CN115612652B - 植物乳杆菌as21及其在预防溃疡性结肠炎中的应用 - Google Patents
植物乳杆菌as21及其在预防溃疡性结肠炎中的应用 Download PDFInfo
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Abstract
本发明公开了一株植物乳杆菌及其应用,属于微生物领域,具体涉及植物乳杆菌AS21及其在预防溃疡性结肠炎中的应用。本发明提供的植物乳杆菌保藏编号为CGMCC No.24968,分类命名为植物乳杆菌Lactobacillus plantarum,于2022年5月26日保藏于中国普通微生物菌种保藏管理中心。本发明提供的植物乳杆菌具有极高的抗氧化性,并对结肠炎具有一定改善作用,能够用于食品、保健品和药品。
Description
技术领域
本发明属于微生物领域,具体涉及植物乳杆菌AS21及其在预防溃疡性结肠炎中的应用。
背景技术
炎症性肠病(IBD)是一种难以治愈的慢性非特异性疾病,包括克罗恩病(CD)和溃疡性结肠炎(UC)。临床IBD的症状多种多样,包括腹痛、便血、腹泻、体重减轻和胃肠道穿孔等,极大地影响了患者的生活质量。UC是IBD的主要形式,因为它的反复发作和长期治疗已发展成为一种全球性疾病。目前,大多数可用的IBD疗法涉及全身抑制免疫系统和手术,但并不能使所有患者受益。因此,寻找IBD病理下更有效的治疗策略成为研究热点。氧化应激与肠道组织的许多病理变化和疾病活动密切相关。实验模型和临床研究的累积数据表明,氧化应激参与并促进IBD的发展。氧化应激与炎症反应密切相关,包括促使肠道屏障中细胞因子的过度释放导致肠道组织损伤。肠道屏障受损使炎症因子和自由基进入血液,进一步导致对其他器官的病理反应,诱发脑神经炎症和氧化损伤。越来越多的证据表明抗氧化疗法可以缓解和治疗IBD。此外,调节氧化防御系统和抑制ROS的产生是IBD的潜在治疗选择。最近的研究表明,肠道微生物环境的平衡在调节结肠炎症和氧化损伤方面起着至关重要的作用,UC患者和小鼠模型中肠道微生物和微生物代谢物的组成发生了巨大变化。
益生菌是对宿主有益的活的微生物,近年来引起了IBD研究的广泛关注。乳酸菌(Lactic acid bacteria,LAB)是最著名的益生菌,通常用作各种疾病的营养、功能食品和辅助药物。目前的研究发现,一些LAB菌株具有抗氧化酶和氧化损伤修复系统相关的强大氧化还原系统,这有助于它们在氧化应激下生存。适当摄入LAB可以增加宿主体内抗氧化酶的活性和抗氧化物质的含量。一些商业菌株如乳酸杆菌和双歧杆菌已被用于缓解UC患者的临床症状。此外,补充乳酸菌可以通过平衡肠道微生物环境来调节结肠炎症和氧化损伤。由于不同菌株的作用和功能存在差异,需要更多的证据来证明LAB作为体内抗氧化剂的能力和作用机制。然而,具有抗氧化特性的LAB菌株对抗氧化和抗炎功能、调节肠道微生物群和屏障的机制尚不清楚。在青藏高原极端环境下更有可能筛选出具有高抗氧化能力的特殊功能乳酸菌,对维持人体健康具有十分重要的意义。
发明内容
为了解决上述问题,本发明从传统发酵牦牛奶中分离出具有高抗氧化活性的植物乳杆菌AS21,并验证了它在DSS(葡聚糖硫酸钠)诱导的结肠炎小鼠模型中对肠道微生物群、肠道屏障功能、氧化应激和炎症的改善作用。
一方面,本发明提供了一株具有高抗氧化活性的植物乳杆菌。
所述的植物乳杆菌为植物乳杆菌AS21,保藏编号为CGMCC No.24968,分类命名为植物乳杆菌
