CN111826312A - 一株可缓解苯并芘暴露的鼠李糖乳杆菌及其应用 - Google Patents
一株可缓解苯并芘暴露的鼠李糖乳杆菌及其应用 Download PDFInfo
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Abstract
本发明公开了一株可缓解苯并芘暴露的鼠李糖乳杆菌及其应用,属于微生物技术领域以及医药技术领域。本发明提供了一株鼠李糖乳杆菌(Lactobacillus rhamnosus)CCFM1125,此鼠李糖乳杆菌CCFM1125能够缓解苯并芘暴露,具体体现在:此鼠李糖乳杆菌CCFM1125能够显著降低苯并芘暴露小鼠粪便中苯并芘代谢物3‑羟基苯并芘的含量,可见,鼠李糖乳杆菌(Lactobacillus rhamnosus)CCFM1125在制备预防和/或治疗苯并芘暴露的产品(如食品或药品等)中具有巨大的应用前景。
Description
技术领域
本发明涉及一株可缓解苯并芘暴露的鼠李糖乳杆菌及其应用,属于微生物技术领域以及医药技术领域。
背景技术
苯并芘(benzopyrene)是一种由5个苯环构成的多环芳烃,其分子式为C20H12,分子量为252.30,常温下为无色至淡黄色针状晶体(纯品),性质稳定,沸点310~312℃,熔点178℃,不溶于水,微溶于乙醇、甲醇,溶于苯、甲苯、二甲苯、氯仿、乙醚、丙酮等有机溶剂。
1933年,英国科学家J.W.Cook等人首次从沥青中分离得到苯并芘纯品,并通过动物实验证明了苯并芘可诱导小鼠产生了皮肤癌,由此,苯并芘被确认为是第一个化学环境致癌物,长期暴露于含有苯并芘的环境无疑会对人体造成极大的危害(具体可见参考文献:Cancer Letters,2004,207:157-163.)。
与此同时,苯并芘在环境中是广泛存在的,其来源主要有两个方面:一是工业生产和生活过程中煤炭、石油和天然气等燃料不完全燃烧产生的废气,包括汽车尾气、橡胶生产以及吸烟产生的烟气等,通过对水源、大气和土壤的污染进入到蔬菜、水果、粮食、水产品和肉类等人类赖以生存的食物中;二是食物在熏制、烘烤和煎炸过程中,脂肪、胆固醇、蛋白质和碳水化合物等在高温条件下会发生热裂解反应,再经过环化和聚合反应就能够形成包括苯并芘在内的多环芳烃类物质,尤其是当食品在烟熏和烘烤过程中发生焦糊现象时,苯并芘的生成量将会比普通食物增加10~20倍。可见,人们在日常生活中几乎每时每刻都暴露于含有苯并芘的环境中。因此,急需找到一种可有效缓解苯并芘暴露的药物。
发明内容
[技术问题]
本发明要解决的技术问题是提供一株可缓解苯并芘暴露的鼠李糖乳杆菌(Lactobacillus rhamnosus)。
[技术方案]
为解决上述问题,本发明提供了一株鼠李糖乳杆菌(Lactobacillus rhamnosus)CCFM1125,所述鼠李糖乳杆菌CCFM1125保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61019,保藏日期为2020年05月06日。
所述鼠李糖乳杆菌CCFM1125来源于新疆省昌吉军户农场的人群肠道内容物样本,该菌株经测序分析,其16S rDNA序列如SEQ ID NO.1所示,将测序得到的序列在GeneBank中进行核酸序列比对,结果显示菌株为鼠李糖乳杆菌,命名为鼠李糖乳杆菌CCFM1125。
所述鼠李糖乳杆菌CCFM1125的形态特征为:短杆菌,无芽孢。
所述鼠李糖乳杆菌CCFM1125的菌落特征为:乳白色圆形凸起,表面光滑。
本发明还提供了上述鼠李糖乳杆菌在制备预防和/或治疗苯并芘暴露的药品中的应用。
本发明的一种实施方式中,所述药品中,上述鼠李糖乳杆菌CCFM1125的活菌数为不低于1×106CFU/mL或1×106CFU/g。
本发明的一种实施方式中,所述药品含有上述鼠李糖乳杆菌CCFM1125、药物载体和/或药用辅料。
