CN114933993B - 一种解木聚糖拟杆菌及其复合制剂在缓解溃疡性结肠炎中的应用 - Google Patents
一种解木聚糖拟杆菌及其复合制剂在缓解溃疡性结肠炎中的应用 Download PDFInfo
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Abstract
本发明公开了一种解木聚糖拟杆菌及其复合制剂在缓解溃疡性结肠炎中的应用,属于微生物技术领域。本发明的解木聚糖拟杆菌NSP003能够快速利用果胶,并且解木聚糖拟杆菌NSP003及其复合制剂能够改善溃疡性结肠炎小鼠的疾病活动指数及脾脏肿大,维持肠道屏障完整,减少氧化应激水平,显著降低结肠炎临床诊断标志物的水平,效果优于临床药物美沙拉嗪。本发明所述的解木聚糖拟杆菌NSP003及其复合制剂用于制备缓解溃疡性结肠炎的食品、药物组合物、保健品或饲料添加剂,具有非常广泛的应用前景。
Description
技术领域
本发明涉及一种解木聚糖拟杆菌及其复合制剂在缓解溃疡性结肠炎中的应用,属于微生物技术领域。
背景技术
炎症性肠病(Inflammatory bowel disease,IBD)通常指由遗传易感性、肠道微生物组的改变、先天和适应性免疫系统的缺陷以及不同环境暴露等各种原因引起的结肠炎症性病变,主要包括溃疡性结肠炎(Ulcerative colitis,UC)和克罗恩病(Crohn's disease,CD),在临床上主要表现为腹泻、腹痛、黏液便及脓血便。目前,IBD已成为21世纪世界范围内面临的公共卫生挑战之一,在全球各地区的发病率呈上升趋势。据相关研究预测到2030年,我国城市地区的人口比例将达到60%,IBD的人口预计超过1.4亿,由此可能成为我国一个巨大的医疗保健负担。
IBD的临床表现可能因疾病表型的多样性而高度可变,至今仍缺乏有效的治疗手段。目前已知的针对溃疡性结肠炎的治疗方法主要是柳氮磺吡啶、美沙拉嗪等药物,其中的有效成分是5-氨基水杨酸,能够通过抑制结肠黏膜释放的白三烯和清除活性氧等,减少巨噬细胞和中性粒细胞释放与分泌促炎因子并减少TNF-α的产生,进而减轻对肠道黏膜的损害,使肠道功能得到改善。但这些药物治疗结肠炎的方法均存在诸多副作用,且具有高复发率。因此更安全有效的治疗方法亟需开发。
研究表明膳食纤维如果胶、菊粉、β-葡聚糖等能够通过缓解结肠萎缩、增强肠道屏障、调节炎症因子、降低氧化应激水平、维持肠道微生态平衡等方面缓解肠道炎症(LinlinFan,et al.Preventive effects of pectin with various degrees of esterificationon ulcerative colitis in mice[J].Food&Function,2020,11,2886-2897)。然而,因个体差异等原因,膳食纤维干预可能导致少数腹胀、腹鸣等不适的症状。因而通过挖掘益生菌种及其与膳食纤维的组合物,能够为缓解溃疡性结肠炎食品、药物组合物、保健品或饲料添加剂的开发提供新思路。
拟杆菌是人体肠道共生菌中含量最高的一类革兰氏阴性菌,具有大量碳水化合物降解基因,环境适应力强,并且与宿主的免疫、代谢及神经系统均存在紧密联系。目前已有观点认为拟杆菌可能成为“新一代益生菌”。例如,解木聚糖拟杆菌(Bacteroidesxylanisolvens)DSM23964能够激活TFα特异性免疫球蛋白M,经欧盟委员会(EU)2015/1291号实施决定,批准解木聚糖拟杆菌DSM23964可作为灭活发酵乳制品的发酵剂投放市场,表明了解木聚糖拟杆菌的应用潜力。
国内针对拟杆菌的研究大都集中在脆弱拟杆菌(Bacteroides fragilis)、吉氏副拟杆菌(Parabacteroides distasonis)等常见菌种,例如,专利CN111718869B公开了一株脆弱拟杆菌(Bacteroides fragilis)CCFM1122,能够促进肠道短链脂肪酸生成从而缓解内毒素引起的炎症;专利CN106389478B公开了一株脆弱拟杆菌(Bacteroides fragilis)ZY-312对于治疗和/或预防肥胖症或糖尿病具有很好的效果,不产生耐药性,且安全无毒,不含肠毒素基因;专利CN112410242A公开了一株吉氏副拟杆菌(Parabacteroides distasonis)DXBHYZ1,能够抑制结直肠癌细胞系增殖。
而目前,由于膳食纤维干预可能导致少数腹胀、腹鸣等不适的症状。因此亟需一株既能够降解纤维素缓解腹胀、腹鸣症状,又能够缓解溃疡性结肠炎症状的菌株。
发明内容
本发明总结现有技术的基础尝试,通过大量实验研究,筛选出了一种能够快速降解果胶并且具有缓解溃疡性结肠炎的解木聚糖拟杆菌,并证明该菌株以及菌株与果胶的复合剂在动物模型中可改善溃疡性结肠炎小鼠疾病活动指数及脾脏肿大,同时维持肠道屏障完整,降低氧化应激水平,缓解全身性及器官组织氧化损伤。进一步考察了其在食品、药物组合物、保健品或饲料添加剂中的应用,在溃疡性结肠炎的干预方面具有重要意义和广阔前景。
