CN116218735A - 一种单形拟杆菌菌株及其培养方法和应用 - Google Patents
一种单形拟杆菌菌株及其培养方法和应用 Download PDFInfo
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Abstract
本发明属于肠道微生物技术领域,尤其涉及一种单形拟杆菌菌株F18‑22及其培养方法和在制备治疗炎症性肠病药物中的应用。所述单形拟杆菌菌株为单形拟杆菌菌株F18‑22(Bacteroide uniformis F18‑22),该菌株分离于健康人体肠道并保藏于中国典型微生物保藏中心,保藏编号为CCTCC NO:M 20221994。该菌株能够避免结直肠黏膜的完整性受到破坏,并对已有的肠道粘膜损伤进行修复,进而改善便稀和便血情况,缓解因结直肠损伤造成体重快速下降,最终实现对于炎症性肠病的治疗。
Description
技术领域
本发明属于肠道微生物技术领域,尤其涉及一种单形拟杆菌菌株F18-22及其培养方法和在制备治疗炎症性肠病药物中的应用。
背景技术
炎症性肠病(IBD)包括溃疡性结肠炎(UC)和克罗恩病(CD)。目前,全球范围内约有680万人受到炎症性肠病的影响(PMID:31648971)。炎症性肠病的发生发展与宿主的遗传背景、生存环境、饮食营养、免疫失衡和肠道菌群紊乱密切相关(PMID:26907531)。研究表明,肠道菌群紊乱可以导致炎症性肠病,因此,药物可以通过靶向肠道菌群,改善肠道菌群紊乱达到治疗炎症性肠病的目的(PMID:32076145)。
拟杆菌是人体肠道重要的共生菌,目前,已报道的从人体肠道中分离得到的拟杆菌有34种(PMID:30716990)。研究表明,多形拟杆菌(Bacteroides thetaiotaomicron)、卵形拟杆菌(Bacteroides ovatus)、普通拟杆菌(Bacteroides vulgatus)和解木聚糖拟杆菌(Bacteroides xylanisolvens)均具有缓解溃疡性结肠炎的功能(PMID:30215718;中国发明专利CN202210548538.0)。单形拟杆菌(Bacteroides uniformis)是Eggerth AH等人1933年首次从人体粪便样品中分离得到的拟杆菌属新物种,是一种严格厌氧的革兰氏阴性菌(PMID:16559622)。研究表明,口服单形拟杆菌具有良好的安全性并且其能够通过调节机体的代谢功能治疗肥胖(PMID:33499721;PMID:26784747)。此外,最新研究发现,单形拟杆菌还能够通过调控“脑-肠轴”缓解抑郁症(PMID:35143877)。作为“新一代益生菌”,单形拟杆菌在治疗代谢性疾病及神经系统疾病方面具有良好的开发应用前景。然而,目前尚未有关于单形拟杆菌抗溃疡性结肠炎的报道。
发明内容
本发明的提供一种单形拟杆菌菌株及其培养方法和应用,具体为单形拟杆菌菌株F18-22(Bacteroide uniformis F18-22)在制备治疗炎症性肠病药物中的应用。
本发明的技术方案是:
一种单形拟杆菌菌株,所述单形拟杆菌菌株为单形拟杆菌菌株F18-22(Bacteroide uniformis F18-22),该菌株分离于健康人体肠道,该菌株保藏于中国典型微生物保藏中心,保藏编号为CCTCC NO:M 20221994。
上述的单形拟杆菌菌株的培养方法,包括以下步骤:
1)将所述单形拟杆菌菌株F18-22活化培养获得种子液;
2)将步骤1)所述种子液接种至发酵培养基中进行厌氧发酵培养40~60h。
