CN113041266A - 一株改善银屑病样小鼠病理特征的干酪乳杆菌及其应用 - Google Patents
一株改善银屑病样小鼠病理特征的干酪乳杆菌及其应用 Download PDFInfo
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- CN113041266A CN113041266A CN202110291624.3A CN202110291624A CN113041266A CN 113041266 A CN113041266 A CN 113041266A CN 202110291624 A CN202110291624 A CN 202110291624A CN 113041266 A CN113041266 A CN 113041266A
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Abstract
本发明公开了一株改善银屑病样小鼠病理特征的干酪乳杆菌及其应用，属于微生物技术领域以及医药技术领域。本发明提供了干酪乳杆菌(Lactobacillus casei)CCFM1074作为口服制剂或口服制剂的主要成分，改善银屑病样小鼠病理特征，及缓解银屑病方面的新用途。该菌株可通过内用口服的形式使银屑病样病变皮肤情况好转、银屑病样皮肤表皮层增厚被抑制、病变耳朵增厚被抑制，并使银屑病样皮肤中IL‑23水平降低39.1％，IL‑17水平降低20.2％，结肠内容物中乙酸水平升高75.2％，在制备预防和/或治疗银屑病的产品(如食品、药品或保健食品等)中，具有巨大的应用前景。
Description
技术领域
本发明涉及一株改善银屑病样小鼠病理特征的干酪乳杆菌及其应用，属于微生物技术领域以及医药技术领域。
背景技术
银屑病是一种免疫介导的慢性复发性红斑鳞屑性皮肤病，最常见于皮肤和关节，但也与心血管、代谢和神经精神作用有关。银屑病影响全球人口约2％，但在不同种族中的患病率存在着明显差异。银屑病的症状发作到诊断的均值时间为2年，平均诊断年龄为34岁。在纬度较高的国家银屑病的患病率似乎更高。作为一种皮肤病，银屑病可表现为多种表型，包括斑块型银屑病(又称寻常型银屑病)、点状银屑病、逆型银屑病、脓疱性银屑病、掌跖牛皮癣和红皮病性银屑病。同一个体可能出现多种表型，其中寻常型银屑病是最常见的。所有表型共有的症状包括皮肤上层的大小不一的丘疹和红斑，并且表面还会覆盖着银白色鳞屑，且多产生于头皮、手脚伸侧易被他人看见的部位，影响患者社交生活，也给患者的心理带来了极大的压力。
银屑病的病因虽然进行过许多研究，但至今尚不十分清楚。目前认为，银屑病的发生不是单一的原因，而是涉及遗传、环境、免疫等多方面。遗传方面，目前研究认为银屑病是多基因遗传疾病，约30％的患者具有家族史。环境方面，受潮、感染、外伤、饮酒、进食鱼虾、精神紧张及吸烟与银屑病发病有关。免疫方面，银屑病最初被描述为一种“Th1”疾病，因为患者皮肤中发现的促炎细胞因子是由T辅助型1(Th1)细胞产生的，如白细胞介素-1(IL-1)、肿瘤坏死因子α(TNF-α)和干扰素γ(INF-γ)等。然而，越来越多的研究证明了CD4阳性辅助性T淋巴细胞17(Th17)细胞在银屑病病理机制中起到了关键作用，由Th17细胞产生的细胞因子，如IL-17和IL-22也在病变皮肤中被发现。银屑病患者体内Th细胞产生的细胞因子混合物作用于真皮和表皮细胞，并改变角质形成细胞和其他细胞的基因表达和成熟。除此之外，银屑病患者的调节性T细胞数量也减少了，这使得银屑病皮肤中的过度免疫反应不易被调节。银屑病目前没有彻底治愈的医疗方法和药物，并且大多伴有副作用，因此银屑病的治疗仍然是一个世界性医疗难题。