本发明提供了一株解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003,已于2021年1月19日保藏于广东省微生物菌种保藏中心,保藏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:61441。
所述解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003是从来源于江西南昌地区的健康人体粪便样本中分离得到的,该菌株经测序分析,其16S rDNA序列如SEQ IDNO.1所示,并将测序得到的序列在NCBI中进行核酸序列比对,结果显示,与解木聚糖拟杆菌属的核酸序列相似度高达99%,因此,该菌株为解木聚糖拟杆菌(Bacteroidesxylanisolvens),命名为:解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003。
所述的解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003具有下列性质:
菌体特征:革兰氏阴性杆状细菌,无孢子,无鞭毛,宽约0.9-1.2μm,长约3-8μm。
菌落特征:在培养基上形成明显地菌落,直径在0.3-2mm之间,正面形态圆形,中间凸起,边缘整齐,微白,不透明,表面湿润光滑。
生长特性:该菌株为严格厌氧菌,对氧气敏感,在温度35-38℃生长最佳,最适生长pH值为6.6-7.0,在含有葡萄糖的培养基中生长良好,16-24h可进入对数后期或稳定前期。
本发明还提供了一种复合制剂,其特征在于,所述复合制剂中含有上述解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003及益生元。
在本发明的一种实施方式中,所述复合制剂中,解木聚糖拟杆菌(Bacteroidesxylanisolvens)NSP003的活菌数不低于1×106CFU/mL或1×106CFU/g。
在本发明的一种实施方式中,所述复合制剂中,所述益生元为果胶,所述果胶的酯化度低于50%,并且所述果胶的添加量不低于复合制剂总质量的20%。
本发明还提供了一种微生物菌剂,所述微生物菌剂含有上述解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003或其发酵液,或其提取物。
在本发明的一种实施方式中,所述微生物菌剂中,解木聚糖拟杆菌(Bacteroidesxylanisolvens)NSP003的活菌数不低于1×106CFU/mL或1×106CFU/g。
本发明还提供了一种产品,所述产品中含有上述解木聚糖拟杆菌(Bacteroidesxylanisolvens)NSP003,或含有上述微生物菌剂,或含有上述复合制剂。
在本发明的一种实施方式中,所述产品中,解木聚糖拟杆菌(Bacteroidesxylanisolvens)NSP003的活菌数不低于1×106CFU/mL或1×106CFU/g。
在本发明的一种实施方式中,所述产品中,含有上述复合制剂,所述复合制剂中的益生元为果胶,所述果胶的酯化度低于50%,并且所述果胶的添加量不低于复合制剂总质量的20%。
在本发明的一种实施方式中,所述产品为食品、药物组合物、保健品或饲料添加剂。
在本发明的一种实施方式中,所述食品、药物组合物、保健品或饲料添加剂,含有上述微生物菌剂或复合制剂以及可接受的载体和/或辅料。
在本发明的一种实施方式中,所述可接受的辅料,包括一种或多种通常使用的增稠剂、抗氧化剂、酸碱调节剂、乳化剂、防腐剂、填充剂、粘合剂、润湿剂、崩解剂、润滑剂及矫味剂等。
本发明的一种实施方式中,所述填充剂为淀粉、蔗糖、乳糖、硫酸钙和/或微晶纤维素。
在本发明的一种实施方式中,所述粘合剂为纤维素衍生物、藻酸盐、明胶和/或聚乙烯吡咯烷酮。
在本发明的一种实施方式中,所述润湿剂为水、乙醇、淀粉和/或糖浆。
在本发明的一种实施方式中,所述崩解剂为羧甲基淀粉钠、羧丙纤维素、交联羧甲基纤维素、琼脂、碳酸钙和/或碳酸氢钠。
在本发明的一种实施方式中,所述润滑剂为滑石粉、硬脂酸钙、硬脂酸镁、微粉硅胶和/或聚乙二醇。
在本发明的一种实施方式中,所述矫味剂为单糖浆、蔗糖、卵磷脂、橙皮糖浆、樱桃糖浆、柠檬、茴香、薄荷油、海藻酸钠、阿拉伯胶、明胶、甲基纤维素、羧甲基纤维素钠、柠檬酸、酒石酸和/或碳酸氢钠。
在本发明的一种实施方式中,所述食品、药物组合物、保健品或饲料添加剂的剂型包括颗粒剂、胶囊剂、片剂、丸剂或口服液等剂型。
本发明还提供了上述解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003、上述微生物菌剂或复合制剂在预防和/或治疗溃疡性结肠炎方面的应用。
本发明还提供了上述解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003,或上述复合制剂,或上述微生物菌剂在制备预防和/或治疗溃疡性结肠炎的产品中的应用。
