CN112546074A - 一株能够抑制IL-23、Th17轴相关炎症因子释放的短双歧杆菌及其应用 - Google Patents
一株能够抑制IL-23、Th17轴相关炎症因子释放的短双歧杆菌及其应用 Download PDFInfo
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Abstract
本发明公开了一株能够抑制IL‑23、Th17轴相关炎症因子释放的短双歧杆菌及其应用,属于微生物技术领域以及医药技术领域。本发明提供了短双歧杆菌(Bifidobacterium breve)CCFM1078在抑制IL‑23/Th17轴相关炎症因子释放,及缓解银屑病方面的新用途。本发明的短双歧杆菌CCFM1078,能够缓解银屑病样小鼠病变皮肤、抑制皮肤表皮层增厚,使皮肤中IL‑23水平、IL‑22水平和IL‑17水平分别降低20.3%、22.0%和18.3%;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IL-23、Th17轴相关炎症因子释放的短双歧杆菌及其应用,属于微生物技术领域以及医药技术领域。
背景技术
辅助性T细胞17(T helper cell 17,Th17)是一种新发现的能够分泌白介素-17(interleukin-17,IL-17)的T细胞亚群,在自身免疫性疾病和机体防御反应中具有重要的意义。初始CD4+T细胞接受抗原刺激后,在不同的条件下可分化成不同亚型的T细胞,执行不同的功能。其分化方向受抗原的性质、局部环境中的激素及细胞因子等多种因素的调控,其中细胞因子的种类和细胞因子之间的平衡对Th细胞的分化具有重要的调节作用。IL-23属于IL-12家族成员,为异二聚体,包括IL-23特异性的p19亚单位和与IL-12共享的p40亚单位。IL-23受体相应的也为异二聚体,由IL-12Rβ1和IL-23R两个亚单位组成。IL-23R主要表达在T细胞、自然杀伤T细胞、单核细胞和树突细胞。IL-23在Th17细胞的分化中具有重要作用,可直接诱导原始T细胞产生IL-22和IL-17。IL-23/Th17轴参与了多种疾病的发生,包括银屑病、关节炎、炎性肠病等。
银屑病是一种慢性的、遗传和环境共同诱发、免疫介导的炎症性疾病,无传染性,易复发,可能会随着年龄的增长而病情逐渐恶化,或者严重程度上时好时坏,有的病例甚至终身不愈。世界卫生组织2016年发布的银屑病全球患病率为0.09%-11.43%。在中国,银屑病患病率小于0.5%。高纬度国家的银屑病患病率似乎更高,这可能与纬度不同引起的紫外线强度不同有关。相比儿童,银屑病更易在成年人中发病。虽然可在任意年龄阶段发病,但在18-39岁或50-69岁之间的发病率最高。银屑病发病机制尚未完全明确。免疫相关细胞释放的促炎性细胞因子增多以及先天性和适应性免疫系统的慢性激活被认为是导致多个组织和器官长期受损的机制。目前多种银屑病相关的异常已被报道,包括抗原提呈、NF-κB信号通路的激活、辅助性T细胞群(尤其是IL-17的主要来源的TH17细胞)的分化和IL-17反应的增强,这些异常促进了宿主的免疫应答和免疫细胞的浸润。刘爱民、乔菊、郭庆浩等均报道了在银屑病患者血清内IL-23、IL-22、IL-17表达水平升高。
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。银屑病的治疗方法取决于几个因素。早期多采用局部治疗,病变的区域决定了局部治疗的配方和剂量,皮质类固醇是目前局部治疗银屑病的主要方法。当传统治疗方案疗效不佳时,可考虑应用生物制剂。近年来,多种高效生物制剂可用来治疗中、重度银屑病,主要包括肿瘤坏死因子(TNF)抑制剂、T细胞调节剂、IL-12/23p40抑制剂及IL-17A抑制剂。近年来的研究强调了IL-23和IL-17的重要性,因此研究重点偏向了针对IL-23和IL-17的生物制剂。但生物制剂价格高昂。
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。国外相关研究也证实了该种可能,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,该戊糖乳杆菌降低小鼠皮肤内IL-23、IL-22、IL-17水平,且小鼠病情得到缓解。David等人发现26个银屑病患者连续口服婴儿双歧杆菌(Bifidobacterium infantis)35624 6周后,患者病情得到缓解。
因此,或许可通过开发能够抑制IL-23/Th17轴相关炎症因子释放的益生菌,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短双歧杆菌(Bifidobacteriumbreve)
CCFM1078在抑制IL-23/Th17轴相关炎症因子释放,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短双歧杆菌CCFM1078在制备抑制IL-23/Th17轴相关炎症因子释放、缓解银屑病的产品中的应用,所述短双歧杆菌CCFM1078已于2020年5月6日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No.61011,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,所述产品包含食品、药品或保健食品。
在一种实施方式中,所述产品中短双歧杆菌CCFM1078的活菌数不低于1×106CFU/mL.
