酸奶中嗜热链球菌的分离和性能研究

采用稀释涂布法和平板划线法从酸奶中分离出嗜热链球菌,对其茵落和细胞的形态特征进行观察,并进行糖发酵、生长、耐盐、明胶液化、过氧化氢酶等生理生化鉴定试验．确定其为嗜热链球菌后,以凝固试验和产酸试验相结合研究其性能,从而挑选出性能优良的菌株．共分离出6株菌株,经观察、鉴定,确定其全部为嗜热链球菌,其中优良菌株1株,合格菌株3株,不合格菌株2株．     

两株降解纤维素真菌的分子鉴定及酶活分析

应用刚果红鉴定培养基,从土壤中筛选出两种分解纤维素能力较强的真菌,分别命名为NY01和NY02.滤纸条降解度分析显示,NY01的降解能力高于NY02.应用分子生物学手段,对两株菌rDNA的ITS区域序列分析显示,NY01与木霉的相似性高达99%,NY02与毛霉的相似性高达99%,暗示NY01可能是木霉的一个种,NY02可能是毛霉的一个种.培养基中碳氮比例对两株真菌纤维素降解酶活性的研究显示,在N/C比值较低和较高时,CMC酶和FPA酶活性都低,N/C比值在1∶8时酶活性最高,说明两株真菌降解纤维素的最佳N/C比值是1∶8.     

三七疫霉病拮抗根际土壤细菌的筛选及初步鉴定

为了筛选对环境友好的生防细菌防治三七疫霉病,通过高温处理、稀释涂布法分别从三七健康及患病植株根际土壤分离了21和16株可培养细菌,并以对峙培养法分别筛选到6和7株拮抗菌株,上述13株拮抗菌株经生理生化特征鉴定为芽孢杆菌属。结果表明,健康植株根际土壤有较丰富的可培养细菌,而患病植株根际土壤可培养细菌中的拮抗菌株较丰富,表明健康植株与患病植株根际土壤的拮抗细菌丰度发生了变化。解淀粉芽孢杆菌B1的抑菌带宽达8.5mm,具有一定的潜在生防价值。     

牛蛙源弗氏柠檬酸杆菌的分离鉴定和药敏分析

为了分离和鉴定牛蛙（Rana catesbeiana）养殖环境的致病微生物,并筛选相关的抗菌药物,采用稀释涂布法从牛蛙养殖场的废水中分离获得1株优势菌株W6,并通过革兰氏染色和菌落形态观察、生理生化特性分析、16SrRNA基因序列分析等进行菌株鉴定,随后对该菌进行药敏性分析以及致病性试验．结果显示：该菌株为革兰氏阴性短杆菌,培养特性、生理生化反应与弗氏柠檬酸杆菌基本一致;16SrRNA基因同源性及系统进行分析显示,W6菌株与弗氏柠檬酸杆菌参考菌株同源性大于99%,且为同一进化分支．同时,药敏实验结果表明,该菌株对大多数抗生素具有较强耐药性,仅对呋喃唑酮（痢特灵）、头孢他定高度敏感．中草药水提取物抑菌实验表明,乌梅、五味子、艾叶、黄芩等中草药水提取物对该菌株具有较好的体外抑菌活性．致病性分析表明,该菌对牛蛙具有一定的致病性．     

一株巨大芽孢杆菌的分类鉴定

基于16S rRNA序列分析和生理生化实验,对从伽师瓜病瓜上分离的一株细菌ZP-1进行了分类鉴定.结果表明,该分离菌株ZP-1与Bacillus megaterium strain PAB1C5（EU221343.1）具有99%的相似性.因此,初步推断ZP-1菌株隶属于巨大芽孢杆菌属（Bacillus magaterium）.     