在银屑病的发病机制中，肠道结构和功能之间可能的关系近年来引起了广泛的关注。因此，肠道的改变和银屑病之间的关系似乎变得更强，这表明肠道参与和皮肤疾病之间的病理生理学联系。在健康的肠道菌群中，原始CD4阳性T细胞分化为效应T细胞(Th1、Th2和Th17)和调节性T细胞(Tregs)之间存在平衡。而肠道微生物的紊乱可能通过T细胞的活动引发多种免疫紊乱，并导致炎症。因此，肠道微生物群失调伴随的肠屏障破坏可能是慢性炎症性疾病发展的重要因素。益生菌是通过定殖在人体内，改变宿主某一部位菌群组成的一类对宿主有益的活性微生物。益生菌的代谢产物短链脂肪酸是联系宿主和肠道菌群的重要中介物质，具有生物学效应。如丁酸，可通过促进Treg细胞分化，进而影响Th17/Treg平衡，从而降低IL-17水平。
综上所述，或许可通过开发能够改善银屑病样小鼠病理特征的益生菌，从而解决银屑病难治疗、易反复、副作用大的现状。
发明内容
技术问题：
本发明要解决的技术问题是提供干酪乳杆菌(Lactobacillus casei)CCFM1074作为口服制剂或口服制剂的主要成分，改善银屑病样小鼠病理特征，及缓解银屑病方面的新用途。
技术方案：
本发明的第一个目的是提供干酪乳杆菌CCFM1074在制备在改善银屑病样哺乳动物病理特征、缓解或治疗银屑病的产品中的应用，所述干酪乳杆菌(Lactobacillus casei)CCFM1074已于2019年09月05日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCCNo.60766，保藏地址为广州市先烈中路100号大院59号楼5楼，并记载于公开号为CN110591986A的专利申请文件中。
在一种实施方式中，所述缓解银屑病包括(1)～(4)中至少一种：
(1)缓解皮肤褶皱、鳞屑和/或红斑症状；
(2)抑制皮肤表皮层增厚；
(3)抑制病变耳朵增厚；
(4)降低皮肤中IL-23和/或IL-17水平。
在一种实施方式中，所述产品可摄入哺乳动物胃肠道内，包括但不限于食品、药品或保健食品。
在一种实施方式中，所述产品中干酪乳杆菌CCFM1074的活菌数不低于1×106CFU/mL。
在一种实施方式中，所述药品含有干酪乳杆菌CCFM1074、药物载体和/或药用辅料。
在一种实施方式中，所述药物载体包含微囊、微球、纳米粒以及脂质体。
在一种实施方式中，所述药用辅料包含赋形剂以及附加剂。
在一种实施方式中，所述药用辅料包含抗黏合剂、渗透促进剂、缓冲剂、增塑剂、表面活性剂、消泡剂、增稠剂、包合剂、吸收剂、保湿剂、溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、pH值调节剂、黏合剂、崩解剂、填充剂、润滑剂、润湿剂、整合剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、发泡剂、助悬剂、包衣材料、芳香剂、稀释剂、絮凝剂与反絮凝剂、助滤剂以及释放阻滞剂。
在一种实施方式中，所述附加剂包含微晶纤维素、羟丙基甲基纤维素以及精制卵磷脂。
在一种实施方式中，所述药品的剂型包含颗粒剂、胶囊剂、片剂、丸剂或口服液。
在一种实施方式中，所述食品为含有干酪乳杆菌CCFM1074或其发酵代谢物的食品。
在一种实施方式中，所述食品是使用干酪乳杆菌CCFM1074或含干酪乳杆菌CCFM1074的发酵剂生产得到的乳制品、豆制品或果蔬制品。
在一种实施方式中，所述食品是含有干酪乳杆菌CCFM1074的固体饮料。