在一种实施方式中,所述药品含有短双歧杆菌CCFM1078、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,所述食品为含有短双歧杆菌CCFM1078或其发酵代谢物的食品。
在一种实施方式中,所述食品是使用短双歧杆菌CCFM1078或含短双歧杆菌CCFM1078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,所述食品是含有短双歧杆菌CCFM1078的固体饮料。
在一种实施方式中,所述发酵剂的制备方法为:将短双歧杆菌CCFM1078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,得到短双歧杆菌CCFM1078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%的酵母浸膏溶解于水中制得的。
在一种实施方式中,所述培养基的pH为6.8。
有益效果:本发明提供一株抑制IL-23/Th17轴相关炎症因子释放、进而缓解银屑病的短双歧杆菌CCFM1078,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20.3%;
(4)银屑病样小鼠皮肤中IL-22水平降低22.0%;
(5)银屑病样小鼠皮肤中IL-17水平降低18.3%;
因此,短双歧杆菌CCFM1078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。
生物材料保藏
短双歧杆菌(Bifidobacterium breve)CCFM1078,分类命名为Bifidobacteriumbreve,已于2020年5月6日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCCNo.61011,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: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-22含量。
图5: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。
短双歧杆菌B.breve 1为采用相同方法分离从不同的粪便样品中分离获得的另一菌株。
实施例1:短双歧杆菌的培养
将短双歧杆菌CCFM1078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。
将短双歧杆菌CCFM1078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,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。
实施例2:短双歧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,实验组包括灌胃短双歧杆菌CCFM1078的CCFM1078组和灌胃短双歧杆菌B.breve 1的B.breve 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,CCFM1078组每天灌胃0.2mL活菌数为1×109CFU/mL的CCFM1078菌悬液,B.breve 1组每天灌胃0.2mL活菌数为1×109CFU/mL的B.breve 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。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CCFM1078组较之模型组,背部脱毛区皮肤光滑,且基本无鳞屑不红斑;而B.breve 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,相比于短双歧杆菌B.breve 1,短双歧杆菌(Bifidobacteriumbreve)CCFM1078更能够缓解银屑病小鼠病变皮肤的症状。
实施例3:短双歧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,实验组包括灌胃短双歧杆菌CCFM1078的CCFM1078组和灌胃短双歧杆菌B.breve 1的B.breve 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。第1-3周,实验期间,CCFM1078组每天灌胃0.2mL活菌数为1×109CFU/mL的CCFM1078菌悬液,B.breve 1组每天灌胃0.2mL活菌数为1×109CFU/mL的B.breve 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CCFM1078组较之模型组,角质增厚现象不明显,且炎症现象较轻;而B.breve 1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,相比于短双歧杆菌B.breve 1,短双歧杆菌CCFM1078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:短双歧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,实验组包括灌胃短双歧杆菌CCFM1078的CCFM1078组和灌胃短双歧杆菌B.breve 1的B.breve 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,CCFM1078组每天灌胃0.2mL活菌数为1×109CFU/mL的CCFM1078菌悬液,B.breve 1组每天灌胃0.2mL活菌数为1×109CFU/mL的B.breve 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-23的含量,结果见图3。
