CN111534447A - 约氏乳杆菌及其应用 - Google Patents
约氏乳杆菌及其应用 Download PDFInfo
- Publication number
- CN111534447A CN111534447A CN201911265623.0A CN201911265623A CN111534447A CN 111534447 A CN111534447 A CN 111534447A CN 201911265623 A CN201911265623 A CN 201911265623A CN 111534447 A CN111534447 A CN 111534447A
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- Prior art keywords
- lactobacillus johnsonii
- antioxidant
- bacteria
- strain
- lactobacillus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
本发明涉及微生物技术领域,具体而言,提供了一种约氏乳杆菌及其应用。本发明提供的约氏乳杆菌保藏编号为CCTCC NO:M 2019976。约氏乳杆菌在制备用于调节肠道菌群的产品中的应用和制备抗氧化剂中的应用也属于本申请的保护范围。本发明提供的菌株是一株具有潜在益生特性的优良菌株,可以作为益生菌用于抗菌药物、食品添加剂和动物饲料添加剂中,应用前景十分广阔。
Description
技术领域
本发明涉及微生物技术领域,具体而言,涉及一种约氏乳杆菌及其应用。
背景技术
约氏乳杆菌(Lactobacillus johnsonii,L.johnsonii)分类于乳杆菌属(Lactobacillus)。研究表明,约氏乳杆菌具有良好的益生作用,如能够促进仔猪的生长并降低仔猪腹泻;防治家禽坏死性肠炎、改善肉鸡的生长性能,减少鸡体内病原菌的定植;还可以预防老龄小鼠因蛋白质能量失衡导致的免疫机能障碍,提高小鼠小肠上皮细胞抗原受体的基因表达以及预防肥胖小鼠非酒精性脂肪性肝病;另有研究表明约氏乳杆菌能刺激分泌型免疫球蛋白A(sIgA)的产生以及调节环氧化酶-2(COX-2)、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-4(IL-4)、白细胞介素-6(IL-6)和γ-干扰素(IFN-γ)等细胞因子的水平。欧盟于2010年对约氏乳杆菌进行了安全资格认定(QPS),并批准其在饲料和食品中使用。
近年来,宠物犬的饲养量大幅上升,犬类被视为人类最忠实的伴侣,甚至有人把宠物犬视为重要的家庭成员,随着人们生活水平的提高,宠物犬的健康及生活质量也逐渐受到重视。无毒副作用、无残留的益生菌制剂在犬科动物疾病治疗和预防保健中的应用越来越广泛。在国外,犬用益生菌制剂的核心菌种主要集中于乳酸杆菌、肠球菌、双歧杆菌和芽孢杆菌,但其中犬科动物原籍菌的研究和应用仍较少。从动物自身分离得到的原籍菌能更好的适应同种类动物的胃肠道环境,从而能更好地发挥其益生特性,取得最大化的治疗和保健效果,但犬源约氏乳杆菌的报道较少,市场相应产品更少。
有鉴于此,特提出本发明。
发明内容
本发明的目的在于提供一种约氏乳杆菌及其应用。
为了实现本发明的上述目的,特采用以下技术方案:
一种约氏乳杆菌,所述约氏乳杆菌于2019年11月25日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 2019976。
上述的约氏乳杆菌在调节肠道菌群或制备用于调节肠道菌群的产品中的应用。
一种用于调节肠道菌群的产品,活性成分包括上述的约氏乳杆菌。
进一步地,所述调节肠道菌群包括如下(a)-(c)中任一种:
(a)抑制大肠杆菌;
(b)抑制沙门氏菌;
(c)抑制金黄色葡萄球菌。
上述的约氏乳杆菌在制备抗氧化剂中的应用。
进一步地,所述通过清除自由基实现抗氧化功能;
优选地,所述自由基为DPPH自由基。
一种抗氧化剂,活性成分包括上述的约氏乳杆菌。
进一步地,所述通过清除自由基实现抗氧化功能;
优选地,所述自由基为DPPH自由基。
上述的约氏乳杆菌在备抗菌药物中的应用;