优选的,上述的单形拟杆菌菌株的培养方法,所述发酵培养基,以水为溶剂,包括以下浓度的组分:褐藻寡糖6~10g/L、胰蛋白胨2~4g/L、蛋白胨2~4g/L、酵母提取物2~4g/L、粘液素0.4~0.6g/L、3号胆盐0.3~0.5g/L、L-半胱氨酸盐酸盐0.7~0.9g/L、血红素0.04~0.06g/L、吐温80 0.8~1.2ml/L、氯化钠4~5g/L、氯化钾2~3g/L、氯化镁4~5g/L、氯化钙0.1~0.3g/L、磷酸二氢钾0.3~0.5g/L和微量元素1~3ml/L,所述发酵培养基的pH值为6.4~6.5。
优选的,上述的单形拟杆菌菌株的培养方法,所述褐藻寡糖的分子量为2.0kDa。
优选的,上述的单形拟杆菌菌株的培养方法,步骤2)中所述种子液的接种量为2%~10%,v/v。
一种单形拟杆菌菌株在制备治疗炎症性肠病药物中的应用。
优选的,上述的一种单形拟杆菌菌株在制备治疗炎症性肠病药物中的应用,所述炎症性肠病为由葡聚糖硫酸钠诱导产生。
一种治疗炎症性肠病的药物,所述药物包含单形拟杆菌菌株F18-22。
优选的,上述的一种治疗炎症性肠病的药物,所述治疗炎症性肠病药物剂型为液体制剂,所述药物中单形拟杆菌菌株F18-22的活菌浓度为(1~9)×108CFU/mL。
一种单形拟杆菌菌株在制备食品中的应用。
本发明的优点及有益效果:
本发明提供的单形拟杆菌菌株F18-22(Bacteroide uniformis F18-22),该菌株能够避免结直肠黏膜的完整性受到破坏,并对已有的肠道粘膜损伤进行修复,进而改善便稀和便血情况,缓解因结直肠损伤造成体重快速下降,最终实现对于炎症性肠病的治疗。
附图说明
图1为小鼠体重变化率,
其中,NC:自然生长对照组;MD:葡聚糖硫酸钠(DSS)喂养疾病模型组;BU:单形拟杆菌F18-22灌胃治疗组,*表示与NC组相比有显著差异(P<0.05);**表示与NC组相比有极显著差异(P<0.01);#表示与MD组相比有显著差异(P<0.05);##表示与MD组相比有极显著差异(P<0.01);运用Student t-test进行统计学检验,数据展示方式为Mean±SEM;
图2为各组小鼠结直肠长度对比图,
其中,*表示与NC组相比有显著差异(P<0.05);**表示与NC组相比有极显著差异(P<0.01);#表示与MD组相比有显著差异(P<0.05);##表示与MD组相比有极显著差异(P<0.01)。运用Student t-test进行统计学检验,数据展示方式为Mean±SEM;
图3为各组小鼠结直肠组织形态对比图;
图4为各组小鼠疾病活动指数(Disease activity index,DAI)对比图,
其中,Total:总评分;Stool consistence:粪便粘稠度评分;Occult/grossbleeding:隐匿性或总出血量评分;Body weight:体重评分;*表示与NC组相比有显著差异(P<0.05);**表示与NC组相比有极显著差异(P<0.01);#表示与MD组相比有显著差异(P<0.05);##表示与MD组相比有极显著差异(P<0.01)。按照文献(PMID:34973740)方法进行评分,运用Student t-test进行统计学检验,数据展示方式为Mean±SEM;
图5为各组小鼠结直肠HE染色图,
其中,bar=100μm;
图6为各组小鼠结直肠组织损伤评分图,
其中,*表示与NC组相比有显著差异(P<0.05);**表示与NC组相比有极显著差异(P<0.01);#表示与MD组相比有显著差异(P<0.05);##表示与MD组相比有极显著差异(P<0.01)。按照文献(PMID:34973740)方法进行评分,运用Student t-test进行统计学检验,数据展示方式为Mean±SEM。
具体实施方式
生物保藏说明:
本发明提供的单形拟杆菌菌株F18-22,保藏编号:CCTCC NO:M 20221994;分类命名:拟杆菌属,单形拟杆菌种;拉丁文学名:Bacteroide uniformis F18-22;该菌株保藏于中国典型微生物保藏中心,保藏日期为2022年12月19日,保藏地址为湖北省武汉市武昌区八一路299号武汉大学校内,中国典型培养物保藏中心。