酱油酿造中耐盐性乳酸菌的筛选及分子生物学鉴定

从酱醅中分离筛选出一株耐盐性乳酸菌,在18％、20％NaCl的浓度下都能生长,并产生乳酸。对该菌进行分子生物学鉴定,测得菌株的16S rRNA序列与GenBank登记号为FSB201的嗜盐四联球菌（Tetragenococcus halophlus）具有99％的同源性。再将该16SrRNA序列与乳酸菌分类中17个模式菌株的16SrRNA序列共同创建进化树．发现筛选菌与GenBank登记号为AB106344、AB106343的嗜盐四联球菌亲缘关系最近,证明筛选菌应属四联球菌属中嗜盐四联球菌中的一种。     

草甘膦降解菌C-17的分离鉴定及生物学特性

从福建三农集团污水处理池分离到一株降解草甘膦的菌株C—17,经形态与生理生化实验,C-17初步鉴定为胶红酵母属真菌。在草甘膦基础培养基上,C-17菌株对草甘膦的最高耐受浓度为6000mg/L,草甘膦浓度为3000mg／L时生长量最高。C-17菌株能以草甘膦为唯一氮、磷源,培养7d对草甘膦的降解率为83．6％,培养基中缺乏别的磷源,有利于促进C-17菌株对草甘膦的降解。     

一株互花米草耐重金属内生菌的分离及其特性分析

利用微生物分离培养方法从互花米草根部和叶片获得内生细菌,经重金属筛选,分离到一种耐受重金属Cu2＋、Pb2＋和Cr6＋的细菌．为了进一步鉴定分离菌株,利用 PCR 方法扩增其部分16S rDNA 序列,目标DNA条带约1500 bp,测序和在线分析结果后显示,所测序列均与基因库中已上传的 Lysinibacillus sphaericus菌株16S rDNA 序列（KJ878614．1,JN613478．1）相似性均为100％．初步鉴定该菌为 Lysinibacillus属球形芽孢杆菌（Lysinibacillus sphaericus）,命名为Lysinibacillus sphaericus QZ1-1．对该菌株采用促生长特性分析结果显示,该菌株具有产生 IAA和 ACC的能力,同时该菌能在无氮培养基中生长,具备固氮功能,为日后重金属污染生物修复相关研究提供了良好的目标菌株．     

急性腹泻患者奇异变形杆菌分离鉴定及其生物学特性

目的:对某市医院腹泻门诊收集的一起急性腹泻患者肛拭子样本进行奇异变形杆菌分离鉴定、耐药性与致病作用分析。方法:增菌培养、划线分离,革兰氏染色,镜检观察细菌形态,16S rRNA基因扩增、测序鉴定细菌种类;纸片法进行药物敏感性检测,PCR法筛检毒力基因,结晶紫染色法检测细菌生物被膜形成,小鼠感染试验分析分离株致病力;荧光PCR法分析奇异变形杆菌感染小鼠致不同脏器炎症因子表达水平。结果:成功从腹泻样本中分离获得3株细菌(NB1711、NB1335和NB1336),经菌种鉴定均为奇异变形杆菌。3株细菌对米诺环素、红霉素、多粘菌素等9种药物表现为多重耐药,对头孢唑林、哌拉西林、环丙沙星等敏感。NB1711分离株完整编码8种毒力因子,生物被膜形成能力强,对小鼠致病最高,感染该菌株可显著上调小鼠不同脏器IL-1β、IL-6、TNFα、IFN-γ等炎症因子的表达。结论:奇异变形杆菌感染是引发此次急性腹泻的主要病因,细菌分离株NB1711耐药谱广、毒力强、可形成致密生物被膜,感染小鼠介导形成炎症反应。本研究为进一步探究奇异变形杆菌感染机制及临床用药提供科学依据。     

