CN116676206A - 一株具有良好益生特性的乳明串珠菌及其在制备防治炎症性肠病药物中的应用 - Google Patents
一株具有良好益生特性的乳明串珠菌及其在制备防治炎症性肠病药物中的应用 Download PDFInfo
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- CN116676206A CN116676206A CN202310150690.8A CN202310150690A CN116676206A CN 116676206 A CN116676206 A CN 116676206A CN 202310150690 A CN202310150690 A CN 202310150690A CN 116676206 A CN116676206 A CN 116676206A
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- leuconostoc lactis
- leuconostoc
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- lactis
- inflammatory bowel
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Abstract
本发明涉及微生物技术领域,具体涉及一株具有良好益生特性的乳明串珠菌及其在制备防治炎症性肠病药物中的应用。本发明提供的乳明串珠菌(Leuconostoc lactis)LL271保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.25974。该菌株具有良好的耐酸、耐胆汁酸盐能力、自聚集能力和疏水性以及能够抑制常见人体病原微生物等益生特性;同时能够改善肠道炎症,对于炎症性肠病具有防治功效,且具有较高的生物安全性,可作为益生菌制剂用于缓解炎症性肠病,改善肠道粘膜损伤和结肠炎症,为炎症性肠病的防治提供了新的策略,具有潜在的临床应用前景。
Description
技术领域
本发明涉及微生物技术领域,尤其涉及一株具有良好益生特性的乳明串珠菌及其在制备防治炎症性肠病药物中的应用。
背景技术
炎症性肠病(Inflammatory bowel disease,IBD)是指一组原因不明的肠道炎症性疾病,主要表现为黏膜和全身炎症,分为溃疡性结肠炎(Ulcerative colitis,UC)和克罗恩病(Crohn's disease,CD),发病率逐年增加。临床上经常使用的氨基水杨酸类抗炎药、免疫抑制剂或者结肠切除术等IBD治疗方案会产生各种毒副作用。因此,亟需研发安全有效的替代疗法。
乳明串珠菌(Leuconostoc lactis,L.lactis)隶属于明串珠菌属(Leuconostoc),为革兰氏阳性、圆形球状、无芽胞、兼性厌氧的传统益生菌,多作为发酵剂用于发酵乳制品。目前尚未有关于乳明串珠菌治疗或预防炎症性肠病发生发展的相关报道。
发明内容
本发明的目的是提供一株乳明串珠菌及其应用。
具体地,本发明提供以下技术方案:
本发明提供乳明串珠菌(Leuconostoc lactis)LL271(菌株名称简称为271),该菌株于2022年10月28日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编100101),分类命名为乳明串珠菌Leuconostoc lactis,保藏编号为CGMCC No.25974。
乳明串珠菌(Leuconostoc lactis)LL271分离自健康成人粪便样本,经测序验证,该菌株的16S rDNA的序列如SEQ ID NO.1所示。经体外实验发现该菌株具有良好的耐酸、耐胆汁酸盐、疏水性和自聚集能力以及抑制常见人体病原菌等益生特性,且无溶血活性、无明胶酶活性,对选取的抗生素敏感;通过构建小鼠结肠炎模型证明该菌株对动物无害并具有改善炎症性肠病的功能,可减轻结肠炎小鼠的疾病活动指数(Disease activity index,DAI)和结肠长度的缩短,并降低结肠病理评分,从而降低炎症损伤程度。本发明提供的乳明串珠菌LL271显示出良好益生潜力以及在预防和/或治疗炎症性肠病中的优异应用前景。