本发明中单形拟杆菌菌株F18-22源于人体肠道,16S rDNA的序列如序列表所示。
单形拟杆菌菌株F18-22的培养方法,包括以下步骤:
1)将所述单形拟杆菌菌株F18-22活化培养获得种子液;
2)将所述种子液接种至发酵培养基中进行厌氧发酵培养40~60h。
在本发明中,将所述单形拟杆菌菌株F18-22活化培养获得种子液;本发明对所述活化培养的方法没有特殊限定,采用本领域常规的活化方法即可。在本发明中,所述种子液为对数生长期的菌液。本发明在获得所述种子液后,将所述种子液接种至发酵培养基中进行厌氧发酵培养40~60h。在本发明中,所述厌氧发酵培养优选的在厌氧培养箱中进行,所述种子液的接种量优选为2%~10%(v/v),进一步优选为4%~6%,更进一步优选为5%。在本发明中,所述厌氧发酵培养的温度优选为36~38℃,进一步优选为37℃;所述厌氧发酵培养的时间优选为45~55h,进一步优选为48h。
在本发明中,所述发酵培养基,以水为溶剂,优选的包括以下浓度的组分:褐藻寡糖6~10g/L、胰蛋白胨2~4g/L、蛋白胨2~4g/L、酵母提取物2~4g/L、粘液素0.4~0.6g/L、3号胆盐0.3~0.5g/L、L-半胱氨酸盐酸盐0.7~0.9g/L、血红素0.04~0.06g/L、吐温80 0.8~1.2ml/L、氯化钠4~5g/L、氯化钾2~3g/L、氯化镁4~5g/L、氯化钙0.1~0.3g/L、磷酸二氢钾0.3~0.5g/L、微量元素1~3ml/L,进一步优选的包括以下浓度的组分:褐藻寡糖8g/L、胰蛋白胨3g/L、蛋白胨3g/L、酵母提取物3g/L、粘液素0.5g/L、3号胆盐0.4g/L、L-半胱氨酸盐酸盐0.8g/L、血红素0.05g/L、吐温80 1ml/L、氯化钠4.5g/L、氯化钾2.5g/L、氯化镁4.5g/L、氯化钙0.2g/L、磷酸二氢钾0.4g/L、微量元素2ml/L,所述发酵培养基的pH值优选为6.4~6.5。在本发明中,所述褐藻寡糖的分子量优选为2.0kDa。在本发明中,所述微量元素包括以下浓度的组分:MgSO4·7H2O 3.0g/L、CaCl2·2H2O 0.1g/L、MnC12·4H2O 0.32g/L、FeSO4·7H2O 0.1g/L、CoSO4·7H2O0.18 g/L、ZnSO4·7H2O 0.18g/L、CuSO4·5H2O 0.01g/L、NiCl2·6H2O 0.092g/L。在本发明中,所述发酵培养基优选的置于厌氧培养瓶中充氮气灭菌后使用。
本发明还提供了所述的单形拟杆菌菌株F18-22在制备治疗炎症性肠病的药物中的应用。
在本发明中,所述炎症性肠病为由葡聚糖硫酸钠诱导产生,所述炎症性肠病也包括由其它因素诱导产生。
本发明还提供了一种治疗炎症性肠病的药物,所述药物包含单形拟杆菌菌株F18-22。
在本发明中,所述药物的剂型优选为液体制剂。在本发明中,所述药物中单形拟杆菌菌株F18-22的活菌浓度优选为(1~9)×108CFU/mL。所述药物的溶剂优选为PBS溶液。在本发明中,所述药物优选的灌胃服用,所述药物的用量优选为(1~9)×109CFU/kg。
本发明还提供了所述的单形拟杆菌菌株F18-22在制备食品中的应用。本发明对所述食品的剂型没有特殊限定,采用本领域常规的食品剂型均可,例如固体制剂或液体制剂。在本发明中,当所述食品为固体菌剂时,所述食品通过将单形拟杆菌菌株F18-22的菌液冷冻干燥制备获得。在本发明中,所述食品还包括食品上可接受的辅料,本发明对所述辅料的种类和用量没有特殊限定。
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。
实施例1.
