CN112646744A - 一株罗伊氏乳杆菌在预防和缓解溃疡性结肠炎中的应用 - Google Patents
一株罗伊氏乳杆菌在预防和缓解溃疡性结肠炎中的应用 Download PDFInfo
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- CN112646744A CN112646744A CN202011584808.0A CN202011584808A CN112646744A CN 112646744 A CN112646744 A CN 112646744A CN 202011584808 A CN202011584808 A CN 202011584808A CN 112646744 A CN112646744 A CN 112646744A
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Abstract
本发明公开了一株罗伊氏乳杆菌在预防和缓解溃疡性结肠炎中的应用,属于功能性微生物技术领域。罗伊氏乳杆菌CCFM1135能耐受人体胃肠环境,能够显著降低溃疡性结肠炎患病期间疾病活动指数,改善结肠粘膜损伤,降低MPO活性,减少结肠中促炎因子TNF‑α,IL‑6,IFN‑γ含量,上调结肠紧密连接相关蛋白Claudin‑3、ZO‑1、ZO‑2和Occludin的转录水平,上调结肠抗菌肽Reg3g、Reg3b的转录水平,提高肠道菌群多样性,降低粪便中不动杆菌属的相对丰度。
Description
技术领域
本发明涉及一株罗伊氏乳杆菌在预防和缓解溃疡性结肠炎中的应用,属于功能性微生物技术领域。
背景技术
炎症性肠病(Inflammatory bowel disease,IBD)是一种易复发的胃肠道疾病。炎症性肠病病因不明、病程长、易复发、并发症多且难以治愈。溃疡性结肠炎(ulcerativecolitis,UC)是炎症性肠病的一种,主要临床表现为慢性或亚急性腹泻,黏液脓血便和腹痛。UC是结肠黏膜层和黏膜下层连续性炎症,疾病通常由直肠开始向向全结肠蔓延。UC开始于西方国家,近几十年来,随着经济发展,亚洲、南美和中东等非发达地区也开始出现UC,目前UC已成为全球性疾病。近年来,中国的UC发病率呈快速增长趋势。UC的病程长,易复发,不仅影响患者的生活质量,还增加了患者罹患肠癌的风险。目前UC的发病机制尚不清楚,研究显示其主要与免疫因素、遗传因素和环境因素有关。
UC病因不明,且难以治愈,目前常用的UC治疗药物有三种,氨基水杨酸、激素和免疫抑制剂。但这三种药物对UC的治疗作用较局限且副作用较多。大量研究表明肠上皮正常的细胞间紧密连接相关蛋白保证了肠道屏障的完整性,从而维持正常的肠道功能进而保证人类健康。UC患者肠道内紧密连接蛋白的表达受到影响,进而导致肠道屏障功能的丧失,无法抵御有害菌的侵袭。因此,减轻炎症修复肠道屏障也是缓解UC的一种重要手段。近年来越来越多的研究显示,UC的发病与肠道菌群的异常有直接关系,而随着人们对肠道菌群与肠道疾病相关性的逐步深入了解,人们已经认识到,调节肠道菌群修复肠道屏障是缓解UC的一种重要手段。一些肠道菌可以影响黏蛋白的表达,修复肠道黏液层的厚度。近年来,一些特殊的益生菌菌株被提出在干预UC的过程中能起到有益作用,这种效果在益生菌中不是普遍性的,具有菌株特异性。但是目前还未有相关的能够起到很好预防、调节或治疗作用的益生菌。
发明内容
鉴于目前还未有效果较好的缓解UC的菌株,提出了本发明。
本发明为克服现有技术中存在的不足,本发明提供了一株罗伊氏乳杆菌(Lactobacillus reuteri),于2020年7月24日保藏于广东省微生物菌种保藏中心,地址为广州市先烈中路100号大院59号楼5楼,保藏编号为GDMCC No:61102。
