CN117327632B - 一种动物双歧杆菌及其应用 - Google Patents
一种动物双歧杆菌及其应用 Download PDFInfo
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- CN117327632B CN117327632B CN202311631165.4A CN202311631165A CN117327632B CN 117327632 B CN117327632 B CN 117327632B CN 202311631165 A CN202311631165 A CN 202311631165A CN 117327632 B CN117327632 B CN 117327632B
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- bifidobacterium animalis
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- diarrhea
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- bifidobacterium
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Abstract
本发明属于微生物技术领域,具体涉及一种动物双歧杆菌及其应用。所述动物双歧杆菌Banim‑1选自保藏号为CCTCC NO:M 20231933的动物双歧杆菌Banim‑1,该菌株较为安全,能抑制多种致病菌、抑制促炎因子表达和改善腹泻,具有改善个体胃肠道功能以及预防/治疗腹泻的潜力。
Description
技术领域
本发明属于微生物技术领域,具体涉及一种动物双歧杆菌及其应用。
背景技术
肠道和人类的健康息息相关。它是人体重要的消化吸收场所,营养物质在这里被吸收运往全身,并排出身体代谢产生的毒素和垃圾。肠道也是人体最大的免疫器官,肠道的淋巴组织中分布着人体大部分的免疫细胞,抵抗着外来入侵的细菌和病毒。据世界卫生组织统计,肠胃疾病对人类的威胁触目惊心:全球每年死于肠胃疾病的人数在1000万以上,中国有1/5的人口患有肠胃疾病,居世界首位。
人类的胃肠内有数万亿个微生物,包括细菌、真菌和病毒。它们大多分布在大肠中,数量大致与全身的细胞数相当。在人体肠道细菌中,有10%到20%的细菌是与他人相同的。微生物群影响着人的健康和食欲、体重和心情。近年来,大量益生菌被发现对肠道健康具有好处,它们被用来预防或治疗炎性肠道疾病、肠道易激综合症甚至孤独症等。
多种肠道疾病都表现出炎性症状、粘膜层受损、腹泻或便秘,并以腹泻更为多见。放化疗引起的肠损伤是肠道损伤中较为严重的类型,其中化疗药物引起的腹泻(也称化疗相关性腹泻,CID)对患者影响较大,轻则影响患者生活质量,重则导致肿瘤治疗方案中断,更严重的还会导致患者脱水、感染、休克甚至死亡。化疗药物5-氟尿嘧啶(5-FU)和伊立替康(Irinotecan , CPT-11)引起的腹泻发生率最高,可达80%。
益生菌维护胃肠道健康已经有较长的使用历史,其优点在于安全性较高,比如妈咪爱(枯草杆菌二联活菌)、益菌康(复方嗜酸乳杆菌)、整肠生(地衣芽孢杆菌活菌)、培菲康(双歧杆菌三联活菌)、金双歧(双歧杆菌乳杆菌三联活菌)等。Joanne M Bowen等人(Cancer biology&therapy, 2007, 6(9): 1445-1450)在伊立替康介导的大鼠腹泻模型中证实了含有8株菌的VSL#3可以有效预防伊立替康化疗后的严重腹泻。应用广泛的明星菌株LGG可降低与5-FU化疗相关的严重腹泻和腹部不适(British journal of cancer, 2007, 97(8): 1028-1034)。Zhongyue Ren等(Pharmacological Research, 2022, 184: 106406)发现了一株动物双歧杆菌乳亚种菌株SF可以增强CPT-11的抗肿瘤活性并降低毒副作用。专利CN110938572B公开了一种含有植物乳杆菌、鼠李糖乳杆菌、动物双歧杆菌和嗜酸乳杆菌的益生菌组合物,及其在减轻鼻咽癌放化疗副作用口腔粘膜炎中的用途。但迄今为止仍缺少能够有效预防/治疗化疗相关性腹泻的益生菌产品。