在本发明的一种实施方式中,所述产品中,解木聚糖拟杆菌(Bacteroidesxylanisolvens)NSP003的活菌数不低于1×106CFU/mL或1×106CFU/g。
在本发明的一种实施方式中,所述产品中,含有上述复合制剂,所述复合制剂中的益生元为果胶,所述果胶的酯化度低于50%,并且所述果胶的添加量不低于复合制剂总质量的20%。
在本发明的一种实施方式中,所述产品为食品、药物组合物、保健品或饲料添加剂。
在本发明的一种实施方式中,所述食品、药物组合物、保健品或饲料添加剂为,含有上述微生物菌剂或复合制剂以及可接受的载体和/或辅料。
在本发明的一种实施方式中,所述可接受的辅料,包括一种或多种通常使用的增稠剂、抗氧化剂、酸碱调节剂、乳化剂、防腐剂、填充剂、粘合剂、润湿剂、崩解剂、润滑剂及矫味剂等。
本发明的一种实施方式中,所述填充剂为淀粉、蔗糖、乳糖、硫酸钙和/或微晶纤维素。
在本发明的一种实施方式中,所述粘合剂为纤维素衍生物、藻酸盐、明胶和/或聚乙烯吡咯烷酮。
在本发明的一种实施方式中,所述润湿剂为水、乙醇、淀粉和/或糖浆。
在本发明的一种实施方式中,所述崩解剂为羧甲基淀粉钠、羧丙纤维素、交联羧甲基纤维素、琼脂、碳酸钙和/或碳酸氢钠。
在本发明的一种实施方式中,所述润滑剂为滑石粉、硬脂酸钙、硬脂酸镁、微粉硅胶和/或聚乙二醇。
在本发明的一种实施方式中,所述矫味剂为单糖浆、蔗糖、卵磷脂、橙皮糖浆、樱桃糖浆、柠檬、茴香、薄荷油、海藻酸钠、阿拉伯胶、明胶、甲基纤维素、羧甲基纤维素钠、柠檬酸、酒石酸和/或碳酸氢钠。
在本发明的一种实施方式中,所述食品、药物组合物、保健品或饲料添加剂的剂型包括颗粒剂、胶囊剂、片剂、丸剂或口服液等剂型。
有益效果
本发明筛选出了一株能够快速降解果胶的解木聚糖拟杆菌(Bacteroidesxylanisolvens)NSP003,并且该菌株或在与果胶复配后能够缓解溃疡性结肠炎,具体体现在:
1)降解果胶的效率高于已知具有抑炎作用的脆弱拟杆菌NCTC9343;
2)能够改善溃疡性结肠炎小鼠疾病活动指数及脾脏肿大,缓解炎症;
3)能够降低溃疡性结肠炎小鼠肠道通透性,增加结肠组织中紧密连接蛋白的水平,维持肠道屏障完整;
4)能够降低溃疡性结肠炎小鼠血清中的一氧化氮合成酶(iNOS)的水平,缓解全身性及器官组织氧化损伤。
5)能够降低溃疡性结肠炎临床诊断标志物-结肠内容物中钙防卫蛋白的水平。
本发明的解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003能够快速利用果胶,采用其同果胶进行复配后得到的复合制剂,能够缓解溃疡性结肠炎,效果优于果胶及阳性药物美沙拉嗪,能够避免某些个体对直接补充果胶等膳食纤维容易出现胀气等症状。
因此,本发明的解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003可用于制备能够缓解溃疡性结肠炎的药物组合物、食品、保健品或饲料添加剂,具有非常广泛的应用前景。
生物材料保藏
一株解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003,分类学命名为Bacteroides xylanisolvens,已于2021年1月19日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61441,保藏地址为广州市先烈中路100号大院59号楼5楼,广东省微生物研究所。
附图说明
图1:解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003在固体培养基上的菌落形态。
图2:解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003经革兰氏染色后显微镜下放大1000倍的形态。
图3:解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003在以果胶为唯一碳源培养基中的生长情况。
图4:解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003及其复合制剂干预溃疡性结肠炎小鼠后的疾病活动指数(A)及脾脏指数(B)的变化。
图5:解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003及其复合制剂干预溃疡性结肠炎小鼠后的肠道通透性(A)及结肠中紧密连接蛋白Occludin含量(B)的变化。
图6:解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003及其复合制剂干预溃疡性结肠炎小鼠后血清中一氧化氮合成酶iNOS的含量变化。
