| Dictyophora indusiata, a kind of precious fungi with strong antibacterial activity, has vast prospect to become a natural preservations. In this paper, seven bacterium, tow yeasts and three molds were chosen to investigate the antimicrobial activity of the different ethanol concentration extracts from Dictyophora indusiata. The antibacterial activity of extracts against seven species of bacterium was investigated by agar plate method and the minimum inhibitory concentration was also analyzed using same method. Second, single-factor tests were designed by the volume of extraction and its antibacterial activity as index. Then quadratic regression general rotary unitized was designed according to the single-factor tests. And the optimal process parameters were determined by means of regression analysis. By silica gel column chromatography, thin layer chromatography, tracking antibacterial test, the crude extract was isolated and purified, a monomer compound was obtained for the first time. Based on the nuclear magnetic resonant analysis, the monomer compound was identified as m-trihydroxybenzene.The main conclusions could be summarized as follows:1. The fruit body of Dictyophora indusiata, after pre-treatment, was extracted under different ethanol concentration. Using antibacterial activity as a index, the results showed that the optimal extracting agent was 100% ethanol2. Antibacterial activity of crude extracts against bacterium, mold and yeast was investigated by agar plate method. The results showed that the crude extracts had strong Antibacterial activity to bacteria, but almost not work to mold and yeast.3. Minimum inhibitory concentration of crude extracts against seven species of bacterium was analyzed by agar diffusion plate method, double dilution method and gradient-dilution method. The results showed that the crude extracts had the lowest MIC (30mg/mL) against Salmonella enteritidis, Bacillus thuringiensis and Bacillus subrilis. The results demonstrated that Dictyophora indusiata contains antibacterial substances.4. The optimal extracting parameters were determined by mono-parameter experiment, the results were as follows:extraction time of 2h, extraction temperature of 70℃, solvent/material ration of 25:1. At the same time, antibacterial effects were determined and correlation curves between the volume of extraction and antibacterial effects were established. The result was that antibacterial activity correlated positively with the volume of extraction, so the volume of extraction can be used as an index, instead of antibacterial activity, to measure the extracting effects on the quadratic general rotation design experiment.5. On the basis of mono-parameter experiment, quadratic general rotation design experiment was used to optimize the technological parameters, the regression equation about the relationship between the effect factors (extraction time, extraction temperature, solvent/material ration) and volume of extraction was obtained. By optimizing the regression model of the volume of extraction, the optimum extraction conditions were obtained:extraction time of 2h, extraction temperature of 72℃, solvent/material ration of 25:1. The mathematical model established in this paper was significant, so it could be applied to analyze and predict the extraction technique of antibacterial substances from Dictyophora indusial.6. A monomer compound was isolated from crude extract by extraction,3 times silica gel column chromatography with different mobile phase, reversed-phase silica gel column chromatography, thin layer chromatography and tracking antibacterial test. The compound was identified as m-trihydroxybenzene by NMR analysis.7. The results of agar diffusion plate, in-situ inhibition test, microscope observation and analysis of IC5o showed that the monomer compound has strong inhibition activity against bacteria, the half inhibitory concentration to Slmonella enteritidis and Bcillus megaterium were 51.58ug/mL and 83.06ug/mL, respectively. |
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