| Antimicrobial lipopeptides synthesized nonribosomally by a large multifunctional enzyme complex are produced by Bacillus sp, exhibit significant inhibitory activity on bacteria, fungi, virus and mycoplasmal etc. In addition, lipopeptides are an extraordinarily powerful biosurfactant. Therefore, they have a great number of potential applications in the food, cosmetic industries, plant disease biocontrol, biomedicine, petroleum industry and for the bioremediation of oil-contaminated sites.In order to carry out a deeper and more extensive study on lipopetides, it is necessary for us to construct the preparation methods to obtain the lipopetides with high purity, which can provide the theory basis and the technical support for it’s further application. In this research, the preparation methods of surfactin and fengycin with high purity were constructed respectively, the quantitative detection method of fengycin by HPLC was established as well. Besides, we purified and identified the antibacterium substances with antifungal activity against Aspergillus flavus produced by Bacillus subtilis fmbj and the inhibition effect of purified Bacillomycin D on Aspergillus flavus was studied. The main results are as following:1. The method for gaining surfactin with high purity was constructed. Surfactin was separated and purified from the fermentation broth, acid precipitation, organic extraction, Sephadex LH-20column chromatography and preparative high performance liquid chromatography were used. The results of the antibacterial experiment, hemolysis test and the HPLC analysis of the retention time to constrast with the retention time of the surfactin standard showed that this method was simple and effective to obtain the surfactin with high purity.2. The preparation method and the detection method of fengycin was constructed respectively. Fengycin was separated and purified from the fermentation broth of Bacillus amyloliquefaciens ES-2-4, acid precipitation, organic extraction, column chromatography, preparative high performance liquid chromatography and freeze-drying were used. The results of the antibacterial experiment, the HPLC analysis, LC-MS and MS-MS analysis showed that this method was simple and effective to obtain the fengycin with high purity. A new method of detecting the content of fengycin was established by HPLC with the high purity fengycin we purifed. Fengycin was detected at UV205nm on a chromatographic column of Agilent C18(4.6x250mm×0.5μm), with a mobile phase of acetonitrile(0.1%TFA) and water(0.1%TFA) at a flow rate of0.6mL/min by gradient elution. The results showed that by using this method, the correlation coefficient of integral area and concentration was0.9997within the range of5to1000mg/L, with precision1.7673%, detection limit2mg/L.3. The antifungal substances with antifungal activity against Aspergillus flavus was separated and purified from the fermentation broth of Bacillus subtilis finbj, acid precipitation, organic extraction, Sephadex LH-20column chromatography and preparative high performance liquid chromatography were used. We obtained6purified peaks with antifungal activity against Aspergillus flavus. LC-MS showed that its molecular weight was1030、1044、1058、1072, which are identified as four homologous compound of Bacillomycin D. The preparation method of Bacillomycin D was constructed, the antibacterial spectrum experiment of the purified Bacillomycin D was studied, the results indicated that Bacillomycin D have strong inhibition of all the moulds.4. The inhibition effect of purified Bacillomycin D on Aspergillus flavus was studied. Bacillomycin D could inhibit the hypha growth, spore germination and spore productivity of Aspergillus flavus. The minimal inhibitory concentration (MIC) against Aspergillus flavus spores was0.05mg/mL; the minimal fatal concentration (MFC) against Aspergillus flavus spores was0.2mg/mL; Scanning electron microscopy and Transmission electron microscopy were used to study changes in the morphology and inner structure of Aspergillus flavus after Bacillomycin D treatment. The results indicated Bacillomycin D could injure the cell wall of the hypha and spore and cross the cell membrane. Then, cytoplasms and organelles inside the cell were damaged to form empty hole and exude. |
PDF Full Text Request