CN111876356A - 一种具有抗衰老功能的发酵乳杆菌及其应用 - Google Patents
一种具有抗衰老功能的发酵乳杆菌及其应用 Download PDFInfo
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Abstract
本发明提供一种具有抗衰老功能的发酵乳杆菌及其应用,涉及生物工程技术领域,所述该菌株分离自百岁老人粪便,具有良好的耐酸、耐胆盐、具有较强的DPPH自由基、羟自由基清除能力、较强的Fe2+螯合能力和抗脂质过氧化能力,该菌株运用于SAMP8衰老小鼠可以降低血清中炎症因子IL‑6、IL‑1β和TNF‑α的含量,还增加了小鼠血清中超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH‑PX)活性,降低了血清中脂质过氧化物丙二醛的浓度。本发明所述的发酵乳杆菌对人体衰老方面的应用具有广泛的应用前景。
Description
技术领域
本发明属于生物工程技术领域,主要涉及一种具有抗衰老功能的发酵乳杆菌及其应用。
背景技术
衰老是几乎所有活生物体都会经历的过程,衰老的特征在于随着时间推移,机体细胞、组织、器官功能逐渐衰退,同时伴随着认知和记忆功能减退。在全球范围内,年龄在65岁以上的人口为6.17亿(8.5%),预计到2050年,这一数字将达到16亿。目前关于衰老的机制主要有氧化应激、细胞凋亡、DNA损伤、免疫、炎性衰老等。
最近研究表明,老年人肠道微生物群的多样性降低,有益菌减少而兼性厌氧菌增多。衰老引起的肠道微生物失调伴随着肠道通透性的增加,肠道微生物及其代谢产物进入血液循环,会引发全身性炎症。肠道菌群还可以通过脑-肠轴调控宿主行为,影响宿主的血脑屏障功能以及小胶质细胞成熟等基础性神经发育进程,参与脑功能的调控。肠道菌群参与衰老的进程主要通过调节氧化应激、免疫反应及调节代谢等多种途径。调节并维持老年人的肠道菌群平衡可能是预防和治疗衰老相关疾病及延缓衰老的手段,值得深入探讨和研究。
益生菌作为一类对机体有益的活性微生物,在维持肠道内微生物平衡有很大作用。健康的肠道微生物组可以控制代谢、抵抗感染和炎症、预防自身免疫和癌症,并且在调节脑-肠轴方面起着关键作用。补充益生菌可以通过多种途径提高机体免疫力,提高老年生活质量,在一定程度上可以延缓机体的衰老,增加寿命。发酵乳杆菌属于乳杆菌属,是一种能促进人体健康的常见菌种之一,具有抗氧化活性、降低胆固醇、降血压、调节肠道菌群平衡等作用。
现在市场上的发酵乳杆菌大多从发酵食品中分离所得,而我们认为在百岁老人体内具有更健康的肠道菌群,因此从百岁老人粪便中提取出具有抗衰老作用的发酵乳杆菌非常有意义。
发明内容
本发明的一个目的在于提供了一种耐酸、耐胆盐、能够清除自由基和耐受过氧化氢的抗氧化的发酵乳杆菌(Lactobacillusfermentum)2953菌株。
本发明的另一个目的在于提供一种抗衰老作用的发酵乳杆菌。
为解决上述技术问题,本发明提供了一种干酪乳杆菌制剂,所述发酵乳杆菌株是分离自百岁老人粪便中,具有耐酸、耐胆盐、抗氧化能力的益生菌株。
优选的,所述发酵乳杆菌作为发酵菌种的应用。
优选的,所述发酵乳杆菌制备的发酵剂。
优选的,所述发酵乳杆菌发酵制备的发酵酸奶产品。
优选的,所述发酵乳杆菌菌制备的益生菌菌片。
优选的,所述发酵乳杆菌在抗衰老方面的应用。