图7:解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003及其复合制剂干预溃疡性结肠炎小鼠后结肠内容物中钙防卫蛋白的含量变化。
图8:实施例中动物实验的分组设计及实施方案。
其中,模型组为DSS结肠炎小鼠组,NSP003为DSS+解木聚糖拟杆菌NSP003组,NSP003+果胶为DSS+解木聚糖拟杆菌NSP003+果胶组,果胶为DSS+果胶组,美沙拉嗪为DSS+阳性药物美沙拉嗪组。
“*”表示与模型组具有显著性差异(*:p<0.05;**:p<0.01;***:p<0.001);“#”表示与正常组具有显著性差异(#:p<0.05;##:p<0.01;###:p<0.001)。
具体实施方式
下述实施例中所涉及的Dextran-FITC购自Sigma-Aldrich公司,葡聚糖硫酸钠(DSS)购自MPBio公司,美沙拉嗪颗粒购自法国爱的发制药有限公司。
下述实施例中所涉及的脆弱拟杆菌NCTC9343购自广东省科学院微生物研究所。
下述实施例中所涉及的卵形拟杆菌(Bacteroides ovatus)S2PLA3、粪便拟杆菌(Bacteroides stercoris)S2Bt1、吉氏副拟杆菌(Parabacteroides distasonis)S4Bt9保藏于实验室菌种库中。
下述实施例中所涉及的果胶为酯化度低于50%的果胶,购自CPKelco公司。
下述实施例中所涉及的培养基如下:
脑心浸液BHI液体培养基:脑心浸液BHI液体培养基为青岛海博生物公司产品,将上述产品按说明溶解于蒸馏水中,然后加入半胱氨酸盐酸盐1g/L,维生素K1 0.002g/L,氯化血红素0.01g/L混合均匀,并调整pH至6.8-7.4,121℃灭菌15min后,即得到所述液体培养基。
脑心浸液BHI固体培养基:在脑心浸液BHI液体培养基的基础上,加入1.5%-2%的琼脂,混合均匀,然后调整pH至6.8~7.4,121℃灭菌15min。冷却至50-60℃时,向培养基中加入终浓度为0.0075‰的无菌万古霉素及0.1‰的卡那霉素,即得到所述固体培养基。
拟杆菌液体培养基的制备:配制脑心浸液BHI液体培养基(例如青岛海博生物技术有限公司的产品),溶解于蒸馏水中,并加入半胱氨酸盐酸盐1g/L,维生素K1 0.002g/L,氯化血红素0.01g/L,混合均匀,然后调整其pH为6.8-7.4,115-121℃灭菌15-20min后,即得到所述拟杆菌液体培养基。
拟杆菌固体培养基的制备:按照拟杆菌液体培养基的配方,再加入1.5-2%的琼脂,混合均匀,然后调整其pH为6.8-7.4,115-121℃灭菌15-20min。待冷却至50-60℃时,向培养基中加入终浓度为0.0075‰的无菌万古霉素及0.1‰的卡那霉素,即得到所述拟杆菌固体培养基。
下述实施例中所涉及的溶液的配制方法如下:
3%DSS的灭菌蒸馏水的配制方法为:称取30g的DSS,溶于蒸馏水中,采用高压蒸汽灭菌锅,121℃灭菌15min后,即得到所述3%DSS的灭菌蒸馏水。
下述实施例中所涉及的检测方法如下:
疾病活动指数(DAI)的判断标准如表1所示:
表1疾病活动指数(DAI)评分
注:正常粪便:成形粪便;松散粪便:不黏附于肛门的糊状、半成形粪便;稀便:黏附于肛门的稀水样粪便。
实施例1:解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003的分离筛选
1、样品采集
采集江西南昌地区的健康人体粪便样本,样本置于保藏管中,保存于装有冰袋的保温盒中,带回实验室后迅速置于-80℃冰箱待分离筛选。
2、解木聚糖拟杆菌的分离纯化
(1)粪便样品梯度稀释:在无菌厌氧环境中,取约1g步骤1采集到的粪便样本,加入到9mL生理盐水,得到第一梯度稀释液,吸取1mL第一梯度稀释液于9mL生理盐水,得到第二梯度稀释液,以此类推,共配制9个梯度稀释液;
(2)涂布培养:分别吸取100μL上述所有梯度稀释液分别置于拟杆菌固体培养基上,涂布后至于37℃厌氧条件下培养48h,得到稀释涂布平板;
(3)纯化培养:挑取拟杆菌固体培养基上不同形态的菌落进行划线分离,直至得到边缘整齐,微白,不透明,表面湿润光滑,且形态一致的纯的单菌落(如图1所示);挑取拟杆菌固体培养基上的纯菌落接种于5mL拟杆菌液体培养基中,至于37℃厌氧条件下培养24h,得到纯化培养液。
3、菌种保藏与鉴定
取10μL步骤2获得的纯化培养液在载玻片上进行涂片固定,用草酸铵结晶紫试剂染1分钟后,蒸馏水冲洗;再加碘液覆盖涂面染约1分钟,蒸馏水冲洗;加95%酒精数滴,并轻轻摇动进行脱色,20秒后水洗,吸去水分;用蕃红染色液染1分钟后,蒸馏水冲洗。干燥后镜检。结果如图2所示,发现编号NSP003的菌株为革兰氏阴性杆菌。
4、将步骤2获得的纯化培养液在8000rpm条件下离心10min,弃上清得菌体。用细菌16S rDNA引物(见表2)进行PCR。
表2引物名称
