| Pseudomonas aeruginosa is a kind of antagonistic strain that separated from Hybrid bamboo’s branch, microorganism which located on leave’s surface, inhaibited Hybrid bamboo blight pathogenic bacteria-Arthrinium phaeospermum (Corda)M.B.Elli. This study made Pseudomonas aeruginosa as subject, screened out the optimal culture condition of which could produced antibacterial material, and made a primary research about this material’s physicochemical property, then purified a kind of yellow active substance from the crude antibacterial material, meanwhile, identified the chemical construction with mass spectrum, nuclear magnetic resonance (NMR) and infrared spectrum. The study also did detailed research on active pigment’s stability about six parts:illumination, temperature, pH, oxidizer, reducing agent and heterogeneous metallic ion.This related study can supply strong theory basises for showing the materical’s property and the active material’s uitilization, at the same time, lay a solid foundation for later study. Primary research’s results shows:1. Through monofactorial and orthogonal test, the fermentation medium of antibiotic materials which produced by Pseudomonas aeruginosa was optimized, the analysis results showed that Carbon source, nitrogen source, temperature, pH and inoculum concentration have great effect on antibiotic materials, while inorganic salt and bottle volume exert a small influence. The optimum conditions:sucrose5g, peptone10g, NaCl2.5g, water1000mL, tempreture25℃, initial pH7.5, inoculum concentration1mL, bottle volume125mL in300mL triangular flask, ferment time60h.Majorized culture medium and condition of culture were enchanced the ability of antibiotic materials greatly, meanwhile, bacteriostatic effects were obvious increased.2. The Pseudomonas aeruginosa which trained by optimum condition of culture was oscillating culture (140r/min) and produced crude active material. Through detection, the active ingredient’s molecular weight less-than1000Da; the higher molecular weight proteins’s biological activity of metabolite was less than enough with micromolecule unprotein ingredients. Its crude extract which had biological activity were small polarity matter, had better solubility in organic phase, especially methenyl chloride, so the best extracting agent was methenyl chloride. 3. The Pseudomonas aeruginosa cultured by liquid fermentation and leached by methenyl chloride would got active constituent which can inhibit the Arthrinium phaeospermum’s growth. Chloroform:methanol(30:1) as eluent, crude extact separated by column chromatography on silica gel, then got2kinds of active materials--yellow and green substances, the yellow substance’s activity was stronger than the green’s, the yellow substance separated a kind of purified matter by silica gel column repeatedly, and then through nuclear magnetic resonance, infra-red spectrum, detected this matter was phenazine-compound which had aromatic nucleus and its chemical formula was C14H10O2N2.4. Purified materical had stability research trough6parts-illumination(natural daylight and UV-light), temperature, pH, H2O2, Na2SO3and different kinds of metallic ion. Totally, illumination and temperature had slight effect on active pigment; light concentrate oxidizer and reducing agent had a small effect on active pigment, while heavy concentrate’s effect was contrary; active pigment had max activity when pH is7-8; when pH<=3, active pigment was instability, no bacteriostatic activity allmostly; metallic ion had effect on pigment’s stability in various degree, Na+, K+, Ba2+, Ca2+and Ag+had light effect on pigment’s stability, however, Al3+, Cu2+, Fe3+, Pb2+had a heavy influence, lost all activity nearly. |
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