由图3可知,小鼠灌胃短双歧杆菌CCFM1078后,皮肤中IL-23的含量降低了20.3%,较模型组显著降低(p<0.001),说明本发明中的菌株,能够抑制炎症反应;而小鼠灌胃短双歧杆菌B.breve 1后,皮肤中IL-23的含量仅降低了4.4%,与模型组无显著差异。
以上实验结果表明,短双歧杆菌CCFM1078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,显著优于另外一株短双歧杆菌B.breve 1。
实施例5:短双歧杆菌对银屑病小鼠皮肤中IL-22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,实验组包括灌胃短双歧杆菌CCFM1078的CCFM1078组和灌胃短双歧杆菌B.breve 1的B.breve 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,CCFM1078组每天灌胃0.2mL活菌数为1×109CFU/mL的CCFM1078菌悬液,B.breve 1组每天灌胃0.2mL活菌数为1×109CFU/mL的B.breve 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-22的含量,结果见图4。
由图4可知,小鼠灌胃短双歧杆菌CCFM1078后,皮肤中IL-22的含量降低了22.0%,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;而小鼠灌胃短双歧杆菌B.breve 1后,皮肤中IL-22的含量仅降低了0.5%,与模型组无显著差异。
以上实验结果表明,短双歧杆菌CCFM1078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-22水平,显著优于另外一株短双歧杆菌B.breve 1。
实施例6:短双歧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,实验组包括灌胃短双歧杆菌CCFM1078的CCFM1078组和灌胃短双歧杆菌B.breve 1的B.breve 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,CCFM1078组每天灌胃0.2mL的CCFM1078菌悬液,B.breve 1组每天灌胃0.2mL的B.breve 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5。
由图5可知,小鼠灌胃短双歧杆菌CCFM1078后,皮肤中IL-17的含量降低了18.3%,较模型组显著降低(p<0.01),说明本发明中的菌株,能够抑制炎症反应;而小鼠灌胃短双歧杆菌B.breve 1后,皮肤中IL-17的含量甚至升高了。
以上实验结果表明,短双歧杆菌CCFM1078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,显著优于另外一株短双歧杆菌B.breve 1。
实施例7:含有短双歧杆菌CCFM1078菌粉的制备
将保藏于保菌管的短双歧杆菌CCFM1078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,得到短双歧杆菌CCFM1078菌粉。
实施例8:含有短双歧杆菌CCFM1078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℃,加入短双歧杆菌CCFM1078冻干菌粉,短双歧杆菌CCFM1078冻干菌粉的投料量为109CFU短双歧杆菌CCFM1078/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.短双歧杆菌(Bifidobacterium breve)CCFM1078在制备抑制IL-23/Th17轴相关炎症因子释放、缓解银屑病的产品中的应用,其特征在于,所述短双歧杆菌CCFM1078已于2020年5月6日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No.61011。
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,所述产品包含食品、药品或保健食品。
4.根据权利要求1~3任一所述的应用,其特征在于,所述产品中短双歧杆菌CCFM1078的活菌数不低于1×106CFU/mL或1×106CFU/g。
5.根据权利要求3所述的应用,其特征在于,所述药品含有短双歧杆菌CCFM1078、药物载体和/或药用辅料。
6.根据权利要求5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。
7.根据权利要求3所述的应用,其特征在于,所述食品含有短双歧杆菌CCFM1078或其发酵代谢物。
8.根据权利要求3所述的应用,其特征在于,所述食品是使用短双歧杆菌CCFM1078或含有短双歧杆菌CCFM1078的发酵剂生产得到的乳制品、豆制品或果蔬制品。
9.根据权利要求7所述的应用,其特征在于,所述食品是含有短双歧杆菌CCFM1078的固体饮料。
10.根据权利要求1所述的应用,其特征在于,所述发酵剂按如下方法制备:将短双歧杆菌CCFM1078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;将菌体清洗后用冻干保护剂重悬,得到重悬液;再将重悬液冻干,得到短双歧杆菌CCFM1078的发酵剂。
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