提供了罗伊氏乳杆菌CCFM1135在制备改善溃疡性结肠炎症状的产品中的应用。
在本发明的一种实施方式中,所述改善溃疡性结肠炎症状包括(a)~(f)至少一方面:
(a)改善结肠粘膜损伤;
(b)降低髓过氧化物酶(MPO)活性;
(c)减少结肠中促炎因子的含量;
(d)上调结肠中的抗菌肽的转录水平;
(e)提高肠道菌群多样性;
(f)降低粪便中不动杆菌属菌株的相对丰度。
在本发明的一种实施方式中,所述改善结肠粘膜损伤包括提高结肠中与屏障功能相关蛋白的转录水平,所述蛋白包括Claudin-3、ZO-1、ZO-2和Occludin。
在本发明的一种实施方式中,所述结肠中促炎因子包括TNF-α,IL-6,IFN-γ。
在本发明的一种实施方式中,所述结肠中的抗菌肽包括Reg3g、Reg3b。
在本发明的一种实施方式中,所述改善溃疡性结肠炎症状的产品包括发酵食品,或含有罗伊氏乳杆菌的益生菌制剂,或药物,或药物组合物。
在本发明的一种实施方式中,所述发酵食品为使用所述罗伊氏乳杆菌发酵生产制得,所述发酵食品包括固态食品、液态食品、半固态食品。
在本发明的一种实施方式中,所述发酵食品包括乳制品、豆制品、果蔬制品。
在本发明的一种实施方式中,所述乳制品包括酸奶、奶油、干酪;所述果蔬制品包括黄瓜、胡萝卜、甜菜、芹菜、圆白菜、香蕉、木瓜制品。
在本发明的一种实施方式中,所述益生菌制剂中含有不低于1.0×106cfu/g的罗伊氏乳杆菌。
在本发明的一种实施方式中,所述的药物或药物组合物中的含量为不低于1.0×106cfu/mL或1.0×106cfu/g。
在本发明的一种实施方式中,所述药物或药物组合物还包括药学上可接受的赋型剂;所述药学上可接受的赋型剂是指任何可用于药学领域的稀释剂、辅助剂和/或载体。
本发明还提供制备含有罗伊氏乳杆菌的菌剂的方法。
在本发明的一种实施方式中,罗伊氏乳杆菌的培养基为MRS培养基。
在本发明的一种实施方式中,罗伊氏乳杆菌CCFM1135菌种按照2-4mL/100mL的接种量接种到在119-123℃条件下灭菌15-25min后的培养基中,然后在温度35-39℃厌氧条件下培养18-24h,用pH为7.0-7.4磷酸盐缓冲液清洗2-4次,用所述的保护剂重悬,使菌浓度达到1010CFU/mL;接着,悬浮液在温度37℃厌氧条件下预培养50-70min,再在-15~-20℃预冻8-14h,进行真空冷冻干燥得到所述的发酵菌剂。
在本发明的一种实施方式中,保护剂为100g/L-150g/L脱脂奶粉,和/或100g/L-150g/L麦芽糊精,和/或140g/L-160g/L海藻糖,余量为水的保护剂。
本发明的优点和效果:
本发明罗伊氏乳杆菌CCFM1135对胃肠液具有较好的耐受能力,能够显著降低溃疡性结肠炎患病期间疾病活动指数,改善结肠粘膜损伤,降低MPO活性,减少结肠中促炎因子TNF-α,IL-6,IL-1β,IFN-γ含量,上调结肠紧密连接相关蛋白Claudin-3、ZO-1、ZO-2和Occludin的转录水平,上调结肠抗菌肽Reg3g、Reg3b的转录水平,提高肠道菌群多样性,降低粪便中不动杆菌属的相对丰度,还可用于制备具有预防UC的乳制品、豆制品与果蔬制品,具有非常广泛的应用前景。
生物材料保藏
本发明所提供的罗伊氏乳杆菌Lactobacillus reuteri CCFM1135,分类学命名为Lactobacillus reuteri,于2020年7月24日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61102,保藏地址为广州市先烈中路100号大院59号楼5楼。
