An Endophytic Streptomyces Cavourensis From Camellia Sinensis:Optimization Of Fermentation Condition And Product Purification

The endophytic mycelium is normal flora within the plant tissue,including endophytic fungus, endophytic bacteria and endophytic actinomycetes. Endophytic actinomycetes exist in plants, as important taxa, may produce antibiotics which is different from the those in the soil. And these active substances usually have higher value in use. It would be an important source of new antibiotics.Streptomyces strain BF-01was isolated from colony of endophytic fungi Guignardia mangiferae which was isolated from leaf of tea plant C. sinensis. It has a broad-spectrum antibiotic effect. Established a composite microbial system of Streptomyces strain BF-01and endophytic fungi Guignardia mangiferae to mimicked the endophytic environment of ecological relationships.Determining inhibitory effect of the fermentation of composite microbial system in different time by using Magnaporthe grisea as sensitive fungus. The Fermentation conditions are as follows:inoculum size:7%, rotation speed:180rpm, temperature:28℃. The inhibitory effect of fermentation was best after mixed cultured7days on the basis of Guignardia mangiferae cultured for one day. Its inhibition rate was improved from initial15.68%increased to21.38%, which was increased36.4%. The inhibition rate of fermentation was20.28%after mixed cultured8days, which was increased29.3%.To maximize the antibiotic substances, the experiment optimized the fermentation conditions of the composite microbial system by single factor experiment and the uniform design method biased by the mixed fermentation. Firstly, The single-factor experimental design sifted the flour and peptone as best carbon source and nitrogen source. By uniform design optimization, optimizing the best ratio of carbon source,nitrogen source and other ingredients in medium, then work out a quadratic regression equation with a well degree of fitting. The optimum medium is:millet18.60g/L, flour44.66g/L, peptone13.01g/L, NaCl3.66g/L. Available theoretical inhibition rate of fermentation broth was38.67%after optimization. The inhibition rate improved95.6%compared with the original medium. And the uniform design experimental results are in the range of forecasts. This shows that the a quadratic regression equation obtained through the uniform design model can well account for strain BF-01metabolic processes.Dark brown fermentation broth from amplification fermentation were ultrafilter by ultrafiltration column with pore size for5kD, removing the part of the pigment. The antibiotic effect of ultrafiltration retentate is obvious. It was purified through alcohol precipitation, chloroform extraction, and then obtaining the brown-yellow extract which quality is458mg. After silica gel column chromatography, an high activity component XI was found. After solvent evaporation, pale yellow or brownish-yellow lamellate mixed crystal was collected, which quality is13.5mg.Range of antibiotic activity was determined experimentally on the mixed crystal.(1) The result of antibiotic test showed that mixed crystal have different degrees of inhibition effect on five species of fungus.The highest inhibition rate to Colletotrichum gloeosporioides reached78.19%; Followed by rice grain dry bacteria, the inhibition rate was66.87%. The inhibition effect to Guignardia mangiferae and Trichoderma viride was poorer,and the inhibition rate was only28.40%and21.81%;(2) Mixed crystal composition also has a good antibiotic effect on bacteria. The antibiotic effect were best against E. coli and Pseudomonadaceae. The inhibition diameter were5.3mm and4.5mm respectively. But the inhibition effect to Salmonella sp. was poorer. The inhibition diameter was only1.36mm.A significant difference analysis showed great significant and significant on the effect by SPSS software. In summary, the metabolic component Ⅺ of strain BF-01has a broad-spectrum antibiotic activity.