| This study aimed at separating and screening the broad-spectrum antagonism actinomyces from the silt samples at the bottom of Weishan Lake, and then preliminary researches were carried out for fermentative conditions and active productions. The main methods and results are summarized as follows:1,The research took Gause's Synthetic medium as the separating medium.18 strains of actinomycetes were separated, the spore piles of which had different morphologies and colors. One strain named Actinobacteria 12 was selected after comparison on inhibition activity to some pathogenic fungi and some bacteria using the method of dot and cylinder plate. Its metabolites had strong inhibiting activities. Actinobacteria 12 can inhibit the growth of Staphylococcus aureus, Escherichia coli, Bacillus licheniformis, Trichoderma viride, Aspergillus niger, Penicillium and so on, and the diameterof the bacteriostatic circle are all above 15mm.2,Through observation on substrate mycelium, aerial mycelium, sporothrix, biochemical and physiological characteristics and 16SrDNA sequence analysis, species relationships of Actinobacteria 12 were explored. On Gause's Synthetic medium, Actinobacteria 12 grew well, with gray spore pile. Spore silks were often born on aerial mycelium, which were straight or curve and sometimes in clumps. According to the above characteristics, the strain 12 was preliminary identified as Cinereus of Streptomyces. Actinobacteria 12 was considered very similar with Streptomyces rocher according to the analysis data of 16SrDNA gene sequence and phylogenetic tree. Because of the similarity of 99.7% and difference with Streptomyces rocher, Actinobacteria 12 was preliminary identified as a variant of Streptomyces rocher.3,By means of combinations of single factor-experiment, Plackett-Burman (PB) and Orthogonal experiment, the optimal medium was obtained. The optimal fermentation condition for strain 12 was as follows:in 300 mL flask with 50 mL medium,17h of liquid culture time, inoculumconcentration with 2mL, medium's initial pH at 7.5, ferment for 72h, and its optimum fermentation medium contained (w/V):1% glucose,0.35% NH4Cl,0.6% NaCl,0.2% MgSO4, 0.25% CaCO3,1.25% DouBing powder.4,The stability of the fermented broth of the strain 12 was tested. The results indicated that the active secondary metabolites of strain 12 were stable to acid, temperature and Ultraviolet lamplight. The fermented broth of the strain12 was respectively treated by the pH of 2,4,6,8,10, 12 or by illuminating under Ultraviolet lamplight with 20min,40min, 1h,2h,3h,4h,5h, 6h, and the activity of metabolites was virtually unchanged. When the fermented broth was treated under the temperature below 80℃, the antibacterial activity was virtually unchanged; less than 100℃processing of 1h, antibacterial activity was slight abate; under 121℃processing of 20min, antibacterial activity still existed. As a whole, the stability of the fermented broth of the strain 12 was very good.5,The fermented active substances were expected to be purified using organic solvent extraction, but the allocation rate of the active secondary metabolites was lower in ethyl acetateg and chloroform, and the active metabolites cannot be extracted by petroleum ether, butanol and benzene. Chromatographic separation was processed using the Czech eight-solvent systemthe. The result showed that the Rf value of the fifth and sixth kinds of solvent systems were bigger than that of others. The ligature of Rf value displayed like the sails. That was very similar with Streptomycin, Kanamycin and Jinggangmycin. Thus the active substances may belong to alkaline and water-soluble antibiotic substances. |
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