发明内容
本发明针对现有研究仍存在的不足,提供了一种能够改善受试者肠道不适症状,特别是腹泻症状的益生菌解决方案。
首先,本发明提供了一种动物双歧杆菌(Bifidobacterium animalis)菌株,所述菌株选自保藏号为CCTCC NO:M 20231933的动物双歧杆菌Banim-1。
在一些具体实施方式中,所述菌株的16S rDNA序列如SEQ ID NO.1所示,且所述菌株具有可编码序列如SEQ ID NO.2所示的产乙酸相关酶、SEQ ID NO.3所示的产丙酸相关酶的基因。
其次,本发明还提供了前述动物双歧杆菌(Bifidobacterium animalis)菌株的培养方法,所述方法包括将所述动物双歧杆菌菌株接种至培养基,进行增殖培养,得到增殖的动物双歧杆菌菌株。
再次,本发明还提供了一种食品、保健品或药物组合物,其含有前述的动物双歧杆菌(Bifidobacterium animalis)菌株,或含有通过前述的培养方法得到的动物双歧杆菌(Bifidobacterium animalis)菌株。
在一些具体实施方式中,所述组合物中,动物双歧杆菌(Bifidobacteriumanimalis)菌株作为唯一活性成分。
在一些具体实施方式中,将所述食品、保健品或药物组合物制备成单个制剂。
在一些具体实施方式中,所述单个制剂中含有102~1015 CFU的动物双歧杆菌(Bifidobacterium animalis)菌株。
最后,本发明还提供了前述动物双歧杆菌(Bifidobacterium animalis)菌株在制备微生态制剂中的应用,所述微生态制剂用于改善受试者的肠道健康状况,或治疗/预防腹泻和/或抑制肠道病原菌的繁殖。
在一些具体实施方式中,所述腹泻为化疗相关性腹泻。
在一些具体实施方式中,所述化疗相关性腹泻是活性成分选自如下一种或其组合引起的腹泻:5-氟尿嘧啶、替加氟、5'-2'-脱氧尿苷、卡培他滨、替吉奥、紫杉醇、多西他赛、长春瑞滨、顺铂、卡铂、奈达铂、奥沙利铂、洛铂、环磷酰胺、异环磷酰胺、美法仑、卡莫司汀、伊立替康。
在一些具体实施方式中,所述病原菌选自铜绿假单胞菌、志贺氏菌、小肠结肠炎耶尔森菌和副溶血性弧菌中的一种或其组合。
本发明的动物双歧杆菌具有如下特点:
1)无毒力因子、非溶血,具有较好的安全性;
2)抑制多种致病菌,调节肠道菌群,抑制致病菌生长;
3)抑制肠道促炎因子的表达,减轻肠道炎症反应;
4)在腹泻总分指标中相比模型组动物有显著改善,与阳性对照洛哌丁胺效果相当,而且不存在抑制胃肠道蠕动等的副作用,能满足肿瘤患者需要长期服用的需求。
本发明的菌株保藏信息如下:
菌株名称:动物双歧杆菌(Bifidobacterium animalis)Banim-1
保藏日期:2023年10月18日
保藏单位:中国典型培养物保藏中心(China Center for Type CultureCollection,CCTCC),地址:湖北省武汉市武汉大学,邮编:430072,电话:027-68754052
保藏编号:CCTCC NO:M 20231933。
附图说明
图 1 是本发明的动物双歧杆菌Banim-1菌落形态正面照片;
图 2 是实施例4中动物双歧杆菌Banim-1对多种致病菌的抑菌试验结果;