PCR产物经核酸电泳分析确认后,扩增产物送至公司进行测序,其16S rDNA序列如SEQ ID NO.1所示,测序结果与NCBI数据库中序列进行比对分析;比对结果发现编号NSP003的菌株为解木聚糖拟杆菌(Bacteroides xylanisolvens),命名为:解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003。
实施例2:解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003对果胶的降解作用
1、以果胶为唯一碳源的培养基配制
按比例称取蛋白胨20g/L,酵母提取物5g/L,NaCl 5g/L,K2HPO4 0.05g/L,KH2PO40.05g/L,半胱氨酸盐酸盐1g/L,氯化血红素0.01g/L,维生素K1 2mg/L,果胶5g/L,溶解于蒸馏水中,并调整pH至6.8~7.4,121℃灭菌15min后,即得到所述以果胶为唯一碳源的培养基。
2、实验方法
分别将解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003、脆弱拟杆菌NCTC9343以及实验室菌库中卵形拟杆菌(Bacteroides ovatus)S2PLA3、粪便拟杆菌(Bacteroides stercoris)S2Bt1、吉氏副拟杆菌(Parabacteroides distasonis)S4Bt9在脑心浸液BHI固体培养基上划线,长出单菌落后,挑取单菌落接种至步骤1配制得到的以果胶为唯一碳源的培养基进行适应性生长,在37℃下厌氧培养18~24h,制备得到种子液;
将上述种子液以2%(v/v)的接种量接种至新的步骤1配制得到的以果胶为唯一碳源的培养基中,在37℃下厌氧培养24h,分别在0h和24h测定培养液在λ=600nm波长下的吸光度,计算不同拟杆菌在以果胶为唯一碳源的培养基中的增长量(采用24h测定培养液在λ=600nm波长下的吸光度-0h测定培养液在λ=600nm波长下的吸光度得到的),结果如图3所示。
结果显示,经过24小时的厌氧培养,解木聚糖拟杆菌NSP003的OD600增长量能够达到0.31,显著高于脆弱拟杆菌NCTC9343的OD600增长量0.27、卵形拟杆菌S2PLA3的OD600增长量0.22、粪便拟杆菌S2Bt1的OD600增长量0.16及吉氏副拟杆菌S4Bt9的OD600增长量0.21。
以上结果表明,本发明的解木聚糖拟杆菌NSP003相比其它拟杆菌能够更加快速高效地利用果胶,特别是优于已有大量文献报道具有抑炎作用的脆弱拟杆菌NCTC9343(JuneL.Chan,et al.Non-toxigenic Bacteroides fragilis(NTBF)administration reducesbacteria-driven chronic colitis and tumor development independent ofpolysaccharide A.Mucosal Immunology,2019,12:164-177)。
实施例3:解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003及其复合制剂对溃疡性结肠炎小鼠疾病活动指数及脾脏指数的影响
1、解木聚糖拟杆菌NSP003冻存剂的制备:
(1)菌株培养:将解木聚糖拟杆菌NSP003在脑心浸液BHI固体培养基上划线,长出单菌落后,挑取接种至脑心浸液BHI液体培养基,在37℃下厌氧培养18~24h,制备得到解木聚糖拟杆菌NSP003种子液。
(2)保护剂制备:用蒸馏水配制体积分数为20%~25%的甘油溶液,并加入质量分数为0.1%的半胱氨酸盐酸盐,121℃灭菌15min后,即得到所述保护剂。
(3)保存方法:将步骤(1)制备得到的解木聚糖拟杆菌NSP003种子液在4℃下5000rpm离心10min后,取沉淀为解木聚糖拟杆菌NSP003菌体,将解木聚糖拟杆菌NSP003菌体用无菌PBS溶液(pH 7.2)清洗1~2次后,用步骤(2)制备的保护剂重悬使菌液浓度为5.0×108CFU/mL,即得所述解木聚糖拟杆菌NSP003冻存剂,于-80℃保存备用。
2、果胶溶液的制备
根据小鼠体重称取果胶,溶于水后即为果胶溶液。灌胃剂量为每千克小鼠体重300mg果胶,灌胃体积为0.2mL。
3、美沙拉嗪溶液的制备
根据小鼠体重称取美沙拉嗪颗粒,溶于质量分数为5%的羧甲基纤维素钠水溶液后即为美沙拉嗪溶液。灌胃剂量为每千克小鼠体重300mg美沙拉嗪,灌胃体积为0.2mL。
4、实验方法
取5周龄健康雄性C57BL/6小鼠60只,随机分为六组:正常组、溃疡性结肠炎组(模型组)、DSS+解木聚糖拟杆菌NSP003组(NSP003组)、DSS+解木聚糖拟杆菌NSP003+果胶组(NSP003+果胶组)、DSS+果胶组(果胶组)及DSS+美沙拉嗪(阳性药物对照,美沙拉嗪组),每组含小鼠10只。
小鼠饲养于25±2℃、相对湿度50±5%、12h光照12h黑暗的标准化实验室中,适应性正常喂养7天后开始为期14天的实验,实验流程如图8所示。
