CN112553117B - 一株能够抑制皮肤角质层增厚的罗伊氏乳杆菌及其应用 - Google Patents
一株能够抑制皮肤角质层增厚的罗伊氏乳杆菌及其应用 Download PDFInfo
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Abstract
本发明公开了一株能够抑制皮肤角质层增厚的罗伊氏乳杆菌及其应用,属于微生物技术领域以及医药技术领域。本发明提供了罗伊氏乳杆菌(Lactobacillus reuteri)CCFM1132或含罗伊氏乳杆菌CCFM1132的发酵剂在制备缓解银屑病的产品方面的新用途,该菌株能使银屑病样小鼠病变皮肤情况好转,皮肤表皮层增厚被抑制;银屑病样小鼠皮肤中IL‑23水平降低37.6%、IL‑17水平降低18.5%,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
本发明涉及一株能够抑制皮肤角质层增厚的罗伊氏乳杆菌及其应用,属于微生物技术领域以及医药技术领域。
背景技术
皮肤是包在身体表面,直接同外界环境接触,具有保护、***、调节体温和感受外界刺激等作用的一种器官,是人的身体器官中最大的器官。皮肤分表皮和真皮两层,表皮在皮肤表面。角质层是表皮最外层的部分,主要由10至20层扁平、没有细胞核的死亡细胞组成。当这些细胞脱落时,底下位于基底层的细胞会被推上来,形成新的角质层。角质层的主要作用是保护其皮下组织,防止皮下组织遭受感染,脱水以及抵抗化学和外力所带来的压力。角质层一般介乎10至40微米不等,取决于其对应的身体部份需要多少保护。角质形成细胞是表皮的主要构成细胞,数量占表皮细胞的80%以上,在分化过程中产生角蛋白。根据分化阶段和特点可分为五层,由内至外分别为基底层、棘层、颗粒层、透明层和角质层。
银屑病是一类以表皮异常增殖、红斑鱗屑为临床特征的慢性复发性炎症性皮肤病,其病理改变主要有表皮角质形成细胞的异常增殖与分化、真皮炎症细胞浸润以及血管新生。该病且病程较长,容易复发,甚至有的患者这一生都无法治愈。该病多见于青壮年,发病时身体会出现鳞屑、红斑等现象,多见于头皮,四肢,而且冬天情况会加重。已严重影响到患者的身体健康以及精神状态。银屑病发病原因尚未完全明确。国内外医学界均对其发病原因、病理机制做了大量研究,但至今仍无完整定论。西医学认为,其发病与遗传因素、免疫因素(免疫或炎症介导)、感染因素(细菌、真菌、病毒)、内分泌因素(性激素)、神经精神因素(心理因素)、生活习惯(饮酒、吸烟等)、药物因素、环境因素(受潮、季节气候等)有关。
银屑病目前没有彻底治愈的医疗方法和药物,这仍然是一个世界性医疗难题。早期治疗银屑病主要聚焦于抑制异常增殖的角质层。焦油(Tar)用于治疗皮肤病己有2000年历史。目前,焦油制品己被广泛用于治疗银屑病,异位性皮炎,脂溢性皮炎和痒疹等。其对皮脂腺分泌、表皮基底细胞的有丝***数目和鳞屑的产生均有抑制作用。煤焦油可减少皮脂腺合成皮脂并抑制其有丝***。体外试验表明,0.351ag/ml的煤焦油能抑制角朊细胞的DNA合成,从而延缓表皮细胞核有丝***时间,发挥抗增生作用。但使用焦油制品具有副作用,包括短期副作用和长期副作用。短期使用可因阻塞毛囊而发生毛囊炎,部分人群还可能因此过敏。长期使用可因煤焦油中的多环芳烃(PAHs)而致癌。
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。益生菌在生长过程中产生多种代谢产物调节人体健康。Rather等用沙克乳杆菌(Lactobacillus sakei)proBio-65的乙醇提取物SEL001作用于银屑病样小鼠,发现小鼠角质层的异常增厚被抑制,且病情得到缓解。除此外,将益生菌作用于肠道同样具此功效。Chen等人用戊糖乳杆菌(Lactobacillus pentosus)GMNL-77干预银屑病样小鼠,结果显示,小鼠角质层的异常增厚被抑制,且病情得到缓解。
因此,或许可通过开发能够抑制皮肤角质层增厚的益生菌,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罗伊氏乳杆菌(Lactobacillusreuteri)
CCFM1132在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罗伊氏乳杆菌CCFM1132或含罗伊氏乳杆菌CCFM1132的发酵剂在制备缓解银屑病的产品方面的新用途,所述罗伊氏乳杆菌CCFM1132已于2020年7月22日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No.61093,保藏地址为广州市先烈中路100号大院59号楼5楼。
在一种实施方式中,所述缓解银屑病包括:缓解皮肤褶皱、鳞屑和/或红斑症状,或抑制皮肤角质增厚。
在一种实施方式中,所述产品包含药品。
在一种实施方式中,所述产品中罗伊氏乳杆菌(Lactobacillus reuteri)CCFM1132的活菌数不低于1×106CFU/mL。
在一种实施方式中,所述药品含有罗伊氏乳杆菌(Lactobacillus reuteri)CCFM1132、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,所述发酵剂的制备方法为:将罗伊氏乳杆菌CCFM1132按照占培养基总质量2~4%的接种量接种到培养基中,于35~37℃下培养20~30h,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,得到罗伊氏乳杆菌CCFM1132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脱脂奶粉、麦芽糊精和L-谷氨酸钠;其中脱脂奶粉:麦芽糊精:L-谷氨酸钠=8~10:8~10:1。