单形拟杆菌菌株F18-22的分离鉴定。
(1)培养基的配制
配制VI液体培养基,具体成分如下:褐藻寡糖8g/L、胰蛋白胨3g/L、蛋白胨3g/L、酵母提取物3g/L、粘液素0.5g/L、3号胆盐0.4g/L、L-半胱氨酸盐酸盐0.8g/L、血红素0.05g/L、吐温80 1ml/L、氯化钠4.5g/L、氯化钾2.5g/L、氯化镁4.5g/L、氯化钙0.2g/L、磷酸二氢钾0.4g/L、微量元素2ml/L,微量元素由以下浓度的组分组成:MgSO4·7H2O 3.0g/L、CaCl2·2H2O 0.1g/L、MnC12·4H2O 0.32g/L、FeSO4·7H2O 0.1g/L、CoSO4·7H2O 0.18g/L、ZnSO4·7H2O 0.18g/L、CuSO4·5H2O 0.01g/L、NiCl2·6H2O0.092g/L。溶剂为蒸馏水,pH值为6.4,将培养基灌注至特制厌氧小瓶,充入氮气后封口,高温灭菌。
(2)粪便的前处理
取1名志愿者新鲜粪便,用PBS(pH7.0)配成20%(wt/vol)的悬浮液,充分混匀,后用直径为2mm的金属筛过滤,除去大的食物颗粒,得到粪便PBS溶液。
(3)接种培养
所得粪便PBS溶液接种至高温灭菌后的厌氧小瓶中,37℃,富集培养,24h后采用10倍稀释涂布法涂布平板,涂布量为100μl,平板为VI液体培养基加终浓度2wt%琼脂。平板置于厌氧工作站37℃培养72h后,于厌氧工作站中挑取单菌落至厌氧小瓶液体培养基中进行传代培养和纯化。
(4)菌株的鉴定
a.参考前期文献研究方法(PMID:20495039),菌株F18-22的生理生化特性鉴定如下:
表1菌株F18-22生化特性(其中,-表示阴性,+表示阳性)
b.16S rDNA序列分析
DNA的提取:将步骤(5)得到的菌株F18-22采用德国QIAGEN公司粪便分析试剂盒(Cat No.51604)提取DNA。
16S rDNA全长扩增:
引物序列:
27ff:5’-CAGAGTTTGATCCTGGCT-3’,SEQ ID NO.2
1492r:5’-AGGAGGTGATCCAGCCGCA-3’,SEQ ID No.3
扩增体系:反应体系25μL,DNA模板l00 ng,10×PCRBuffer 2.5μL,dNTP Mix(10mM)0.5μL,10μM上下游引物各0.5μL,Taq酶(5U/μL)0.2μL,加去离子水补足至25μL。
扩增条件:预变性94℃保持5min,循环94℃下35s,55℃下35s,72℃保持1min,运行35个循环,延伸8min。
PCR产物纯化后送至生工生物工程(上海)股份有限公司(上海,中国)进行DNA测序(如SEQ ID No.1所示),将测序结果提交NCBI数据库中进行Blast比对,比对结果显示该菌株与Bacteroides uniformis同源性为100%,根据生理生化特性及16SrDNA序列比对,将菌株F18-22鉴定为单形拟杆菌(Bacteroides uniformis),命名为单形拟杆菌菌株F18-22。
SEQ ID No.1序列为:
acgcgtgcaagtcgaggggcagcatgaacttagcttgctaagtttgatggcgaccggcgcacgggtgagtaacacgtatccaacctgccgatgactcggggatagcctttcgaaagaaagattaatacccgatggcataattcttccgcatggtagaactattaaagaatttcggtcatcgatggggatgcgttccattaggttgttggcggggtaacggcccaccaagccttcgatggataggggttctgagaggaaggtcccccacattggaactgagacacggtccaaactcctacgggaggcagcagtgaggaatattggtcaatggacgagagtctgaaccagccaagtagcgtgaaggatgactgccctatgggttgtaaacttcttttatacgggaataaagtgaggcacgtgtgcctttttgtatgtaccgtatgaataaggatcggctaactccgtgccagcagccgcggtaatacggaggatccgagcgttatccggatttattgggtttaaagggagcgtaggcggacgcttaagtcagttgtgaaagtttgcggctcaaccgtaaaattgcagttgatactgggtgtcttgagtacagtagaggcaggcggaattcgtggtgtagcggtgaaatgcttagatatcacgaagaactccgattgcgaaggcagcttgctggactgtaactgacgctgatgctcgaaagtgtgggtatcaaacaggattagataccctggtagtccacacagtaaacgatgaatactcgctgtttgcgatatacagtaagcggccaagcgaaagcgttaagtattccacctggggagtacgccggcaacggtgaaactcaaaggaattgacgggggcccgcacaagcggaggaacatgtggtttaattcgatgatacgcgaggaaccttacccgggcttgaattgcaactgaatgatgtggagacatgtcagccgcaaggcagttgtgaaggtgctgcatggttgtcgtcagctcgtgccgtgaggtgtcggcttaagtgccataacgagcgcaacccttatcgatagttaccatcaggttatgctggggactctgtcgagactgccgtcgtaagatgtgaggaaggtggggatgacgtcaaatcagcacggcccttacgtccggggctacacacgtgttacaatggggggtacagaaggcagctacacggcgacgtgatgctaatccctaaagcctctctcagttcggattggagtctgcaacccgactccatgaagctggattcgctagtaatcgcgcatcagccacggcgcggtgaatacgttcccgggccttgtacacaccgcccgtcaagccatgaaagccgggggtacctgaagtgcgtaaccgcaaggagcgccctagggtaacttcct
实施例2.
单形拟杆菌菌株F18-22在治疗炎症性肠病中的应用
1、实验材料:实验动物28只C57BL/6小鼠(六周龄,雄性),购自维通利华实验动物科技有限公司(北京)(证书编号SCXK(京)2016-0011);葡聚糖硫酸钠(DSS,CatNo.160110),购自美国MP Biomedicals公司。
实验主要设备:显微镜(奥林巴斯CKX41SF)。
实验菌株来源:按照实例1制备获得。
2、实验方法:
(1)实验动物分组及模型建立
将28只雄性C57BL/6小鼠(体重为25g左右)随机分为3组,即正常对照组(NC,n=8)、模型对照组(MD,n=10)和单形拟杆菌菌株F18-22给药组(BU,n=10)。所有小鼠都饲养在温度约23℃的环境中,光照周期为12h,昼夜交替,自由采食标准饲料和水。经过两周的适应期后,开始造模同时给药。其中NC组正常饮水+PBS(150μL)灌胃6天,MD组饮用2wt%DSS+PBS(150μL)灌胃6天,BU组饮用2wt%DSS+灌胃150μL浓度为7×108CFU/mL的F18-22菌悬液6天。第7天,处死所有小鼠。收集结直肠用于进一步实验,MD组小鼠体重降低百分比较其他组小鼠显著,肠切片的病理评分高于其他组,肠水肿程度也高于其他组则说明该模型建立成功。