优选地,所述菌为细菌或真菌;
优选地,所述细菌为革兰氏阳性细菌或革兰氏阴性细菌;
优选地,所述细菌包括大肠杆菌、沙门氏菌或金黄色葡萄球菌中的至少一种。
上述的约氏乳杆菌在制备食品添加剂或动物饲料添加剂中的应用。
与现有技术相比,本发明的有益效果为:
本发明提供的保藏编号为CCTCC NO:M 2019976的约氏乳杆菌是发明人首次从健康成年蓝狐后肠内容物中分离筛选得到的菌株。通过体外酸耐受和胆盐耐受、黏附能力、抗氧化作用和抑菌试验等检测,证实了该菌株具有良好的胃肠道定植能力,同时还具有抑制病原菌及抗氧化等多种益生功能,可作为益生菌制剂的备选菌株,应用前景十分广阔。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式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,下面描述中的附图是本发明的一些实施方式,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,还可以根据这些附图获得其他的附图。
图1为本发明实施例1中约氏乳杆菌的菌落形态图;
图2为本发明实施例1中约氏乳杆菌的细胞形态图;
图3为本发明实施例3中约氏乳杆菌的温度生长曲线;
图4为本发明实施例4中约氏乳杆菌的产酸速率曲线;
图5为本发明实施例5中约氏乳杆菌对酸度的耐受性;
图6为本发明实施例9中约氏乳杆菌粘附细胞图(1000×)。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限制本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。
除非另有说明,本文中所用的专业与科学术语与本领域熟练人员所熟悉的意义相同。此外,任何与所记载内容相似或均等的方法或材料也可应用于本发明中。
本发明提供的约氏乳杆菌于2019年11月25日保藏于中国典型培养物保藏中心,地址为湖北省武汉市武昌区八一路299号,保藏编号为CCTCC NO:M 2019976,拉丁名:Lactobacillus johnsonii。
乳酸菌进入动物体内的第一道安全屏障是胃酸,在通过小肠时肠道内的胆盐也会通过改变菌体细胞外膜的通透性来抑制乳酸菌的正常生长。食物通过胃的时间一般为1-3h,通过小肠的时间约为1.5h,小肠内的胆盐浓度在0.03%-0.3%范围内波动,因此0.3%胆盐浓度被认为是耐胆盐菌株的筛选临界浓度,益生菌能否顺利的通过胃肠道成功定植并发挥益生作用与菌株对低pH环境、胃蛋白酶、胰蛋白酶及胆盐的耐受能力有关。而菌株的表面疏水性是判断细菌与肠道表皮细胞相互吸附能力的重要指标之一,菌株的自凝聚性能又能影响细菌生物膜的形成,因此表面疏水性和自凝聚性作为乳酸菌的自身特性,被用于间接地评估益生菌的肠道定植能力。
本发明提供的约氏乳杆菌ZJBF004在低pH环境、人工模拟胃肠道环境及胆盐环境中均具有较强的耐受性,再结合自凝聚率、表面疏水性及细胞粘附试验结果可以发现菌株具有很强的胃肠道定植能力,满足在机体内发挥作用的基本要求。
此外,本发明提供的约氏乳杆菌对大肠杆菌、沙门氏菌和金黄色葡萄球菌均具有良好的抑制作用。同时,约氏乳杆菌ZJBF004株发酵上清液的DPPH自由基清除率为31.1%,与其他菌株相比抗氧化性能良好。
约氏乳杆菌在调节肠道菌群或制备用于调节肠道菌群的产品中的应用也属于本发明的保护范围。
本发明还保护一种用于调节肠道菌群的产品,活性成分包括本发明的约氏乳杆菌。例如用于调节肠道菌群的产品具有如下(a)-(c)中任一种功能:(a)抑制大肠杆菌;(b)抑制沙门氏菌;(c)抑制金黄色葡萄球菌。
本发明还保护约氏乳杆菌在制备抗氧化剂中的应用。例如抗氧化剂的功能为清除自由基;自由基例如为DPPH自由基。
本发明还保护一种抗氧化剂,活性成分包括本发明的约氏乳杆菌。例如抗氧化剂的功能为清除自由基;自由基例如为DPPH自由基。
本发明还保护约氏乳杆菌在制备抗菌药物中的应用。
其中,菌为细菌,例如革兰氏阳性细菌或革兰氏阴性细菌,也可为真菌。
进一步地,细菌包括大肠杆菌、沙门氏菌或金黄色葡萄球菌中的至少一种。
进一步地,抗菌药物中除了约氏乳杆菌还包括辅料。辅料的作用可为赋形、充当载体、提高稳定性、增溶、助溶、缓释等等。