图 3是实施例5中各实验组对5-氟尿嘧啶致腹泻小鼠的治疗效果,A是各组D8腹泻评分图;B是各组腹泻总分图;
图4是实施例6中各实验组对5-氟尿嘧啶致腹泻小鼠结肠组织TNF-α 基因表达的影响。
具体实施方式
定义与说明
针对本发明所请求保护的菌株(微生物保藏号为CCTCC NO:M 20231933的动物双歧杆菌菌株,Banim-1),Banim-1株的没有突变的基因组完全相同的、或传代中积累微小突变的,但毒性、免疫原性与生物活性没有实质变化的传代菌株应当视为经过微生物保藏的Banim-1株。并且本发明所请求保护的菌株包括活菌和灭活形式,完整的菌体或其裂解物或其发酵产物等衍生物。
Banim-1株经传代应用不可避免引入微小突变,毒性、免疫原性与生物活性没有实质变化的传代菌株或突变菌株应当属于本发明的贡献范围。毒性、免疫原性与生物活性没有实质变化,包括担不限于,在检测灵敏度、检测限等检测技术的局限性和可接受或不可避免误差的范围内视为毒性、免疫原性与生物活性是相同的。
经常需要用动物测定Banim-1株后代的毒性、免疫原性与生物活性,由于动物品种、年龄、性别、健康状况等体现的差别以及可预期或不可避免的系统误差属于没有实质性变化,属于毒性、免疫原性与生物活性没有实质变化的传代菌株。
Banim-1株传代多次后不可避免引入微小的突变,当突变发生在非编码序列区或者编码区的同义突变或者不影响菌株毒性、免疫原性与生物活性的突变(比如,可能是两个结构域之间的连接氨基酸残基,或者位于蛋白质高级结构内部因不与免疫细胞接触而不影响毒性、免疫原性与生物活性的微小突变的残基),可以合理预期的是当这些微小变化没有明显影响后代毒株的毒性、免疫原性与生物活性的情况下,仍然在本发明的实质技术贡献范围内,这些微小的突变仍属于非实质性突变,应当视为毒性、免疫原性与生物活性没有变化的突变菌株。
本发明Banim-1株的培养基培养传代菌株,可合理预期的是,与其他细菌一样,不可避免引入微小突变,当其毒性、免疫原性与生物活性没有实质性变化时,属于毒性、免疫原性与生物活性没有实质变化的传代菌株或突变菌株。
Banim-1株来自人粪便,必然在不同人体中或环境中有可能在本发明申请日以后分离到并鉴定的与Banim-1株具有共同祖先且与其他已知动物双歧杆菌菌株存在明显生理遗传差别的同源菌株,它们基因组与Banim-1株可能完全相同,也可能有微小的差别。
当这些同源菌株与Banim-1株的差别程度相当于毒性、免疫原性与生物活性没有实质变化的传代菌株或突变菌株与Banim-1株的差别程度时,这些同源菌株与Banim-1株相同或视为毒性、免疫原性与生物活性没有差别,这些同源菌株属于与Banim-1株实质相同的菌株。
本发明所述的组合物含有活性成分动物双歧杆菌Banim-1,以及其他成分,比如不具有生理功效的辅料成分,或者其他功效性成分。功效性成分包括但不限于其他功效菌株,或具有营养、膳食补充作用的营养成分、膳食纤维、益生元成分、后生元成分等。
动物双歧杆菌Banim-1具有抑制病原菌和抗炎功效,因而本发明的动物双歧杆菌Banim-1或含该菌株的组合物可以用于制备食品或保健品,用以改善受试者的肠道健康状况。动物双歧杆菌Banim-1还能改善受试者腹泻症状,特别是对化疗药物引起的严重损伤和腹泻发挥作用,因而本发明的动物双歧杆菌Banim-1或含该菌株的组合物可以用于制备药品,用以预防/治疗普通腹泻或化疗相关性腹泻。
本发明所述的组合物可以制备成任一种便于使用的形式,例如临床或食品中常见的粉剂、片剂、颗粒剂、凝胶剂、胶囊或液体剂。
本发明所述的组合物以能发挥功效的含量(治疗有效量)和频率给予使用对象,推荐单次使用剂量中含有102~1015 CFU、104~1013 CFU或105~1012 CFU的动物双歧杆菌Banim-1。