本发明提供一种菌剂,所述菌剂含有以上所述的乳明串珠菌(Leuconostoclactis)LL271。
优选地,上述菌剂中乳明串珠菌(Leuconostoc lactis)LL271以活菌形式存在。
上述菌剂可以为液体菌剂或固体菌剂。
本发明还提供以上所述的菌剂的制备方法,所述方法包括将乳明串珠菌(Leuconostoc lactis)LL271经培养获得培养物的步骤。
优选地,所述培养使用的培养基为含脱纤维羊血的MRS培养基;所述培养基中,脱纤维羊血的体积百分含量为4–5%。
上述脱纤维羊血具体为脱纤维无菌羊血。
优选地,所述培养为在37℃,5%(v/v)CO2条件下培养。
在本发明的一些实施方式中,乳明串珠菌(Leuconostoc lactis)LL271的培养方法为:使用含5%(v/v)脱纤维无菌羊血的MRS培养基,在37℃,5%(v/v)CO2条件下培养。
上述培养获得的菌液可直接或加入微生物制剂领域允许的辅料制备为液体菌剂,也可收集菌液中的菌体与冻干保护剂等微生物制剂领域允许的辅料混合后经真空冷冻干燥制备为干粉菌剂。
基于乳明串珠菌(Leuconostoc lactis)LL271的功能,本发明提供该菌株的以下应用:
本发明提供所述乳明串珠菌(Leuconostoc lactis)LL271或所述菌剂在制备用于缓解肠道炎症和/或改善肠黏膜损伤的药物中的应用。
本发明提供所述乳明串珠菌(Leuconostoc lactis)LL271或所述菌剂在制备用于预防和/或治疗结肠炎的药物中的应用。
本发明提供所述乳明串珠菌(Leuconostoc lactis)LL271或所述菌剂在制备用于预防和/或治疗炎症性肠病的药物中的应用。
优选地,所述炎症性肠病为溃疡性结肠炎或克罗恩病。
本发明提供所述乳明串珠菌(Leuconostoc lactis)LL271或所述菌剂在抑制病原菌中的应用。
优选地,所述抑制病原菌为非疾病诊断和治疗目的的用途,包括用于抑制环境或食品中的病原菌。
乳明串珠菌(Leuconostoc lactis)LL271同样可以在体内发挥抑制病原菌的作用。
本发明提供所述乳明串珠菌(Leuconostoc lactis)LL271或所述菌剂在制备用于抑制病原菌的药物中的应用。
优选地,本发明中所述的病原菌为人体病原菌,包括但不限于来源于志贺氏菌属(Shigella)、链球菌属(Streptococcus)、沙门氏菌属(Salmonella)、大肠埃希菌属(Escherichia)、葡萄球菌属(Staphylococcus)或李斯特菌属(Listeria)的人体病原菌。
在本发明的一些实施方式中,所述病原菌为福氏志贺菌、猪链球菌、鼠伤寒沙门菌、肠集聚性大肠埃希菌、金黄色葡萄球菌、单核细胞增生李斯特菌和/或肠出血性大肠杆菌。
优选地,以上所述的药物的有效成分含有所述乳明串珠菌(Leuconostoc lactis)LL271或所述菌剂。
所述药物中,乳明串珠菌(Leuconostoc lactis)LL271优选以活菌形式存在。
以上所述的药物中,除含有乳明串珠菌(Leuconostoc lactis)LL271外,还可包含其它对于炎症性肠病具有防治功效的有效成分和/或含有药学领域允许的载体或辅料。
在本发明的一些实施方式中,上述药物的有效成分中含有补充型乳明串珠菌(Leuconostoc lactis)LL271菌液。
上述补充型乳明串珠菌(Leuconostoc lactis)LL271菌液的制备方法包括:将在培养基中活化培养的乳明串珠菌(Leuconostoc lactis)LL271制备为菌悬液。活化培养的时间优选为24–36h。活化培养的条件优选为37℃,5%(v/v)CO2条件下培养。
上述菌悬液可通过将乳明串珠菌(Leuconostoc lactis)LL271菌体以磷酸盐缓冲液(phosphate buffered saline,PBS,1×)等缓冲液悬浮制得。菌悬液的OD600值优选为4–5。
本发明还提供所述乳明串珠菌(Leuconostoc lactis)LL271或所述菌剂在制备食品、保健品或药物中的应用。
本发明提供一种产品,所述产品包含乳明串珠菌(Leuconostoc lactis)LL271。
