Identification and preliminary characterization of non-polyene antibiotics secreted by new strain of actinomycete isolated from sebkha of Kenadsa, Algeria

Objective: To study the antimicrobial activity of actinomycetes isolated from sabkha of Kenadsa and identification of the isolate interesting. Methods: Eighteen strains were isolated, using four culture media from sebkha of Kenadsa(Bechar, Southwestern Algeria). Screening of antimicrobial activity consisted of two steps: in primary screening, antibacterial activity was determined by using the agar plug method against test strains; in secondary screening, better isolate which showed a good activity in the first screening was selected to extract antimicrobial substances. The antimicrobial activities of extracts were evaluated by using Kirby-Bauer disc diffusion method. Partial characterization of antimicrobial products was performed on the basis of chemical revelations, UV-vis spectrometry and infrared spectroscopy. The identification of isolate interesting was performed through morphological, chemical, biochemical and physiological characteristics. Results: All isolates showed antimicrobial activity against at least one microorganism test. One isolate, LAM143 cG 3, was selected for its broad spectrum and high antimicrobial activity. The isolate LAM143 c G3 was identified as Spirillospora sp. The comparison between the species of this genus(Spirillospora rubra and Spirillospora albida) and our isolate indicated the existence of several physiological and biochemical differences which led us to suppose that this was a new member of this genus. Primary characterization of antimicrobial substances produced by the isolate LAM143 c G3 indicated the presence of amines and phenols. The UV–vis spectrum suggested a non-polyenic nature of substances secreted by our isolate, while infrared confirmed the presence of amine groups.Conclusions: The result of the present study revealed that sebkha of Kenadsa was rich in rare actinomycetes, that secreted interesting antimicrobial substance.

Hyoscyamine-producing marine Actinomycetes from Lagos Lagoon sediment

Objective:To isolate and screen Actinoniycetes from Lagos Lagoon soil sediments for production of bioactive metabolites.Methods:Sediment samples were collected from four different locations of Lagos Lagoon and were dried for 2 weeks after which the Actinoniycetes were isolated by serial dilution using the spread plate method on starch casein and Kuster's agar supplemented with 80 ug/mL cycloheximide to prevent fungal growth.The plates were incubated at 28 C for 1-2 weeks.Isolates were selected based on their colonial characteristics as well as their Gram's reaction and subciiltured using the same media for isolation until pure cultures were obtained and incubated at 28 C for 3 d.Thereafter,they were inoculated into starch casein and Kuster's broth media and incubated for 8 d.The secondary metabolites were screened for antimicrobial activity against the following microorganisms:methicillin resistant Staphylococcus aureus.Staphylococcus aureus ATCC 29213.Escherichia coli ATCC 29522.Pseudomonas aeruginosa ATCC 27853.Candida albicans and Enterocolitis faecal is ATCC 29212.Coagulasenegative staphylococci isolated from HIV patients were also used(Staphylococcus warneri.Staphylococcus xylosus and Staphylococcus epidennidis).The antimicrobial metabolites of the Actinoniycetes isolates were identified using gas chromatography(GC).Results:Crude extracts of isolates showed antimicrobial activity against some of the test organisms.The GC data analysis showed the antibiotic profile of these isolates.Conclusions:Analysis of the crude extracts of the isolates using GC method,revealed the presence of antibiotics including an anticholinergic hyoscyamine among other conclusions.

Electrochemical Identification of Yulania spp. by Fingerprinting of Leaves Using Glassy Carbon Electrode

In this communication,we used electrochemical sensor for recording the electrochemical profiles of eleven species of Yulania spp.from leaf extract.Two solvents and two buffer conditions were used for electrochemical fingerprints collection.Their electrochemical fingerprints can be converted to different patterns and consequently for species recognition.The results indicate the pattern recognition is much convenient than that of the recognition of species directly using voltammetric signal.The current information in electrochemical fingerprinting represents the type and amount of electrochemically active molecules,which linked to the genetic differences among the plants.Therefore,the electrochemical fingerprints were applied for further phylogenetic study.The phylogenetic tree deduced from voltametric curves is divided into three main groups.The first clade contains Y.denudate,Liriodendron chinense,Y.cylindrica,Y.biondii,Y.sprengeri.The second clade contains Y.zenii,Y.liliiflora,Y.kobus,and Y.amoena.The third clade contains Y.×soulangeana,Manglietia fordiana and Y.sinostellata.In addition,Y.salicifolia is not in these main clades.The results demonstrate that electrochemical fingerprinting can be used as a com-plementary tool in the study of phylogenetics.