在一种实施方式中,所述培养基是由占培养基总质量10%的脱脂乳、0.5%的葡萄糖、1.5%的胰蛋白胨以及0.3%的酵母浸膏溶解于87.7%的水中制得。
在一种实施方式中,所述培养基的pH为6.8。
有益效果:本发明提供一株能够抑制皮肤角质层增厚、进而缓解银屑病的罗伊氏乳杆菌CCFM1132,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)银屑病样小鼠皮肤中IL-23水平降低37.6%;
(4)银屑病样小鼠皮肤中IL-17水平降低18.5%;
因此,罗伊氏乳杆菌CCFM1132在制备预防和/或治疗银屑病的产品(如药品)中,具有巨大的应用前景。
生物材料保藏
罗伊氏乳杆菌(Lactobacillus reuteri)CCFM1132,分类命名为Lactobacillusreuteri,已于2020年7月22日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61093。
附图说明
图1:不同组别实验小鼠病变皮肤情况。
图2:不同组别实验小鼠皮肤病理切片。
图3:不同组别实验小鼠皮肤中IL-23含量。
图4:不同组别实验小鼠皮肤中IL-17含量。
具体实施方式
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,K2HPO4 2g,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。
罗伊氏乳杆菌(L.reuteri)1为采用相同方法从不同粪便样品中分离获得的菌株。
实施例1:罗伊氏乳杆菌的培养和菌悬液的制备
将罗伊氏乳杆菌(Lactobacillus reuteri)CCFM1132接入mMRS固体培养基中于37℃培养48h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。
将罗伊氏乳杆菌(Lactobacillus reuteri)CCFM1132接入MRS液体培养基中于37℃培养30h后,转入新鲜的MRS液体培养基中,同样条件培养30h,6000×g离心菌体15min,0.9%生理盐水洗涤菌体后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,罗伊氏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。
实施例2:罗伊氏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,实验组包括灌胃罗伊氏乳杆菌CCFM1132的CCFM1132组和灌胃罗伊氏乳杆菌L.reuteri1的L.reuteri 1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和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,CCFM1132组每天灌胃0.2mL(活菌数为1×109CFU/mL)的CCFM1132菌悬液,L.reuteri1组每天灌胃0.2mL(活菌数为1×109CFU mL)的L.reuteri1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;而模型组小鼠背部脱毛区皮肤具有褶皱感,并覆有明显的鳞屑,伴有红斑;而CCFM1132组较之模型组,背部脱毛区皮肤更为光滑,且鳞屑较少没有红斑;而L.reuteri 1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。
以上实验结果表明,相比于罗伊氏乳杆菌L.reuteri 1,罗伊氏乳杆菌CCFM1132更能够缓解银屑病小鼠病变皮肤的症状。
实施例3:罗伊氏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,实验组包括灌胃罗伊氏乳杆菌CCFM1132的CCFM1132组和灌胃罗伊氏乳杆菌L.reuteri1的L.reuteri 1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和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,CCFM1132组每天灌胃0.2mL(活菌数为1×109CFU)的CCFM1132菌悬液,L.reuteri 1组每天灌胃0.2mL(活菌数为1×109CFU)的L.reuteri1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2。
由图2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CCFM1132组较之模型组,角质增厚现象不明显,而L.reuteri 1组小鼠皮肤角质增厚明显。
以上实验结果表明,相比于罗伊氏乳杆菌L.reuteri 1,罗伊氏乳杆菌CCFM1132更能够抑制银屑病小鼠病变皮肤的角质增厚现象。
实施例4:罗伊氏乳杆菌对银屑病小鼠皮肤中IL-23的影响