在一种实施方式中，所述发酵剂的制备方法为：将干酪乳杆菌CCFM1074按照占培养基总质量2～4％的接种量接种到培养基中，于37℃下培养25-30h，得到培养液；将培养液离心，收集菌体；将菌体用pH为7.2的磷酸盐缓冲液清洗3次后用冻干保护剂重悬，得到重悬液；将重悬液采用真空冷冻法进行冻干，得到干酪乳杆菌CCFM1074的发酵剂。
在一种实施方式中，所述冻干保护剂和菌体的质量比为2:1。
在一种实施方式中，所述冻干保护剂含有海藻糖，海藻糖浓度为50～150g/L。
在一种实施方式中，所述培养基为mMRS培养基。
有益效果：
本发明提供一株改善银屑病样小鼠病理特征、进而缓解银屑病的干酪乳杆菌CCFM1074，该菌株可通过内用口服的形式起效，具体体现在：
(1)银屑病样小鼠病变皮肤情况好转；
(2)银屑病样小鼠皮肤表皮层增厚被抑制；
(3)银屑病样小鼠病变耳朵增厚被抑制；
(4)银屑病样小鼠皮肤中IL-23水平降低39.1％；
(5)银屑病样小鼠皮肤中IL-17水平降低20.2％；
(6)银屑病样小鼠结肠内容物中乙酸水平升高75.2％。
因此，干酪乳杆菌CCFM1074在制备预防和/或治疗银屑病的产品(如食品、药品或保健食品等)中，具有巨大的应用前景。
附图说明
图1：不同组别实验小鼠病变皮肤情况。
图2：不同组别实验小鼠病变耳朵增厚情况。
图3：不同组别实验小鼠皮肤病理切片。
图4：不同组别实验小鼠皮肤中IL-23含量。
图5：不同组别实验小鼠皮肤中IL-17含量。
图6：不同组别实验小鼠结肠内容物中乙酸含量。
具体实施方式
关于菌株：
干酪乳杆菌(Lactobacillus casei)CCFM1074已于2019年09月05日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No.60766，保藏地址为广州市先烈中路100号大院59号楼5楼，并记载于公开号为CN110591986A的专利申请文件中，该菌株的16S Rdna序列比对结果与副干酪乳杆菌(Lactobacillus paracasei)，因此，实施例中以同源性更为接近的副干酪乳杆菌CCFM1147作为对照。
副干酪乳杆菌CCFM1147公开于公开号为CN110591986A的专利申请中，该菌株功效为抑制HaCaT细胞过度增殖进而缓解银屑病，即通过外用涂抹的形式起效，但以内用口服的形式并不具有缓解银屑病的功效。
下述实施例中涉及的培养基如下：
mMRS培养基配方(1L)：蛋白胨10g，牛肉膏10g，酵母粉5g，葡萄糖20g，K2HPO42g，柠檬酸二铵2g，乙酸钠2g，吐温80 1mL，MgSO4·7H2O 0.5g，半胱氨酸盐酸盐0.5g，MnSO4·4H2O0.25g，pH为7.2～7.4。
mMRS固体培养基配方(1L)：蛋白胨10g，牛肉膏10g，酵母粉5g，葡萄糖20g，K2HPO42g，柠檬酸二铵2g，乙酸钠2g，吐温80 1mL，MgSO4·7H2O 0.5g，半胱氨酸盐酸盐0.5g，MnSO4·4H2O 0.25g，琼脂20g，pH为7.2～7.4。
下述实施例中涉及的检测方法如下：
活菌数的检测方法：采用国标《GB 4789.35-2016食品安全国家标准食品微生物学检测乳酸菌检测》。
酸度检测方法：采用国标GB 431334-2010。
实施例1：干酪乳杆菌和副干酪乳杆菌的培养
将干酪乳杆菌CCFM1074接入MRS固体培养基中于37℃培养24h后，观察其菌落并在显微镜下对其菌体进行观察，发现其菌落乳白色、不规则型、圆形凸起、光滑，其菌体形状呈轻微不规则、圆形末端的弯曲杆菌，通常单个、成对和小簇存在。
