CN117264841A - 一株分离自鳗鲡肠道的地衣芽孢杆菌ajq05及其应用 - Google Patents
一株分离自鳗鲡肠道的地衣芽孢杆菌ajq05及其应用 Download PDFInfo
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Abstract
本发明公开了一株分离自鳗鲡肠道的地衣芽孢杆菌（Bacillus licheniformis）AJQ05及其应用。所述菌株已于2023年7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 20231382，保藏地址为湖北省武汉市武昌区八一路299号。地衣芽孢杆菌AJQ05具产4种胞外酶能力；耐受性强；在空间位点竞争上占优势，与病菌竞争黏附时，对其有较高的抑制黏附率；对水产行业常见的30种抗生素表现为敏感和中度敏感。因此该菌株可以应用于制备水产动物饲料添加剂和养殖水体添加剂，应用于生产蛋白酶、淀粉酶、脂肪酶、纤维素酶。利用本发明的地衣芽孢杆菌，为准确研究地衣芽孢杆菌的生物学特性奠定基础，明确该菌株的应用范围。
Description
技术领域
本发明属于微生物技术领域，具体涉及一株地衣芽孢杆菌AJQ05及其应用。
背景技术
鳗鲡是我国重要的水产养殖品种之一，主要养殖品种有日本鳗鲡(Anguillajaponica)和欧洲鳗鲡(Anguillaanguilla)。近年来，由于过渡捕捞、环境污染等，鳗鲡苗种资源，尤其是日本鳗鲡和欧洲鳗鲡苗种资源急剧减少。导致鳗鲡养殖存在这些问题的主要原因有：①消化能力较弱，在水温过高或过低时，易造成消化不良，引起肠炎病；②鳗鲡皮肤较薄，细菌和寄生虫易入侵，养殖病害多；③鳗鲡对药物更加敏感，一旦发生病害，比较难以控制，导致养殖成活率低。
地衣芽孢杆菌(Bacilluslicheniformis)是一种广泛分布好氧型革兰氏阳性杆状细菌，具良好的产蛋白酶、脂肪酶、纤维素酶、淀粉酶等胞外酶能力。通过与病原菌竞争营养和生存空间，产生抑制病原菌生长的代谢产物，分泌溶解病原菌细胞壁或细胞膜的溶菌物质，促进生长、诱导产生抗性，从而发挥其防病治病作用。地衣芽孢杆菌不污染环境、对人和动物无害、环境适应性强、能产生抗逆性强的芽孢、极易分离培养。近年来，地衣芽孢杆菌作为生态制剂、饲料添加剂广泛应用于水产养殖业中，它和其他种类的芽孢杆菌、光合细菌等有益微生物共同作用，可有效地改善养殖水质，促进水产养殖生物的生长和发育，但国内学者的研究涉及日本鳗鲡肠道原籍地衣芽孢杆菌的应用研究较少。
本发明采用生长曲线、产胞外酶能力、耐受能力、黏附能力等实验筛选出生物学特性良好的日本鳗鲡肠道原籍菌株，为促进日本鳗鲡生长、提高抗病力等方面提供具高效益生作用的菌种参考菌株。
发明内容
本发明的目的在于提供一株地衣芽孢杆菌AJQ05及其应用。
为实现上述目的，本发明采用如下技术方案：
一株地衣芽孢杆菌，所述菌株分类命名为地衣芽孢杆菌(Bacilluslicheniformis)AJQ05，已于2023年7月25日保藏于中国典型培养物保藏中心，保藏编号为CCTCCNO：M20231382，保藏地址为湖北省武汉市武昌区八一路299号。
一种菌剂，所述菌剂的活性成分包括上述的地衣芽孢杆菌。
上述的地衣芽孢杆菌或菌剂在制备抑制水产病原菌黏附能力的药物中的应用，所述水产病原菌为变形假单胞菌、迟钝爱德华菌、哈维氏弧菌、鳗弧菌、嗜水气单胞菌。
上述的地衣芽孢杆菌在制备水产动物饲料添加剂中的应用，所述水产动物为日本鳗鲡。