本发明最后保护约氏乳杆菌在制备食品添加剂或动物饲料添加剂中的应用。
下面通过具体的实施例进一步说明本发明,但是,应当理解为,这些实施例仅仅是用于更详细地说明之用,而不应理解为用于以任何形式限制本发明。
实施例1蓝狐源约氏乳杆菌的分离鉴定
1.1分离用培养基:MRS培养基,培养基成分如下:
pH 6.2-6.5,121℃灭菌30min。
1.2分离方法:
(1)样品的采集与微生物分离:选择中国农业科学院特产研究所毛皮动物饲养基地的健康成年蓝狐,采取后肠内容物,迅速放入装有液体培养基的灭菌螺口具盖试管中,带回实验室后立即进行梯度稀释,每梯度三个平板,倾注法分离培养,于37℃倒置培养。
(2)分离纯化:取以上长有各种细菌的平板,选取乳酸菌单菌落,采用连续划线法进行分离纯化。将有不同菌落形态的单菌落均进行纯化。重复3-4次,镜检,直至获得纯种。
(3)观察菌落特征,利用革兰氏染色、镜检,观察细菌形态。
(4)DNA提取按照大连宝生物公司DNA提取试剂盒(akaRa 2.0)说明书方法提取细菌DNA,利用细菌的通用引物:27f,1492r对所获得乳酸菌保守序列进行扩增并测序,引物合成及测序送检上海生工生物工程技术服务有限公司完成,对16S rRNA序列进行同源性比较。16S rDNA的扩增体系如下,扩增体系的总体积为50μl:
ddH<sub>2</sub>O | 22μl |
Taq mix | 25μl |
DNA plate | 1μl |
Primer F | 1μl |
Primer R | 1μl |
PCR反应程序:94℃预变性5min,94℃变性30s,52℃退火45s,72℃延伸1min 30s,25个循环,72℃延伸8-10min,4℃保存。
1.3约氏乳杆菌的分离筛选鉴定:
经形态学观察,显微镜检,酶学分析以及碳水化合物利用实验,结合16S rRNA测序比对,鉴定为约氏乳杆菌,命名为ZJBF004,16S rRNA基因序列如SEQ ID NO.1所示。菌株培养24h后,菌落中间凸起,边缘整齐,表面光滑;兼性厌氧,革兰氏阳性,细胞呈杆状。ZJBF004的菌落形态如图1所示,显微镜检形态如图2所示。
ZJBF004的16S rRNA基因序列:
TTGGTCCTACCTTAGACGGCTGACTCCTATAAAGGTTATCCCACCGGCTTTGGGTGTTACAGACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAACGGCTTTAAGAGATCCGCTTGCCTTCGCAGGTTCGCTTCTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGATGGCAACTAATGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTCAGCGTCCCCGAAGGGAACACCTAATCTCTTAGGTTTGCACTGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTcCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGTTCAACAGTTTCTGATGCAATTCTCCGGTTGAGCCGAAGGCTTTCACATCAGACTTATTGAACCGCCTGCACTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTAAGTAATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCTTTCTTCACTACCAACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATTGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATGCACCGCAGGTCCATCCAAGAGTGATAGCAGAACCATCTTTCAAACTCTAGACATGCGTCTAGTGTTGTTATCCGGTATTAGCATCTGTTTCCAGGTGTTATCCCAGTCTCTTGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTCGCTTGTATCTAGTTTCATTTAGTGCAAGCACTAAAATCATCTAGGCAAGCTCGCTCGACTGCATTATTAGGCA(SEQ ID NO.1)。