本发明的一种实施方式中,所述药物载体包含微囊、微球、纳米粒和/或脂质体。
本发明的一种实施方式中,所述药用辅料包含赋形剂和/或附加剂。
本发明的一种实施方式中,所述赋形剂包含溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、吸收剂、稀释剂、絮凝剂、反絮凝剂、助滤剂和/或释放阻滞剂。
本发明的一种实施方式中,所述附加剂包含微晶纤维素、羟丙基甲基纤维素和/或精制卵磷脂。
本发明的一种实施方式中,所述药品的剂型为粉剂、颗粒剂、胶囊剂、片剂、丸剂或口服液。
本发明还提供了一种的产品,所述产品含有上述鼠李糖乳杆菌CCFM1125。
本发明的一种实施方式中,所述产品中,上述鼠李糖乳杆菌CCFM1125的活菌数为不低于1×106CFU/mL或1×106CFU/g。
本发明的一种实施方式中,所述产品为食品、药品或苯并芘清除剂。
本发明的一种实施方式中,所述药品含有上述鼠李糖乳杆菌CCFM1125、药物载体和/或药用辅料。
本发明的一种实施方式中,所述药物载体包含微囊、微球、纳米粒和/或脂质体。
本发明的一种实施方式中,所述药用辅料包含赋形剂和/或附加剂。
本发明的一种实施方式中,所述赋形剂包含溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、吸收剂、稀释剂、絮凝剂、反絮凝剂、助滤剂和/或释放阻滞剂。
本发明的一种实施方式中,所述附加剂包含微晶纤维素、羟丙基甲基纤维素和/或精制卵磷脂。
本发明的一种实施方式中,所述药品的剂型为粉剂、颗粒剂、胶囊剂、片剂、丸剂或口服液。
本发明的一种实施方式中,所述食品为保健食品;或所述食品为使用含有上述鼠李糖乳杆菌CCFM1125的发酵剂生产得到的乳制品、豆制品或果蔬制品;或所述食品为含有上述鼠李糖乳杆菌CCFM1125的饮料或零食。
本发明的一种实施方式中,所述发酵剂的制备方法为将上述鼠李糖乳杆菌CCFM1125接种到培养基中进行培养,得到培养液;将培养液离心,得到菌体;将菌体用生理盐水或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,得到发酵剂。
在本发明的一种实施方式中,所述冻干保护剂包含100g/L脱脂奶粉、30mL/L甘油、100g/L麦芽糊精、150g/L海藻糖和10g/L L-谷氨酸钠。
本发明还提供了上述鼠李糖乳杆菌或上述产品在清除苯并芘中的应用。
有益效果:
(1)本发明提供了一株鼠李糖乳杆菌(Lactobacillus rhamnosus)CCFM1125,此鼠李糖乳杆菌CCFM1125能够缓解苯并芘暴露,具体体现在:此鼠李糖乳杆菌CCFM1125能够显著降低苯并芘暴露小鼠粪便中苯并芘代谢物3-羟基苯并芘的含量,可见,鼠李糖乳杆菌(Lactobacillus rhamnosus)CCFM1125在制备预防和/或治疗苯并芘暴露的产品(如食品或药品等)中具有巨大的应用前景。
(2)本发明提供了一株鼠李糖乳杆菌(Lactobacillus rhamnosus)CCFM1125,此鼠李糖乳杆菌CCFM1125能够清除苯并芘,具体体现在:将此鼠李糖乳杆菌CCFM1125添加至含有苯并芘的二甲基亚砜(DMSO)中振荡培养2h,即可使二甲基亚砜(DMSO)中苯并芘的清除率达60%以上,可见,鼠李糖乳杆菌(Lactobacillus rhamnosus)CCFM1125在制备苯并芘清除剂中具有巨大的应用前景。
生物材料保藏
一株鼠李糖乳杆菌(Lactobacillus rhamnosus)CCFM1125,分类学命名为Lactobacillus rhamnosus,已于2020年05月06日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61019,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:不同鼠李糖乳杆菌清除二甲基亚砜(DMSO)中苯并芘的清除率。
