CN116622585A - 动物双歧杆菌乳亚种bl03在制备抗氧化和抗衰老产品中的应用 - Google Patents
动物双歧杆菌乳亚种bl03在制备抗氧化和抗衰老产品中的应用 Download PDFInfo
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Abstract
本发明公开了动物双歧杆菌乳亚种BL03在制备抗氧化和抗衰老产品中的应用,属于微生物技术领域。本发明公开的动物双歧杆菌乳亚种BL03在斑马鱼氧化应激模型中具有显著降低斑马鱼体内ROS水平,和显著提高斑马鱼体内SOD活性的潜能,此为利用动物双歧杆菌乳亚种BL03开发抗氧化的益生菌制剂提供理论参考和指导依据。
Description
技术领域
本发明涉及微生物技术领域,更具体的说是涉及动物双歧杆菌乳亚种BL03在制备抗氧化和抗衰老产品中的应用。
背景技术
活性氧(ROS)是细胞有氧代谢的副产物,在细胞生命周期中发挥着重要作用。低浓度的ROS可作为细胞内的关键信号分子以调节细胞的生长、增殖和分化。然而,ROS的积累会严重破坏细胞生物大分子如蛋白质、脂质和DNA等,导致多种慢性疾病,包括动脉粥样硬化、关节炎、糖尿病、神经退行性疾病、老化、炎症肠病等。大量实验研究表明,抗氧化剂或自由基清除剂有助于避免ROS的积累,对氧化损伤具有保护作用,均可有效控制这些疾病的发生,发挥良好的预防作用。但大多数化学合成或植物提取的抗氧化药物因具有潜在的不良反应而不推荐长期使用。与传统抗氧化药物相比,具有抗氧化功效的益生菌因其副作用小并且具有其他益生功效等特点,而被广泛关注。
因此,提供动物双歧杆菌乳亚种BL03在制备抗氧化和抗衰老产品中的应用是本领域技术人员亟需解决的问题。
发明内容
有鉴于此,本发明提供了动物双歧杆菌乳亚种BL03在制备抗氧化和抗衰老产品中的应用。
甲萘醌是一种氧化剂,通过细胞内还原酶系统(微粒体的P450还原酶和线粒体的呼吸链还原酶),产生不稳定的半醌,进入氧化还原循环,产生活性氧族。用甲萘醌可诱导斑马鱼建立氧化应激模型。
经过特异性荧光染色(呈绿色,主要定位于细胞核和线粒体),发生氧化应激反应斑马鱼全身明显比正常斑马鱼绿很多,可以用荧光显微镜下观察到斑马鱼体内的活性氧含量。
为了实现上述目的,本发明采用如下技术方案:
动物双歧杆菌乳亚种(Bifidobacterium animalis subsp.lactis)BL03在制备抗氧化和抗衰老产品中的应用,所述动物双歧杆菌乳亚种BL03的保藏编号为CGMCC No.23451(参见专利号202211551619.2)。
进一步,所述的动物双歧杆菌乳亚种BL03在制备降低体内ROS水平,提高体内SOD活性产品中的应用。
进一步,所述动物双歧杆菌乳亚种BL03为菌悬液。
动物双歧杆菌乳亚种BL03在体内氧化应激模型中均能显著降低斑马鱼体内ROS水平和显著提高斑马鱼体内SOD活性,增强机体清除自由基的能力,表现出良好抗氧化、抗衰老的作用。
经由上述的技术方案可知,与现有技术相比,本发明公开提供了动物双歧杆菌乳亚种BL03在制备抗氧化和抗衰老产品中的应用,动物双歧杆菌乳亚种BL03在斑马鱼氧化应激模型中具有显著降低斑马鱼体内ROS水平,和显著提高斑马鱼体内SOD活性的潜能,此为利用动物双歧杆菌乳亚种BL03开发抗氧化的益生菌制剂提供理论参考和指导依据。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1附图为本发明动物双歧杆菌乳亚种BL03对甲萘醌诱导斑马鱼氧化应激模型中ROS水平影响的直观图;
其中,A:正常组;B:模型组;C:阳性对照组;D:1×106CFU/mL动物双歧杆菌乳亚种27536;E:1×106CFU/mL动物双歧杆菌乳亚种BL03;
图2附图为本发明动物双歧杆菌乳亚种BL03对甲萘醌诱导斑马鱼氧化应激模型中ROS水平影响的统计图;
图3附图为本发明动物双歧杆菌乳亚种BL03对甲萘醌诱导斑马鱼氧化应激模型中SOD活性的影响。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