将约氏乳杆菌ZJBF004在中国典型培养物保藏中心进行保藏,保藏编号为CCTCCNO:M 2019976。
实施例2生理生化特性
取复苏后的菌株培养24h,参照《伯杰氏细菌鉴定手册》、《常见细菌系统鉴定手册》和《乳酸细菌分类鉴定及实验方法》进行乳酸菌生理生化特性鉴定,明胶试验、V-P试验、MR试验、糖发酵试验等采用细菌微量生化反应鉴定管进行鉴定。
菌株ZJBF004的部分生理生化鉴定结果如表1所示。约氏乳杆菌ZJBF004可利用葡萄糖、果糖、半乳糖等单糖,可利用纤维二糖、麦芽糖、蔗糖、乳糖等二糖,以及七叶苷,具有蛋白酶及淀粉酶活性,在对菌株进行培养时可用碳源的选择更加丰富。
表1约氏乳杆菌ZJBF004生理生化特性
注:+:阳性;-:阴性。
实施例3不同温度生长试验
将培养24h的新鲜菌液以2.0%的接种量接种于MRS液体培养基中,分别于20℃、37℃和55℃恒温厌氧培养。分别于0、1、2、3、4、5、6、8、10、12、14、24、36、48h测定其OD600值,每组3个平行。测定完成后取3组平行试验结果计算平均值,根据计算结果绘制生长曲线图,结果如图3所示。由图可知,约氏乳杆菌ZJBF004接种于MRS液体培养基后,在37℃获得最快生长速度。接种后0-2h生长缓慢,处于生长滞缓区;2h后菌液吸光度值迅速升高,进入对数生长期;10h后菌液吸光度趋于平缓,菌株达到生长稳定期,该时期菌株活力强,活菌数量达到高峰。以上情况表明,该菌株在37℃培养条件下,营养充分时能较为迅速的进入对数生长期,从而在条件适宜时的微生物竞争中更容易成为优势菌株。
实施例4产酸性能测定
将培养24h的新鲜菌液以2.0%的接种量接种于MRS液体培养基中,37℃恒温厌氧培养。分别于0、2、4、6、8、10、12、14、24h利用pH计测定培养液pH值,根据测定结果绘制产酸性能曲线,结果如图4所示。由图可知,培养液的pH变化趋势与生长曲线相一致,约氏乳杆菌ZJBF004接种后2h内pH下降缓慢,从6.22下降至6.16;2h后进入对数生长期,菌液pH快速下降至4.10;10h后菌液pH变化趋于平缓,稳定在3.92左右。
该菌株在进入对数生长期后产酸能力较强,能使环境pH快速下降到4.0以下,低pH能够激活胃蛋白酶而达到促进胃肠道蠕动、增强营养物质消化吸收的作用。
实施例5耐酸特性试验
将培养24h的新鲜菌液以2.0%的接种量接种于初始pH分别为1.0、2.0、3.0、4.0、5.0、6.0、7.0的MRS液体培养基中,37℃恒温厌氧培养。分别于0、6、12、24h测定其OD600值,每组3个平行。测定完成后取3组平行试验结果计算平均值,根据计算结果绘制曲线,结果如图5所示。由图可知,菌株ZJBF004在pH≤3.0时生长完全被抑制,随培养时间延长,OD值未见变化。在pH=4.0时,菌株能缓慢生长,但OD值明显低于pH=5.0、6.0、7.0时。在pH≥5.0时,菌株ZJBF004均能正常生长,但在到达稳定期时OD值存在一定差别,其中OD值pH=7.0>pH=6.0>pH=5.0,活菌数与OD值变化趋势相一致。
实施例6人工胃肠液耐受性试验
人工胃液和人工肠液的配置参考2010年版《中国药典》。
取培养24h的新鲜菌液,于4℃,8000rpm离心10min,弃去上清液后加入等体积的生理盐水重悬,取重悬后的菌悬液1mL分别加入1mL经0.2μm无菌滤膜过滤处理后的人工胃液和人工肠液,37℃恒温厌氧培养。分别于0h和3h时取100μL培养液梯度稀释后涂布,用平板计数法计算活菌数,以0h的活菌数为对照计算存活率,测定菌株对人工胃肠液的耐受性,结果如表2所示。由表2可知,约氏乳杆菌ZJBF004在人工胃肠液中具有很强的存活能力。接种于人工胃液3h后菌株的存活率为95.39%,活菌数约为7.35×107CFU/mL;接种于人工肠液3h后菌株的存活率为95.28%,活菌数约为2.98×107CFU/mL。
表2约氏乳杆菌ZJBF004在人工胃肠液中的存活率