附图说明
图1是罗伊氏乳杆菌CCFM1135的菌落形态图;
图2是罗伊氏乳杆菌CCFM1135对UC小鼠结肠结肠粘膜损伤的影响图;
图3是罗伊氏乳杆菌CCFM1135对UC小鼠结肠组织病理评分和结肠MPO活性的影响图;
图4是罗伊氏乳杆菌CCFM1135对UC小鼠结肠中促炎因子含量的影响图;
图5是罗伊氏乳杆菌CCFM1135对UC小鼠结肠紧密连接相关蛋白转录水平的影响图;
图6是罗伊氏乳杆菌CCFM1135对UC小鼠结肠抗菌肽Reg3g和Reg3b转录水平的影响图;
图7是罗伊氏乳杆菌CCFM1135对UC小鼠肠道菌群多样性的影响图;
图8是罗伊氏乳杆菌CCFM1135对对UC小鼠粪便中不动杆菌属相对丰度的影响图。
具体实施方案
罗伊氏乳杆菌CCFM1135具有下述生物学特性:
(1)菌体特征:呈革兰氏染色阳性,不形成孢子,不运动的细菌。
(2)菌落特征:有氧或厌氧培养36小时形成明显的菌落,直径在0.5-2mm之间,正面形态圆形,侧面形态呈突起状,边缘不齐,乳白色,不透明,表面湿润光滑,不产生色素,参见附图1。
(3)生长特性:在37℃恒温有氧或厌氧的条件下,在mMRS培养基中培养约15小时达到对数末期。
(4)对模拟胃肠液具有较好的耐受能力。
本菌株罗伊氏乳杆菌CCFM1135的获得方法为:
(1)取1g来自健康人群的新鲜粪便。将样品在含有山梨醇GM17培养基中35℃富集12h;
(2)将富集样品进行梯度稀释后涂布于添加了0.02%嗅甲酚紫的GM17固体平板上,培养24~48h;
(3)选取变色圈明显,并且符合乳酸菌目基本形态的单菌落进行平板划线纯化,筛选分离出乳酸菌;
(4)将上述单菌落培养于液体GM17培养液中培养24h后进行革兰氏染色,选取革兰氏阳性菌进行后续试验。
mMRS培养基:MRS培养基+0.05%半胱氨酸盐酸盐。
罗伊氏乳杆菌FCQHC8L6:与本发明的菌株罗伊氏乳杆菌CCFM1135采用相同的方法从粪便中筛选得到。
实施例1:罗伊氏乳杆菌CCFM1135对模拟胃肠液具有良好的耐受性
将冷冻保存的罗伊氏乳杆菌CCFM1135接种于mMRS培养基中,在温度37℃厌氧培养48h,再经mMRS培养液传代培养2~3次后,取1mL罗伊氏乳杆菌CCFM1135的培养液,与9.0mLpH 2.5人工模拟胃液(含1%胃蛋白酶、pH=2.5的mMRS培养基)混合,并在37℃下厌氧培养,分别在0h、0.5h、1h、2h和3h时取样,用mMRS琼脂培养基浇注培养进行平板菌落计数,测定活菌数并计算其存活率(见表1)。
取1mL罗伊氏乳杆菌CCFM1135的培养液加入9mL人工模拟肠液(含0.3%牛胆盐、1%胰蛋白酶、pH=8.0的mMRS培养基)中,在37℃下厌氧培养,分别在0h、0.5h、1h、2h、3h和4h时取样,用mMRS琼脂培养基浇注培养进行平板菌落计数,测定活菌数并计算其存活率。存活率是在该培养液中在取样时的活菌数对数值与在第0h时活菌数对数值之比,以%表示(见表2)。
实验结果如表1和表2所示。结果表明,罗伊氏乳杆菌CCFM1135对人工胃肠液具有较好的耐受性。
表1罗伊氏乳杆菌CCFM1135在人工模拟胃液中的耐受性
表2罗伊氏乳杆菌CCFM1135在人工模拟肠液中的耐受性
实施例2:罗伊氏乳杆菌CCFM1135对C57BL/6J小鼠无毒副作用
将罗伊氏乳杆菌CCFM1135菌体重悬于3mg/100mL(w/v)的蔗糖溶液中,制成浓度为5.0×109CFU/mL的菌悬液。取体重14-16g左右的健康雄性C57BL/6J小鼠6只,适应环境一周后,每日给予该浓度菌悬液灌胃一次(每次灌胃0.2mL),观察一周,记录死亡和体重情况。