本发明提供的具有抗衰老功能的发酵乳酸菌(Lactobacillusfermentum)2953菌株分离自百岁老人粪便,该菌株经菌种测序分析,其序列如SEQIDNO.1所示,将得到的序列经NCBI核酸序列对比,结果显示菌株为发酵乳杆菌,命名为发酵乳杆菌(Lactobacillusfermentum)2953。
本发明所涉及的所述发酵乳杆菌,它可以降低SAMP8小鼠的衰老评分,缓解衰老小鼠的记忆障碍,降低小鼠血清中的炎症因子IL-6、IL-1β和TNF-α的含量,还增加了小鼠血清中超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-PX)活性,降低了血清中脂质过氧化物丙二醛的浓度。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为本发明发酵乳杆菌的耐酸测定结果示意图;
图2为本发明发酵乳杆菌的耐胆盐测定结果示意图;
图3为本发明发酵乳杆菌的抗氧化测定结果示意图;
图4为本发明发酵乳杆菌制剂对SAMP8小鼠旷场实验结果示意图;
图5为本发明发酵乳杆菌制剂对SAMP8小鼠巴恩斯迷宫实验结果示意图;
图6为本发明发酵乳杆菌制剂对SAMP8小鼠血清中炎症因子IL-6、IL-1β和TNF-α的影响。
图7为本发明发酵乳杆菌制剂对SAMP8小鼠血清中SOD的影响;
图8为本发明发酵乳杆菌制剂对SAMP8小鼠血清中GSH-Px的影响;
图9为本发明发酵乳杆菌制剂对SAMP8小鼠血清中MDA的影响;
图10为本发明试管编号图;
图11为本发明酶促反应图。
具体实施方式
应该指出,以下详细说明都是示例性的,旨在对本发明提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
下述实施例中所用的材料、试剂等,如无特殊要求均从商业途径获得。
实施例1:发酵乳杆菌的获得及其鉴定
一、发酵乳杆菌的分离纯化
百岁老人的粪便采用30%的甘油保存,使用无菌PBS(1%,w/v)稀释,均匀涂布于MRS琼脂平板上,37℃静置培养48h后,根据菌落形态,在MRS琼脂平板上反复划线纯化,直到固体培养基上长出纯单一菌落,将分离的乳酸菌连续传代3次,然后以30%甘油与菌液1:1混合保存于-80℃。
二、发酵乳杆菌的鉴定
将挑选的菌落连续培养后,取部分菌液离心,按照天根细菌DNA提取试剂盒说明书提取DNA,然后送于公司(上海生工),由公司进行16SrDNA序列扩增、纯化,然后测序。
实施例2:发酵乳杆菌的耐酸实验
取100ul的发酵乳杆菌,加入5ml的对应的液体培养基,37℃培养箱培养24h。取100ul的菌液,用PBS缓冲液梯度稀释101,103,105倍,(取100ul加入到900ul的PBS缓冲液,再从中取10ul加入到990ul的PBS中,再从中取10ul加入到990ul的PBS中),2500rpm离心4min,弃掉上清,加入pH为2、3、4、5、7的PBS缓冲液,放置4小时,混匀取10ul涂板,37℃培养箱培养过夜。活菌计数。结果如图1所示。
实施例3:发酵乳杆菌的耐胆盐实验
取100ul的发酵乳杆菌,加入5ml的对应的液体培养基,37℃培养箱培养24h。分别取100ul的菌液,接种至0%胆盐、0.1%胆盐、0.2%胆盐和0.3%胆盐的MRS培养基中,37℃培养24h,用PBS缓冲液梯度稀释102,104,106,108倍,混匀取10uL涂板,37℃培养箱培养过夜。活菌计数。结果如图2所示。