本发明所述腹泻是指排便次数明显超过平时习惯(>3次/d),粪质稀薄,含水量增加(>85%),大便可伴有黏液、脓血或未消化的食物的一种临床症状。腹泻是指排便次数明显超过平时习惯(>3次/d),粪质稀薄,含水量增加(>85%),大便可伴有黏液、脓血或未消化的食物的一种临床症状,在本发明中其含义覆盖抗生素相关性腹泻、化疗相关性腹泻、功能性腹泻、感染性腹泻等。
本发明中的特定温度参数,如无特殊说明,应理解为恒温处理,并允许在一定温度区间内存在变动。如在±5℃、±4℃、±3℃、±2℃、±1℃的范围内波动。
辅料成分包括药物载体和赋型剂。药物载体是指不对受试者引起显著刺激且不消除所施用的益生菌的生物活性及特性的药学载体。药学上可接受的载体可增强或稳定组合物,或可用于促进组合物的制备。药学上可接受的载体可包括溶剂、分散介质、涂层、表面活性剂、抗氧化剂、等渗剂、吸收延迟剂、盐、药物稳定剂、结合剂、赋形剂、崩解剂、润滑剂、甜味剂、调味剂、染料等及其组合,正如本领域技术人员已知的(参见例如Remington'sPharmaceutical Sciences,第18版MackPrinting Company,1990,第1289-1329页)。除非常规载体与活性成分不相容,否则考虑将其用于治疗性或药物组合物中。载体可经选择以使受试者的不利副作用降至最低和/或使活性成分的失活降至最低。
赋型剂是指添加至药物组合物中以使药物具有一定形状或一定浓度的物质。例如无菌水、生理盐水、聚亚烷基二醇(诸如聚乙二醇)、植物油或氢化萘、碳酸氢钙、磷酸钙、多种糖、各种类型淀粉、纤维素衍生物、明胶等。
下面将结合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,本领域技术人员在没有做出创造性劳动的前提下所获得的所有其它实施例,均应属于本发明保护的范围。
以下实施例中所用到的培养基配制方法如下:
YCFA液体培养基的配制(1L):称取酪蛋白10.0 g,酵母提取物2.5 g,MgSO4·7H2O0.45 mL(10%的母液),10 mg/mL的CaCl2 溶液0.45 mL,TE141 10 mL,K2HPO40.45 g,KH2PO40.45 g,NaCl 0.90 g,溶解于适量的蒸馏水中,加热煮沸后停止加热。培养基冷却过程中,先分批次向3.2 mL的VFA-mix中添加NaOH调节pH至中性,待培养基冷却至室温后添加到培养基中,随后添加一水合半胱氨酸盐酸盐0.5 g,0.1% 刃天青1 mL搅拌均匀,NaOH调节pH至中性,二次加热煮沸后,维持微沸状态20 min左右停止加热,N2置换降温并分装,121℃高温湿热灭菌30 min,阴凉、干燥处存放,备用。
TE141的配制:称取Nitrilotriacetic acid 1.50 g加入到200 mL纯水中,加入适量NaOH至溶液变澄清,之后加水800 mL,用50% HCl调节pH值为5.5,之后依次称取MgSO4·7H2O 3.00 g,MnSO4·H2O 0.50 g,NaCl 1.00 g,FeSO4·7H2O 0.10 g,CoSO4·7H2O 0.18g,CaCl2·2H2O 0.10 g,ZnSO4·7H2O 0.18 g,CuSO4·5H2O 0.006 g,KAl(SO4)2·12H2O0.02 g,H3BO30.01 g,Na2MoO4·2H2O 0.01 g,NiCl2·6H2O 0.03 g,Na2SeO3·5H2O 0.03 mL(10 mg/mL母液),Na2WO4·2H2O 0.03 mL(10 mg/mL母液)加入上述试液中,加入过程不断搅拌,保持溶液澄清,备用。
VFA-mix的配制:量取乙酸90 mL,丙酸30 mL,正戊酸10 mL,异丁酸10 mL,丁酸10mL混匀备用,使用前用5 M 浓度 NaOH调至中性。