Antimethicilin resistance agents from marine actinomycetes from soil sediments of Lagos Lagoon

Objective:To evaluate the isolation of actinomycetes strains with potential for producing antimicrobials with high methicilin resistance capability.Methods:The soil samples were collected from four different locations of Lagos lagoon.The Actinomycetes were isolated from the samples by serial dilution using spread plate method.Isolates were selected based on their cultural characteristics as well as their Gram reaction and phenotypically and molecularly characterized Streptomyces sp.Isolates were inoculated in starch casein and Kuster’s broth media and secondary metabolites were screened for antimicrobial activity against the following microorganisms:methicillin resistant Staphylococcus aureus,Staphylococcus aureus ATCC 29213,Escherichia coli ATCC 29522,Pseudomonas aeruginosa ATCC 27853,Candida albicans,Enterococcus faecalis ATCC 29212.Coagulase-negative staphylococci isolated from HIV patients were also used(Staphylococcus warneri,Staphylococcus xylosus and Staphylococcus epidermidis).The antimicrobial metabolites of the isolates were identified using gas chromatography-mass spectrometer.Results:Extracts from isolates ULS12 and ULS13 showed antimicrobial activity against methicillin resistant Staphylococcus aureus while ULK3 inhibited Candida albicans only.The gas chromatography-mass spectrometer data analysis showed the antibiotic profile of these isolates.Conclusions:The isolates ULS12 and ULS13 were found to display the highest antimicrobial activity against the test organisms and could be a potential source of new antibiotics.

延边州晒烟放线菌分离鉴定与系统发育多样性分析

为了获取不同环境和生态位对晒烟可培养放线菌的影响,从延边州龙井、图们和延吉3个不同地点的晒烟生长区取样,采用纯培养的方法,从烟草非根际土、根际土、根、茎、叶中分离晒烟放线菌。根据菌体形态和16S rDNA部分序列测定及GenBank-BLAST同源性分析进行菌种鉴定,共得到链霉菌属和红球菌属放线菌11株,且延吉晒烟样品分离出的菌株数量多于龙井和图们。对烟草不同部位的放线菌进行对比分析发现,分离到的放线菌数量由多到少依次为根、茎、叶。该研究结果有利于进一步解决晒烟种植过程中的适应性和安全性问题,并为农业可持续发展奠定基础。

酱香型白酒酿造过程中高温放线菌的筛选及其特性

为探究白酒酿造过程中高温放线菌的特性,利用传统方法从酱香型白酒酿造过程中筛选得到3株高温放线菌,经形态学、16S rDNA生物学鉴定、生理生化及特性分析,确定分离纯化出的F-40号菌株为白色链霉(Streptomyces albus)、F-41号菌株为橄榄链霉菌(Streptomyces olivaceus)、F-42号菌株为植物内生糖多孢菌(Saccharopolyspora endophytica)。F-40号菌株的最适生长温度为30℃,最适生长pH8,最大耐乙醇浓度为9%;F-41号菌株的最适生长温度为50℃,最适生长pH7,最大耐乙醇浓度为6%;F-42号菌株的最适生长温度为40℃,最适生长pH6,最大耐乙醇浓度为6%。3株放线菌代谢产生其各自的特征性风味物质,F-40号菌株可代谢产生反-萘烷醇,F-41号菌株可代谢产生小分子醇类及内酯,F-42号菌株可代谢产生不饱和醛类,且均可代谢产生较多的醇类及醛酮类物质,如2-庚酮、苯乙醇等,有助于酱酒风味的形成。