上述的地衣芽孢杆菌在制备水产动物养殖水体添加剂中的应用，所述水产动物为日本鳗鲡。
上述的地衣芽孢杆菌在生产蛋白酶、淀粉酶、脂肪酶、纤维素酶中的应用。
上述的地衣芽孢杆菌在研究地衣芽孢杆菌产胞外酶能力、抗生素耐药性、耐pH能力、耐胆盐能力、耐NaCl能力、耐胃液能力、耐肠液能力、黏附能力、抗水产病原菌黏附能力中的应用。
与现有技术相比，本发明的有益效果为：
本发明的地衣芽孢杆菌(Bacilluslicheniformis)AJQ05具产4种胞外酶能力；耐受性强；在空间位点竞争上占优势，与病菌竞争黏附时，对其有较高的抑制黏附率；对水产行业常见的30种抗生素表现为敏感和中度敏感。利用本发明的地衣芽孢杆菌，为准确研究地衣芽孢杆菌的生物学特性奠定基础，明确该菌株的应用范围。
附图说明
图1为地衣芽孢杆菌AJQ05形态图。
图2为地衣芽孢杆菌AJQ05革兰氏染色图。
图3为地衣芽孢杆菌AJQ05的16SrDNA电泳图。
图4为地衣芽孢杆菌AJQ05生长曲线图。
图5为地衣芽孢杆菌AJQ05对绵羊血红细胞的溶血图。
图6为地衣芽孢杆菌AJQ05产蛋白酶能力图。
图7为地衣芽孢杆菌AJQ05产淀粉酶能力图。
图8为地衣芽孢杆菌AJQ05产脂肪酶能力图。
图9为地衣芽孢杆菌AJQ05产纤维素酶能力图。
图10为地衣芽孢杆菌AJQ05对pH的耐受能力图。
图11为地衣芽孢杆菌AJQ05对胆盐的耐受能力图。
图12为地衣芽孢杆菌AJQ05对NaCl的耐受能力图。
图13为地衣芽孢杆菌AJQ05对人工胃液的耐受能力图。
图14为地衣芽孢杆菌AJQ05对人工肠液的耐受能力图。
图15为地衣芽孢杆菌AJQ05体外黏附能力图。
图16为地衣芽孢杆菌AJQ05对5株致病菌竞争抑制黏附能力图。
图17为地衣芽孢杆菌AJQ05对5株致病菌取代抑制黏附能力图。
图18为地衣芽孢杆菌AJQ05对5株致病菌排斥抑制黏附能力图。
具体实施方式
为了更好地理解本发明，下面结合实例和附图对本发明做进一步的详细说明，但应当理解下述实例不是对本发明保护范围的限制，任何在本发明基础上做出的改变和变化，都在本发明的保护范围之内。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均为市售所得。
实施例1
本实施例提供地衣芽孢杆菌AJQ05的分离与鉴定。
1.1地衣芽孢杆菌AJQ05菌株来源将健康日本鳗鲡解剖，无菌取其肠道，研磨后60℃水浴加热1h涂布，挑选大小、颜色不同的菌落进行分离纯化，获得菌株AJQ05。
1.2地衣芽孢杆菌AJQ05的鉴定
形态学鉴定：
将处于对数生长期，且菌落大小稳定的单菌落进行状态描述，主要包括菌落的大小、颜色、透明度、湿润度、菌落表面形态、菌落边缘状态；挑取单菌落涂片，经革兰氏染色后采用光学显微镜观察菌体的形态。
如图1所示，菌株AJQ05在LB培养基上的菌落形态为：菌落形态不规则、白色、中间有白色凸起圆点，表面褶皱有干燥。如图2所示，菌株AJQ05为革兰氏阳性菌，是一种杆状细菌，形态呈长线形、椭圆形。
16SrDNA序列同源性分析：
使用诺唯赞Biospin细菌基因组DNA提取试剂盒提取菌株AJQ05基因组DNA，具体操作步骤子详见其说明书。使用诺唯赞2×MaxMasterMix(DyePlus)进行PCR扩增。
表1PCR扩增体系
表2PCR循环条件
表3引物序列信息
PCR产物电泳结果：
使用1％浓度的琼脂糖凝胶电泳，电压120V，电泳20min，每孔上样1μL，电泳图谱见图3。如图3所示，PCR产物电泳结果表明细菌的16SrDNA片段扩增成功。