原始样本保护剂配置(1 L):称取Na2HPO4·12H2O 3.85g,KH2PO40.27 g,NaCl 8.00g,加入适量蒸馏水充分溶解后,加入200 mL丙三醇,加热煮沸后,通N2冷却至室温。然后加入1.00 g半胱氨酸盐和0.1%的刃天青1 mL,充分溶解后利用5 M 浓度 NaOH溶液调至pH为中性,二次煮沸至保护剂颜色恢复无色状态,继续维持加热20 min以上,通N2冷却至室温后分装,121℃高温湿热灭菌30 min,阴凉、干燥处存放。
厌氧三混液体培养基(BHI+MRS+改良GAM)的配制:称取BHI肉汤粉末(青岛海博生物技术有限公司,HB8297-5)19.25 g,MRS肉汤粉末(广东环凯生物科技有限公司,027312)13.5 g,改良 GAM肉汤粉末(青岛海博生物技术有限公司,HB8518-3)15 g,溶解于1 L的蒸馏水中,N2置换除氧并分装,121℃高温湿热灭菌30 min,阴凉、干燥处存放。
厌氧PBS的配制:称取磷酸二氢钾0.27 g,磷酸氢二钠1.42 g,氯化钠8 g,氯化钾0.2 g,溶解于1 L的蒸馏水中,加热煮沸,冷却至室温加入0.55 g半胱氨酸盐酸盐,搅拌溶解后调节pH至6.5,装上定量分液器并通N2,加热至沸腾,在微沸状态下保持30 min,冷却后分装为400 mL/瓶,121℃高温湿热灭菌30 min,阴凉、干燥处存放,备用。
TSB(胰蛋白胨大豆肉汤,青岛海博生物技术有限公司,HB4114)、TSA(胰蛋白胨大豆琼脂,青岛海博生物技术有限公司,HB4138)培养基的配制均按照说明书进行称量溶解,121℃高温湿热灭菌30 min,阴凉、干燥处存放。
菌粉制备培养基配制:
Bifidobacterium animalisBanim-1:称取无水葡萄糖20 g,大豆蛋白胨10g,酵母浸粉5 g,乙酸钠5 g,磷酸二氢钾4.5 g,磷酸氢二钠4.7 g,硫酸镁0.1 g,硫酸锰0.045 g,吐温80 1 g,一水合半胱氨酸盐酸盐0.5 g,溶解于1 L的蒸馏水中,N2置换除氧,分装,121℃灭菌15 min。阴凉、干燥处存放。
Bifidobacterium animalisBanim-2:称取无水葡萄糖12 g,大豆胨 5 g,大豆蛋白胨5 g,大豆蛋白胨5 g,酵母浸粉5 g,乙酸钠5 g,磷酸二氢钾4.5 g,磷酸氢二钠4.7 g,硫酸镁0.1 g,硫酸锰0.045 g,吐温80 1 g,一水合半胱氨酸盐酸盐0.5 g,溶解于1 L的蒸馏水中,N2置换除氧,分装,121 ℃灭菌15 min。阴凉、干燥处存放。
实施例1动物双歧杆菌的分离鉴定
动物双歧杆菌Bifidobacterium animalisBanim-1的筛选分离方法如下:
(1)采集健康志愿者的新鲜粪便样本,称取1~2 g样本于50 mL离心管中,按照1:10比例加入原始样本保护剂,充分震荡,重悬样本。在N2保护下,经纱布过滤重悬菌液至50 mL离心管中,然后传递进入手套箱进行分装。分装时使用2 mL螺帽管,每支1 mL,分装后贴好标签套袋抽真空,于-80℃冰箱保存备用。
(2)分离时取1支冻存的样本管,传递进手套箱进行解冻。待样本解冻后用移液器取0.5 mL菌悬液于4.5 mL厌氧PBS中振荡混匀,梯度稀释至10-6,取适当梯度菌液与YCFA培养基混匀后分布到384孔板中,37℃厌氧培养9天。提供OD监测,挑取已生长孔位菌液转接到新的384孔板中,一式两份,培养48 h后,一份利用MALDI-TOF-MS检测,初步对分离菌株进行分类,另一份根据质谱结果,再次转接到96孔板中,一式两份,培养48 h后一个板进行16SrDNA基因扩增并送至北京擎科生物科技有限公司成都分公司测序,另一板按1:1加入50%的甘油混合均匀临时保藏,待PCR结果确认后使用。