实施例7胆盐耐受性试验
取培养24h的新鲜菌液200μL加入胆盐浓度为0.3%的MRS液体培养基中,37℃恒温厌氧培养。分别于0h和6h时取100μL培养液梯度稀释后涂布,用平板计数法计算活菌数,以0h的活菌数为对照计算存活率,测定菌株对胆盐的耐受性,结果如表3所示。由表3可知,菌株ZJBF004对胆盐具有较强的耐受性。接种于胆盐浓度为0.3%的MRS液体培养基中6h后菌株的存活率为65.39%,活菌数约为1.375×105CFU/mL。
表3约氏乳杆菌ZJBF004在牛磺胆酸钠溶液中的存活率
实施例8表面疏水性测定和自凝聚性测定
表面疏水性能:
采用细菌碳烃化合物黏着法(bacterium adhesion to hydrocarbons,BATH),通过乳酸菌对碳烃化合物的亲和力来反应菌株表面的疏水性能。取培养24h的新鲜菌液用PBS洗涤3次后调节菌液OD600=0.25±0.05(A0)备用,取3mL上述菌液与等体积二甲苯混合,涡旋振荡3min,在37℃下孵育1h,吸取水相,测量OD600(At)。重复三次试验,取平均值。疏水率计算公式如下:
疏水率(CSH)=[(A0-At)/A0]×100% (1)
式(1)中:At为t时刻的吸光度,A0为0时的吸光度。
自凝聚性能:
取培养24h的新鲜菌液,于4℃,8000rpm离心10min,取沉淀,用PBS洗涤3次后加入2倍体积的PBS重悬备用,取2mL上述菌液,涡旋振荡10s,测量OD600(A0)。37℃静置2h,小心吸取上清液,测量OD600(At)。重复三次试验,取平均值。自凝聚率计算公式如下:
自凝聚率=[(A0-At)/A0]×100% (2)
式(2)中:At为t时刻的吸光度,A0为0时的吸光度。
细菌根据表面疏水性定义标准,一般设定CSH%>50%为高度疏水,CSH%介于20%和50%为中度疏水,CSH%<20%为非疏水。自凝聚能力一般分为:低(16%-35%),中等(35%-50%)和高(50%以上)三类。由表4可知,约氏乳杆菌ZJBF004具有中度疏水性,但是自凝聚性能较低,低于16%的最低界定值。
表4约氏乳杆菌ZJBF004的表面疏水率及自凝聚率
实施例9细胞粘附试验
将蓝狐小肠上皮细胞(本实验室)按常规传代方法培养于DMEM培养液中,10%CO2培养箱37℃培养,每2d换液一次。将培养好的细胞制成细胞悬液(5×104个/mL),向已放置玻璃爬片的24孔板中加入2mL细胞悬液,10%CO2培养箱37℃培养。待玻璃爬片上形成单层细胞后,吸出孔内旧的细胞培养液,用PBS清洗3次后每孔加入1mL培养24h的新鲜菌液(菌液浓度调节为1×108CFU/mL)和1mL细胞培养液,37℃厌氧培养2h。吸出孔内的混合液体,用PBS漂洗3次后每孔加入适量的高聚甲醛固定30min。吸出孔内的高聚甲醛,用PBS漂洗3次后进行革兰氏染色,显微镜下每孔随机计数30个视野,每个视野选择单个分布的细胞,通过计数黏附在30个细胞上的菌体个数,来判断黏附能力的大小。黏附细胞图如图6所示(1000×),约氏乳杆菌ZJBF004对蓝狐肠上皮细胞粘附性在18.67±3.12个/细胞,较一般的乳酸菌的粘附率强。
实施例10抑菌试验
菌株的抑菌性能采用牛津杯琼脂扩散法进行测定。取培养24h的新鲜菌液,于4℃,8000rpm离心10min取上清液备用,分别取200μL大肠杆菌、沙门氏菌和金黄色葡萄球菌新鲜菌液均匀涂布于LB固体培养基上。待菌液吸收完全后,在每个平板上成品字形放置3个牛津杯,每个牛津杯内加入200μL上清液,平板于37℃恒温培养箱正置放置培养,24h后观察是否有抑菌圈形成,并精确量取抑菌圈直径。
牛津杯直径为8mm,故设定抑菌圈直径大于9mm为具有抑菌性,小于等于9mm视为无抑菌性。由表5可知,菌株ZJBF004的发酵上清液对大肠杆菌、沙门氏菌、金黄色葡萄球菌均有较好的抑制作用,对金黄色葡萄球菌的抑菌活性最强,抑菌圈直径大于24mm;其次是沙门氏菌,抑菌圈直径大于16mm;再次是大肠杆菌抑菌圈直径也在11~13mm范围内。
表5约氏乳杆菌ZJBF004的抑菌活性
菌株 | 抑菌活性 |
大肠杆菌 | + |
沙门氏菌 | ++ |
金黄色葡萄球菌 | +++ |
注:抑菌圈直径(mm):+++:21-26mm;++:15-20mm;+:9-14mm;-:没有抑菌圈。