具体过程如下:
正常组:第1-14天,自由饮用灭菌蒸馏水;
干预治疗实验过程:
溃疡性结肠炎组(模型组):第1~7天,自由饮用灭菌蒸馏水,第8~14天,自由饮用含3%DSS的灭菌蒸馏水;
DSS+解木聚糖拟杆菌NSP003(NSP003组):第1~7天,自由饮用灭菌蒸馏水,第8~14天,自由饮用含3%DSS的灭菌蒸馏水;并且,第1~14天每天灌胃一次0.2mL步骤1制备的解木聚糖拟杆菌NSP003冻存剂(菌浓为5.0×108CFU/mL);
DSS+解木聚糖拟杆菌NSP003+果胶组(NSP003+果胶组):第1~7天,自由饮用灭菌蒸馏水,第8~14天,自由饮用含3%DSS的灭菌蒸馏水,并且,第1~14天每天灌胃一次0.2mL步骤1制备的解木聚糖拟杆菌NSP003冻存剂(菌浓为5.0×108CFU/mL)和0.2mL步骤2制备的果胶溶液(300mg/kg bw);
DSS+果胶组(果胶组):第1~7天,自由饮用灭菌蒸馏水,第8~14天,自由饮用含3%DSS的灭菌蒸馏水,并且,第1~14天每天灌胃一次0.2mL步骤2制备的果胶溶液(300mg/kg bw);
DSS+美沙拉嗪(阳性药物对照,美沙拉嗪组):第1~7天,自由饮用灭菌蒸馏水,第8~14天,自由饮用含3%DSS的灭菌蒸馏水,并且,第1~14天每天灌胃一次0.2mL步骤3制备的美沙拉嗪溶液(300mg/kg bw)。
5、实验结束后,处死所有小鼠,收集脾脏组织,称取脾脏组织的重量,根据其占据小鼠体重的百分数,换算脾脏指数,实验结果如图4所示。
图4A显示,模型组小鼠连续7天饮用含有3%DSS的灭菌蒸馏水后,小鼠的DAI值升至2.76(p<0.001)。而空白组小鼠的DAI值为0。
经解木聚糖拟杆菌NSP003干预后,小鼠DAI指数明显降低至2.40;NSP003+果胶组小鼠的DAI值降至2.24(p<0.01),低于阳性药物美沙拉嗪组小鼠的DAI值(2.38)。
图4B显示,模型组小鼠的脾脏肿大,脾脏指数从2.77(正常组)升至8.67(p<0.001)。经过解木聚糖拟杆菌NSP003干预后脾脏指数显著下降至6.13(p<0.01),NSP003+果胶组小鼠的脾脏指数下降至4.81(p<0.001),低于单独果胶干预的果胶组小鼠的脾脏指数(5.59)。
以上结果表明,本发明的解木聚糖拟杆菌NSP003及其与果胶的复合物均能够降低溃疡性结肠炎小鼠的疾病活动指数,缓解脾脏异常肿大,并且解木聚糖拟杆菌NSP003与果胶复配制剂的效果优于果胶。
实施例4:解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003及其复合制剂对溃疡性结肠炎小鼠肠道屏障的影响
具体实施方式同实施例3,区别在于,实验结束后,分别将正常组、溃疡性结肠炎组(模型组)、DSS+解木聚糖拟杆菌NSP003组(NSP003组)、DSS+解木聚糖拟杆菌NSP003+果胶组(NSP003+果胶)、DSS+果胶组(果胶组)及DSS+美沙拉嗪(阳性药物对照,美沙拉嗪组)小鼠在处死前6h开始禁食,处死前4h灌胃600mg/kg小鼠体重的Dextran-FITC,之后处死所有小鼠,收集血清及结肠组织样本。
用荧光分光光度计测定血清中的FITC含量,激发光485nm,发射光535nm。
采用ELISA试剂盒(南京森贝伽生物科技有限公司的产品)测定结肠组织中紧密连接蛋白(Occludin)的含量。实验结果如图5所示。
如图5A所示,模型组小鼠连续饮用7天含3%DSS的灭菌蒸馏水使得小鼠血清中的Dextran-FITC含量从0.06mg/L(正常组)显著升高至1.09mg/L(p<0.001)。
经解木聚糖拟杆菌NSP003干预后,小鼠血清中的Dextran-FITC含量明显降低至0.63mg/L,低于阳性药物美沙拉嗪组小鼠血清中的Dextran-FITC含量(0.78mg/L);NSP003+果胶组小鼠血清中的Dextran-FITC含量降至0.43mg/L(p<0.05),低于单独果胶干预的果胶组小鼠血清中的Dextran-FITC含量(0.48mg/L)及阳性药物美沙拉嗪组小鼠血清中的Dextran-FITC含量(0.78mg/L)。
同时,如图5B所示,模型组小鼠结肠中的紧密连接蛋白(Occludin)含量从110.64pg/mL(正常组)显著降至75.14pg/mL(p<0.001)。
NSP003组小鼠结肠中的紧密连接蛋白(Occludin)含量为93.80pg/mL(p<0.01),高于阳性药物美沙拉嗪组小鼠结肠中的紧密连接蛋白(Occludin)含量(87.38pg/mL);NSP003+果胶组小鼠结肠中的紧密连接蛋白(Occludin)含量为103.76pg/mL(p<0.001),高于单独果胶干预的果胶组小鼠结肠中的紧密连接蛋白(Occludin)含量(101.11pg/mL)及阳性药物美沙拉嗪组小鼠(87.38pg/mL)。