(3)对16S rDNA测序结果进行分析,将序列与NCBI nt库做比对,结果显示有两株菌与动物双歧杆菌的序列相似性最高,均为100%。由此初步鉴定两株菌均为Bifidobacterium animalis,分别命名为Banim-1和Banim-2。其中,动物双歧杆菌Banim-1其菌落形态呈白色不透明状圆形菌落,中间凸起、表面光滑湿润,正面照片见图1,该菌株已于2023年10月18日保藏于中国典型培养物保藏中心(CCTCC),该菌株保藏号为:CCTCC NO:M20231933。
实施例2 动物双歧杆菌的全基因组分析
将动物双歧杆菌Banim-1按2%接种量接种至5 mL厌氧三混液体培养中,培养至对数生长后期,提取菌株全基因组DNA,利用Illumina高通量测序平台NovaSeq 6000进行全基因组测序。组装及注释后,将蛋白序列输入毒力基因库Virulence Factor Databases(VFDB)进行毒力因子分析。结果显示,该菌不具有毒力因子,基因组层面安全。
利用平均核苷酸相似度(Average Nucleotide Identity,ANI)进行菌株的新颖性分析。通过在Genbank中进行搜索,从中找到了125个已公开的Bifidobacterium animalis全基因组,通过Snippy(v4.6.0)分析发现,与本动物双歧杆菌Banim-1全基因组最相近的菌株GCA_900157135.1相比,两个菌株间SNP(单核苷酸多样性,single nucleotidepolymorphism)位点有3个,DEL(Deletion,Del)位点有1个。动物双歧杆菌Banim-1的16SrDNA序列如SEQ ID NO .1所示。
通过emapper-2.1.9对全基因组序列进行注释,进一步发现动物双歧杆菌Banim-1具有可编码序列如SEQ ID NO:2所示的产乙酸相关酶、序列如SEQ ID NO:3所示的产丙酸相关酶的基因。
实施例3 动物双歧杆菌的溶血试验
将保藏的动物双歧杆菌Banim-1按2%接种量接种至5 mL厌氧三混液体培养基中,以粪肠球菌(β溶血,CICC23658,购自至中国工业微生物菌种保藏管理中心)作为阳性对照,以空白培养基作为阴性对照。所有菌株均在厌氧三混培养基中37℃厌氧培养至对数生长后期,得到活化菌株。各取2.5 μL活化菌株接种至哥伦比亚血平板(上海科玛嘉微生物技术有限公司)上,每组设置3个平行。于37℃厌氧培养48 h后进行观察,阳性菌株菌落周围形成界限明显、完全透明的溶血环,为β溶血;动物双歧杆菌Banim-1菌落周围的培养基没有变化,为γ溶血,即不溶血,因此,对人体施用没有溶血风险。
实施例4 动物双歧杆菌对致病菌的抑菌能力
选取4种常见的导致腹泻的致病菌进行抑菌能力检测,致病菌株来源信息如下:
表1 致病菌来源信息
菌株名称 | 菌株保藏号 | 菌株保藏单位 |
铜绿假单胞菌 | CMCC(B)10104 | 中国食品药品检定研究院 |
志贺氏菌 | CMCC(B)51252 | 中国食品药品检定研究院 |
小肠结肠炎耶尔森菌 | CMCC(B)52204 | 中国食品药品检定研究院 |
副溶血性弧菌 | ATCC 17802 | 美国微生物菌株保藏中心 |
Banim-1发酵液获得:Banim-1经活化后,按2%的接种量接种于厌氧三混液体培养基中37℃厌氧培养48 h,获得发酵液。
病原菌制备与涂布:铜绿假单胞菌、志贺氏菌、小肠结肠炎耶尔森菌和副溶血性弧菌经TSB肉汤培养基活化后,于TSB肉汤培养基中稀释50倍达到合适浓度,取0.2 mL稀释的菌液于TSA固体培养基上涂布。
Banim-1与病原菌共培:放置3支灭菌牛津杯于涂布好的病原菌平板上,在牛津杯中加入0.2 mL Banim-1发酵液。放入培养盒,平皿正置培养24 h,用游标卡尺测量抑菌圈大小,并计算平均值。
