Isolation And Identification Of One Antimicrobial Marine Actinomycete And Elementary Study On Its Active Component

This thesis was aimed to isolate actinomycetes from the sea mud samples,which came from Liaoning,Shandong,Zhejiang,Fujian and Guangxi province.To screen strains producing antibacterial product,we use agar diffusion method and flat hole-trap method against Aeromonas hydrophila,Aeromonas punctata f.instestinalis,Vibrio anguillarum,Aeromonas salmonida,Vibrio Harvey,Eschenchia coli and Staphlococcus aureus at primary screening phase,and use excavating-aperture bioassay test at examination phase.One high antibacterial strain FA-F-5 was choosen.The conclusions were gained as below by the polyphasic taxonomy,the biotherapy test,the effect of fermentation liquid on Carassius auratus' immune function and preliminary separation and purification of active component:1 The Seperation and Identification of FA-F-5:16S rDNA gene sequence of the strain FA-F-5 was determined.The sequence was compared with 16SrDNA gene sequences in data of GenBank,the similarity between the strain FA-F-5 and Streptomyces fradiae was 99.5%.The result indicated that the strain FA-F-5 had a near system development with Streptomyces fradiae by constructing a system evolution tree of 16S rDNA.Binding morphological characteristics,cultural charachteristics and physic-biochemical charachteristics,the strain FA-F-5 was belonged to Streptomyces fradiae.2 The Biotherapy Test of FA-F-5:The Carassius auratus infected by Aeromonas hydrophila were fed with the food containing the active component.When the dose of the active component reached 370μg/kg·d,the bacterial hemorhage disease was cured,which indicated the antibacterial effct of active component in living body was good.Meanwhile the Carassius auratus were fed with the food containing five and ten times dose of the best cural dose active component,there is no toxicological phenomenon on the surface and viscer,which indicated the active component had no acute toxicity to organism.3 The Effect of the Condensed Fermentive Liquor on the Carassius auratus Immune Function:The Carassius auratus was fed with condensed fermentive liquor.To study the effect of the condensed fermentive liquor on the Carassius auratus' immune function,agglutinating antibody titer and bactericidal activity of phagocytes were checked on 0day,7 day and 14 day respectively.The result showed that the agglutinating antibody titer and bactericidal activity of phagocytes of the tested group was significantly different((P＜0.01) from the control group,which indicated the active component can improve the immune function of the Carassius auratus.4 The Optimization of Fermentive Conditions:Fermentive conditions optimization test of the strain FA-F-5 was done by orthogonal design.The optimum medium for FA-F-5 was maltose 15g/L,soybean 25 g/L,,CaCO₃ 1 g/L,KH₂PO₄ 0.5 g/L,MgSO₄·7H₂O 0.5 g/L,salt 10 g/L. The optimum fermentive conditions was temperature 25℃,initial pH value 7,inoculation volume 125 mL/L,medium volume 100 mL/L and seed age 48 h.When the fermentive time was 96h,the strongest potency of fermentation liquid was 2158 IU/mL and 61.43μg/mL equivalenting to Gentamycin and Ciprofloxacin respectively.5.The Stability of the Active Component:The fermentive liquor was dealed with different temperature,pH,light and storage time.The results of the stability test indicated that active substance of fermentation liquid of the strain FA-F-5 was highly stable for hyperthermy,pH,light and storage.6 Preliminary Separation and Purification of Active Component:By means of 732 ion exchage chromatography,polyamide column chromatography and alcohol precipitation, the crude crystal of antibiotic from fermentation liquid of the strain FA-F-5 was obtained.TLC spray reagents of bismuth potassium iodide indicated the crude crystal of antibiotics belonged to the aminocyclitols antibiotic.Some antibacterial actinomycetes were gained in this experiment,which was important means for development and utilization of microorganism resourses.Classification status of the strain FA-F-5,bioavailability of fermentation liquid,fermentive conditions optimization and initial separation and purification of active component were studied,which would settle foundation for exploration and industrialization product.