图2:不同组别苯并芘暴露小鼠粪便中苯并芘代谢物3-羟基苯并芘的含量。
具体实施方式
下述实施例中涉及的SPF级8周龄雄性BALB/C小鼠购自斯莱克实验动物有限公司;下述实施例中涉及的脱脂乳购自福麦斯生物技术有限公司;下述实施例中涉及的玉米油购自阿拉丁生化科技股份有限公司;下述实施例中涉及的脱脂奶粉购自纽瑞兹食品有限公司;下述实施例中涉及的苯并芘标准品购自美国sigma公司;下述实施例中涉及的二甲基亚砜(DMSO)购自国药集团化学试剂有限公司。
下述实施例中涉及的培养基如下:
LFMATA固体培养基(g/L):蛋白胨10g/L、牛肉膏10g/L、酵母粉5g/L、吐温1mL/L、磷酸氢二钾3g/L、乙酸钠2g/L、柠檬酸二铵2g/L、七水硫酸镁0.1g/L、一水硫酸锰0.05g/L、碳源20g/L、万古霉素20×10-3g/L、链霉素0.256g/L、庆大霉素6.4×10-2g/L、L-半胱氨酸0.5g/L、琼脂18g/L。
MRS固体培养基(g/L):蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO40.05 g/L、吐温801mL/L、琼脂20g/L、半胱氨酸氨酸盐0.5g/L,pH为6.8。
MRS液体培养基(g/L):蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO40.05 g/L、吐温801mL/L、半胱氨酸氨酸盐0.5g/L,pH为6.8。
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》。
下述实施例中涉及的鼠李糖乳杆菌菌体和菌悬液的制备方法如下:
将鼠李糖乳杆菌菌液划线于MRS固体培养基上,37℃条件下培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于MRS液体培养基中,37℃条件下培养18h,得到菌液;将菌液经5000g离心15min,得到鼠李糖乳杆菌菌体;将鼠李糖乳杆菌菌体经生理盐水洗涤3次后重悬于浓度为130g/L的脱脂奶粉溶液中至菌浓度为1.5×1010CFU/mL,得到菌悬液,将菌悬液于-80℃下保存待用。
实施例1:鼠李糖乳杆菌CCFM1125的获取
具体步骤如下:
1、分离纯化
(1)稀释涂布:以来源于新疆省昌吉军户农场的人群肠道内容物为样本,吸取0.5g保存于30%(v/v)甘油中的样本在无菌环境下加入装有4.5mL生理盐水的10mL离心管中,得到10-1稀释液,重复上述稀释步骤,依次得到10-2、10-3、10-4、10-5、10-6稀释液;
(2)涂布培养:分别吸取100μL步骤(1)获得的10-4、10-5、10-6三个梯度稀释液涂布于LFMATA固体培养基上,37℃培养48h,得到稀释涂布平板;
(3)一级纯化培养:取步骤(2)获得的菌落数在30~300区间的稀释涂布平板,在每个稀释涂布平板上随机挑选10个乳白色或白色,表面光滑,边缘整齐,大小不一的单菌落在MRS固体培养基上划线,37℃培养48h,得到单菌落;
(4)二级纯化培养:取步骤(3)获得的单菌落分别接种至MRS液体培养基中,37℃培养20h,得到菌液。
2、菌种鉴定
将分离纯化的各菌液所对应的各菌株编号后,参照教科书《微生物学》(沈萍,陈向东主编)中所记载的步骤进行菌株鉴定、革兰氏染色、生理生化等实验,选取具有鼠李糖乳杆菌典型特征的菌株,经实验获得一株菌,将此菌株命名为CCFM1125;
其中,菌株鉴定过程如下:
提取CCFM1125的基因组,将CCFM1125的16S rDNA进行扩增和测序(由上海生工生物工程股份有限公司完成),将测序分析得到的CCFM1125的16S rDNA序列(CCFM1125的16SrDNA序列如SEQ ID NO.1所示)在GenBank中进行比对,结果显示此菌株确为鼠李糖乳杆菌,命名为鼠李糖乳杆菌(Lactobacillus rhamnosus)CCFM1125;
鼠李糖乳杆菌(Lactobacillus rhamnosus)CCFM1125的形态特征为:短杆菌,无芽孢;
鼠李糖乳杆菌(Lactobacillus rhamnosus)CCFM1125的菌落特征为:乳白色圆形凸起,表面光滑;
鼠李糖乳杆菌(Lactobacillus rhamnosus)CCFM1125的生理生化特征为:革兰阳性厌氧菌,无质粒;不能利用乳糖,但可代谢单糖;对青霉素、氯林可霉素、红霉素、万古霉素、四环素和氯霉素敏感。
实施例2:鼠李糖乳杆菌CCFM1125对苯并芘的清除作用
具体步骤如下:
准确称取10mg的苯并芘标准品溶于1mL二甲基亚砜中,充分混匀溶解后使用0.22μm微孔滤膜进行过滤除菌,得到浓度为10mg/mL的苯并芘标准品母液;准确称取10mg/mL的苯并芘标准品母液100μL用二甲基亚砜定容至100mL,得到浓度为10μg/mL的苯并芘标准品母液;以不添加鼠李糖乳杆菌菌体的浓度为10μg/mL的苯并芘标准品母液作为空白对照,将3×109CFU的鼠李糖乳杆菌CCFM492菌体、鼠李糖乳杆菌FS7-5菌体、鼠李糖乳杆菌CCFM1125(实施例1筛选所得)、鼠李糖乳杆菌JS-WX-24-1菌体、鼠李糖乳杆菌FAHWH26-L1菌体、鼠李糖乳杆菌FJSYC4-L1菌体、鼠李糖乳杆菌FFJND15-L1菌体、鼠李糖乳杆菌CCFM237菌体、鼠李糖乳杆菌FAHWH26-L9菌体、鼠李糖乳杆菌FHNFQ4-L1菌体、鼠李糖乳杆菌FJSYC4-L5菌体、鼠李糖乳杆菌FHNFQ14-L7菌体、鼠李糖乳杆菌F-F-J-L-Y-7-L1菌体、鼠李糖乳杆菌FZJHZD11L1菌体依次分别与1mL浓度为10μg/mL的苯并芘标准品母液混合后于37℃、150r/min的条件下共培养2h,得到培养液1~14;将培养液1~212000rpm离心10min,取上清用0.22μm微孔滤膜进行过滤除菌,得到滤液1~14;其中,鼠李糖乳杆菌CCFM492、鼠李糖乳杆菌FS7-5、鼠李糖乳杆菌JS-WX-24-1、鼠李糖乳杆菌FAHWH26-L1、鼠李糖乳杆菌FJSYC4-L1、鼠李糖乳杆菌FFJND15-L1、鼠李糖乳杆菌CCFM237、鼠李糖乳杆菌FAHWH26-L9、鼠李糖乳杆菌FHNFQ4-L1、鼠李糖乳杆菌FJSYC4-L5、鼠李糖乳杆菌FHNFQ14-L7、鼠李糖乳杆菌F-F-J-L-Y-7-L1、鼠李糖乳杆菌FZJHZD11L1均是从人或动物粪便以及发酵制品中筛选得到的。
通过HPLC法检测滤液1~14中苯并芘的含量,并根据公式:清除率(%)=[(空白对照中苯并芘的含量-滤液中苯并芘的含量)/空白对照中苯并芘的含量]×100%,计算滤液1~14中苯并芘的清除率(检测结果见图1);其中,HPLC法的检测条件为:色谱柱WatersAtlantis C18,150mm×4.6mm×5μm;流动相为乙腈:水=88:12,流速1mL/min,检测器为荧光检测器,检测波长406nm,上样量为20μL,苯并芘的出峰时间为12~14min。
由图1可知,滤液3中苯并芘的含量为2.47μg、苯并芘的清除率为65.3%。可见,鼠李糖乳杆菌(Lactobacillus rhamnosus)CCFM1125可有效清除二甲基亚砜中的苯并芘。
实施例3:鼠李糖乳杆菌CCFM1125对苯并芘暴露小鼠的影响
具体步骤如下:
取24只SPF级8周龄雄性BALB/C小鼠,随机分为3组,每组8只,3组分别为:载体对照组、模型组和CCFM1125干预组。所有小鼠饲养于25±2℃、相对湿50±5%、12h光照/12h黑暗的标准化实验室中,适应性喂养一周后开始实验。