将干酪乳杆菌CCFM1074接入MRS液体培养基中于37℃培养24h后，转入新鲜的MRS液体培养基中，同样条件培养12h，6000×g离心菌体15min，用0.9％生理盐水洗涤菌体后于6000×g再次离心10min，收集菌体，用30％(m/v)蔗糖溶液重悬，冻存于80℃待用。
可选地，干酪乳杆菌用于给小鼠灌胃时，从-80℃取出后离心弃上清，用生理盐水重悬得到灌胃用的菌悬液。
副干酪乳杆菌CCFM1147的培养方法和菌液制备方法同干酪乳杆菌CCFM1074。
实施例2：干酪乳杆菌对银屑病小鼠病变皮肤的缓解作用
将6-8周龄的SPF级BALB/c雌性小鼠分成4组，分别为正常组、模型组和实验组，其中，实验组包括灌胃干酪乳杆菌CCFM1074的CCFM1074组和灌胃副干酪乳杆菌CCFM1147的CCFM1147组，每组6只，饲养在江南大学实验动物中心，普通饲料喂养，恒定温度21-26℃，湿度40-70％，噪声小于等于60dB，动物照度15-20LX(所有动物实验程序皆由江南大学动物福利与伦理管理委员会进行审查并批准)。
实验周期共计3周，第3周进行造模，造模前一天对小鼠背部进行脱毛处理，面积约为2.5cm×2.5cm。造模期间每天对模型组和实验组小鼠背部脱毛区涂抹咪喹莫特乳膏，耳部10mg，背部62.5mg，正常组仅涂抹等量的凡士林。实验期间，CCFM1074组每天灌胃0.2mL活菌数为1×109CFU/mL的CCFM1074菌悬液，CCFM1147组每天灌胃0.2mL活菌数为1×109CFU/mL的CCFM1147菌悬液，正常组和模型组仅灌胃等量的无菌生理盐水作为对照，所有分组均为自由饮水和摄食。造模期间每天对小鼠背部情况拍照记录，于第4周第1天处死小鼠，结果见图1。
由图1可知，正常组小鼠背部脱毛区皮肤光滑，无鳞屑无红斑；而模型组小鼠于第三天背部脱毛区皮肤出现鳞屑，并且逐渐加厚，出现红斑，且症状日渐加重；而CCFM1074组较之模型组，背部脱毛区皮肤光滑，且基本无鳞屑和红斑；而CCFM1147组小鼠背部脱毛区皮肤如模型组一样，具有褶皱感并覆有明显鳞屑和红斑。
以上实验结果表明，相比于副干酪乳杆菌CCFM1147，干酪乳杆菌(Lactobacilluscasei)CCFM1074更能够缓解银屑病小鼠病变皮肤的症状。
实施例3：干酪乳杆菌对银屑病小鼠病变耳朵增厚的缓解作用
将6-8周龄的SPF级BALB/c雌性小鼠分成4组，分别为正常组、模型组和实验组，其中，实验组包括灌胃干酪乳杆菌CCFM1074的CCFM1074组和灌胃副干酪乳杆菌CCFM1147的CCFM1147组，每组6只，饲养在江南大学实验动物中心，普通饲料喂养，恒定温度21-26℃，湿度40-70％，噪声小于等于60dB，动物照度15-20LX(所有动物实验程序皆由江南大学动物福利与伦理管理委员会进行审查并批准)。
实验周期共计3周，第3周进行造模，造模前一天对小鼠背部进行脱毛处理，面积约为2.5cm×2.5cm。造模期间每天对模型组和实验组小鼠耳部脱毛区涂抹咪喹莫特乳膏，耳部10mg，背部62.5mg，正常组仅涂抹等量的凡士林。实验期间，CCFM1074组每天灌胃0.2mL活菌数为1×109CFU/mL的CCFM1074菌悬液，CCFM1147组每天灌胃0.2mL活菌数为1×109CFU/mL的CCFM1147菌悬液，正常组和模型组仅灌胃等量的无菌生理盐水作为对照，所有分组均为自由饮水和摄食。造模期间每天用电子游标卡尺测量小鼠耳朵厚度，于第4周第1天处死小鼠。结果见图2。