测序及比对结果：
PCR产物电泳后送生物技术公司进行测序，测序结果如SEQ ID NO.1所示。将所得的序列结果在GenBank数据库中进行比对，发现菌株AJQ05与地衣芽孢杆菌(Bacilluslicheniformis)相似性达99％。
由形态特征和16SrDNA序列同源性分析结果可知,菌株AJQ05为地衣芽孢杆菌(Bacilluslicheniformis)的新株系。
实施例2
本实施例提供地衣芽孢杆菌AJQ05的生长曲线。
将地衣芽孢杆菌AJQ05于LB液体培养基中培养至OD600＝0.5左右，用PBS缓冲液稀释至OD600＝0.1±0.01，再逐步稀释104倍。设10个重复，酶标条每孔加入200μL稀释好的菌液；对照组为10个孔的200μLLB液体培养基。利用多功能微孔板检测仪进行测定(参数为：T＝28℃，Time＝48h，Space＝1h，Wavelength＝600nm)。
如图4所示，OD600从4h后开始增加，OD600最高在1.5左右，20-24h后进入生长平台期。
实施例3
本实施例提供地衣芽孢杆菌AJQ05的溶血性试验。
在添加1％脱纤维绵羊血的血平板上打孔，取10μL地衣芽孢杆菌AJQ05菌液滴至孔中，三个平行。设阴性对照，滴加10μLLB肉汤培养基，设阳性对照，滴加10μLTrionX-100。28℃培养24h后进行观察。具有溶血性的菌落周围会产生透明的溶血圈，根据溶血圈判断菌株是否具有溶血活性。(α溶血：草绿色；β溶血：有透明圈；γ溶血：无溶血现象)如图5所示，地衣芽孢杆菌AJQ05在血平板上呈γ溶血，即无溶血现象。
实施例4
本实施例提供地衣芽孢杆菌AJQ05产胞外酶能力试验。
产酶筛选培养基：
蛋白酶筛选培养基：牛肉膏0.3g，蛋白胨1.0g，2％脱脂奶粉，NaCl0.5g，琼脂1.6g，H2O100mL。
淀粉酶筛选培养基：牛肉膏0.3g，蛋白胨1.0g，可溶性淀粉1.0g，NaCl0.5g，琼脂1.6g，H2O100mL。
脂肪酶筛选培养基：蛋白胨1.0g，CaCl·H2O0.01g，Tween-801.0mL，琼脂1.6g，H2O100mL。
纤维素酶筛选培养基：牛肉膏0.3g，蛋白胨1.0g，羧甲基纤维素钠(CMC-Na)2g，琼脂1.6g，H2O100mL。
在不同培养基上用100μL枪头打孔，打孔完用无菌针头将琼脂孔中的培养基挑出，每个板打3个孔。将地衣芽孢杆菌AJQ05于LB液体培养基中过夜培养，用PBS缓冲液稀释至OD600＝0.3，将稀释后的菌液各取10μL加至琼脂孔中，静置摆放1h后放入28℃恒温箱中培养48h，观察有无透明溶解环。在培养48h的淀粉酶筛选培养基琼脂孔中滴加10μL卢戈氏碘液，室温染色15min，观察有无透明溶解环和白色沉淀环；在纤维素酶筛选培养基平板上滴加刚果红染液，室温染色30min，观察有无透明溶解环。用菌落计数仪测量溶解环和白色沉淀环直径。
如图6、图7、图8、图9所示，蛋白酶、淀粉酶、脂肪酶、纤维素酶筛选培养基上均出现透明圈和白色沉淀环，表明地衣芽孢杆菌AJQ05具有产多种酶能力，产蛋白酶水解圈直径为1.96±0.14mm，产淀粉酶水解圈直径为2.12±0.13mm，产脂肪酶白色沉淀环直径为2.16±0.03mm，产纤维素酶水解圈直径为2.27±0.11mm。
实施例5
本实施例提供地衣芽孢杆菌AJQ05的药敏试验。
采用纸片琼脂扩散法(K-B)法进行药物敏感性检测。选择常见的33种药敏纸片(杭州微生物试剂有限公司)：青霉素、苯唑西林、氨苄西林、羧苄西林、哌拉西林、头孢氨苄、头孢唑啉、头孢拉定、头孢呋辛、头孢他啶、头孢曲松、头孢哌酮、丁胺卡那、庆大霉素、卡那霉素、新霉素、四环素、多西环素、米诺环素、红霉素、麦迪霉素、诺氟沙星、氧氟沙星、环丙沙星、万古霉素、多粘菌素B、复方新诺明、呋喃唑酮、氯霉素、克林霉素、强力霉素、恩诺沙星。将地衣芽孢杆菌AJQ05接种于LB液体培养基中，使菌液OD600达到0.3，取1mL菌液，12000rpm离心1min，用PBS重悬菌体，取100μL重悬菌液均匀涂布于LB固体培养基上，用灭菌过的镊子分别夹取33种药敏纸片放于涂过菌液的平板表面，放入28℃生化培养箱中培养24h，观察是否产生抑菌圈并测量抑菌圈直径。
由表4可知，地衣芽孢杆菌AJQ5对青霉素、苯唑西林、氨苄西林、羧苄西林等30种抗生素表现为敏感和中毒敏感，对四环素、万古霉素、复方新诺明表现为耐药。
表4药敏试验结果
注：S表示敏感；L表示中度敏感；R表示耐药；0表示无抑菌圈，即耐药。
实施例6
本实施例提供地衣芽孢杆菌AJQ05的耐受性试验。
耐pH试验：
将地衣芽孢杆菌AJQ05接种于LB液体培养基中，使菌株OD600达到0.6，取1mL菌液，12000rpm离心1min，用PBS重悬菌体。准备条件培养基，用稀盐酸和氢氧化钠分别将液体LB培养基pH调至2、4、6、7、8、10、12，121℃灭菌，取出培养基，待降至室温使用。将菌液按1:50的比例接种于上述条件培养基中，28℃摇床220rpm培养24h，培养结束后测OD600，各梯度测三个平行。
如图10所示，地衣芽孢杆菌AJQ05的pH适应范围在6-8，pH＝7时OD600最高。
耐胆盐试验：
将地衣芽孢杆菌AJQ05接种于LB液体培养基中，使菌株OD600达到0.6，取1mL菌液，12000rpm离心1min，用PBS重悬菌体。准备条件培养基，在灭菌后的液体LB培养基中分别加入0g/L、0.15g/L、0.3g/L、0.45g/L、0.6g/L胆盐。将菌液按1：50的比例接种于上述条件培养基中，28℃摇床220rpm培养4h，每小时测一次OD600，各梯度测三个平行。
如图11所示，胆盐浓度为0.15g/L时，OD600随时间增长；胆盐浓度为0.3-0.6g/L时，OD600几乎不变，表明地衣芽孢杆菌AJQ05耐胆盐能力强。
耐NaCl实验：
将地衣芽孢杆菌AJQ05接种于LB液体培养基中，使菌株OD600达到0.6，取1mL菌液，12000rpm离心1min，用PBS重悬菌体。准备条件培养基，用稀盐酸和氢氧化钠分别将液体LB培养基NaCl浓度调至0.5％、1.5％、2.5％、3.5％、4.5％、5.5％、6.5％，121℃灭菌，取出培养基，待降至室温使用。将菌液按1:50的比例接种于上述条件培养基中，28℃摇床220rpm培养24h，结束后测OD600，各个梯度测三个平行。
如图12所示，地衣芽孢杆菌AJQ05对NaCl的适应范围在0.5％-6.5％之间，适应范围广，耐受性强。
实施例7
本实施例提供地衣芽孢杆菌AJQ05对模拟消化道的耐受力试验。
模拟人工胃液耐受试验：
人工胃液：称取LB肉汤300mL，将pH调至2、3、4，将胃蛋白酶浓度调至1g/L，混匀后过滤除菌，分装于无菌试管中。将配置好的人工胃液分装于试管中，每管5mL；取OD600＝1.0的地衣芽孢杆菌AJQ05新鲜菌液1mL，按1：50的比例分别接种于pH为2、3、4的人工胃液中，充分混匀后放于28℃生化培养箱静置培养，人工胃液处理0min的菌液作为对照，分别于培养后的0-4h取样测OD600。