这些试验结果列于表3中。这些结果表明,喂食浓度5.0×109CFU/mL的罗伊氏乳杆菌CCFM1135未对小鼠造成明显影响,体重无显著变化,无死亡现象产生。小鼠外观无明显病理症状。
表3小鼠体重的变化及死亡情况
时间(天) | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
体重(g) | 20.13±0.42 | 20.42±0.37 | 20.70±0.19 | 20.92±0.31 | 21.24±0.25 | 21.59±0.34 | 21.96±0.28 |
死亡情况 | - | - | - | - | - | - | - |
注:-:小鼠无死亡
实施例3:罗伊氏乳杆菌CCFM1135对UC小鼠疾病症状的影响
取体重14-16g的健康雄性C57BL/6J小鼠30只,适应环境1周,每组6只小鼠,随机分为5组:空白组、模型组、药物组、罗伊氏乳杆菌CCFM1135干预组(CCFM1135)、罗伊氏乳杆菌FCQHC8L6对照组(FCQHC8L6)。灌胃菌悬液的剂量为5.0×109CFU/mL,重悬于3%(w/v)的蔗糖溶液中,每日灌胃0.2mL。第5周在饮水中添加2.5%(w/v)的葡聚糖硫酸钠(dextransulphate sodium,DSS)持续7天以诱导小鼠结肠炎。实验动物分组及处理方法见表4。
表4动物实验设计
第五周,即造模期间(DSS处理),每天定时称量小鼠体重并计算其变化的百分比。此外,每日观察小鼠的粪便性状,将其分为三个等级:第一,正常小鼠的粪便成型,且为颗粒状;第二,粪便粘度增加且易散,但不粘附于***则为“松散”;第三,粪便呈稀水样或不成形且粘附于***,即为“稀便”。同时,使用匹拉米洞法测定小鼠粪便隐血情况,若小鼠粪便含有肉眼可见的红褐色或鲜红色血液,则判定为肉眼便血。最后,根据小鼠体重、粪便性状和便血情况,计算得出小鼠的疾病活动指数(Disease activity index,DAI),具体评分项目的得分如表5。
表5动物疾病活动指数(DAI)评分***
表6小鼠造模期间疾病活动指数
实验结果如表6所示。如表6,使用DSS造模期间,UC模型组小鼠的疾病活动指数显著上升,模型组小鼠体重从第5天开始显著下降,在第七天时体重下降率接近10%,并在第7天时增长至2.72±0.14,而罗伊氏乳杆菌CCFM1135干预后显著抑制了疾病活动指数的上升,即CCFM1135干预可减少UC小鼠的体重下降,并改善粪便性状和便血情况,这表明本发明筛选的罗伊氏乳杆菌CCFM1135具有缓解UC小鼠疾病活动的功能。
实施例4:罗伊氏乳杆菌CCFM1135可改善小鼠结肠粘膜损伤
C57BL/6J小鼠分组、造模及处理方法同实施例3。
在第36天处死小鼠后,收集小鼠结肠组织,按照MPO试剂盒说明书测定结肠MPO活性,制作结肠石蜡切片并进行HE染色,实验步骤为:
(1)取距***1cm处的一段远端结肠1cm用4%多聚甲醛固定48h;
(2)将固定好的结肠组织流水冲洗8h后进行脱水,样品依次经70%、80%、90%各级乙醇溶液脱水,各30min,再放入95%、100%各2次,每次20min;
(3)将结肠样品放入1∶1的二甲苯和酒精混合液中15min,然后放入二甲苯I和二甲苯II各15min;
(4)将结肠组织转移至二甲苯和石蜡各半的混合液15min,再放入石蜡Ⅰ、石蜡Ⅱ透蜡各1h,温度保持60℃;
(5)用莱卡石蜡包埋机将结肠包埋于重新融化的蜡块中;
(6)用组织切片机对包埋好的组织切片,厚度为5μm;
(7)粘片后晾干,置于62℃烘箱中1h。
HE染色过程如下:
(1)石蜡切片经二甲苯Ⅰ、Ⅱ脱蜡各5min,然后放入100%、95%、90%、80%、70%各级酒精溶液中各3-5min,再放入蒸馏水中3min;
(2)用苏木精染色20s;
(3)蒸馏水洗去未结合的苏木精;
(4)伊红染色2S,依次进入95%乙醇Ⅰ、Ⅱ,70%乙醇中,并快速拿出,然后进入80%乙醇中50~55s,进入无水乙醇中2min;
(5)切片放入1:1的二甲苯和酒精混合液中1min,接着放入二甲苯Ⅰ、Ⅱ中各2~3min;
(6)用中性树胶封片。
实验结果如图2和图3所示。由图2可见,模型组小鼠的结肠可见大量黏膜上皮细胞变性、坏死,杯状细胞数量明显减少和肠隐窝损伤,炎细胞浸润等病理变化,其结肠组织损伤评分显著高于空白对照组(图3),且结肠MPO值显著升高(图3)。灌胃罗伊氏乳杆菌CCFM1135显著改善了UC小鼠结肠粘膜损伤,且结肠结构接近于空白组(图2),显著降低结肠组织损伤评分(图3)。此外,CCFM1135处理显著降低了UC小鼠的MPO活性并接近于空白对照组(图3)。说明罗伊氏乳杆菌CCFM1135能够显著改善UC小鼠的结肠粘膜损伤,且效果优于药物美沙拉嗪和罗伊氏乳杆菌FCQHC8L6。
实施例5:罗伊氏乳杆菌CCFM1135可显著降低结肠中促炎因子含量
C57BL/6J小鼠分组、造模及处理方法同实施例3。
在第36天处死小鼠后,收集小鼠结肠组织。小鼠结肠组织按1:9比例加入预冷PBS缓冲液进行组织研磨,12000×g,离心15min,取上清,分别按照TNF-α、IL-1β、IL-6和IFN-γ酶联免疫试剂盒的检测方法测定结肠中TNF-α、IL-1β、IL-6和IFN-γ的含量。
实验结果如图4所示,模型组小鼠结肠中促炎因子TNF-α、IL-6和IFN-γ含量显著增高,灌胃罗伊氏乳杆菌CCFM1135显著降低了小鼠结肠中促炎因子TNF-α、IL-6和IFN-γ含量,且效果优于药物美沙拉嗪和罗伊氏乳杆菌FCQHC8L6。
实施例6:罗伊氏乳杆菌CCFM1135可增强肠道屏障功能
C57BL/6J小鼠分组、造模及处理方法同实施例3。
在第36天处死小鼠后,收集小鼠结肠组织测定结肠中紧密连接相关蛋白Claudin-3、ZO-1、ZO-2和Occludin的转录水平。测定方法如下:合成Claudin-3、ZO-1、ZO-2、Occludin和GAPDH的引物序列,引物信息如表6。取小鼠同部位的结肠组织1cm,迅速放入液氮中,置于负80℃冰箱冻存,取出冻存结肠组织放入已加入1mL TRIzol和3粒锆珠的1.5mL无酶离心管中,用组织研磨匀浆机充分匀浆,室温静置5min。加入0.2mL氯仿,剧烈振荡30s,静置10min。接着以12000g,4℃离心15min。小心吸取上层水相至一新的无酶1.5mL离心管中,加入等体积异丙醇,上下轻轻颠倒混匀,室温静置10min。接着以12000g,4℃离心15min。弃去上清,加入1mL预冷75%乙醇,轻弹洗涤沉淀。以12000g,4℃离心5min,小心吸取弃去上清,于超净台内吹干沉淀,加入50μL无酶超纯水溶解RNA。使用微量分光光度计测定提取的RNA浓度,OD260/OD280在1.9~2.0之间的质量合格。以提取质量合格的总RNA为模板,按照反转录试剂盒说明书的步骤合成cDNA。反转录得到的cDNA进行q RT-PCR检测,PCR体系为:5μL SYBRGreen Supermix,3μL去离子水,0.5μL上游引物(10μmol/L),0.5ul下游引物(10μmol/L)和1μL的cDNA模板(100ng/μL)。QPCR运行程序设定:94℃,2min,(94℃,30s;61℃,30s;72℃,20s)39个循环;目的基因经过Real-time PCR检测后,以GAPDH为内参基因,采用2-△△CT法进行相对基因表达分析。