一种斜纹夜蛾新型病原菌的分离与鉴定

采用10倍系列稀释法从罹病斜纹夜蛾虫尸内分离出球状病原细菌IAM1218菌株,并按Kcoh规则进行侵染试验,同时,采用菌液PCR法扩增菌株的16S rDNA序列,在GenBank数据库中进行同源序列搜索,通过Bootstrap analysis法进行1000次重复抽样分析和Neighbor-Joining法构建分支系统树分析等,结果表明,该菌株各浓度处理的斜纹夜蛾幼虫死亡率均较高,最低达(83.22±4.72)%,LT50值最高为(24.50±1.08)h;罹病幼虫体色变黑,体壁易破裂流出粘稠状物质;IAM1218菌株(GenBank No.KC790981)和酪黄肠球菌Enterococcus casseliflavus的16S rDNA区序列完全相同,相似性为100%,且二者聚类在一组,概率100%。综合分析确定该菌株为酪黄肠球菌Enterococcus casseliflavus,属于一种新型的斜纹夜蛾致病病原细菌。这为斜纹夜蛾的生物防治提供了新的技术途径。     

Cloning and Efficient Expression ofBacillus sp. BH072 tasAGene inEscherichia coli

The Bacillus strain BH072 isolated from a honey sample showed strong antifungal activity against phytopathogen. Gene cloning test demonstrated that the strain had a tas A gene encoding an antifungal Tas A protein. Although the wild strain simultaneously produced various antifungal substances, only the physicochemical property and antifungal activity of Tas A protein were unclear due to the difficulty in extraction. In this study, tas A gene encoding the protein from Bacillus sp. BH072 was amplified by using the polymerase chain reaction(PCR) method and cloned into p ET 28a(+) vector, and then expressed in host cells Escherichia coli BL21(DE3). The expressed proteins were collected by centrifugation and ultrasonic treatment, and then purified by using nickel-nitrilotriacetic acid(Ni-NTA)metal affinity column and dialysis methods. The result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) test showed that an expected protein band appeared with a size of 31 k Da. The expressed products possessed antifungal activity against the phytopathogenic indicator strain Botrytis cinerea. A genetically engineered strain tas A of E. coli was established in this study which can efficiently express Tas A protein.     

紫外线复合Nd:YAG激光对Bacillus sp.产脂肪酶的诱变

以Bacillus sp．为出发菌株，经过紫外线诱变，筛选出较稳定的菌株UV-4，其产脂肪酶活力较出发菌株提高了28％；UV-4经过YAG激光诱变处理，得到变株Y-1，其产酶能力在此基础上又提高了41％，且产酶水平较为稳定。     

原油降解菌的筛选及其降解性能研究

从茂名炼油厂附近长期被石油污染的土壤中分离出2株能够以原油为惟一碳源和能源的原油降解菌株,经鉴定分属假单胞菌属和芽孢杆菌属。进一步研究了2株菌原油降解特性及其影响因素,结果表明：2株菌5d原油平均降解率分别为42．8％和44．7％;在pH接近7．0,NaCI的质量浓度为10g/L,表面活性剂吐温-80的质量浓度为40mg／L时降解效果最佳。     

江苏省苏北地区土壤苏云金杆菌及其杀虫特性

根据不同的土壤类型,从江苏省苏北地区6市采集土样422份,分离出苏云金杆菌76株.血清学分析表明,它们分属于H_3、H_7、H_10、H_21和H_43型,另一株为自凝菌.通过对76个分离株的生物毒力测定,筛选出4林对赤松毛虫、小菜蛾、玉米螟等具有高毒力的菌株,为开发高效苏云金杆菌杀虫剂提供了重要的菌种资源.     

一株高生物量酵母菌株的鉴定

从土壤中分离到一株高生物量酵母菌株,经形态特征及12项生理生化特性的检测。以N．J．W．Kreger-van Rij和J．A．Barnett等的酵母分类系统为依据进行了分类鉴定,初步鉴定为丝孢酵母属（Trichosporon）个种。     