由图2可知，正常组小鼠耳朵厚度始终低于0.2mm；而模型组小鼠耳朵厚度日渐加厚，于第七天超过0.4mm；而CCFM1074组较之模型组，耳朵厚度增厚较少，始终低于0.3mm；而CCFM1147组小鼠耳朵厚度如模型组一样，于第七天超过0.4mm。
以上实验结果表明，相比于副干酪乳杆菌CCFM1147，干酪乳杆菌(Lactobacilluscasei)CCFM1074更能够缓解银屑病小鼠病变耳朵增厚。
实施例4：干酪乳杆菌对银屑病小鼠角质增厚的抑制作用
将6-8周龄的SPF级BALB/c雌性小鼠分成4组，分别为正常组、模型组和实验组，其中，实验组包括灌胃干酪乳杆菌CCFM1074的CCFM1074组和灌胃副干酪乳杆菌CCFM1147的CCFM1147组，每组6只，饲养在江南大学实验动物中心，普通饲料喂养，恒定温度21-26℃，湿度40-70％，噪声小于等于60dB，动物照度15-20LX(所有动物实验程序皆由江南大学动物福利与伦理管理委员会进行审查并批准)。
实验周期共计3周，第3周进行造模，造模前一天对小鼠背部进行脱毛处理，面积约为2.5cm×2.5cm。造模期间每天对模型组和实验组小鼠背部脱毛区涂抹咪喹莫特乳膏，耳部10mg，背部62.5mg，正常组仅涂抹等量的凡士林。实验期间，CCFM1074组每天灌胃0.2mL活菌数为1×109CFU/mL的CCFM1074菌悬液，CCFM1147组每天灌胃0.2mL活菌数为1×109CFU/mL的CCFM1147菌悬液，正常组和模型组仅灌胃等量的无菌生理盐水作为对照，所有分组均为自由饮水和摄食。于第4周第1天处死小鼠，取小鼠背部脱毛区部分皮肤制作病理学切片，进而进行组织病理学分析。结果见图3。
由图3可知，空白组小鼠皮肤表皮层仅由一层或两层细胞组成，表皮各层未见炎症反应；而模型组小鼠角质明显增厚，并伴有严重的炎症；而CCFM1074组较之模型组，角质增厚现象不明显，且炎症现象较轻；而CCFM1147组小鼠皮肤角质增厚明显，且炎症现象严重。
以上实验结果表明，相比于副干酪乳杆菌CCFM1147，干酪乳杆菌(Lactobacilluscasei)CCFM1074更能够抑制银屑病小鼠病变皮肤的角质增厚现象。
实施例5：干酪乳杆菌对银屑病小鼠皮肤中IL-23水平的影响
造模方法同实施例4，,取小鼠背部脱毛区部分皮肤于-80℃保存，通过ELISA试剂盒检测皮肤中IL-23的含量，结果见图4。
由图4可知，小鼠灌胃干酪乳杆菌CCFM1074后，皮肤中IL-23的含量降低了39.1％，较模型组显著降低(p＜0.0001)，说明本发明中的菌株，能够抑制炎症反应；而小鼠灌胃副干酪乳杆菌CCFM1147后，皮肤中IL-23的含量并未降低，与模型组无显著差异。
以上实验结果表明，干酪乳杆菌CCFM1074能显著下调银屑病小鼠中典型上调促炎因子至正常水平，尤其是降低IL-23水平，显著优于另外一株副干酪乳杆菌CCFM1147。
实施例6：干酪乳杆菌对银屑病小鼠皮肤中IL-17水平的影响
造模方法同实施例4，取小鼠背部脱毛区部分皮肤于-80℃保存，通过ELISA试剂盒检测皮肤中IL-23的含量，结果见图5。
由图5可知，小鼠灌胃干酪乳杆菌CCFM1074后，皮肤中IL-17的含量降低了20.2％，较模型组显著降低(p＜0.05)，说明本发明中的菌株，能够抑制炎症反应；而小鼠灌胃副干酪乳杆菌CCFM1147后，皮肤中IL-17的含量甚至显著升高了。
以上实验结果表明，干酪乳杆菌CCFM1074能显著下调银屑病小鼠中典型上调促炎因子至正常水平，尤其是降低IL-17水平，显著优于另外一株副干酪乳杆菌CCFM1147。
实施例7：干酪乳杆菌对银屑病小鼠结肠内容物中乙酸含量的影响