统计结果如图2所示:Banim-1菌对铜绿假单胞菌、志贺氏菌、小肠结肠炎耶尔森菌和副溶血性弧菌有抑菌能力。
实施例5 动物双歧杆菌对5-FU致腹泻小鼠的治疗效果
冻干保护剂配置:
A液:蔗糖8 g,海藻糖8 g,纯化水44 g;115℃灭菌20 min。
B液:谷氨酸钠2 g,精氨酸盐酸盐2 g,纯化水16 g;115℃灭菌20 min。
C液:维生素C钠4 g,纯化水16 g。过滤除菌备用。
使用时体积比按A:B:C=6:2:2混合。
菌粉制备:将保藏的动物双歧杆菌Banim-1和Banim-2按10%接种量分别接种至各自的菌粉制备培养基,37℃、90 rpm厌氧培养6~10 h,得到一级种子液(OD600值≥ 1.0)。随后,按1.5%接种量转接至菌粉制备培养基,37℃、90 rpm厌氧培养10~14 h,得到二级种子液(OD600值≥1.0)。按1.5%接种量将二级种子液用蠕动泵泵入发酵罐中,设置发酵参数(37℃、pH 5.1、100 rpm、0.06 MPa),发酵培养。发酵菌液OD600值≥1.8或OD600值增长≤0.1时停止发酵,设置发酵温度为20℃,离心收集菌体。按菌泥和冻干保护剂重量比1:1~1:2添加冻干保护剂,混匀乳化菌泥。乳化后的菌悬液,放入降温至-40℃的冷冻干燥机的板层上冻干,粉碎后即得菌粉。动物给药前使用0.2 mL生理盐水将1×109CFU菌粉配置成菌悬液。
试验动物:25只SPF级雄性Balb/c小鼠,体重18~22 g,购自北京维通利华实验动物技术有限公司,饲养于SPF级动物房。
试验设计:采用5-FU(购自天津金耀药业有限公司,规格10 mL/支,0.25 g/10 mL)溶液诱导小鼠化疗相关性腹泻模型,根据小鼠初始体重随机分为5组,分别为正常对照组、模型对照组、阳性对照洛哌丁胺组和动物双歧杆菌Banim-1组、动物双歧杆菌Banim-2组,每组5只。
总体实验周期为9天,记为D1-D9。D3进行5-FU单次造模处理,除正常对照组腹腔注射生理盐水外,其他组别均进行5-FU单次腹腔注射造模处理,造模剂量按体重给药(350mg/kg)。
所有组别给药方式为灌胃,正常对照组、模型对照组灌胃冻干保护剂;连续灌胃5天(D1-D5);阳性对照组连续灌胃洛哌丁胺(购自西安杨森制药有限公司,LFJ8684)9天(D1-D9),剂量为20 mg/kg;Banim-1组和Banim-2组连续灌胃5天(D1-D5),按1×109CFU/只的剂量灌胃动物双歧杆菌Banim-1菌悬液和Banim-2菌悬液。在D5给药结束后,连续观察4天。具体实验分组和给药方案见表2。
表2 实验分组和给药方案
组别 | 数量 | 造模剂 | 造模剂量 | 受试物 | 给药体积 | 给药剂量 | 给药天数 |
正常对照组 | 5 | 生理盐水 | / | 冻干保护剂 | 0.2 mL/只 | / | 5 d |
模型对照组 | 5 | 5-FU | 350mg/kg | 冻干保护剂 | 0.2 mL/只 | / | 5 d |
洛哌丁胺 | 5 | 5-FU | 350mg/kg | 洛哌丁胺 | 10 mL/kg | 20 mg/kg | 9 d |
Banim-1 | 5 | 5-FU | 350mg/kg | Banim-1 | 0.2 mL/只 | 1×109CFU/只 | 5 d |
Banim-2 | 5 | 5-FU | 350mg/kg | Banim-2 | 0.2 mL/只 | 1×109CFU/只 | 5 d |
注:5-FU:5-氟尿嘧啶;CFU: colony forming unit 菌落形成单位;d: 天
腹泻观察与评分:将小鼠置于垫有洁净滤纸的小鼠笼内,每笼1只。粪便硬,正常便视为0分;轻度、轻微湿便或软便视为1分;中度,湿便、粪便不成形且肛周不洁视为2分;重度,稀便且严重肛周不洁视为3分。实验周期内,每天对小鼠粪便进行观察、评分,腹泻总分为每天腹泻评分的总和。