南极钩虾Gammarussp.肠道共附生菌的分离及StreptomycesxiamenensisYF008的化学成分研究

南极极端生境中蕴含着发掘特殊生物活性天然产物的巨大潜力。对采自南极海域水深6000m海区钩虾(Gammarussp.)的肠道共附生微生物进行分离,并对一株有价值菌株的代谢产物进行研究。采用稀释涂布法进行菌株分离,利用形态学观察及16SrRNA测序进行菌株种属鉴定;综合运用薄层色谱、正相硅胶柱色谱、凝胶柱色谱、反相ODS柱色谱和半制备高效液相(HPLC)等色谱手段分离纯化得到单体化合物,采用波谱学和质谱技术鉴定其化学结构。从钩虾中分离得到17株菌,并将其从分类学角度归属于9个属,其中细菌11株,放线菌6株。从放线菌StreptomycesxiamenensisYF008的发酵产物中分离得到8个单体化合物,分别鉴定为:1(10)E,5E-germacradiene-2,11-diol(1),N-[2-(4-hydroxyphenyl)]ethylacetamide(2),环(L-苯丙氨酸-甘氨酸)(3),环(L-苯丙氨酸-L-丙氨酸)(4),环(D-苯丙氨酸-L-异亮氨酸)(5),环(L-羟脯氨酸-L-苯丙氨酸)(6),环(D-亮氨酸-L-脯氨酸)(7),环(L-苯丙氨酸-L-脯氨酸)(8)。化合物1、3~6系首次从该菌中分离得到,且化合物1为特殊的十元环germacrane型倍半萜类化合物。

海洋链霉菌MA12的次级代谢产物及抗真菌活性研究

对一株海洋放线菌Streptomyces sp.MA12进行次级代谢产物的研究和体外抗真菌活性测试,采用MCI中压柱色谱、硅胶柱色谱、SephadexLH-20凝胶柱色谱及半制备高效液相色谱(HPLC)等方法对发酵产物进行分离和纯化,综合运用质谱(MS)和核磁共振(NMR)等波谱学方法进行结构解析和鉴定。从放线菌Streptomyces sp.MA12发酵产物中分离鉴定出3种化合物,分别是trienomycin A(化合物1)、trienomycin B(化合物2)和ansatrienin B(化合物3)。抗菌活性测试发现,化合物3对3株白色念珠菌(标准敏感白色念珠菌SC5314,临床分离耐氟康唑白色念珠菌901,实验室基因编辑耐氟康唑白色念珠菌816)有明显抑制作用,MIC值分别为4、8、4μg·mL^(-1)。从该海洋放线菌中发现了安莎三烯类化合物,并首次报道了其对氟康唑耐药真菌的抑制活性,研究结果丰富了海洋微生物的次级代谢产物种类,可为新型抗真菌药物的研发提供科学参考。

寡营养型类节杆菌OM7的分离鉴定及固沙特性

生物土壤结皮能有效改善沙漠土壤特性,防治荒漠化。土壤微生物尤其是寡营养型微生物是生物结皮形成过程中不可或缺的重要角色。在毛乌素沙地苔藓结皮样品中分离筛选得到寡营养型固沙菌株OM7,并通过生理生化和16S rRNA基因进行鉴定,对菌株OM7的固沙特性进行初步研究,以期为沙漠生态治理提供优良菌种资源。在淀粉稀释度为1/40的高氏一号培养基中进行发酵培养,在沙罐中均匀施洒菌液后通过测定结皮厚度和抗压强度来研究菌株OM7的固沙能力。初步鉴定菌株OM7为类节杆菌OM7(Paenarthrobacter sp.OM7),为寡营养型类节杆菌的首次报道。类节杆菌OM7在1/40高氏一号培养基中培养3天的发酵液,于沙罐内接种菌剂培养物后在1个月内所形成结皮最厚为1.83 mm,抗压强度最大为10.48 N。寡营养型类节杆菌OM7具有高效固沙能力,适应养分贫瘠的环境,使其在沙漠生态治理中更具优势。菌株OM7的固沙能力与其生长状态呈正相关,发酵时间的选取对于固沙效果至关重要。