实施例4:发酵乳杆菌的抗氧化实验
溶液配制:MRS液体培养基、去离子水、0.2mmol/LDPPH甲醇溶液、2mmol/L硫酸亚铁溶液、6mmol/L过氧化氢溶液、6mmol/L水杨酸溶液、150mmol/LpH=8.0的Tris-HCL溶液、1.2mmol/L邻苯三酚溶液、0.4%的硫酸亚铁溶液、1%的VC,0.2mol/L的氢氧化钠溶液、10%的三氯乙酸溶液、0.1%邻二氮菲溶液、pH=6.6的磷酸缓冲液、1%铁氰化钾溶液和0.1%的三氯化铁溶液。
灭菌:以上的溶液
活化:取100uL的短乳杆菌液加到装有5mL的MRS培养基的玻璃试剂管内,37℃培养箱培养过夜。第二天,离心,取上清液。
①DPPH自由基清除能力的测定,
取2mL的上清液加到玻璃试管内,再加2mL配制好的DPPH甲醇溶液,黑暗下室温反应30分钟,离心取上清。另一根玻璃试管内加入2mL的去离子水和2mL的DPPH甲醇溶液作为对照。517nm测OD值。记录对照组和实验组数据。
DPPH自由基清除率=[1-A517(样品)/A517(空白)]×100%
②羟基自由基清除能力
取1mL的细菌上清液加到玻璃试管内,加入1mL的硫酸亚铁溶液,1mL的过氧化氢溶液,1mL的水杨酸溶液,静置30分钟。另一根玻璃管内加入上述溶液,并加入1mL的去离子水。30分钟后,510nm处测吸光度。记录对照组和实验组数据。
羟基自由基清除率=[1-A510(样品)/A510(空白)]×100%
③超氧自由基清除能力
取0.5mL细菌上清液加到玻璃试管1号内,加入2mL的Tris-HCL溶液,1mL邻苯三酚溶液,室温反应30min,2号试管加0.5mL的去离子水,1mL邻苯三酚和2mL的2mLTris-HCL溶液,3号试管加1.5mL的去离子水,2mLTris-HCL溶液,4号试管加1mL的去离子水,0.5mL细菌上清和2mLTris-HCL溶液。
超氧自由基清除率=[1-(A11-A10)/(A01-A00)]×100%
A00:不含样品和邻苯三酚(3);A01:不含样品含邻苯三酚(2)
A10:含样品不含邻苯三酚(4);A11:含样品和邻苯三酚(1)
④对亚铁离子的螯合能力
取0.5mL细菌上清液加到玻璃管内,加入0.1mL的硫酸亚铁溶液混合均匀,再加入0.1mL的VC和1mL的氢氧化钠溶液,37度烘箱内反应20分钟,然后加入三氯乙酸,离心去除蛋白,4℃环境下6000rpm离心10min。取0.4mL的上清,加入4mL邻二氮菲。另一根试管加0.5mL的水,其他相同。室温反应10分钟,536nm处测吸光度。记录对照组和实验组数据。
Fe2+的螯合能力=(A空白-A样/A空白)×100%
⑤总还原力测定
取1mL的细菌上清液,加入1mL的磷酸缓冲液和1mL的铁氰化钾,混匀,50℃下保温2min,加入1mL的三氯乙酸,震荡混匀后取1mL的混合液,加入4mL的去离子水和0.4mL的三氯化铁溶液,静置10min。另一根试管加1mL的去离子水,其他加的试剂和处理情况相同。静置后拿去测吸光度,用去离子水作为空白。700nm测吸光度,记录对照组和实验组数据。结果如图1所示。
实施例5:发酵乳杆菌对小鼠的抗衰应用
A、实验菌株的制备:将发酵乳杆菌按1%(v/v)接种于MRS培养基,37℃培养过夜,至OD600=0.6,8000rpm离心3min收集菌体,用无菌生理盐水清洗2次,再用含0.1%的明胶生理盐水重悬,调整菌数为1×109cfu/mL。