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,实验组包括灌胃罗伊氏乳杆菌CCFM1132的CCFM1132组和灌胃罗伊氏乳杆菌L.reuteri1的L.reuteri 1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和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,CCFM1132组每天灌胃0.2mL(活菌数为1×109CFU/mL)的CCFM1132菌悬液,L.reuteri1组每天灌胃0.2mL(活菌数为1×109CFU/mL)的L.reuteri1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于-80℃保存,通过ELISA试剂盒检测皮肤中IL-23的含量,结果见图3。
由图3可知,小鼠灌胃罗伊氏乳杆菌CCFM1132后,皮肤中IL-23的含量降低了37.6%,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;而小鼠灌胃罗伊氏乳杆菌L.reuteri1后,皮肤中IL-23的含量仅降低了12.4%。
以上实验结果表明,罗伊氏乳杆菌(Lactobacillus reuteri)CCFM1132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,显著优于另外一株罗伊氏乳杆菌L.reuteri 1。
实施例5:罗伊氏乳杆菌对银屑病小鼠皮肤中IL-17的影响
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,实验组包括灌胃罗伊氏乳杆菌CCFM1132的CCFM1132组和灌胃罗伊氏乳杆菌L.reuteri1的L.reuteri 1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和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,CCFM1132组每天灌胃0.2mL(活菌数为1×109CFU/mL)的CCFM1132菌悬液,L.reuteri1组每天灌胃0.2mL(活菌数为1×109CFU/mL)的L.reuteri 1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于-80℃保存,通过ELISA试剂盒检测皮肤中IL-17的含量,结果见图4。
由图4可知,小鼠灌胃罗伊氏乳杆菌CCFM1132后,皮肤中IL-17的含量降低了18.5%,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;而小鼠灌胃罗伊氏乳杆菌L.reuteri 1后,皮肤中IL-17的含量仅降低了9.7%,于模型组无显著差异。
以上实验结果表明,罗伊氏乳杆菌CCFM1132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,显著优于另外一株罗伊氏乳杆菌L.reuteri 1。
实施例6:含有罗伊氏乳杆菌CCFM1132菌粉的制备
将保藏于保菌管的罗伊氏乳杆菌CCFM1132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次后用海藻糖浓度为100g/L的海藻糖冻干保护剂重悬,并控制冻干保护剂和菌体的质量比为2:1,得到重悬液;将重悬液-80℃预冷1.5h后立即转移至冷冻干燥机干燥24h,得到罗伊氏乳杆菌(Lactobacillus reuteri)CCFM1132菌粉。
实施例7:含有罗伊氏乳杆菌CCFM1132固体饮料的制备
将实施例6制备的含有109CFU的罗伊氏乳杆菌CCFM1132菌粉同麦芽糊精以2:1的比例混合,得到富含罗伊氏乳杆菌CCFM1132的固体饮料。
实施例8:含有罗伊氏乳杆菌CCFM1132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后保加利亚乳杆菌和嗜热链球菌的菌体浓度控制为105CFU/g和107CFU/g,之后进行调配;将发酵产物冷却至37℃,加入罗伊氏乳杆菌CCFM1132冻干菌粉,投料量为109CFU/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 (6)
1.罗伊氏乳杆菌(Lactobacillus reuteri)CCFM1132或含罗伊氏乳杆菌CCFM1132的发酵剂在制备缓解银屑病的药品中的应用,其特征在于,所述罗伊氏乳杆菌CCFM1132已于2020年7月22日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No.61093。
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:缓解皮肤褶皱、鳞屑和/或红斑症状,或抑制皮肤角质增厚。
3.根据权利要求1所述的应用,其特征在于,所述药品中罗伊氏乳杆菌CCFM1132的活菌数不低于1×106CFU/mL或1×106CFU/g。
4.根据权利要求1所述的应用,其特征在于,所述药品含有罗伊氏乳杆菌CCFM1132、药物载体和/或药用辅料。
5.根据权利要求4所述的应用,其特征在于,所述药物载体包含微囊、微球、纳米粒以及脂质体;所述药用辅料包含赋形剂以及附加剂。
6.根据权利要求1所述的应用,其特征在于,所述发酵剂是按如下方法制备:将罗伊氏乳杆菌CCFM1132接种到培养基中,于35~37℃下培养20~30h,得到培养液;将培养液离心,收集菌体;将菌体洗涤后用冻干保护剂重悬,得到重悬液;再将重悬液采用真空冷冻法冻干,获得含罗伊氏乳杆菌CCFM1132的发酵剂。
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