实验共35天:实验期间,载体对照组小鼠按照50mg/kg.bw的剂量每天灌胃玉米油,并且,按照0.2mL/只的剂量每天灌胃浓度为130g/L的脱脂奶粉溶液;模型组小鼠按照50mg/kg.bw的剂量每天灌胃含有苯并芘溶液(苯并芘溶液是通过将苯并芘溶于玉米油得到的),并且,按照0.2mL/只的剂量每天灌胃浓度为130g/L的脱脂奶粉溶液;CCFM1125干预组小鼠按照50mg/kg.bw的剂量每天灌胃含有苯并芘溶液(苯并芘溶液是通过将苯并芘溶于玉米油得到的),并且,按照0.2mL/只的剂量每天灌胃鼠李糖乳杆菌CCFM1125菌悬液。
灌胃结束后处死所有小鼠,收集小鼠的粪便和组织,检测小鼠粪便中3-羟基苯并芘的含量(检测结果见图2);其中,3-羟基苯并芘的含量通过HPLC法测定,检测条件为:色谱柱WatersAtlantis C18,150mm×4.6mm×5μm;流动相为甲醇:水=97:3(pH 4.5),流速0.5mL/min,检测器为荧光检测器,检测波长450nm,上样量为20μL,3-羟基苯并芘的出峰时间为9~11min。
由图2可知,载体对照组小鼠粪便中3-羟基苯并芘的含量为0ng/g干粪便、模型组小鼠粪便中3-羟基苯并芘的含量为268845ng/g干粪便、CCFM1125干预组小鼠粪便中3-羟基苯并芘的含量为144048ng/g干粪便。可见,鼠李糖乳杆菌(Lactobacillus rhamnosus)CCFM1125可有效降低苯并芘暴露小鼠粪便中苯并芘代谢物3-羟基苯并芘的含量。
实施例4:鼠李糖乳杆菌CCFM1125的应用
具体步骤如下:
鼠李糖乳杆菌CCFM1125可用于制备菌粉,菌粉的具体制备过程如下:
鼠李糖乳杆菌CCFM1125划线于MRS固体培养基上,37℃条件下培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于培养基中,37℃条件下培养18h,得到菌液;将菌液经5000g离心15min,得到菌泥;将菌泥用生理盐水清洗3次后用保护剂重悬至浓度为1×1010CFU/mL,得到菌悬液;将菌悬液在温度37℃下预培养60min后冻干,得到鼠李糖乳杆菌CCFM1125菌粉;
其中,培养基的制备方法为:使用以培养基总重量计87.7%的水将10%酶水解脱脂乳、0.5%葡萄糖、1.5%胰蛋白胨与0.3%酵母浸膏溶解,然后调整其pH为6.8,得到培养基;
保护剂的成分包含:100g/L脱脂奶粉、30mL/L甘油、100g/L麦芽糊精、150g/L海藻糖和10g/L L-谷氨酸钠。
实施例5:鼠李糖乳杆菌CCFM1125的应用
具体步骤如下:
鼠李糖乳杆菌CCFM1125可用于制备牛乳,发酵乳的具体制备过程如下:
鼠李糖乳杆菌CCFM1125划线于MRS固体培养基上,37℃条件下培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于培养基中,37℃条件下培养18h,得到菌液;将菌液经5000g离心15min,得到菌泥;将菌泥用生理盐水清洗3次后用保护剂重悬至浓度为1×1010CFU/mL,得到菌悬液;将菌悬液在温度37℃下预培养60min后冻干,得到鼠李糖乳杆菌CCFM1125菌粉;
其中,培养基的制备方法为:使用以培养基总重量计87.7%的水将10%酶水解脱脂乳、0.5%葡萄糖、1.5%胰蛋白胨与0.3%酵母浸膏溶解,然后调整其pH为6.8,得到培养基;
保护剂的成分包含:100g/L脱脂奶粉、30mL/L甘油、100g/L麦芽糊精、150g/L海藻糖和10g/L L-谷氨酸钠。
将脱脂奶在95℃热杀菌20min后冷却至4℃,得到原料;在原料中添加鼠李糖乳杆菌CCFM1125菌粉至浓度为不低于1×106CFU/mL,得到牛乳。
实施例6:鼠李糖乳杆菌CCFM1125的应用
具体步骤如下:
鼠李糖乳杆菌CCFM1125可用于制备豆奶,豆奶的具体制备过程如下:
鼠李糖乳杆菌CCFM1125划线于MRS固体培养基上,37℃条件下培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于培养基中,37℃条件下培养18h,得到菌液;将菌液经5000g离心15min,得到菌泥;将菌泥用生理盐水清洗3次后用保护剂重悬至浓度为1×1010CFU/mL,得到菌悬液;将菌悬液在温度37℃下预培养60min后冻干,得到鼠李糖乳杆菌CCFM1125菌粉;
其中,培养基的制备方法为:使用以培养基总重量计87.7%的水将10%酶水解脱脂乳、0.5%葡萄糖、1.5%胰蛋白胨与0.3%酵母浸膏溶解,然后调整其pH为6.8,得到培养基;
保护剂的成分包含:100g/L脱脂奶粉、30mL/L甘油、100g/L麦芽糊精、150g/L海藻糖和10g/L L-谷氨酸钠。
将大豆在温度80℃下浸泡2h后去除大豆皮,得到去皮大豆;将去皮大豆沥去浸泡水后加沸水磨浆,得到豆浆;将豆浆在高于80℃的温度条件下保温12min,得到熟豆浆;将熟豆浆用150目筛网过滤后离心分离,得到粗豆奶;将粗豆奶加热到温度140~150℃后迅速导入真空冷却室进行抽真空,使得粗豆奶中的异味物质随着水蒸汽迅速排出,得到熟豆奶;将熟豆奶降温至约37℃后在熟豆奶中添加鼠李糖乳杆菌CCFM1125菌粉至浓度为不低于1×106CFU/mL,得到豆奶。
实施例7:鼠李糖乳杆菌CCFM1125的应用
具体步骤如下:
鼠李糖乳杆菌CCFM1125可用于制备果蔬饮料,果蔬饮料的具体制备过程如下:
鼠李糖乳杆菌CCFM1125划线于MRS固体培养基上,37℃条件下培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于培养基中,37℃条件下培养18h,得到菌液;将菌液经5000g离心15min,得到菌泥;将菌泥用生理盐水清洗3次后用保护剂重悬至浓度为1×1010CFU/mL,得到菌悬液;将菌悬液在温度37℃下预培养60min后冻干,得到鼠李糖乳杆菌CCFM1125菌粉;
其中,培养基的制备方法为:使用以培养基总重量计87.7%的水将10%酶水解脱脂乳、0.5%葡萄糖、1.5%胰蛋白胨与0.3%酵母浸膏溶解,然后调整其pH为6.8,得到培养基;
保护剂的成分包含:100g/L脱脂奶粉、30mL/L甘油、100g/L麦芽糊精、150g/L海藻糖和10g/L L-谷氨酸钠。
将新鲜水果和蔬菜洗净后榨汁,得到果蔬汁;将果蔬汁在温度140℃下高温热杀菌2秒,得到杀菌后的果蔬汁;将杀菌后的果蔬汁降温至约37℃后在杀菌后的果蔬汁中添加鼠李糖乳杆菌CCFM1125菌粉至浓度为不低于1×106CFU/mL,得到果蔬饮料。
实施例8:鼠李糖乳杆菌CCFM1125的应用
具体步骤如下:
鼠李糖乳杆菌CCFM1125可用于制备胶囊制品,胶囊制品的具体制备过程如下
鼠李糖乳杆菌CCFM1125划线于MRS固体培养基上,37℃条件下培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于培养基中,37℃条件下培养18h,得到菌液;将菌液经5000g离心15min,得到菌泥;将菌泥用生理盐水清洗3次后用保护剂重悬至浓度为1×1010CFU/mL,得到菌悬液;菌悬液添加至浓度为30g/L的海藻酸钠溶液中至浓度为2×109CFU/mL后,充分搅拌,使得鼠李糖乳杆菌CCFM1125的细胞均匀地分散于海藻酸钠溶液中,得到混合液;将混合液挤压到浓度为20g/L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;
其中,培养基的制备方法为:使用以培养基总重量计87.7%的水将10%酶水解脱脂乳、0.5%葡萄糖、1.5%胰蛋白胨与0.3%酵母浸膏溶解,然后调整其pH为6.8,得到培养基。
实施例9:鼠李糖乳杆菌CCFM1125的应用
具体步骤如下:
鼠李糖乳杆菌CCFM1125可用于制备发酵乳,发酵乳的具体制备过程如下:
鼠李糖乳杆菌CCFM1125划线于MRS固体培养基上,37℃条件下培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于培养基中,37℃条件下培养18h,得到菌液;将菌液经5000g离心15min,得到菌泥;将菌泥用生理盐水清洗3次后用保护剂重悬至浓度为1×1010CFU/mL,得到菌悬液;将菌悬液在温度37℃下预培养60min后冻干,得到鼠李糖乳杆菌CCFM1125菌粉;
其中,培养基的制备方法为:使用以培养基总重量计87.7%的水将10%酶水解脱脂乳、0.5%葡萄糖、1.5%胰蛋白胨与0.3%酵母浸膏溶解,然后调整其pH为6.8,得到培养基;
保护剂的成分包含:100g/L脱脂奶粉、30mL/L甘油、100g/L麦芽糊精、150g/L海藻糖和10g/L L-谷氨酸钠。
将鼠李糖乳杆菌CCFM1125菌粉与商业干粉发酵剂保加利亚乳杆菌和商业干粉发酵剂嗜热链球菌按照质量比1:1:1的比例混合,得到发酵剂;将糖添加至鲜奶中至浓度为50g/L,得到混合液;将混合液在65℃、20MPa的条件下进行均质后在95℃下保温杀菌5min,得到发酵原料;将发酵原料降温至35℃后以0.03%(v/v)的接种量将发酵剂接种至发酵原料中,于35℃下保温发酵16h,得到发酵乳;将发酵乳于42℃下放置4h进行凝乳后,在4℃下冷藏24h进行后熟,得到发酵乳成品。
实施例10:鼠李糖乳杆菌CCFM1125的应用
具体步骤如下:
鼠李糖乳杆菌CCFM1125可用于制备片剂,片剂的具体制备过程如下:
鼠李糖乳杆菌CCFM1125划线于MRS固体培养基上,37℃条件下培养48h,得到单菌落;挑取单菌落接种于MRS液体培养基中,37℃条件下培养18h进行活化,连续活化两代,得到活化液;将活化液按2%(v/v)的接种量接种于培养基中,37℃条件下培养18h,得到菌液;将菌液经5000g离心15min,得到菌泥;将菌泥用生理盐水清洗3次后用保护剂重悬至浓度为1×1010CFU/mL,得到菌悬液;将菌悬液在温度37℃下预培养60min后冻干,得到鼠李糖乳杆菌CCFM1125菌粉;
其中,培养基的制备方法为:使用以培养基总重量计87.7%的水将10%酶水解脱脂乳、0.5%葡萄糖、1.5%胰蛋白胨与0.3%酵母浸膏溶解,然后调整其pH为6.8,得到培养基;
保护剂的成分包含:100g/L脱脂奶粉、30mL/L甘油、100g/L麦芽糊精、150g/L海藻糖和10g/L L-谷氨酸钠。
称取鼠李糖乳杆菌CCFM1125菌粉25.7重量份、淀粉55.0重量份、纤维素衍生物4.5重量份、羧甲基淀粉钠12.0重量份、滑石粉0.8重量份、蔗糖1.0重量份与水1.0重量份,得到原材料;将原材料混合,得到湿颗粒;将湿颗粒用中南制药机械厂的压片机进行压片后使用青州市益康中药机械有限公司的小型药物干燥机进行干燥,得到片剂。
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。
序列表
<110> 江南大学
<120> 一株可缓解苯并芘暴露的鼠李糖乳杆菌及其应用
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 967
<212> DNA
<213> 鼠李糖乳杆菌
<400> 1
atgccaggcg cagctataat gcagtcgacg agttctgatt attgaaaggt gcttgcatct 60
tgatttaatt ttgaacgagt ggcggacggg tgagtaacac gtgggtaacc tgcccttaag 120
tgggggataa catttggaaa cagatgctaa taccgcataa atccaagaac cgcatggttc 180