Lactobacillus plantarum,于2022年5月26日保藏于中国普通微生物菌种保藏管理中心,保藏地址为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。
所述的植物乳杆菌筛选自青藏高原传统发酵牦牛乳。所述的植物乳杆菌的形态特征为:亮白色圆形菌落,菌落饱满凸起,边缘光滑。
另一方面,本发明提供了一种微生物培养物。
所述的微生物培养物通过前述的植物乳杆菌制备。具体地,所述的微生物培养物为将前述的植物乳杆菌接种于培养基制备。
所述的培养基可以是固体培养基,液体培养基或半固体培养基。优选地,所述的微生物培养物为发酵液,为前述植物乳杆菌接种于液体培养基培养制备得到。
再一方面,本发明提供了一种益生菌冻干粉。
所述的益生菌冻干粉中包括前述的植物乳杆菌。所述的益生菌冻干粉可以由前述植物乳杆菌的培养物冻干获得。
优选地,所述的益生菌冻干粉可以由前述的植物乳杆菌的发酵液冻干获得,更优选为发酵液沉淀冻干获得。
又一方面,本发明提供了一种微生物菌剂。
所述的微生物菌剂中包括前述的植物乳杆菌。
具体地,所述的微生物菌剂包括前述的植物乳杆菌菌体和其他辅料。所述的其他辅料可以是赋形剂、吸附剂等。
又一方面,本发明提供了前述的植物乳杆菌的培养方法。
所述的培养方法中包括:将前述植物乳杆菌接种于培养基后培养。
所述的培养基可以是固体培养基,液体培养基或半固体培养基。
又一方面,本发明提供了前述的植物乳杆菌或微生物培养物或益生菌冻干粉或微生物菌剂在制备用于结肠炎治疗或预防或预后的药物中的应用。
优选地,所述的结肠炎为溃疡型结肠炎。
所述的药物中包括前述的植物乳杆菌或微生物培养物或益生菌冻干粉或微生物菌剂。所述的药物中还包括其他药学上可接受的载体或赋形剂。
又一方面,本发明提供了前述的植物乳杆菌或微生物培养物或益生菌冻干粉或微生物菌剂在制备化妆品中的应用。
优选地,所述的化妆品用于抗氧化。
又一方面,本发明提供了一种用于结肠炎治疗或预防或预后的药物。
所述的药物中包括前述的植物乳杆菌或微生物培养物或益生菌冻干粉或微生物菌剂。
又一方面,本发明提供了一种化妆品。
所述的化妆品中包括前述的植物乳杆菌或微生物培养物或益生菌冻干粉或微生物菌剂。
所述的化妆品中还可能包括本领域常用的辅料中的一种或多种,如:保湿剂、柔润剂、乳化剂、调色剂、芳香剂、油性原料、表面活性剂、稳定剂、增稠剂、粘结剂、防腐剂等。
优选地,所述的化妆品具有抗氧化和清除自由基的功效。
本发明的有益效果:
(1)本发明提供的植物乳杆菌具有较强的超氧化物歧化酶和谷胱甘肽歧化酶活性,在胆盐耐受能力、抑菌能力、疏水能力以及模拟胃肠道的体外试验中都表现较好的益生特性,能够应用在制备药品中;
(2)本发明提供的植物乳杆菌可改善肠道菌群,防止短链脂肪酸的减少,减轻炎症因子以及氧化损伤的产生,进而防止肠道组织形态、小鼠肠道黏液层、肠道紧密连接性的损伤,改善小鼠肠病理现象,缓减由DSS引起的小鼠结肠炎;
(3)本发明提供的高抗氧化活性的植物乳杆菌能够有效提高线虫寿命,增加线虫体内抗氧化及含量,降低血清中丙二醛(MDA)的含量,能够应用在药品中,具有抗氧化和清除自由基的能力。
保藏说明
保藏编号:CGMCC No.24968;
分类命名:植物乳杆菌
Lactobacillus plantarum;
保藏时间:2022年5月26日;
保藏单位:中国普通微生物菌种保藏管理中心;
保藏单位简称:CGMCC;
保藏地址: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。
附图说明
图1为本发明实施例1中植物乳杆菌AS21对DSS诱导小鼠体重和DAI评分的影响。