(2)病理组织学检查
处死小鼠后,取下端结直肠0.5cm,用4%多聚甲醛溶液固定。然后,石蜡包埋前用无水乙醇洗涤,5μm切片,苏木精伊红染色,以检查结直肠上皮层的损伤情况。切片样品在显微镜(奥林巴斯CKX41SF)下观察。
3、实验结果与分析
步骤1,每日称量小鼠体重,计算绘制体重变化图,结果如表2所示。
表2小鼠体重变化图
如图1所示:NC组在实验期间体重相对稳定,MD组小鼠在2wt%DSS诱导的第1天,体重略有上升。随后2天体重下降不明显,第4天后,经DSS诱导的各组小鼠体重呈现明显下降趋势,摄食量和饮水量降低,同时出现粪便变软,稀便甚至便血。停止DSS诱导后体重持续下降,最低下降约17%。NC组与MD组体重变化比具有显著差异。与MD组相比,BU组小鼠体重下降缓慢,同时便稀便血状况明显好转。BU组体重变化与MD组相比,在第4天、第5天和第6天具有极显著差异(P<0.01),在第7天具有显著差异(P<0.05),表明BU组的小鼠体重得到明显恢复,接近正常小鼠的生长状态。
步骤2,解剖小鼠后取整段盲肠与结直肠,测量其长度,并绘制图像。
如图2和表3所示,与NC组相比,MD组的结直肠长度显著降低(P<0.01)。与MD组相比,BU组的结直肠长度明显增加,同时BU组结直肠长度与MD组存在显著差异(P<0.05),和NC组存在极显著差异(P<0.01),表明BU组的小鼠结直肠损伤得到明显好转。
表3各组小鼠结直肠长度对比图
组别 | 结直肠长度(cm) | 显著性 |
NC | 9.58±1.08 | |
MD | 6.26±0.30 | ** |
BU | 7.08±0.2 | **# |
步骤3,在步骤2测量后将各组代表小鼠的整段盲肠与结直肠拍照,组合。图3为各组小鼠肠段示意图,可看出MD组小鼠肠段明显缩短,而BU组小鼠结直肠长度明显延长,与结直肠长度统计结果一致。
步骤4,结合步骤1中的小鼠体重变化,以及每日观察检测的粪便粘稠度及便血情况,测定小鼠的疾病活动指数(DAI)并绘制图像。
结果如图4和表4所示,正常对照组的DAI数值保持在0-1左右,表明小鼠体重几乎未出现下降,粪便硬度正常,无便稀和便血情况。而MD组小鼠DAI评分较高,其体重下降幅度较大,粪便较软,便稀便血情况严重,与NC组相比各项均存在极显著差异。相比MD组,BU组小鼠体重下降缓慢,便稀便血状况得到明显改善。BU组小鼠的各项评分与MD组相比均达到显著或极显著差异,说明BU可以缓解DSS诱导所造成的肠炎,改善肠炎症状。
表4各组小鼠疾病活动指数(Disease activity index,DAI)对比
步骤5,取小鼠结直肠远端约0.5cm长度的结直肠组织,固定在4%多聚甲醛固定溶液中,石蜡包埋前用无水乙醇洗涤,5μm切片,苏木精伊红染色,后置于显微镜下镜检并绘制图像。
如图5所示,NC组小鼠具有完整的肠上皮结构,隐窝清晰,杯状细胞明显且完整,没有炎症性细胞浸润或粘膜损坏。MD组小鼠由于DSS的毒性作用出现较为明显的结直肠炎症现象,其上皮细胞完整性丧失,同时可见炎症性细胞浸润,杯状细胞缺失,隐窝损坏等现象。与疾病小鼠相比,BU治疗可以缓解小鼠的肠道黏膜损伤,使肠道上皮细胞保持完整,减轻DSS造成的炎症细胞浸润以及其对杯状细胞及隐窝的破坏。
步骤6,通过步骤4计算小鼠结直肠组织损伤评分图。