窖泥中放线菌的分离鉴定及生理特性研究

为探索窖泥中放线菌在窖池中的性能,本研究以浓香型白酒窖泥为菌源,采用梯度稀释平板法从中分离出两株放线菌菌株,对目标菌株进行多相分类鉴定,包括16S rRNA基因序列分析、形态观察及部分生理代谢特性分析。本研究通过筛选培养获得两株放线菌菌株(X,Z)后的初步鉴定结果为:菌株X和菌株Z分别为产灰链霉菌(Streptomyces griseus)以及德氏链霉菌(Streptomyces drozdowiczii)。通过分析实验结论得知,两株菌最适生长pH值为6.5,最适生长温度区间均在25~30℃;菌株X可耐受乙醇浓度为10%,菌株Z可耐受乙醇浓度为8%;菌株X在液态环境下的对数生长期为12~48 h,菌株Z在液态环境下的对数生长期为18~48 h,两菌株稳定期均在48 h后;通过测试发现两株放线菌均能产生淀粉酶,且菌株X产淀粉酶能力明显强于菌株Z,结果表明窖泥中的放线菌株拥有分解淀粉的能力,可以为其他需葡萄糖微生物提供必要的营养物质;菌X和菌Z均无产抗生素能力或产抗生素能力极低,表明在窖泥环境中,放线菌可能无需产生抗生素也能和其他微生物共生长。

一株拮抗番茄叶霉病菌的放线菌筛选、鉴定及发酵条件研究

菌株xjy是一株从新疆棉田土壤中筛选出的对番茄叶霉病菌(Fulviafulva)有较强拮抗作用的放线菌,其对23种常见植物病原真菌和6种细菌有较好的抑制效果,抗菌谱广。其形态特征、培养特性、生理生化特性、细胞壁组分与放线菌中的淡紫灰链霉菌(Streptomyces lavendulae)相同,16SrDNA序列同源性达99.6%。研究发现该菌的摇瓶发酵配方和培养条件为:大豆粉2%、葡萄糖2%、NaCl0.8%、CaCO30.2%、(NH4)2SO40.32%,起始pH7.0、28℃、180r/mim、摇瓶培养6d,这些结果为该菌株今后的应用、抗生素的分离提纯和产业化提供了实验依据。

植物内生放线菌Lj20的鉴定及其抗真菌物质的合成

【目的】菌株Lj20是从辣椒植株根部分离得到的一株有抗真菌活性的植物内生放线菌。为了进一步开发利用这一放线菌,对其进行了鉴定及抗菌活性物质的研究。【方法】根据Lj20的形态特征、培养特征、生理生化特征、细胞壁组分和16S rDNA序列对其进行鉴定。结合GC-MS分析,合成了代谢产物中所含的抗真菌活性物质,并用菌丝生长抑制法测定其生物活性。【结果】Lj20菌株属于链霉菌属,与娄彻氏链霉菌(Streptomyces rochei)极为相似。代谢产物中含有2,6-二叔丁基对甲酚和3,5-二叔丁基-4-羟基-苯甲醚。两种化合物对番茄灰霉病菌的EC50值分别为237.04mg/L和186.48mg/L。【结论】菌株Lj20鉴定为娄彻氏链霉菌(Streptomyces rochei)。2,6-二叔丁基对甲酚和3,5-二叔丁基-4-羟基-苯甲醚对番茄灰霉病菌都有较强的抑制作用。

产杀虫活性物质海洋放线菌的筛选和初步鉴定

从浙江宁波海域的滩涂泥样中分离到54株海洋放线菌,用卤虫液体测定法进行了杀虫活性菌株的筛选,结果显示多株放线菌具有较高的杀虫活性,菌株XS902,XS904,BL803,BL807对卤虫的ID50高达100以上。同时研究了XS904活性菌株的形态培养特征和生理生化特征,初步鉴定该菌株属于链霉菌属。

拮抗放线菌LJ50和MJ52的分离与初步鉴定

采用常规方法从3种蔬菜地土壤中筛选出2株对棉花黄萎病菌具有拮抗作用的放线菌LJ50和MJ52,通过传统的形态特征和部分生理生化测定,这2株拮抗放线菌属链霉菌属的白孢类群.离体条件下这2株拮抗放线菌的发酵液对棉花黄萎病菌、苹果轮纹病菌、黄瓜炭疽病菌、棉花立枯病菌、番茄灰霉病菌、小麦赤霉病菌、黄瓜枯萎病菌、西瓜枯萎病菌等8种病原真菌均具有抑制作用.