表7引物序列
实验结果如附图5所示。由图5可以看出,模型组小鼠结肠中四种主要紧密连接蛋白Claudin-3、ZO-1、ZO-2和Occludin均发生了显著下调,灌胃罗伊氏乳杆菌CCFM1135显著提高了结肠中四种紧密连接相关蛋白的转录水平,说明罗伊氏乳杆菌CCFM1135能够增强UC小鼠的肠道屏障功能。
实施例7:罗伊氏乳杆菌CCFM1135可提高UC小鼠结肠抗菌肽Reg3b和Reg3g的转录水平
C57BL/6J小鼠分组、造模及处理方法同实施例3。
在第36天处死小鼠后,收集小鼠结肠组织测定结肠中抗菌肽Reg3g、Reg3b的转录水平。测定方法如下:合成Reg3g、Reg3b和GAPDH的引物序列,引物信息如表8。取小鼠同部位的结肠组织1cm,迅速放入液氮中,置于负80℃冰箱冻存,取出冻存结肠组织放入已加入1mLTRIzol和3粒锆珠的1.5mL无酶离心管中,用组织研磨匀浆机充分匀浆,室温静置5min。加入0.2mL氯仿,剧烈振荡30s,静置10min。接着以12000g,4℃离心15min。小心吸取上层水相至一新的无酶1.5mL离心管中,加入等体积异丙醇,上下轻轻颠倒混匀,室温静置10min。接着以12000g,4℃离心15min。弃去上清,加入1mL预冷75%乙醇,轻弹洗涤沉淀。以12000g,4℃离心5min,小心吸取弃去上清,于超净台内吹干沉淀,加入50μL无酶超纯水溶解RNA。使用微量分光光度计测定提取的RNA浓度,OD260/OD280在1.9~2.0之间的质量合格。以提取质量合格的总RNA为模板,按照反转录试剂盒说明书的步骤合成cDNA。反转录得到的cDNA进行qRT-PCR检测,PCR体系为:5μL SYBR Green Supermix,3μL去离子水,0.5μL上游引物(10μmol/L),0.5μL下游引物(10μmol/L)和1μL的cDNA模板(100ng/μL)。QPCR运行程序设定:94℃,2min,(94℃,30s;61℃,30s;72℃,20s)39个循环;目的基因经过Real-time PCR检测后,以GAPDH为内参基因,采用2-△△CT法进行相对基因表达分析。
实验结果如附图6所示。由图6可以看出,罗伊氏乳杆菌CCFM1135干预后显著提高了结肠中抗菌肽Reg3g、Reg3b的转录水平。其中,CCFM1135对Reg3b相对mRNA表达水平的上调作用约为空白组的6倍,对Reg3g的上调作用也接近空白组的6倍。
Reg3g-F:ATGCTTCCCCGTATAACCATCA,SEQ ID NO.11,
Reg3g-R:GGCCATATCTGCATCATACCAG,SEQ ID NO.12,
Reg3b-F:ACTCCCTGAAGAATATACCCTCC,SEQ ID NO.13,
Reg3b-R:CGCTATTGAGCACAGATACGAG,SEQ ID NO.14,
GAPDH-F:TCAAGAAGGTGGTGAAGCAG,SEQ ID NO.15,
GAPDH-R:AAGGTGGAAGAGTGGGAGTTG,SEQ ID NO.16。
实施例8:罗伊氏乳杆菌CCFM1135能够提高UC小鼠肠道菌群多样性
C57BL/6J小鼠分组、造模及处理方法同实施例3。
试验末期收集小鼠新鲜粪便冻存于-80℃,用DNA快速提取试剂盒按说明书方法步骤提取粪便中的DNA,对样本16S rDNA的V3-V4区进行PCR扩增,用胶回收试剂盒对扩增片段进行纯化回收,在Illumina miseq pe300平台进行高通量测序,采用QIIME2分析平台对扩增子进行分析。
PCR引物:
F:5’-AYTGGGYDTAAAGNG-3’,SEQ ID NO.17;