优选地,所述产品为食品、保健品或药物。
本发明的有益效果在于:本发明提供的乳明串珠菌(Leuconostoc lactis)LL271具有良好的耐酸、耐胆汁酸盐能力、自聚集能力和疏水性以及能够抑制常见人体病原微生物等益生特性;同时能够改善肠道炎症,对于炎症性肠病具有防治功效,且具有较高的生物安全性,可作为益生菌制剂用于缓解炎症性肠病,改善肠道粘膜损伤和结肠炎症,为炎症性肠病的防治提供了新的策略,具有潜在的临床应用前景。
经C57BL/6小鼠结肠炎模型实验证实,采用乳明串珠菌(Leuconostoc lactis)LL271菌悬液进行7天预灌胃处理,菌株对实验动物无明显毒副作用;正式干预7天后,发现小鼠腹泻及血便等症状较模型组明显减轻,DAI显著下降,结肠缩短症状以及表型明显改善,减轻了结肠炎小鼠结肠的病理损伤,降低了结肠组织病理学评分,证明乳明串珠菌(Leuconostoc lactis)LL271可改善结肠炎小鼠的肠黏膜损伤及炎症反应。
附图说明
为了更清楚地说明本发明或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明实施例2中乳明串珠菌LL271的明胶酶活性检测结果;其中,金黄色葡萄球菌25923代表金黄色葡萄球菌ATCC25923,LL271代表乳明串珠菌LL271(即乳明串珠菌CGMCC No.25974)。
图2为本发明实施例2中乳明串珠菌LL271对常见人体病原微生物的抑菌圈图;其中,福氏志贺菌301代表福氏志贺菌2a 301、猪链球菌43765代表猪链球菌ATCC43765、鼠伤寒沙门菌14028代表鼠伤寒沙门菌ATCC14028、肠集聚性大肠埃希菌24186代表肠集聚性大肠埃希菌CICC24186、金黄色葡萄球菌25923代表金黄色葡萄球菌ATCC25923、单核细胞增生李斯特菌BAA-679代表单核细胞增生李斯特菌ATCCBAA-679、肠出血性大肠杆菌43895代表肠出血性大肠杆菌ATCC43895。
图3为本发明实施例3中各组小鼠14d体重变化趋势。
图4为本发明实施例3中DSS干预期间各组小鼠DAI统计分析图。
图5为本发明实施例3中各组小鼠结肠长度及表型分析。
图6为本发明实施例3中各组小鼠结肠黏膜组织的HE染色及病理评分统计柱状图。
图3-6中,数值均以均数±标准差展示;*为P<0.05,**为P<0.01,***为P<0.001。其中,图3-6中,NC代表对照组,DSS代表模型组,LL271代表CGMCC No.25974干预组,n=8。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合本发明中的附图,对本发明中的技术方案进行清楚、完整地描述,显然,所描述的实施例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作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乳明串珠菌菌株的分离和鉴定
1、菌株分离
取适量冻存于-80℃冰箱中的健康人粪便样本置于冰上缓慢解冻,用PBS梯度稀释后,移取不同稀释度的样品悬浮液100μL至MRS+5%(v/v)脱纤维无菌羊血培养基,均匀涂布,置于37℃,5%(v/v)CO2条件下培养2-4天,挑取乳白色不透明单菌落进行传代纯化3次,纯化菌株可用于实验或冷冻保藏。
本发明针对分离到的一株乳明串珠菌(命名为乳明串珠菌LL271)进行耐酸和耐胆汁酸盐能力、抗生素敏感性、抑菌、自聚集能力和疏水性以及改善炎症性肠病的功能评价(所有评价方法参考实施例2-3)。
2、菌种保藏
将容量为2mL的保菌管中加入1.5mL保菌液,经121℃,15min高压灭菌处理后以备使用;将乳明串珠菌LL271在MRS+5%(v/v)脱纤维无菌羊血的固体培养基上连续传代3次后,用无菌接种环刮取适量菌体至保菌管中并充分融入保菌液,混匀后于-80℃冻存。其中,保菌液为脑心浸液(Brain Heart Infusion,BHI)液体培养基+20%(v/v)甘油。
3、菌株16S rRNA基因鉴定