实验结果如图3所示,与模型对照组相比,洛哌丁胺能够显著降低模型小鼠的D8腹泻评分和腹泻总分。Banim-1组与模型对照组相比,D8腹泻评分和腹泻总分均显著降低(P<0.05)(图3A、3B),与洛哌丁胺给药组改善效果相当。而Banim-2组与模型对照组相比未见显著差异。
结果表明本发明的双歧杆菌Banim-1能够显著改善化疗药物5-FU导致的腹泻症状,对腹泻的治疗效果优于Banim-2。
实施例6 动物双歧杆菌对5-FU致腹泻小鼠结肠组织炎症因子的影响
实施例5中的动物实验结束后,采集小鼠中段结肠,保存于-80 ℃冰箱。按照试剂说明书提取各组小鼠结肠组织总RNA,并反转录成cDNA,保存于-20 ℃备用。用qRT-PCR检测各组小鼠结肠炎症因子TNF-α的mRNA基因相对转录水平(引物序列见表3)。反应程序:95 ℃3 min,95 ℃ 20 s,60 ℃ 45 s,72 ℃ 20 s,共39个循环。采用2-ΔΔCT法进行分析,通过SPSS 24.0统计软件对数据进行显著性分析。
表3 qRT-PCR引物信息
基因 | 引物 |
Tumor necrosis factor (TNF-α) | Forward: 5’-CTGTAGCCCACGTCGTAGC-3’Reverse: 5’-TTGAGATCCATGCCGTTG-3’ |
检测结果见图4,与正常对照组相比,模型对照组TNF-α的mRNA相对转录水平显著升高(P<0.01);Banim-1给药后可显著降低TNF-α的mRNA相对转录水平(P<0.05);Banim-2给药后TNF-α的mRNA相对转录水平与模型对照组并没有显著差异。
由此可见,本发明的双歧杆菌Banim-1能够改善腹泻症状,对化疗相关性腹泻具有明显的治疗效果,且显著优于同种属的Banim-2。
Claims (7)
1. 一种动物双歧杆菌(Bifidobacterium animalis)菌株,所述菌株选自保藏号为CCTCC NO:M 20231933 的动物双歧杆菌Banim-1。
2. 如权利要求1 所述的动物双歧杆菌(Bifidobacterium animalis)菌株的培养方法,其特征在于,将所述动物双歧杆菌菌株接种至培养基,进行增殖培养,得到增殖的动物双歧杆菌菌株。
3. 一种食品、保健品或药物组合物, 含有权利要求1 所述的动物双歧杆菌(Bifidobacterium animalis)菌株,或含有通过权利要求2 所述的培养方法得到的动物双歧杆菌(Bifidobacterium animalis)菌株。
4. 根据权利要求3所述的食品、保健品或药物组合物,其特征在于,所述动物双歧杆菌(Bifidobacterium animalis)菌株作为唯一活性成分。
5. 根据权利要求4 所述的食品、保健品或药物组合物,其特征在于,组合物的单个制剂中含有102~1015 CFU 的动物双歧杆菌(Bifidobacterium animalis)菌株。
6. 权利要求1 所述的动物双歧杆菌(Bifidobacterium animalis)菌株在制备微生态制剂中的应用,所述微生态制剂用于治疗/预防腹泻和/或抑制肠道病原菌的繁殖,所述腹泻为化疗相关性腹泻,所述病原菌选自铜绿假单胞菌、志贺氏菌、小肠结肠炎耶尔森菌和副溶血性弧菌中的一种或其组合。
7. 根据权利要求6 所述的应用,其特征在于,所述化疗相关性腹泻是活性成分选自如下一种或其组合引起的腹泻:5-氟尿嘧啶、替加氟、5'-2'-脱氧尿苷、卡培他滨、替吉奥、紫杉醇、多西他赛、长春瑞滨、顺铂、卡铂、奈达铂、奥沙利铂、洛铂、环磷酰胺、异环磷酰胺、美法仑、卡莫司汀、伊立替康。
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