Cloning and efficient expression of <Emphasis Type="Italic">Bacillus sp.</Emphasis> BH072 <Emphasis Type="Italic">tasA</Emphasis> gene in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The Bacillus strain BH072 isolated from a honey sample showed strong antifungal activity against phytopathogen. Gene cloning test demonstrated that the strain had a tasA gene encoding an antifungal TasA protein. Although the wild strain simultaneously produced various antifungal substances, only the physicochemical property and antifungal activity of TasA protein were unclear due to the difficulty in extraction. In this study, tasA gene encoding the protein from Bacillus sp. BH072 was amplified by using the polymerase chain reaction (PCR) method and cloned into pET 28a (+) vector, and then expressed in host cells Escherichia coli BL21 (DE3). The expressed proteins were collected by centrifugation and ultrasonic treatment, and then purified by using nickel-nitrilotriacetic acid (Ni-NTA) metal affinity column and dialysis methods. The result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) test showed that an expected protein band appeared with a size of 31 kDa. The expressed products possessed antifungal activity against the phytopathogenic indicator strain Botrytis cinerea. A genetically engineered strain tasA of E. coli was established in this study which can efficiently express Tas A protein.     

Isolation of Secondary Metabolites with Antimicrobial Activities from Bacillus amyloliquefaciens LWYZ003

The strain LWYZ003, which can restrain multiple pathogens, was screened from the sediment of the ocean and identified as Bacillus amyloliquefaciens. Large-scale fermentation and modern chromatographic separation technologies(macroporous resin column chromatography, silica gel column chromatography, thin-layer chromatography and high-performance liquid chromatography) were used to separate antimicrobial products from the fermentation broth of marine-derived Bacillus amyloliquefaciens LWYZ003. Bioactive-guided separation was used in the term of seeking antimicrobial products from the secondary metabolites of Bacillus amyloliquefaciens LWYZ003. As a result, two natural products cycloheximide( 1) and trehalose( 2) were obtained. Their structures were elucidated by Fourier transform infrared spectroscopy, high-resolution mass spectrometry, ~1 H and ~(13) C nuclear magnetic resonance analysis. In the cylinder plate method, compound 1 exhibited stronger antimicrobial activities than compound 2 against Micrococcus luteus, and also exhibited wider antimicrobial spectrum than compound 2. In conclusion, isolation of bioactive secondary metabolites from marine Bacillus sp. has enormous potentials in finding suitable antibiotics to inhibit multiple pathogens.     

淀粉酶产生菌的分离筛选

从大米厂、淀粉厂周边环境土壤中筛选到三株产淀粉酶能力较强的菌株Jz1、Jz2、Jz3。经形态特征及染色等初步鉴定,菌株Jz1和Jz3为芽孢杆菌属(Bacillus),菌株Jz2为短小杆菌属(Curtobacterium)。采用YoungJ.Y00改良法测定此三株菌株的淀粉酶活力分别为47.29 U/mL、48.48 U/mL、49.74 U/mL,具有较好的应用潜力。     

细菌Bacillus sp. NS-3和灰葡萄孢对松材线虫的诱引

通过＂水琼脂平板移行法＂和＂玻管法＂,研究了松材线虫拮抗细菌Bacillus sp.NS-3和真菌灰葡萄孢引诱线虫的能力.结果表明：初始随着作用时间的延长,细菌引诱线虫能力增加,NS-3在72小时（3天）达到对线虫的最大吸引,而随着作用时间的继续延长,细菌吸引线虫能力下降,最后细菌NS-3对线虫的吸引力为负值,从而说明线虫对致病细菌NS-3有趋避行为;真菌灰葡萄孢对松材线虫的诱引能力比较稳定,线虫对真菌没有趋避行为.进一步研究发现,枯木浸出液诱引线虫能力最大,其次是菌株NS-3的发酵液,然后是灰葡萄孢真菌的发酵液.而且,枯木浸出液和灰葡萄孢真菌的发酵液煮沸后诱引线虫能力均有所下降,但差值不大,说明枯木和真菌中对松材线虫起诱引作用的物质并没有因高温而完全失去;而细菌菌株NS-3的发酵液的发酵液煮沸后诱引线虫能力急剧下降,从而推测细菌中诱引线虫因子为挥发性的物质.