如图13所示，与对照相比，人工胃液pH为2、3、4时，OD600在4h内几乎不变。
模拟人工肠液耐受试验：
人工肠液：称取1.36gKH2PO4加入200mLLB肉汤，使用NaOH溶液将pH调至6.8，加入0.42g胰蛋白酶，混匀后过滤除菌，分装于无菌试管中。将配置好的人工肠液分装于试管中，每管5mL；取OD600＝1.0的地衣芽孢杆菌AJQ05新鲜菌液1mL，按1：50的比例接种于人工肠液中，充分混匀后放于28℃生化培养箱静置培养，人工肠液处理0min的菌液作为对照，分别于培养后的0-6h取样测OD600。
如图14所示，与对照相比，试验组OD600增长较缓慢，但呈上升趋势。表明地衣芽孢杆菌AJQ05能在人工肠液中生长。
实施例8
本实施例提供地衣芽孢杆菌AJQ05的体外黏附试验。
日本鳗鲡(Anguilla japonica)黏液及蛋白标准曲线制作：
用玻片刮取日本鳗鲡表皮黏液，置于4℃冰箱过夜，后4℃、12000×g离心30min，取上清液依次用0.1μm滤膜过滤除菌。用全式金Protein Quantitative Kit(Bradford)试剂盒测定蛋白标准曲线并将黏液蛋白浓度调至1mg/mL，分装后置于-20℃保存备用。
黏附观察：
(1)吸取20μL日本鳗鲡表皮黏液(蛋白浓度1mg/mL)至载玻片上，涂抹均匀，静置过夜；
(2)吸取200μL4％甲醇至过夜的载玻片上，涂抹均匀，固定2h；
(3)将地衣芽孢杆菌AJQ05菌液调至OD600＝0.3；
(4)向载玻片滴加200μL地衣芽孢杆菌AJQ05菌液，3个重复，于37℃生化培养箱静置2h；
(5)用PBS冲洗玻片5次，风干；
(6)吸取200μL4％甲醇固定至彻底风干；
(7)向玻片滴加200μL0.1％结晶紫溶液，涂抹均匀，静置2min；
(8)用PBS顺向冲洗玻片；
(9)光学显微镜200倍下观察黏附结果。
如图15所示，200×下，地衣芽孢杆菌AJQ05黏附数量为281个。
实施例10
本实施例提供地衣芽孢杆菌AJQ05对5株病菌的黏附抑制黏附试验。
竞争抑制黏附试验：
吸取20μL日本鳗鲡表皮黏液(蛋白浓度1mg/mL)至载玻片上，涂抹均匀，静置过夜；吸取200μL4％甲醇至过夜的载玻片上，涂抹均匀，固定2h；将菌液调至OD600＝0.3。向甲醇固定后的载玻片滴加100μL致病菌菌液和100μL地衣芽孢杆菌AJQ05菌液，3个重复，于37℃生化培养箱静置2h。用PBS冲洗玻片5次，风干；吸取200μL4％甲醇固定至彻底风干；革兰氏染色；光学显微镜200倍下观察黏附结果。
取代抑制黏附试验：
吸取20μL日本鳗鲡表皮黏液(蛋白浓度1mg/mL)至载玻片上，涂抹均匀，静置过夜；吸取200μL4％甲醇至过夜的载玻片上，涂抹均匀，固定2h；将菌液调至OD600＝0.3。向甲醇固定后的载玻片滴加100μL致病菌菌液，3个重复，于37℃生化培养箱静置1h，后取出载玻片，用PBS冲洗玻片5次，向载玻片滴加100μL地衣芽孢杆菌AJQ05菌液，于37℃生化培养箱静置1h。用PBS冲洗玻片5次，风干；吸取200μL4％甲醇固定至彻底风干；革兰氏染色；光学显微镜200倍下观察黏附结果。
排斥抑制黏附试验：
吸取20μL日本鳗鲡表皮黏液(蛋白浓度1mg/mL)至载玻片上，涂抹均匀，静置过夜；吸取200μL4％甲醇至过夜的载玻片上，涂抹均匀，固定2h；将菌液调至OD600＝0.3。向甲醇固定后的载玻片滴加100μL地衣芽孢杆菌AJQ05菌液，3个重复，于37℃生化培养箱静置1h，取出载玻片，用PBS冲洗玻片5次，向载玻片滴加100μL致病菌菌液，于37℃生化培养箱静置1h。用PBS冲洗玻片5次，风干；吸取200μL4％甲醇固定至彻底风干；革兰氏染色；光学显微镜200倍下观察黏附结果。