图2为植物乳杆菌AS21对DSS诱导小鼠结肠长度的影响。
图3为植物乳杆菌AS21对DSS诱导小鼠组织病理学变化的影响。
图4为植物乳杆菌AS21对DSS诱导小鼠肠道粘液层的影响。
图5为植物乳杆菌AS21对DSS诱导的结肠炎小鼠肠道屏障完整性的影响。
图6为植物乳杆菌AS21对DSS诱导小鼠结肠氧化应激和炎症反应的影响。
图7为植物乳杆菌AS21对DSS诱导小鼠肠道内容物的影响。
图8为植物乳杆菌AS21对DSS诱导小鼠肠道菌群的影响。
图9为植物乳杆菌AS21对DSS诱导小鼠肠道菌群门水平相对丰度的影响。
图10为植物乳杆菌AS21对DSS诱导小鼠肠道菌群属水平相对丰度的影响。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
基础实验例植物乳杆菌的筛选
本发明的植物乳杆菌分离自青藏高原传统发酵牦牛乳;经高H2O2耐受能力,DPPH自由基清除能力,羟自由基(OH自由基)清除能力,超氧阴离子(O2-)清除能力,还原能力,总抗氧化能力(T-AOC)和超氧化物歧化酶(SOD)活性的测定;乳酸菌饲喂秀丽隐杆线虫的自然寿命,运动能力,抗氧化剂和抗氧化损伤能力,自由基随年龄积累情况的检测,和线虫在热应激和H2O2应激情况下的寿命的测定,综合筛选出植物乳杆菌AS21。所述植物乳杆菌具有良好的H2O2耐受能力,DPPH自由基清除能力,羟自由基(OH自由基)清除能力,超氧阴离子(O2-)清除能力,还原能力,总抗氧化能力(T-AOC)和超氧化物歧化酶(SOD)活性;可延长秀丽隐杆线虫的自然寿命,增强线虫运动能力,增强线虫在热应激和H2O2应激情况下的寿命,减少线虫自由基随年龄的积累,增强线虫的抗氧化剂和抗氧化损伤能力具有高抗氧化活性。
本发明筛选的乳酸菌菌种AS21,经测序,与数据库比对后确认为植物乳杆菌
Lactobacillus plantarum,于2022年5月26日保藏于中国普通微生物菌种保藏管理中心,保藏编号为CGMCC No.24968。
本发明的乳酸菌菌种AS21具有以下特征:亮白色的圆形菌落,菌落饱满凸起,边缘光滑。
实施例1植物乳杆菌AS21对结肠炎小鼠模型的影响
1.饲喂菌株制备
1 mL乳酸菌菌液添加至10 mL的MRS培养基,37℃培养12 h后全部加入1 L的MRS培养基中,37℃培养12 h后,5000 g离心5 min,收集菌泥至无菌培养皿中。将菌泥添加适量保护剂(脱脂乳8%,蔗糖10%,丙三醇3%,山梨醇3%,105℃灭菌15 min)后,在-40℃下冷冻干燥,研磨成菌粉。取0.1 g菌粉至10 mL生理盐水中,混匀后取1 mL液体至10 mL新的生理盐水中记为梯度1,在梯度1的液体基础上进行梯度稀释,每个梯度都是由上一个梯度的液体中取1mL液体至10 mL新的生理盐水中,依次重复到梯度2-9后,每个梯度各自取100 μL混悬液在MRS固体培养基上涂布,37℃培养24 h后对平板上的菌落进行计数,每个梯度3个平行。通过计算得到所制备的菌粉每克活菌数约为1.0×1011CFU,菌粉保存在4℃冰箱备用。
2.DSS结肠炎小鼠模型建立(菌株饲喂模式)
无特定病原体(SPF)级C57BL / 6J小鼠(雌性,7周龄,16-18 g,购自中国农业科学院兰州兽医研究所实验动物中心)在SPF级动物房适应2周后,将30只小鼠随机分为三组,每组10只。
将乳酸菌粉悬浮于PBS缓冲液中用于灌胃,采用DSS(MW = 36 - 50 kDa;MPBiomedicals, LLC, Solon, OH, USA)对小鼠进行溃疡性结肠炎造模。
各组具体处理方式如下:
正常对照组(NC):第0-14天灌胃PBS缓冲液(200μL);
DSS诱导组(DSS):第0-14天灌胃PBS缓冲液(200μL);第7-14天补充含3% DSS的水;
DSS+乳酸菌组(DSS+LA):第0-14天灌胃200μL菌液(1.0×109CFU);第7-14天补充含3% DSS的水。
检测项目:(1)DSS造模期间,每天定时称量小鼠体重,记录小鼠粪便,计算小鼠体重与初始体重百分比,并用于对小鼠进行疾病活动指数评分。疾病活动指数(Diseaseactivity index, DAI)评分系统是在以往报道的基础上,根据本研究的实际情况进行了一些调整,如表1所示,DAI评分为体重减轻、腹泻和便血的总和,最高为10分。
表1 疾病活动指数评分表
(2)第14天时,对小鼠实施安乐死取样,测量结肠长度,取少量结肠用于切片染色,剩余结肠组织冷冻于-80°C用于后续实验,用于结肠组织病理学分析。
处死小鼠后,使用冰冷的磷酸盐缓冲盐水清洗远端结肠,并使用直尺测量。
(3)取远端结肠(距肛门约2 cm)用4%多聚甲醛固定,乙醇脱水,石蜡包埋,制成5 μm冠状切片,脱蜡并用苏木精和伊红(H&E)染色。最后,使用光学显微镜获得结肠形态,并参照表2,根据炎症细胞浸润、组织损伤和上皮损伤的程度对组织损伤程度进行评分。
表2组织损伤评分
(4)肠道粘液层观察使用阿辛蓝/核固红染色,观察肠道黏膜组织。将结肠组织垂直包埋在石蜡中并切成5μm的切片。切片用阿辛蓝/核固红染色,用光学显微镜观察结肠组织切片。
(5)评价肠道屏障完整性:使用免疫组化法对肠道组织紧密连接蛋白进行染色分析,制备组织切片,用二甲苯脱蜡,然后在分级酒精系列中水合。组织切片置于盛满柠檬酸(pH 6.0)抗原修复液的高压锅内进行抗原修复,喷气计时2.5 min,自然冷却后将玻片置于PBS(pH 7.4)中在脱色摇床上晃动洗涤3次,每次5 min。切片放入3%甲醇过氧化氢溶液,室温避光孵育20 min,将玻片置于 PBS(pH 7.4)中在脱色摇床上晃动洗涤3次,每次5 min以消除内源性过氧化物酶。切片稍甩干后用组化笔在组织周围画圈(防止抗体流走),在圈内滴加用5% BSA,室温封闭1 h。轻轻甩掉封闭液,在切片上滴加1:200的Occludin和Claudin-1的抗体(市购),切片平放于湿盒内4摄氏度孵育过夜。玻片置于 PBS(pH 7.4)中在脱色摇床上晃动洗涤3次,每次8 min。切片稍甩干后在圈内滴加生物素偶联的二抗,覆盖组织,室温孵育50 min。之后玻片置于 PBS(pH 7.4)中在脱色摇床上晃动洗涤3次,每次 8 min。切片稍甩干后在圈内滴加HRP标记的链霉卵白素的三抗,覆盖组织,室温孵育50 min。之后玻片置于 PBS(pH7.4)中在脱色摇床上晃动洗涤3次,每次8 min。玻片置于 PBS(pH7.4)中在脱色摇床上晃动洗涤3次,每次5 min。切片稍甩干后在圈内滴加新鲜配制的显色液,复染细胞核后脱水封片。用光学显微镜观察染色的组织,评估组织紧密连接蛋白的变化。
(6)氧化应激和炎症反应:使用商品化ELISA试剂盒检测结肠中的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽(GSH)、氧化型谷胱甘肽(GSSG)、丙二醛(MDA)、肿瘤坏死因子-α(TNF-α)、白细胞介素1β(IL-1β)、白细胞介素6(IL -6)和结肠中的干扰素-γ(IFN-γ)细胞因子。结肠中的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽(GSH)、氧化型谷胱甘肽(GSSG)和丙二醛(MDA)水平检测方法严格按照制造商的指南执行(南京建成科技有限公司)。商品化ELISA试剂盒(上海江莱生物科技有限公司)用于测定肿瘤坏死因子-α(TNF-α)、白细胞介素1β(IL-1β)、白细胞介素6(IL -6)和结肠中的干扰素-γ(IFN-γ)细胞因子。样品处理和测试程序严格按照制造商的说明执行。