实施例11抗生素敏感性试验
菌株的抗生素敏感性采用纸片扩散法进行测定。
取培养24h的新鲜菌液200μL均匀涂布于MRS固体培养基上,采用纸片扩散法测定菌株对青霉素、头孢孟多、四环素、阿奇霉素、环丙沙星、万古霉素、氯霉素、克林霉素、庆大霉素、甲氧苄啶、甲硝唑和磷霉素等12种抗生素的敏感性。
药敏片直径为6mm,故设抑菌圈直径大于7mm的为敏感,小于等于7mm视为不敏感。由表6可知,菌株ZJBF004对各种常见抗生素的敏感性不同。对头孢孟多敏感性强,抑菌圈直径大于27mm;其次是青霉素,抑菌圈直径大于23mm;对氯霉素中度敏感,抑菌圈直径大于18mm;对四环素、万古霉素敏感性弱,抑菌圈直径在8-13mm范围内;而对阿奇霉素、环丙沙星、克林霉素、庆大霉素、甲氧苄啶、甲硝唑和磷霉素则不敏感。
表6约氏乳杆菌ZJBF004对常用抗生素的敏感性
注:抑菌圈直径(mm):+++:23-30mm;++:15-22mm;+:7-14mm;-:没有抑菌圈。
实施例12自由基清除试验
取培养24h的新鲜菌液,于4℃,8000rpm离心10min,取沉淀,用PBS洗涤3次后加入等体积的生理盐水重悬作为样品备用。取2mL样品加入2mL 0.2mmol/L DPPH的无水乙醇溶液,充分混匀,放置于37℃恒温培养箱中避光反应1h,然后经过8000rpm离心10min,取上清液,于517nm处测定吸光度。用等体积的生理盐水代替样品溶液作为对照组,同时以等体积的生理盐水和无水乙醇的混合液作为空白调零。
本实验对菌株ZJBF004菌体的DPPH清除能力进行了测定,DPPH自由基清除率的计算公式如下:
DPPH自由基清除率(%)=[1-(A样品-A空白)/A对照]×100%
根据测定结果换算得到菌株ZJBF004的DPPH自由基清除率为31.1%。
实施例13安全性试验
将体重22-27g的昆明系雌性小白鼠8只随机分为2组,实验组一次性腹腔注射0.3mL的新鲜菌液(菌液浓度调节为1×109CFU/mL),对照组腹腔注射等量的生理盐水,连续观察7d,记录观察期内小鼠的死亡情况,观察结束后剖检观察各组小鼠的内脏中毒情况。
临床观察所有小鼠精神状况良好,均能正常采食及饮水,粪便未见异常,试验期间未有试验小鼠死亡。试验结束后剖检观察其内脏均未见异常病理组织学变化及细菌易位,说明菌株针对小鼠临床应用无毒副作用。
尽管已用具体实施例来说明和描述了本发明,然而应意识到,在不背离本发明的精神和范围的情况下可以作出许多其它的更改和修改。因此,这意味着在所附权利要求中包括属于本发明范围内的所有这些变化和修改。
SEQUENCE LISTING
<110> 中国农业科学院特产研究所
<120> 约氏乳杆菌及其应用
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1465
<212> DNA
<213> 约氏乳杆菌16s rRNA
<400> 1
ttggtcctac cttagacggc tgactcctat aaaggttatc ccaccggctt tgggtgttac 60
agactctcat ggtgtgacgg gcggtgtgta caaggcccgg gaacgtattc accgcggcgt 120
gctgatccgc gattactagc gattccagct tcgtgtaggc gagttgcagc ctacagtccg 180
aactgagaac ggctttaaga gatccgcttg ccttcgcagg ttcgcttctc gttgtaccgt 240
ccattgtagc acgtgtgtag cccaggtcat aaggggcatg atgacttgac gtcatcccca 300
ccttcctccg gtttgtcacc ggcagtctca ttagagtgcc caacttaatg atggcaacta 360
atgacaaggg ttgcgctcgt tgcgggactt aacccaacat ctcacgacac gagctgacga 420
cagccatgca ccacctgtct cagcgtcccc gaagggaaca cctaatctct taggtttgca 480