以上结果表明,本发明的解木聚糖拟杆菌NSP003及其与果胶的复合物均能够降低溃疡性结肠炎小鼠的肠道通透性,上调紧密连接蛋白的水平,维持肠道屏障完整,并且解木聚糖拟杆菌NSP003的效果优于临床药物,解木聚糖拟杆菌NSP003与果胶复配制剂的效果优于果胶及临床药物。
实施例5:解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003及其复合制剂对溃疡性结肠炎小鼠氧化应激水平的影响。
具体实施方式同实验例3,在实验后处死全部小鼠,收集血清及结肠组织样本。
采用ELISA试剂盒(南京森贝伽生物科技有限公司的产品)分别测定血清中一氧化氮合成酶iNOS含量,实验结果如图6所示。
结果显示,模型组小鼠血清中iNOS的含量从2.40μmol/L(正常组)显著上升至5.93μmol/L(p<0.001)。
经解木聚糖拟杆菌NSP003干预后,血清中iNOS的含量降低至4.04μmol/L(p<0.001)。
NSP003+果胶组小鼠血清中iNOS的含量为3.22μmol/L(p<0.001),低于单独果胶干预的果胶组小鼠血清中iNOS的含量(3.33μmol/L)及阳性药物美沙拉嗪组小鼠血清中iNOS的含量(3.36μmol/L)。
以上结果表明,本发明的解木聚糖拟杆菌NSP003及其与果胶的复合物均能够降低溃疡性结肠炎小鼠的氧化应激水平,从而缓解结肠炎相关的全身性及组织器官氧化损伤,并且解木聚糖拟杆菌NSP003的效果优于临床药物,解木聚糖拟杆菌NSP003与果胶复配制剂的效果优于果胶及临床药物。
实施例6:解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003及其复合制剂对溃疡性结肠炎诊断标志物的影响。
具体实施方式同实验例3,在实验后处死全部小鼠,收集结肠内容物样本,采用ELISA试剂盒(安迪生物科技有限公司)其中钙防卫蛋白的含量,实验结果如图7所示。
结果显示,模型组小鼠结肠内容物中的钙防卫蛋白水平从3.37pg/mL(正常组)显著上升18.89pg/mL(p<0.001)。
NSP003+果胶组小鼠结肠内容物中的钙防卫蛋白水平显著降低为10.78pg/mL(p<0.01),低于单独果胶干预的果胶组小鼠(11.07pg/mL)及阳性药物美沙拉嗪组小鼠(16.56pg/mL)。
以上结果表明,本发明的解木聚糖拟杆菌NSP003与果胶的复合物能够降低溃疡性结肠炎临床诊断标志物水平,并且效果优于果胶及临床药物。
实施例7:解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003菌剂的应用
解木聚糖拟杆菌NSP003菌剂可用于制备胶囊制品,胶囊制品的具体制备过程如下:
(1)将实施例1获得的解木聚糖拟杆菌NSP003纯化后的培养液以3%(v/v)的接种量接种到培养基中,37℃培养36h,6000rpm离心20min,收集菌泥;使用无菌水对菌泥进行清洗后,用脱脂乳重悬至浓度为2×1010CFU/mL,得到悬浮液;
(2)将步骤(1)制得的悬浮液添加至浓度为3%的海藻酸钠溶液中至浓度为2×109CFU/mL后,充分搅拌,使得解木聚糖拟杆菌NSP003的细胞均匀地分散于海藻酸钠溶液中,得到混合液;将混合液挤压到质量根数为2%的氯化钙溶液中形成胶粒;待形成的胶粒静止固化30min后,过滤收集胶粒;将收集得到的胶粒进行冷冻干燥48h,得到粉剂;将粉剂装入到药用胶囊中,得到胶囊制品。
实施例8:解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003复合制剂的应用
解木聚糖拟杆菌NSP003复合制剂可用于制备胶囊制品,胶囊制品的具体制备过程如下:
(1)将实施例1获得的解木聚糖拟杆菌NSP003纯化后的培养液以3%(v/v)的接种量接种到培养基中,37℃培养36h,6000rpm离心20min,收集菌泥;使用无菌水对菌泥进行清洗后,用脱脂乳重悬至浓度为2×1010CFU/mL,得到悬浮液;
(2)将步骤(1)制得的悬浮液添加至质量分数为3%的海藻酸钠溶液中至浓度为2×109CFU/mL后,充分搅拌,使得解木聚糖拟杆菌NSP003的细胞均匀地分散于海藻酸钠溶液中,得到混合液;将混合液挤压到质量分数为2%的氯化钙溶液中形成胶粒;待形成的胶粒静止固化30min后,过滤收集胶粒;将收集得到的胶粒进行冷冻干燥48h,得到粉剂;
(3)将步骤(2)制得的粉剂与果胶粉末一起装入到药用胶囊中,得到胶囊制品。
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。
SEQUENCE LISTING
<110> 南昌大学
<120> 一种解木聚糖拟杆菌及其复合制剂在缓解溃疡性结肠炎中的应用
<130> BAA211684A
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1183
<212> DNA
<213> 人工序列
<400> 1