(7)肠道内容物(小鼠粪便)短链脂肪酸分析使用气相检测小鼠结肠中短链脂肪酸(SCFAs),采用外标法计算各短链脂肪酸的浓度。将小鼠粪便(每个样品200 mg)置于2 mL离心管中,然后加入500 μL饱和氯化钠溶液,4℃ 振摇3 min,室温静置30 min。然后,加入硫酸(40 µL,10%)进行酸化,涡旋1 min。向管中加入800 µL乙醚,然后在4℃下涡旋1 min,然后在12000×g(15 min,4℃)下离心;随后,加入0.25 g无水硫酸钠后,将800 µL上乙醚清液加入新的离心管中。将样品静置10 min,以12000×g(15 min,4℃)离心,取上清液经0.22μm孔径过滤器过滤后置于气相瓶中。通过GC(Thermo Fisher Scientific,MA,USA)测量样品中 SCFAs(乙酸盐、丙酸盐、丁酸盐、异丁酸盐、戊酸盐和异戊酸盐)的浓度。GC使用Rtx-Wax色谱柱,柱长30 m,内径2.25μm;载气为氦气,流速为2 mL/min;进样量为1 μL,分流比为10:1。注射温度设定为240℃,按以下程序升温:初始温度为100℃,以7.5℃/min 升温至140℃,然后以60℃/min升温至200℃,保持3 min,电离温度220℃,分析采用全扫描模式。采用外标法计算各短链脂肪酸的浓度。
(8)肠道菌群分析使用16SrRNA检测小鼠结肠微生物组,分析主要由专业测序公司完成,具体步骤和方法如下:
使用商业EZNA Stool DNA Kit (Omega, Norcross, GA, USA) 提取结肠内容物的总基因组DNA。使用NanoDrop ND-2000分光光度计(Thermo Fisher Scientific,Waltham,MA,USA)、Qubit 3.0 荧光计(Life Technologies,CA,USA)和琼脂糖凝胶电泳分别测量提取的DNA的数量和质量。
使用正向引物27F (5'-AGGRGTTYGATYMTGGCTCAG-3') 和反向引物1492R (5'-RGYTACCTTGTTACGACTT-3') 对细菌全长16S rRNA进行PCR(聚合酶链反应)扩增。样品特异性16 bp条形码被整合到引物中进行多重测序。PCR扩增子用Agencourt AMPure珠(BeckmanCoulter,Indianapolis,IN,USA)纯化,并使用PicoGreen dsDNA Assay试剂盒(Invitrogen,Carlsbad,CA,USA)进行定量。然后使用 SMRTbell Template Prep Kit 1.0-SPv3 将样本用于生成文库,并使用武汉弗雷泽根生物信息学有限公司(中国武汉)的PacBio平台和DNA/聚合酶Binging Kit 3.0(PacBio)进行测序。
实验结果:
(1)小鼠体重均值如图1中的A所示,小鼠饮用含有3%的DSS水一周后(即实验的第14天),出现明显的急性结肠炎症状,其中包括体重减轻(P<0.01),植物乳杆菌AS21灌胃缓解了DSS处理引起的体重减轻(P<0.01);DAI评分均值如图1中的B所示,口服3%的DSS饮用水一周后,小鼠疾病活动指数(DAI)增加(P<0.01),植物乳杆菌AS21灌胃减轻了DSS引起的小鼠腹泻和便血症状(DAI值降低,P<0.01);