B、实验动物及分组治疗:将16周龄的快速衰老小鼠SAMP8雄性小鼠10只和5只同月龄的雄性SAMR1小鼠(购买自北京大学医学部实验动物中心),适应性饲养一周,标记称重后开始实验,SAMR1为对照组(连续灌胃含0.1%明胶生理盐水治疗,n=5),SAMP8分为2组,分别为衰老组(连续灌胃含0.1%明胶生理盐水治疗,n=5)和发酵乳杆菌组(连续灌胃发酵乳杆菌1×109cfu/只/天,n=5)。在评估衰老情况、旷场实验以及巴恩斯迷宫实验后,收集小鼠血清,并处死小鼠。
C、小鼠血清的提取:小鼠麻醉后取血约1ml,3500rpm离心15min,取上层血清测定生化指标。
D、小鼠老化度评分:从反应性、被动性逃避反应、皮毛光泽、皮毛粗糙度、脱毛程度、皮肤溃疡、眼周损害、角膜浑浊、角膜溃疡、白内障、脊柱后凸等11项指标对各组动物进行客观评分。每项指标划分为4~5级不等,标定分值。动物积分越高表明其老化度越高。
E、旷场试验:测试箱为50cm×50cm×40cm的塑料箱,底部黑色,四壁为白色。设定ANY-MAZE采集软件的实验参数。采集时间为5min。采集指标选择穿越格子的总次数以及移动总距离。实验开始时将每只小鼠面朝箱壁放入测试箱内任一角落,记录随后5min内小鼠的自发活动。每次测试结束后清除小鼠排泄物,使用70%的酒精擦拭箱底及四壁,待酒精挥干后进行下一只鼠测试。结果如图4所示。
F、巴恩斯迷宫实验:实验开始前一天,将动物单个从目标洞置于目标箱内适应4min。将动物置于迷宫中央的塑料圆桶(直径20cm,高27cm)内限制活动5s。移开圆桶,启动计时器,实验者在挡帘后进行观察。动物四肢均进入目标箱,则计为一次逃避(escape),并让动物在箱内停留30s。每一动物一次最多观察4min。在此期间如果动物仍然找不到目标箱,则将动物从迷宫移开,放入目标箱内并停留30s。利用这一间隙清洁迷宫。动物每天训练两次,连续5-6d。从第二次训练开始,每次训练之前将迷宫随机转动一至数个洞的位置,但目标箱始终固定在同一方位。这样做的目的是防止动物依靠气味、而非凭借记忆来确定目标洞的位置。实验记录以下参数:探究任何一个洞的潜伏期、到达目标箱的潜伏期和每只动物的错误次数(一次错误定义为动物把头伸向或探究任何一个非目标洞,包括专注于探究同一个非目标洞)。结果如图5所示。
G、小鼠血清炎症因子的测定:
(1)试剂准备:使用前将所有的试剂和标本缓慢均衡至室温(18-25℃);按照说明书所示配置好标准品、检测溶液A、检测溶液B、洗涤液和底物溶液。
(2)加样:分别设标准孔、待测样品孔、空白孔。设标准孔7孔,依次加入100μL不同浓度的标准品。空白孔加100μL标准品稀释液,余孔加待测样品100μL,酶标板加上覆膜,37℃温育1小时。
(3)弃去液体,甩干,不用洗涤。
(4)每孔加检测溶液A工作液100μL(临用前配制),酶标板覆膜,37℃温育1小时。
(5)弃去孔内液体,每孔用350μL的洗涤液洗涤,浸泡1-2分钟,在吸水纸上轻拍酶标板来移除孔内所有液体。重复洗板3次。最后一次洗涤后,吸取或倒出剩余的洗涤缓冲液,将酶标板倒扣在吸水纸上,将残留在孔内的液体全部吸干。
(6)每孔加检测溶液B工作液(临用前配制)100μL,酶标板覆膜,37℃温育30分钟。
(7)弃去孔内液体,甩干,洗板5次,方法同步骤(5)。
(8)每孔加TMB底物溶液90μL,酶标板覆膜,37℃避光显色(反应时间15分钟)。
(9)每孔加终止溶液50μL,终止反应,此时蓝色立转黄色。