使用Genomic DNA纯化试剂盒提取该分离菌株基因组DNA,用16S rRNA基因通用引物(27F,SEQ ID NO.2:AGAGTTTGATCCTGGCTCA;1492R,SEQ ID NO.3:AAGTCGTAACAAGGTAGCCGT)扩增乳明串珠菌LL271的16S rRNA基因,对PCR扩增产物进行测序,将测序所得序列提交至NCBI在线数据库(https://www.ncbi.nlm.nih.gov/)进行同源性比对,进行初步鉴定。PCR反应的条件和PCR体系(30μL)的组成如下:
PCR反应体系(30μL):Premix Taq,15μL;ddH2O,12μL;上下游引物(27F和1492R),各1μL;基因组DNA,1μL。
PCR扩增条件:预变性94℃,5min;94℃变性、0.5min;55℃退火、0.5min;72℃延伸、0.5min;30个循环;72℃,1min。
序列比对结果显示,待测菌株LL271与乳明串珠菌模式株KCKC3528的序列一致性为99.65%,超过种间阈值98.75%,因此判定该分离菌株为乳明串珠菌菌株。乳明串珠菌LL271(简称乳明串珠菌271)已于2022年10月28日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编100101),分类命名为乳明串珠菌Leuconostoc lactis,保藏编号为CGMCCNo.25974。
实施例2乳明串珠菌CGMCC No.25974的特性分析
1、乳明串珠菌CGMCC No.25974的生化特性
采用法国梅里埃公司生产的“BioMerieux”细菌系统糖发酵反应鉴定卡API 50CH。简言之,将菌株CGMCC No.25974对数期培养物混悬于50CHL培养基,按说明书调至一定的麦氏浊度培养36h后的糖发酵结果见表1,可知乳明串珠菌CGMCC No.25974能够发酵包括半乳糖在内的12种糖。
表1菌株CGMCC No.25974的糖发酵反应(50CHL)鉴定图谱
注:+为阳性;–为阴性;w为弱阳性。
2、乳明串珠菌CGMCC No.25974的溶血活性测定
将活化三代并生长至对数生长期的乳明串珠菌CGMCC No.25974三区划线接种于MRS血琼脂培养基,培养24h后,观察菌落形态和溶血现象。溶血分为α-溶血(菌落周围出现草绿色区域)、β-溶血(菌落周围出现透明区域)、γ-溶血(不会引起溶血)。结果显示菌株CGMCC No.25974无溶血活性。
3、乳明串珠菌CGMCC No.25974的明胶酶活性测定
将上述2中CGMCC No.25974菌体用PBS悬浮成菌悬液,调至麦氏浊度4.0(OD4.0),取10μL接种于MRS+3%(w/v)明胶琼脂培养基表面,培养24h。随后将饱和硫酸铵均匀平铺至培养基表面,观察菌落周围是否出现透明晕圈。其中,金黄色葡萄球菌ATCC25923为明胶酶阳性对照(透明晕圈)。结果如图1所示,菌株CGMCC No.25974无明胶酶活性。
4、乳明串珠菌CGMCC No.25974的抗菌药物敏感性测定(E-test法)
采用临床和实验室标准协会(Clinical and Laboratory StandardsAssociation,CLSI)所制定抗菌药物敏感性测试性能标准(2018版),选取明串珠菌属推荐抗菌药物青霉素、氨苄霉素、氯霉素、米诺环素。简言之,将上述3中的CGMCC No.25974菌悬液调至麦氏浊度0.5(OD0.5),均匀涂布于CAMHB+5%(v/v)脱纤维马血培养基表面,并将E-test条置于培养基表面,培养24h后读取最小抑制浓度(Minimal InhibitoryConcentration,MIC)值。肺炎链球菌ATCC49619作为质量控制菌株。结果如表2所示,菌株CGMCC No.25974对四种抗生素均敏感。
表2E-test法检测菌株CGMCC No.25974的抗菌药物敏感性
5、乳明串珠菌CGMCC No.25974对酸和胆汁酸盐的耐受性
将上述3中的CGMCC No.25974菌悬液分别接种于含3g/L胃蛋白酶、pH 3.0的MRS液体培养基和含0.3%(w/v)胆汁酸盐的MRS液体培养基,在37℃环境中孵育3h。取0h和3h培养液,梯度稀释后接种于MRS琼脂培养基,计算0h活菌总数(N0)和3h活菌总数(N1),根据公式1计算菌株在酸和胆汁酸盐环境中的存活率。