cgatggcttt accctaggac gctccttgcg gttacgtact tcaggtaccc ccggctttca 60
tggcttgacg ggcggtgtgt acaaggcccg ggaacgtatt caccgcgcca tggctgatgc 120
gcgattacta gcgaatccag cttcacgaag tcgggttgca gacttcgatc cgaactgaga 180
gaggtttttg ggattagcat cctgtcgcca ggtagctgcc ttctgtaccc cccattgtaa 240
cacgtgtgta gccccggacg taagggccgt gctgatttga cgtcatcccc accttcctca 300
catcttacga cggcagtctc tctagagtcc tcagcatgac ctgttagtaa ctaaagataa 360
gggttgcgct cgttatggca cttaagccga cacctcacgg cacgagctga cgacaaccat 420
gcagcacctt cacatttgcc ttgcggctat actgtttcca atatattcaa atgcaattta 480
agcccgggta aggttcctcg cgtatcatcg aattaaacca catgttcctc cgcttgtgcg 540
ggcccccgtc aattcctttg agtttcaccg ttgccggcgt actccccagg tggaatactt 600
aatgctttcg cttggccgct tactgtatat cgcaaacagc gagtattcat cgtttactgt 660
gtggactacc agggtatcta atcctgtttg atacccacac tttcgagcat cagtgtcagt 720
tgcagtctag tgagctgcct tcgcaatcgg agttcttcgt gatatctaag catttcaccg 780
ctacaccacg aattccgccc acctctactg tactcaagac tgccagtttc aactgcaatt 840
ttacggttga gccgcaaact ttcacaactg acttaacaat ccacctacgc tccctttaaa 900
cccaataaat cccggataac gctcggatcc tccgtattac cgcggctgct ggcacggagt 960
tagccgatcc ttattcatat ggtacataca aatcccacac gtggatactt tattcccata 1020
taaagagttt acacccatag ggcagtcatc ttcacgctac ttggctgatc agactctcgt 1080
cattgaccat attcctcact gctgcctccc gtagagtttg accgtgttct cagtcatgtg 1140
ggactcctct cagaacccct atcatcgtag tctggtggcc gta 1183
Claims (8)
1.一株解木聚糖拟杆菌(Bacteroides xylanisolvens)NSP003,已于2021年1月19日保藏于广东省微生物菌种保藏中心,保藏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: 61441。
2.一种复合制剂,其特征在于,所述复合制剂中含有权利要求1所述的解木聚糖拟杆菌NSP003及益生元;所述益生元为果胶。
3.如权利要求2所述的复合制剂,其特征在于,所述的复合制剂中,解木聚糖拟杆菌NSP003的活菌数不低于1×106 CFU/mL或1×106 CFU/g;所述果胶的酯化度低于50%,所述果胶的添加量不少于总质量的20%。
4.一种包含权利要求1所述的解木聚糖拟杆菌NSP003,或权利要求2-3任一项所述的复合制剂的产品。
5.如权利要求4所述的产品,其特征在于,所述产品为微生物菌剂、食品、药物组合物、保健品或饲料添加剂。
6.如权利要求4或5所述的产品,其特征在于,所述产品中,解木聚糖拟杆菌NSP003的活菌数不低于1×106 CFU/mL或1×106 CFU/g。
7.权利要求1所述的解木聚糖拟杆菌NSP003,或权利要求2或3所述的复合制剂在制备预防和/或治疗溃疡性结肠炎的产品中的应用。
8.如权利要求7所述的应用,其特征在于,所述产品为药物组合物或饲料添加剂。
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"微生态制剂与炎症性肠病";张峰睿等;《世界华人消化杂志》;第21卷(第27期);第2792-2801页 * |
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