(2)小鼠结肠长度测量示意图如图2中的A所示;结肠长度均值如图2中的B所示,植物乳杆菌AS21可以显著改善DSS处理小鼠的结肠萎缩情况(P<0.05);
(3)远端结肠切片苏木精和伊红染色结果如图3中的A所示,组织评分均值如图3中的B所示,结果表明植物乳杆菌AS21显著减轻了DSS诱导小鼠的结肠溃疡、隐窝受损以及中性粒细胞和单核细胞浸润等肠道组织破坏情况(P<0.01)。
(4)肠道粘液层观察结果见图4,结果表明,植物乳杆菌AS21的摄入抑制了DSS引起的肠道黏膜损伤,减少了杯状细胞的损失(P<0.01),保护了小鼠肠黏膜层。
(5)肠道屏障完整性检测结果如图5所示,结果表明,植物乳杆菌AS21摄入减轻了DSS引起的小鼠肠道跨膜蛋白数量减少,减轻了小鼠肠道屏障完整性的降低(P<0.01)。
(6)氧化应激和炎症反应结果如图6,结果表明,植物乳杆菌AS21摄入减轻了DSS治疗小鼠结肠中氧化应激水平以及炎症因子反应。特别地,植物乳杆菌AS21摄入改善了DSS诱导的结肠中SOD活性的降低(P<0.01),降低了GSSG含量(P<0.01),增加了GSH与GSSG的比率(P<0.05),抑制了结肠中的MDA含量(P<0.01)。显著下调DSS诱导的结肠组织中细胞因子包括TNF-α、IL-1β、IL-6 和IFN-γ的水平(P<0.01)。
(7)小鼠结肠中短链脂肪酸检测结果如图7所示,结果表明,植物乳杆菌AS21摄入改善DSS诱导小鼠的短链脂肪酸含量降低。特别地,显著恢复了DSS引起的乙酸(P<0.01)、丙酸盐(P<0.05)和丁酸盐(P<0.05)含量的降低。
(8)肠道菌群测序分析结果如图8-10所示,图8中的A为各组小鼠肠道菌群PcoA 分析结果,B-E为特定菌群相对丰度统计结果。结果表明,植物乳杆菌AS21摄入减少了DSS引起有害菌丰度,保护肠道有益共生菌丰度。特别地,显著提升了DSS诱导的厚壁菌门富集度的降低(P<0.01),显著抑制了DSS诱导的变形菌门(P<0.01)和去铁细菌门(P<0.01)富集度的增加。
综上,本发明提供的植物乳杆菌AS21对DSS诱导小鼠溃疡性结肠炎有缓解作用;能较好地维持小鼠体重,减轻病理学评分;能有效保护结肠组织、黏膜屏障和肠道紧密连接性;通过增加肠道抗氧化剂和减少炎症因子降低肠道氧化应激反应和炎症;通过保护肠道中短链脂肪酸含量和肠道菌群组成,维持肠道稳态。产品无毒副作用,可作为药品保护肠道组织和稳态。
Claims (6)
1.一株具有高抗氧化活性的植物乳杆菌,其特征在于,保藏编号为CGMCC No.24968,分类命名为植物乳杆菌Lactobacillus plantarum。
2.一种微生物培养物,其特征在于,通过培养权利要求1所述的植物乳杆菌制备。
3.一种益生菌冻干粉,其特征在于,包括权利要求1所述的植物乳杆菌。
4.一种微生物菌剂,其特征在于,包括权利要求1所述的植物乳杆菌。
5.权利要求1所述的植物乳杆菌或权利要求2所述的微生物培养物或权利要求3所述的益生菌冻干粉或权利要求4所述的微生物菌剂在制备用于溃疡性结肠炎治疗或预防的药物中的应用。
6.一种用于溃疡性结肠炎治疗或预防的药物,其特征在于,包括权利要求1所述的植物乳杆菌或权利要求2所述的微生物培养物或权利要求3所述的益生菌冻干粉或权利要求4所述的微生物菌剂。
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