc 660
atccaccgtt tacggcgtgg actaccaggg tatctaatcc tgttcgctcc cacgctttcg 720
ctcctcagcg tcagtaacgg ccaagactgc cttcgccatt gtgtcttccg attctactca 780
ttccaccgtt cgcggaattc agctccctac gcatcagccg ccgtaccggg cgatcacgta 840
agcgatg 847
<210> 2
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
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<212> DNA
<213> 人工序列(artificial sequence)
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tacggctacc ttgttacgac tt 22
Claims (10)
1.一种长双歧杆菌ME-875,其特征在于,所述长双歧杆菌的保藏编号为CGMCCNo.23903。
2.如权利要求1所述的长双歧杆菌ME-875,其特征在于,所述长双歧杆菌的16S rRNA序列如SEQ ID NO.1所示。
3.权利要求1或2所述的长双歧杆菌ME-875在制备提高细胞生长率、预防或治疗与细胞生长障碍有关的疾病的产品中的应用。
4.如权利要求3所述的应用,其特征在于,所述产品包括固体或液体发酵剂、益生菌固体饮料、食品、药品、功能性保健食品。
5.如权利要求3或4所示的应用,其特征在于,所述的活菌数量≥1×106CFU/g。
6.一种用于提高细胞生长率、预防或治疗与细胞生长障碍有关的疾病的产品,其特征在于,所述产品包含权利要求1-5任一项所述的长双歧杆菌ME-875。
7.如权利要求6所述的产品,其特征在于,所述产品包括固体或液体发酵剂、益生菌固体饮料、食品、药品、功能性保健食品。
8.如权利要求6或7所示的产品,其特征在于,所述的活菌数量≥1×106CFU/g。
9.如权利要求6或7所示的产品,其特征在于,所述产品还包括药用辅料。
10.如权利要求9所述的产品,其特征在于,所述药用辅料可选自抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、润湿剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏合剂、整合剂、渗透促进剂、pH值调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂与反絮凝剂、助滤剂以及释放阻滞剂中的至少一种。
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