R:5’-TACNVGGGTATCTAATCC-3’,SEQ ID NO.18。
实验结果如附图7所示。由实验结果可以看出,与空白组相比,模型组小鼠Shannon指数显著降低,即肠道菌群多样性显著下降。罗伊氏乳杆菌CCFM1135干预后显著提高shannon指数,说明罗伊氏乳杆菌CCFM1135干预后显著提高了UC小鼠肠道菌群多样性。
实施例9:罗伊氏乳杆菌CCFM1135能够降低UC小鼠粪便中不动杆菌属的相对丰度
C57BL/6J小鼠分组、造模及处理方法同实施例3。
试验末期收集小鼠新鲜粪便冻存于-80℃,用DNA快速提取试剂盒按说明书方法步骤提取粪便中的DNA,对样本16S rDNA的V3-V4区进行PCR扩增(采用SEQ ID NO.17和18),用胶回收试剂盒对扩增片段进行纯化回收,在Illumina miseq pe300平台进行高通量测序,采用QIIME2分析平台对扩增子进行分析。
实验结果如附图8所示。由实验结果可以看出,与空白组相比,模型组小鼠粪便中致病菌属不动杆菌属(Acinetobacter)的相对丰度显著上升。罗伊氏乳杆菌CCFM1135干预后显著降低了不动杆菌属的相对丰度。
实施例10:罗伊氏乳杆菌CCFM1135在制备发酵食品中的应用
罗伊氏乳杆菌CCFM1135菌种按照2-4mL/100mL的接种量接种到在119-123℃条件下灭菌15-25min后的培养基中,然后在温度35-39℃厌氧条件下培养18-24h,用pH为7.0-7.4磷酸盐缓冲液清洗2-4次,用所述的保护剂重悬,使菌浓度达到1010CFU/mL;接着,悬浮液在温度37℃厌氧条件下预培养50-70min,再在-15~-20℃预冻8-14h,进行真空冷冻干燥得到所述的发酵菌剂;其中所用的保护剂为100g/L-150g/L脱脂奶粉,和/或100g/L-150g/L麦芽糊精,和/或140g/L-160g/L海藻糖,余量为水的保护剂。
选用新鲜的水果和蔬菜洗净后榨汁,接着进行高温瞬间灭菌,在温度140℃下高温热杀菌2秒后,立即降温至37℃,再接入本发明制备的罗伊氏乳杆菌CCFM1135发酵菌剂,使其浓度达到1.0×106CFU/mL以上,在温度4℃下冷藏保存,于是得到含有本发明罗伊氏乳杆菌CCFM1135活菌的果蔬饮料。
本发明的罗伊氏乳杆菌CCFM1135还能制备其他发酵食品,其中发酵食品包括乳制品、豆制品、果蔬制品,乳制品包括酸奶、奶油、干酪;果蔬制品包括黄瓜、胡萝卜、甜菜、芹菜、圆白菜、香蕉、木瓜制品。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
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Claims (10)
1.罗伊氏乳杆菌(Lactobacillus reuteri),于2020年7月24日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61102。
2.权利要求1所述罗伊氏乳杆菌在制备改善溃疡性结肠炎症状的产品中的应用。
3.如权利要求2所述的应用,其特征在于,所述改善溃疡性结肠炎症状包括(a)~(f)至少一方面:
(a)改善结肠粘膜损伤;
(b)降低髓过氧化物酶活性;
(c)减少结肠中促炎因子的含量;
(d)上调结肠中的抗菌肽的转录水平;
(e)提高肠道菌群多样性;
(f)降低粪便中不动杆菌属菌株的相对丰度。
4.如权利要求3所述的应用,其特征在于,所述改善结肠粘膜损伤包括提高结肠中与屏障功能相关蛋白的转录水平,所述蛋白包括Claudin-3、ZO-1、ZO-2和Occludin。