西瓜根际枯萎病拮抗放线菌的筛选及鉴定

从西瓜根际分离获得了32个放线菌分离物,通过离体拮抗试验,筛选出3株对西瓜枯萎病有良好拮抗效果的放线菌,编号为XF9、XF18和XF22。通过对其进行形态学观察、生理生化测定以及16S rDNA序列分析,初步确定XF9为灰色链霉菌(Streptomyces griseus),XF18为淡紫灰链霉菌(Streptomyces lavendulae),XF22为脱叶链霉菌(Streptomyces exfoliates)。XF9、XF18和XF2216S rDNA序列的GenBank登录号分别为EU723881、EU723880和EU723879。

海洋来源的放线菌3295代谢产物的结构鉴定及抗肿瘤活性(Ⅰ)

目的 对海洋来源的放线菌3295代谢产物中的抗肿瘤活性成分进行分离和鉴定。方法 利用细胞周期抑制为抗肿瘤活性指标,采用Sephadex LH-20,硅胶和HPLC等柱色谱技术对3295活性菌株发酵物进行活性追踪分离,根据理化性质和光谱方法进行化学结构鉴定,用流式细胞术评价化合物的抗肿瘤活性。结果与结论从其代谢产物中分离得到1个具有抗肿瘤活性的化合物Ⅰ,结构鉴定为邻苯二甲酸二丁酯(dibutylphthalate);化合物Ⅰ对小鼠乳腺癌温敏型tsFT210细胞具有G0／G1期细胞周期抑制作用。

一株具有广谱抗菌活性小单孢菌的分离和鉴定

从南昌瑶湖的农田土壤样品中分离到一株具有广谱抗菌活性的稀有放线菌。通过形态特征、培养特征、生理生化特性、细胞化学成分及16SrRNA基因序列等多相分类特征研究,将该稀有放线菌鉴定为炭样小单孢菌。

一株抗根结线虫放线菌的筛选与鉴定

从海南省东寨港红树林底泥中分离获得具有抗线虫活性的放线菌菌株HA11097。将该菌株发酵液稀释20倍和40倍时,抗根结线虫校正死亡率分别为54.2%和46.7%。16S rRNA基因序列分析表明,菌株HA11097属于链霉菌属(Streptomyces)中的成员,与S.aculeolatus的相似性最高,为98.9%。二者在系统发育树上处于同一分支,亲缘关系最近,DNA-DNA杂交值为94.4%。该菌的形态特征、培养特征以及生理生化特征与S.aculeolatus也基本一致。综合以上结果,鉴定菌株HA11097为针孢链霉菌Streptomyces aculeolatus,首次报道了其抗线虫活性。

海洋放线菌Y12-26代谢产物中bafilomycins分离纯化及结构鉴定

目的了解海洋放线菌Y12-26的次级代谢产物中对稻瘟病菌(Pyricularia oryzae)等植物病原真菌具有较好的拮抗活性的具体化合物。方法以P.oryzae指示菌进行活性跟踪,对其代谢物进行分离提取,通过有机溶剂萃取、硅胶柱层析、TLC和HPLC制备等方法分离得到活性化合物,并通过UV、MS、NMR等方法进行结构鉴定。结果从海洋放线菌Y12-26发酵物中分离得到了3个大环内酯类活性化合物Y12-26-A2a、A3a和A3b,确定Y12-26-A2a分子式为C35H56O8,与bafilomycin D结构相同;Y12-26-A3a分子式为C35H58O9,与bafilomycin A1结构相同;Y12-26-A3b为新结构化合物,分子式为C35H58O8,命名为bafilomycin K。结论化合物Y12-26-A2a、A3a和A3b对P.oryzae的MIC分别为125、16和31μg/mL。

放线菌BOS-009的分离、拮抗活性及鉴定

对从长白山不同海拔环境中分离筛选出可以产生抗菌活性物质的BOS-009放线菌菌株进行了研究,测定了该菌的抗菌谱,发现该菌株对多种植物病原性真菌及细菌有拮抗作用。通过形态特征、培养特征观察,生理生化特性及抑菌谱等系列比较,发现该菌株与链霉菌属的除虫链霉菌(Streptomyces avermitilis)的特征基本相符;通过16srDAN序列比对,发现该菌株16srDAN与除虫链霉菌的16srDAN同源性达99%。根据多项分类原则和系统进化树的构建分析,将该菌株暂归入除虫链霉菌类。