还原型谷胱甘肽(GSH)、甲萘醌、二甲基亚砜(DMSO)均购自上海源叶生物科技有限公司;2',7'-二氯二氢荧光素二乙酸酯(DCFH-DA)和超氧化物歧化酶(SOD)检测试剂盒均购自Sigma-Aldrich公司;动物双歧杆菌乳亚种27536(ATCC 27536)购自北京百欧博伟生物技术有限公司。
实施例1动物双歧杆菌乳亚种BL03菌悬液(菌体)的制备
将动物双歧杆菌乳亚种BL03活化培养后接种于BS液体培养基中,37℃培养24h后,4℃,6000r/min离心10min,得到菌体沉淀;菌体沉淀经PBS两次洗涤后,将菌体用PBS重新悬浮,调整细胞浓度为1×106CFU/mL得到菌悬液(菌体)。
实施例2动物双歧杆菌乳亚种27536菌悬液(菌体)的制备
将动物双歧杆菌乳亚种27536活化培养后接种于BS液体培养基中,37℃培养24h后,4℃,6000r/min离心10min,得到菌体沉淀;菌体沉淀经PBS两次洗涤后,将菌体用PBS重新悬浮,调整细胞浓度为1×106CFU/mL得到菌悬液(菌体)。
实施例3动物双歧杆菌乳亚种BL03对斑马鱼氧化应激模型中ROS水平的影响
挑选发育至4dpf(dayspostfertilization)的健康野生型AB系斑马鱼置于6孔细胞培养板中,每孔20条鱼。实验设置正常组、模型组、阳性对照组(GSH)、动物双歧杆菌乳亚种27536干预组、动物双歧杆菌乳亚种BL03干预组。正常组和模型组均加入PBS,阳性对照组加入GSH溶液(100μM),动物双歧杆菌乳亚种27536干预组(1×106CFU/mL)加入1×106CFU/m动物双歧杆菌乳亚种27536;动物双歧杆菌乳亚种BL03干预组(1×106CFU/mL)加入1×106CFU/mL动物双歧杆菌乳亚种BL03,每孔2.5mL,28℃孵育,每24h后更换新溶液;孵育48h后,正常组加入2.5mL PBS(1%DMSO),模型组、阳性对照组、动物双歧杆菌乳亚种27536干预组、动物双歧杆菌乳亚种BL03干预组分别加入6μM甲萘醌(先用DMSO将甲萘醌配制为600μM储备溶液,再用PBS稀释为6μM),每孔2.5mL;28℃孵育24h后,弃上述溶液,再用PBS将上述斑马鱼洗涤3次,加入20μg/mL DCFH-DA溶液,每孔3mL,28℃避光孵育1h后,用PBS将上述斑马鱼洗涤3次,置于荧光显微镜下观察斑马鱼体内荧光强度并拍照记录。使用Image J软件对斑马鱼体内荧光强度(S)进行定量统计分析。斑马鱼体内ROS水平计算如下:
采用SPSS 19.0软件统计处理数据,实验数据均用x±SEM数据表示,用T-检验分析,与正常组相比:###P<0.005,与模型组相比:*P<0.05,***P<0.005。
结果见图1、2;由图1和图2可知,斑马鱼体内绿色荧光的强弱反映ROS水平的高低;与正常组相比,模型组斑马鱼体内绿色荧光强度增强,表明模型组斑马鱼体内ROS水平升高;同时,与正常组(100.00±5.88%)相比,模型组斑马鱼体内ROS水平(196.31±8.34%)显著升高(p<0.005),表明本次斑马鱼氧化应激模型建立成功。
与模型组相比,阳性对照组(GSH)斑马鱼体内绿色荧光强度减弱,表明GSH在甲萘醌诱导斑马鱼氧化应激模型中,可降低斑马鱼体内ROS水平;同时,阳性对照组斑马鱼体内ROS水平为108.83±6.63%,与模型组(196.31±8.34%)相比差异性显著(P<0.005);因此,GSH具有明显的抗氧化作用,与临床结果一致。动物双歧杆菌乳亚种27536干预组(1×106CFU/mL)斑马鱼体内ROS水平为168.77±9.09%,与模型组(196.31±8.34%)相比较差异性显著(P<0.05)。另外,与模型组相比,动物双歧杆菌乳亚种BL03斑马鱼体内绿色荧光强度减弱,表明动物双歧杆菌乳亚种BL03在甲萘醌诱导斑马鱼氧化应激模型中,可降低斑马鱼体内ROS水平;同时动物双歧杆菌乳亚种BL03干预组(1×106CFU/mL)斑马鱼体内ROS水平为122.79±6.49%,与模型组(196.31±8.34%)相比较差异性显著(P<0.005)。因此,上述结果表明,在相同的浓度时,动物双歧杆菌乳亚种BL03在体内氧化应激模型中对斑马鱼体内ROS水平的降低作用强于动物双歧杆菌乳亚种27536,表现出良好抗氧化、抗衰老的作用。