ctggatgtca agacctggta aggttcttcg cgttgcttcg aattaaacca catgctccac 540
cgcttgtgcg ggcccccgtc aattcctttg agtttcaacc ttgcggtcgt actccccagg 600
cggagtgctt aatgcgttag ctgcagcact gagaggcgga aacctcccaa cacttagcac 660
tcatcgttta cggcatggac taccagggta tctaatcctg ttcgctaccc atgctttcga 720
gcctcagcgt cagttgcaga ccagagagcc gccttcgcca ctggtgttct tccatatatc 780
tacgcattcc accgctacac atggagttcc actctcctct tctgcactca agttcaacag 840
tttctgatgc aattctccgg ttgagccgaa ggctttcaca tcagacttat tgaaccgcct 900
gcactcgctt tacgcccaat aaatccggac aacgcttgcc acctacgtat taccgcggct 960
gctggcacgt agttagccgt gactttctaa gtaattaccg tcaaataaag gccagttact 1020
acctctatct ttcttcacta ccaacagagc tttacgagcc gaaacccttc ttcactcacg 1080
cggcgttgct ccatcagact ttcgtccatt gtggaagatt ccctactgct gcctcccgta 1140
ggagtttggg ccgtgtctca gtcccaatgt ggccgatcag tctctcaact cggctatgca 1200
tcattgcctt ggtaagccgt taccttacca actagctaat gcaccgcagg tccatccaag 1260
agtgatagca gaaccatctt tcaaactcta gacatgcgtc tagtgttgtt atccggtatt 1320
agcatctgtt tccaggtgtt atcccagtct cttgggcagg ttacccacgt gttactcacc 1380
cgtccgccgc tcgcttgtat ctagtttcat ttagtgcaag cactaaaatc atctaggcaa 1440
gctcgctcga ctgcattatt aggca 1465
Claims (10)
1.一种约氏乳杆菌,其特征在于,所述约氏乳杆菌于2019年11月25日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 2019976。
2.权利要求1所述的约氏乳杆菌在调节肠道菌群或制备用于调节肠道菌群的产品中的应用。
3.一种用于调节肠道菌群的产品,其特征在于,活性成分包括权利要求1所述的约氏乳杆菌。
4.根据权利要求3所述的产品,其特征在于,所述调节肠道菌群包括如下(a)-(c)中任一种:
(a)抑制大肠杆菌;
(b)抑制沙门氏菌;
(c)抑制金黄色葡萄球菌。
5.权利要求1所述的约氏乳杆菌在制备抗氧化剂中的应用。
6.根据权利要求5所述的应用,其特征在于,所述抗氧化剂通过清除自由基实现抗氧化功能;
优选地,所述自由基为DPPH自由基。
7.一种抗氧化剂,其特征在于,活性成分包括权利要求1所述的约氏乳杆菌。
8.根据权利要求7所述的抗氧化剂,其特征在于,所述抗氧化剂通过清除自由基实现抗氧化功能;
优选地,所述自由基为DPPH自由基。
9.权利要求1所述的约氏乳杆菌在制备抗菌药物中的应用;
优选地,所述菌为细菌或真菌;
优选地,所述细菌为革兰氏阳性细菌或革兰氏阴性细菌;
优选地,所述细菌包括大肠杆菌、沙门氏菌或金黄色葡萄球菌中的至少一种。
10.权利要求1所述的约氏乳杆菌在制备食品添加剂或动物饲料添加剂中的应用。
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