(10)在确保酶标板底无水滴及孔内无气泡后,立即用酶标仪在450nm波长测量各孔的光密度值(O.D.值)。
(11)使用CurveExpert软件计算相应浓度。结果如图6所示。
G、小鼠血清SOD活力的测定:
(1)分光光度计预热30min以上,调节波长至560nm,蒸馏水调零。
(2)测定前将试剂一、三和五37℃水浴5min以上。
(3)把试管编号,分别编为测定管、对照管、空白管1和空白管2;试管编号后按下表加入试剂:(4)SOD活性计算:
①抑制百分率的计算:抑制百分率=(ΔA空白-ΔA测定)÷ΔA空白×100%
②SOD酶活性单位:在上述黄嘌呤氧化酶藕联反应体系中抑制百分率为50%时,反应体系中的SOD酶活力定义为一个酶活力单位。
③SOD酶活性计算:血清(浆)SOD活性(U/mL)=[抑制百分率÷(1-抑制百分率)×V反总]÷V样×F=11.4×抑制百分率÷(1-抑制百分率)×F。结果如图7所示。
H、小鼠血清GSH-Px的测定:
(1)酶促反应。
(2)血清中GSH—PX酶活力的计算:规定每O.1ml血清在37。C反应5分钟,扣除非酶促反应作用,使反应体系中GSH浓度降低19mol/L为一个酶活力单位。
血清GSH-PX活力=(非酶管OD值.酶管OD值)÷(标准管OD值-空白管OD值)×标准品浓度(20umol/L)×稀释倍数(6)×样本测试前稀释倍数。结果如图8所示。
I、小鼠血清MDA的测定:采用硫代巴比妥法检测血浆中MDA的浓度,操作步骤如下:
(1)把试管编号,分别编为标准管、标准空白管、测定管和测定空白管;试管编号后按下表加入试剂:
(2)漩涡混匀器混匀,试管口用保鲜薄膜扎紧,用针头刺一小孔,95℃水浴40min;
(3)取出后流水冷却,然后4000r/min,离心10min,使沉淀完全;
(4)取上清,吸取上清比色时用移液器吸取上清加入比色皿中,尽量避免倾倒,以免沉淀进入比色皿,影响吸光度;
(5)把比色皿置于532nm处,1cm光径,蒸馏水调零,测各管吸光度值;
(6)计算血浆中MDA的含量:血浆中MDA含量(ng/ml)=(测定管吸光度-测定空白管吸光度)/(标准管吸光度-标准空白管吸光度)×标准品浓度(10ng/ml)×样品测试前稀释倍数。结果如图9所示。
以上的仅是本发明的优选实施方式,应当指出,对于本领域的普通技术人员来说,在不脱离本发明创造构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。
Claims (6)
1.一种具有抗衰老功能的发酵乳杆菌及其应用,其特征在于:所述发酵乳杆菌株是分离自百岁老人粪便中,具有耐酸、耐胆盐、抗氧化能力的益生菌株。
2.根据权利要求1所述的一种具有抗衰老功能的发酵乳杆菌及其应用,其特征在于:所述发酵乳杆菌作为发酵菌种的应用。
3.根据权利要求1所述的一种具有抗衰老功能的发酵乳杆菌及其应用,其特征在于:所述发酵乳杆菌制备的发酵剂。
4.根据权利要求1所述的一种具有抗衰老功能的发酵乳杆菌及其应用,其特征在于:所述发酵乳杆菌发酵制备的发酵酸奶产品。
5.根据权利要求1所述的一种具有抗衰老功能的发酵乳杆菌及其应用,其特征在于:所述发酵乳杆菌菌制备的益生菌菌片。
6.根据权利要求1所述的一种具有抗衰老功能的发酵乳杆菌及其应用,其特征在于:所述发酵乳杆菌在抗衰老方面的应用。
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