ttggctgaaa gatggcgtaa gctatcgctt ttggatggac ccgcggcgta ttagctagtt 240
ggtgaggtaa cggctcacca aggcaatgat acgtagccga actgagaggt tgatcggcca 300
cattgggact gagacacggc ccaaactcct acgggaggca gcagtaggga atcttccaca 360
atggacgcaa gtctgatgga gcaacgccgc gtgagtgaag aaggctttcg ggtcgtaaaa 420
ctctgttgtt ggagaagaat ggtcggcaga gtaactgttg tcggcgtgac ggtatccaac 480
cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgtta 540
tccggattta ttgggcgtaa agcgagcgca ggcggttttt taagtctgat gtgaaagccc 600
tcggcttaac cgaggaagtg catcggaaac tgggaaactt gagtgcagaa gaggacagtg 660
gaactccatg tgtagcggtg aaatgcgtag atatatggaa gaacaccagt ggcgaaggcg 720
gctgtctggt ctgtaactga cgctgaggct cgaaagcatg ggtagcgaac aggattagat 780
accctggtag tccatgccgt aaacgatgaa tgctaggtgt tggagggttt ccgcccttca 840
gtgccgcagc taacgcatta agcattccgc ctggggagta cgaccgcaaa ggttgaaact 900
caaaggaatt gacaggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg 960
cgaagaa 967
Claims (10)
1.一株鼠李糖乳杆菌(Lactobacillus rhamnosus),其特征在于,所述鼠李糖乳杆菌保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61019,保藏日期为2020年05月06日。
2.权利要求1所述鼠李糖乳杆菌在制备预防和/或治疗苯并芘暴露的药品中的应用。
3.一种产品,其特征在于,所述产品含有权利要求1所述鼠李糖乳杆菌。
4.如权利要求3所述的产品,其特征在于,所述产品中,权利要求1所述鼠李糖乳杆菌的活菌数为不低于1×106CFU/mL或1×106CFU/g。
5.如权利要求3或4所述的产品,其特征在于,所述产品包含食品、药品或苯并芘清除剂。
6.如权利要求5所述的产品,其特征在于,所述药品含有权利要求1所述鼠李糖乳杆菌、药物载体和/或药用辅料。
7.如权利要求6所述的产品,其特征在于,所述药物载体包含微囊、微球、纳米粒和/或脂质体。
8.如权利要求6或7所述的产品,其特征在于,所述药用辅料包含赋形剂和/或附加剂。
9.如权利要求5-8任一项所述的产品,其特征在于,所述药品的剂型为粉剂、颗粒剂、胶囊剂、片剂、丸剂或口服液。
10.权利要求1所述鼠李糖乳杆菌或权利要求5-8任一项所述的产品在清除苯并芘中的应用。
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CN113151116A (zh) * | 2021-05-24 | 2021-07-23 | 江南大学 | 一株可清除壬基酚的植物乳杆菌及其应用 |
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