5.如权利要求4所述的应用,其特征在于,所述结肠中促炎因子包括TNF-α,IL-6,IFN-γ。
6.如权利要求5所述的应用,其特征在于,所述结肠中的抗菌肽包括Reg3g、Reg3b。
7.如权利要求2所述的应用,其特征在于,所述产品包括发酵食品,或含有罗伊氏乳杆菌的益生菌制剂,或药物,或药物组合物。
8.如权利要求7所述的应用,其特征在于,所述发酵食品为使用所述罗伊氏乳杆菌发酵生产制得,所述发酵食品包括固态食品、液态食品、半固态食品。
9.如权利要求8所述的应用,其特征在于:所述发酵食品包括乳制品、豆制品、果蔬制品,所述乳制品包括酸奶、奶油、干酪;所述果蔬制品包括黄瓜、胡萝卜、甜菜、芹菜、圆白菜、香蕉、木瓜制品。
10.如权利要求9的应用,其特征在于,所述的药物或药物组合物中的含量为不低于1.0×106cfu/mL或1.0×106cfu/g。
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CN110591945A (zh) * | 2019-09-06 | 2019-12-20 | 吉林大学 | 一株用于预防溃疡性结肠炎的优良罗伊氏乳杆菌 |
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CN110591945A (zh) * | 2019-09-06 | 2019-12-20 | 吉林大学 | 一株用于预防溃疡性结肠炎的优良罗伊氏乳杆菌 |
CN113209282A (zh) * | 2021-05-05 | 2021-08-06 | 浙江大学 | 维持肠道阿克曼式菌丰度的抗菌肽的用途 |
CN113209282B (zh) * | 2021-05-05 | 2022-05-27 | 浙江大学 | 维持肠道阿克曼式菌丰度的抗菌肽的用途 |
CN113398171A (zh) * | 2021-07-07 | 2021-09-17 | 北京浩鼎瑞生物科技有限公司 | 一种含白头翁提取物的药物组合物及其应用 |
CN113403231A (zh) * | 2021-07-08 | 2021-09-17 | 江南大学 | 一株可干预代谢综合征的罗伊氏乳杆菌ccfm1178及应用 |
CN113430153A (zh) * | 2021-08-10 | 2021-09-24 | 浙江大学 | 降血压的罗伊氏乳杆菌zjuids09及其应用 |
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CN114404459B (zh) * | 2022-01-27 | 2023-09-12 | 江南大学 | 罗伊氏乳杆菌ccfm1135在降低血浆氧化三甲胺上的应用 |
CN115040551A (zh) * | 2022-06-08 | 2022-09-13 | 华南农业大学 | 一株调控宿主褪黑素水平的罗伊氏乳酸杆菌ln0214的应用 |
CN117982541A (zh) * | 2024-02-07 | 2024-05-07 | 中山大学肿瘤防治中心(中山大学附属肿瘤医院、中山大学肿瘤研究所) | 中华伊格尔兹氏菌在制备防治炎症性肠病和结直肠癌的产品中的应用 |
CN117982541B (zh) * | 2024-02-07 | 2024-07-23 | 中山大学肿瘤防治中心(中山大学附属肿瘤医院、中山大学肿瘤研究所) | 中华伊格尔兹氏菌在制备防治炎症性肠病和结直肠癌的产品中的应用 |
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