实施例4动物双歧杆菌乳亚种BL03对斑马鱼氧化应激模型中SOD活性的影响
挑选发育至4dpf(days post fertilization)的健康野生型AB系斑马鱼置于6孔细胞培养板中,每孔20条鱼。实验设置正常组、模型组、阳性对照组(GSH)、动物双歧杆菌乳亚种27536干预组、动物双歧杆菌乳亚种BL03干预组,每组3个复孔。正常组和模型组均加入PBS,阳性对照组加入GSH溶液(100μM),动物双歧杆菌乳亚种27536干预组(1×106CFU/mL)加入1×106CFU/mL动物双歧杆菌乳亚种27536;动物双歧杆菌乳亚种BL03干预组(1×106CFU/mL)加入1×106CFU/mL动物双歧杆菌乳亚种BL03,每孔2.5mL,28℃孵育,每24h后更换新溶液;孵育48h后,正常组加入2.5mL PBS(1%DMSO),模型组、阳性对照组、动物双歧杆菌乳亚种27536干预组、动物双歧杆菌乳亚种BL03干预组分别加入6μM甲萘醌(先用DMSO将甲萘醌配制为600μM储备溶液,再用PBS稀释为6μM),每孔2.5mL;28℃孵育24h后,弃上述溶液,再用PBS将上述斑马鱼洗涤3次,收集斑马鱼至1.5mL离心管,每管50mg斑马鱼,每个实验组收集6管;将离心管中的水吸干后,加入250μL缓冲液(超氧化物歧化酶(SOD)检测试剂盒缓冲溶液)。用S-18KS手持微量电动组织匀浆器将斑马鱼匀浆破碎,直至无明显组织碎块,15000×g,4℃离心15min,收集上清液。使用超氧化物歧化酶(SOD)检测试剂盒(Sigma-Aldrich公司)检测各组的SOD活性。
采用SPSS 19.0软件统计处理数据,实验数据均用x±SEM数据表示,用T-检验分析,与正常组相比:###P<0.005,与模型组相比:**P<0.01,***P<0.005。
结果见图3;由图3可知,与正常组(2.92±0.19U/mg)相比,模型组斑马鱼体内SOD活性(0.91±0.06U/mg)显著降低(p<0.005),表明本次斑马鱼氧化应激模型建立成功。
阳性对照组斑马鱼体内SOD活性为2.46±0.14U/mg,与模型组(0.91±0.06U/mg)相比差异性显著(P<0.005),表明GSH具有明显的抗氧化作用,与临床结果一致。动物双歧杆菌乳亚种27536干预组(1×106CFU/mL)斑马鱼体内SOD活性为1.27±0.07U/mg,与模型组(0.91±0.06U/mg)相比较差异性显著(P<0.01)。另外,动物双歧杆菌乳亚种BL03干预组(1×106CFU/mL)斑马鱼体内SOD活性为1.97±0.07U/mg,与模型组(0.91±0.06U/mg)相比较差异性显著(P<0.005)。因此,上述结果表明,在相同的浓度时,动物双歧杆菌乳亚种BL03在体内氧化应激模型中对斑马鱼体内SOD活性的提高作用强于动物双歧杆菌乳亚种27536,即增强机体清除自由基的能力,表现出良好抗氧化、抗衰老的作用。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (4)
1.动物双歧杆菌乳亚种(Bifidobacteriumanimalissubsp.lactis)BL03在制备抗氧化和抗衰老产品中的应用,其特征在于,所述动物双歧杆菌乳亚种BL03的保藏编号为CGMCCNo.23451。
2.根据权利要求1所述的动物双歧杆菌乳亚种BL03在制备抗氧化和抗衰老产品中的应用,其特征在于,所述动物双歧杆菌乳亚种BL03为菌悬液。
3.权利要求1中所述的动物双歧杆菌乳亚种BL03在制备降低体内ROS水平,提高体内SOD活性产品中的应用。
4.根据权利要求3所述的动物双歧杆菌乳亚种BL03在制备降低体内ROS水平,提高体内SOD活性产品中的应用,其特征在于,所述动物双歧杆菌乳亚种BL03为菌悬液。
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