存活率(%)=log CFU N1/log CFU N0(公式1)
结果如表3所示,菌株CGMCC No.25974在pH 3.0和含0.3%胆汁酸盐的MRS培养基中的存活率分别为87.23%和76.00%。
6、乳明串珠菌CGMCC No.25974的粘附相关实验
自聚集能力:将上述3中的CGMCC No.25974菌悬液调至麦氏浊度1.0,记为OD0;培养24h后,测定其麦氏浊度,记为OD1,根据公式2计算自聚集能力。结果如表3所示,菌株CGMCC No.25974的自聚集能力平均为95.00%。
菌株表面疏水性:将上述3中的CGMCC No.25974菌悬液调至麦氏浊度1.0,记为OD0;加入等体积的二甲苯,剧烈混匀2min,培养1h后除去有机相,测定其水相麦氏浊度记为OD1,根据公式2计算表面疏水性。结果如表3所示,菌株CGMCC No.25974的疏水性平均为90.00%。
自聚集/疏水性(%)=[(OD0-OD1)/OD0]×100%(公式2)
对人结肠癌细胞HT-29细胞的粘附作用:将1×105cell/mL HT-29细胞接种于24孔培养板(内置细胞爬片),培养过夜后,无血清DMEM(1×)培养液洗细胞三次,加入1×107CFU/mL CGMCC No.25974菌悬液,不加菌液的孔设为对照组。培养3h后,用上述培养液洗细胞三次,然后甲醇固定、吉姆萨染色和封片。在光学正置显微镜1000倍视野下观察菌株CGMCC No.25974粘附情况,计算粘附指数(Adhesive index),即随机从显微镜下选择10个视野计算HT-29细胞粘附的平均细菌数。其中,粘附指数<1为弱粘附;<50为中等粘附;>50为强粘附。结果如表3所示,菌株CGMCC No.25974的粘附指数平均为24.91CFU/cell。
表3菌株CGMCC No.25974对酸和胆汁酸盐耐受性及细胞粘附作用
注:数值为均数±标准差。
7、乳明串珠菌CGMCC No.25974抑制常见人体致病菌的能力测定
将福氏志贺菌2a 301、猪链球菌ATCC43765、鼠伤寒沙门菌ATCC14028、肠集聚性大肠埃希菌CICC24186、金黄色葡萄球菌ATCC25923、单核细胞增生李斯特菌ATCCBAA-679、肠出血性大肠杆菌ATCC43895和菌株CGMCC No.25974调至108CFU/mL,并用棉签蘸取适量致病菌菌悬液均匀涂布于MRS琼脂培养基表面,随后取15μL CGMCC No.25974菌悬液接种于培养基表面培养24h测量抑菌圈大小,即抑菌圈以距离菌落边缘的距离计量。结果如图2和表4所示,CGMCC No.25974对所检测的所有人体致病菌均有抑制作用,其中对福氏志贺菌抑制作用最强。
表4CGMCC No.25974对常见人体病原菌的抑制作用
注:数值以均数±标准差展示。
实施例3乳明串珠菌CGMCC No.25974的抗炎功能评价
采用C57BL/6小鼠构建的DSS结肠炎模型,予以CGMCC No.25974菌悬液灌胃干预(予以200μL/只灌胃,菌量约3×108CFU)。实验设计:6周龄实验小鼠在25℃、12h光/暗循环的SPF环境适应3d,予以普通啮齿类动物饲料和灭菌水喂养。随机分成3组(n=8),即对照组(NC组)、模型组(DSS组)和CGMCC No.25974干预组(LL271组)。第-7d~6d,LL271组和DSS组小鼠每天分别灌胃200μL CGMCC No.25974和PBS菌悬液。第0d~6d,DSS组和LL271组饮用3%(w/v)DSS。每日记录小鼠的体重、粪便性状和潜血状况,并根据表5中所示的评分标准、根据公式3计算疾病活动指数(Disease activity index,DAI)。实验结束后,颈椎脱臼处死小鼠,分离结肠,观察结肠表型和测量长度,拍照记录,并用4%多聚甲醛固定远端结肠组织进行H&E染色。病理评分标准如表6所示。
表5DAI评分标准
注:/表示无此项评分。DAI=体重下降评分+粪便性状评分+潜血状况评分(公式3)。
表6病理评分标准