图16、图17、图18分别为地衣芽孢杆菌AJQ05对5株致病菌的竞争、取代、排斥抑制黏附实验结果，A-E分别表示变形假单胞菌(Pseudomonasplecoglossicida)、迟钝爱德华菌(Edwardsiellatarda)、哈维氏弧菌(Vibrioharveyi)、鳗弧菌(Vibrioanguillarum)、嗜水气单胞菌(Aeromonashydrophila)与地衣芽孢杆菌的作用结果。结果表明，地衣芽孢杆菌AJQ05对5株致病菌都有不同程度的抑制效果，其中不同抑制黏附方式对不同的致病菌抑制黏附效果不同。
如表5所示，地衣芽孢杆菌AJQ03与哈维氏弧菌、鳗弧菌、迟钝爱德华菌竞争抑制黏附效果较好，黏附抑制率分别为86.0％、65.1％、82.4％；与哈维氏弧菌、鳗弧菌、迟钝爱德华菌取代抑制黏附效果较好，黏附抑制率分别为97.4％、88.7％、78.9％；与鳗弧菌、迟钝爱德华菌排斥黏附效果较好，黏附抑制率分别为74.8％、78.8％。
表5AJQ05对5株病原菌的黏附抑制率
综上所述，地衣芽孢杆菌(Bacillussubtilis)AJQ05具产4种胞外酶能力；耐受性强；在空间位点竞争上占优势，与病菌竞争黏附时，对其有较高的抑制黏附率；对水产行业常见的30种抗生素表现为敏感和中度敏感。利用本发明的地衣芽孢杆菌AJQ05，为准确研究地衣芽孢杆菌的生物学特性奠定基础，明确该菌株的应用范围。
以上描述对本发明的具体示例性实施方案的描述是为了说明和例证的目的。这些描述并非想将本发明限定为所公开的精确形式，根据上述实施例，可以进行很多改变和变化，对示例性实施例进行选择和描述的目的在于解释本发明的特定原理以及实际应用。熟悉本领域的技术人员在依照本发明所作的等效的修饰以及变化，都应当涵盖在本发明的权利要求所保护的范围内。
Claims (10)
1.一株地衣芽孢杆菌，其特征在于：所述菌株分类命名为地衣芽孢杆菌（Bacillus licheniformis）AJQ05，已于2023年7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 20231382，保藏地址为湖北省武汉市武昌区八一路299号。
2.一种菌剂，其特征在于：所述菌剂的活性成分包括权利要求1所述的地衣芽孢杆菌。
3.权利要求1所述的地衣芽孢杆菌或权利要求2所述的菌剂在制备抑制水产病原菌黏附能力的药物中的应用。
4.根据权利要求3所述的应用，其特征在于：所述水产病原菌为变形假单胞菌、迟钝爱德华菌、哈维氏弧菌、鳗弧菌、嗜水气单胞菌。
5.权利要求1所述的地衣芽孢杆菌在制备水产动物饲料添加剂中的应用。
6.根据权利要求5所述的应用，其特征在于：所述水产动物为日本鳗鲡。
7.权利要求1所述的地衣芽孢杆菌在制备水产动物养殖水体添加剂中的应用。
8.根据权利要求7所述的应用，其特征在于：所述水产动物为日本鳗鲡。
9.权利要求1所述的地衣芽孢杆菌在生产蛋白酶、淀粉酶、脂肪酶、纤维素酶中的应用。
10.权利要求1所述的地衣芽孢杆菌在研究地衣芽孢杆菌产胞外酶能力、抗生素耐药性、耐pH能力、耐胆盐能力、耐NaCl能力、耐胃液能力、耐肠液能力、黏附能力、抗水产病原菌黏附能力中的应用。
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