如图6和表5所示,NC组小鼠结直肠组织损伤评分较低,说明小鼠结直肠组织几乎无损伤,而MD组评分较高,小鼠结直肠组织损伤严重,BU组评分在0.9左右,可见BU组结直肠组织损伤减缓,受到保护或有所恢复,MD同NC组以及BU组存在极显著差异(P<0.01)。
表5各组小鼠结直肠组织损伤评分图
组别 | 结直肠组织损伤评分 | 显著性 |
NC | 0.125±0.125 | |
MD | 3±0.25 | ** |
BU | 0.9±0.17 | ## |
本发明中DSS诱导的炎症性肠病模型小鼠后期精神萎靡,毛发凌乱,体重降低严重,出现便稀和便血状况。通过各项评分和HE染色观察结直肠组织等可知DSS的毒害作用使小鼠肠道上皮细胞完整性受损,并造成结直肠黏膜损伤和炎性细胞浸润。单形拟杆菌菌株F18-22给药组小鼠各项肠道炎症情况均有显著改善,其体重下降趋势得到有效逆转,便稀便血情况较轻,肠道上皮细胞完整,结直肠内炎性细胞较少,肠道炎症情况得到明显改善。综上所述,本发明提供的单形拟杆菌菌株F18-22能够显著改善DSS诱导的疾病小鼠的炎症性肠病症状,该菌株能够避免结直肠黏膜的完整性受到破坏,并对已有的肠道粘膜损伤进行保护,进而改善便稀和便血情况,避免因结直肠损伤造成体重快速下降,最终实现对于炎症性肠病的治疗。以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种单形拟杆菌菌株,其特征在于,所述单形拟杆菌菌株为单形拟杆菌菌株F18-22(Bacteroide uniformis F18-22),该菌株分离于健康人体肠道,该菌株保藏于中国典型微生物保藏中心,保藏编号为CCTCC NO:M 20221994。
2.一种权利要求1所述的单形拟杆菌菌株的培养方法,其特征在于,包括以下步骤:
1)将所述单形拟杆菌菌株F18-22活化培养获得种子液;
2)将步骤1)所述种子液接种至发酵培养基中进行厌氧发酵培养40~60h。
3.根据权利要求2所述的单形拟杆菌菌株的培养方法,其特征在于,所述发酵培养基,以水为溶剂,包括以下浓度的组分:褐藻寡糖6~10g/L、胰蛋白胨2~4g/L、蛋白胨2~4g/L、酵母提取物2~4g/L、粘液素0.4~0.6g/L、3号胆盐0.3~0.5g/L、L-半胱氨酸盐酸盐0.7~0.9g/L、血红素0.04~0.06g/L、吐温80 0.8~1.2ml/L、氯化钠4~5g/L、氯化钾2~3g/L、氯化镁4~5g/L、氯化钙0.1~0.3g/L、磷酸二氢钾0.3~0.5g/L和微量元素1~3ml/L,所述发酵培养基的pH值为6.4~6.5。
4.根据权利要求2所述的单形拟杆菌菌株的培养方法,其特征在于,所述褐藻寡糖的分子量为2.0kDa。
5.根据权利要求2~4任一权利要求所述的单形拟杆菌菌株的培养方法,其特征在于,步骤2)中所述种子液的接种量为2%~10%,v/v。
6.一种权利要求1所述的单形拟杆菌菌株在制备治疗炎症性肠病药物中的应用。
7.根据权利要求6所述的单形拟杆菌菌株在制备治疗炎症性肠病药物中的应用,其特征在于,所述炎症性肠病为由葡聚糖硫酸钠诱导产生。
8.一种治疗炎症性肠病的药物,其特征在于,所述药物包含单形拟杆菌菌株F18-22。
9.根据权利要求8所述的一种治疗炎症性肠病的药物,其特征在于,所述治疗炎症性肠病药物剂型为液体制剂,所述药物中单形拟杆菌菌株F18-22的活菌浓度为(1~9)×108CFU/mL。
10.一种权利要求1所述的单形拟杆菌菌株在制备食品中的应用。
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