结果如表7、表8和表9以及图3、图4、图5、图6所示,菌株CGMCC No.25974对实验动物无明显毒副作用,且能有效缓解小鼠急性结肠炎症状。表7和图3显示,予以CGMCCNo.25974菌悬液预灌胃7d(-7~-1d)未发现小鼠死亡情况,每日称重发现小鼠平均增长体重较为稳定,活动性及精神状态良好,干预组与对照组体重变化无差异;与NC组相比,饮用DSS干预后(即0~6d)DSS组和LL271组出现了不同程度的体重减轻,DSS组小鼠体重第4d起显著降低(P<0.05),这一现象持续到实验结束;而菌株CGMCC No.25974可减缓小鼠体重的下降,从第6d开始小鼠体重减轻相比DSS组已有所缓解(P<0.05)。表8和图4显示,与NC组相比,DSS组小鼠DAI评分第2d起显著升高(P<0.05),这一现象持续到实验结束;而菌株CGMCCNo.25974可降低小鼠DAI评分,从第3d起DAI评分显著低于DSS组(P<0.05),这一现象持续到实验结束。
表7各组小鼠每日体重统计表
注:与DSS组相比,*,P<0.05;***,P<0.001。
表8各组小鼠DAI得分表
注:与DSS组相比,*,P<0.05;**,P<0.01;***,P<0.001。
此外,图5和表9结果显示,DSS组和LL271组结肠长度与NC组相比缩短,且LL271组结肠显著长于DSS组(P<0.001);同时,NC组小鼠结肠弹性较好,肠内粪便呈颗粒状;DSS组小鼠结肠弹性较差,肠内无粪便或血便且明显水肿;LL271组较DSS组小鼠结肠弹性有明显恢复,肠内粪便可见颗粒状且无明显水肿和血便。组织病理学结果图6和表9显示,相较于NC组,LL271和DSS组病理评分显著升高,但LL271组病理评分显著低于DSS组(P<0.01)。其中,NC组小鼠结肠组织结构完整,未见明显的炎性改变;DSS组小鼠结肠组织可见较重的病理损伤,可见弥漫性黏膜溃疡,黏膜上皮及隐窝结构消失,被增生的结缔组织替代,杯状细胞数量重度减少;炎症反应重度,侵及黏膜下层,可见大量的淋巴细胞浸润;黏膜下层可见多灶性水肿,结缔组织疏松,并伴有少量淋巴细胞浸润;LL271组小鼠结肠组织可见较轻的病理损伤,可见小灶性黏膜坏死,杯状细胞数量轻度减少,炎症反应较轻,可见少量的淋巴细胞浸润。
表9各组小鼠结肠长度及病理评分表
注:与DSS组相比,**,P<0.01;***,P<0.001。
最后应说明的是:以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (10)
1.乳明串珠菌(Leuconostoc lactis)LL271,其特征在于,其保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.25974。
2.一种菌剂,其特征在于,所述菌剂含有权利要求1所述的乳明串珠菌(Leuconostoclactis)LL271。
3.权利要求2所述的菌剂的制备方法,其特征在于,所述方法包括将所述乳明串珠菌(Leuconostoc lactis)LL271经培养获得培养物的步骤。
4.根据权利要求3所述的制备方法,其特征在于,所述培养使用的培养基为含脱纤维羊血的MRS培养基;
所述培养基中,脱纤维羊血的体积百分含量为4-5%。
5.权利要求1所述的乳明串珠菌(Leuconostoc lactis)LL271或权利要求2所述的菌剂在制备用于缓解肠道炎症和/或改善肠黏膜损伤的药物中的应用。
6.权利要求1所述的乳明串珠菌(Leuconostoc lactis)LL271或权利要求2所述的菌剂在制备用于预防和/或治疗结肠炎的药物中的应用。
7.权利要求1所述的乳明串珠菌(Leuconostoc lactis)LL271或权利要求2所述的菌剂在制备用于预防和/或治疗炎症性肠病的药物中的应用。
8.权利要求1所述的乳明串珠菌(Leuconostoc lactis)LL271或权利要求2所述的菌剂在抑制病原菌中的应用。
9.权利要求1所述的乳明串珠菌(Leuconostoc lactis)LL271或权利要求2所述的菌剂在制备用于抑制病原菌的药物中的应用。
10.根据权利要求5~9任一项所述的应用,其特征在于,所述药物的有效成分含有权利要求1所述的乳明串珠菌(Leuconostoc lactis)LL271或权利要求2所述的菌剂。
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