造模方法同实施例4，取小鼠结肠内容物50mg于-80℃保存，使用气相色谱-质谱联用仪检测结肠内容物中短链脂肪酸的含量，结果见图6。
由图6可知，小鼠灌胃干酪乳杆菌CCFM1074后，结肠内容物中乙酸的含量升高了75.2％，较模型组显著降低(p＜0.0001)，说明干酪乳杆菌CCFM1074，能够提升肠道内短链脂肪酸水平；而小鼠灌胃副干酪乳杆菌CCFM1147后，结肠内容物中乙酸的含量无明显变化。
以上实验结果表明，干酪乳杆菌CCFM1074能显著提升银屑病小鼠中肠道内短链脂肪酸水平，尤其是乙酸，显著优于另外一株副干酪乳杆菌CCFM1147。
实施例8：含有干酪乳杆菌CCFM1074菌粉的制备
将保藏于保菌管的干酪乳杆菌CCFM1074的种子液按照占培养基总质量3％的接种量接种到mMRS培养基中，于37℃下培养30h，得到菌株培养液；将菌株培养液离心，收集菌体；将菌体用pH为7.2的磷酸盐缓冲液清洗3次后，将海藻糖浓度为100g/L的海藻糖溶液作为冻干保护剂重悬菌体，并控制冻干保护剂溶液和菌体的质量比为2:1，得到重悬液；将重悬液在-80℃预冷1.5h后立即转移至冷冻干燥机干燥24h，得到干酪乳杆菌CCFM1074菌粉。
实施例9：含有干酪乳杆菌CCFM1074酸奶的制备
将奶粉、菊粉、甜菊糖、水按重量比20：5：5：75进行混料，均质，制成发酵原材料；以121℃超高温灭菌300s杀菌，然后冷却到42℃，接种保加利亚乳杆菌和嗜热链球菌的混合菌粉，在42℃下发酵12h后，使保加利亚乳杆菌和嗜热链球菌的菌体浓度达105CFU/mL和107CFU/mL，之后进行调配；将发酵产物冷却至37℃，加入干酪乳杆菌CCFM1074冻干菌粉，干酪乳杆菌CCFM1074冻干菌粉的投料量为109CFU干酪乳杆菌CCFM1074/ml酸奶，搅拌，罐装，在4℃下保存2天自然完成后熟，制成益生菌酸奶。
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。
Claims (10)
1.干酪乳杆菌CCFM1074在制备在改善银屑病样哺乳动物病理特征、缓解或治疗银屑病的产品中的应用；所述干酪乳杆菌CCFM1074已于2019年09月05日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No.60766。
2.根据权利要求1所述的应用，其特征在于，所述缓解银屑病包括(1)～(4)中至少一种：
(1)缓解皮肤褶皱、鳞屑和/或红斑症状；
(2)抑制皮肤表皮层增厚；
(3)抑制病变耳朵增厚；
(4)降低皮肤中IL-23和/或IL-17水平。
3.根据权利要求1或2所述的应用，其特征在于，所述产品可摄入哺乳动物胃肠道内，包括但不限于食品、药品或保健食品。
4.根据权利要求3所述的应用，其特征在于，所述产品中干酪乳杆菌CCFM1074的活菌数不低于1×106CFU/mL。
5.根据权利要求3或4所述的应用，其特征在于，所述产品为含干酪乳杆菌CCFM1074的菌粉。
6.根据权利要求3所述的应用，其特征在于，所述药品含有干酪乳杆菌CCFM1074、药物载体和/或药用辅料。
7.根据权利要求6所述的应用，其特征在于，所述药物载体包含微囊、微球、纳米粒或脂质体。
8.根据权利要求6所述的应用，其特征在于，所述药用辅料包括赋形剂和/或附加剂。
9.根据权利要求3所述的应用，其特征在于，所述食品是含有干酪乳杆菌CCFM1074或其发酵代谢物的食品。
10.根据权利要求9所述的应用，其特征在于，所述食品为酸奶。
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