Preliminary Study On Anti-candida Albicans Activity By Lipopeptide Produced By Bacillus Amyloliquefaciens

Antimicrobial substances of the secondary metabolites produced by Bacillus sppwere lipopeptides. Generally, they include surfactin, iturin and fengycin. They wereable to kill bacteria, fungi, virus and mycoplasma. So these substances can be appliedto control the diseases caused by fungi or bacteria. Lipopeptide is composed oflipophilic-hydroxyl-fatty acid chain and hydrophilic peptide chain. Because of itsstructure, lipopeptides have a wide range of applications in the food industry,pharmaceuticals industry and agricultural production.In this study, the anti-CA strain with the strongest anti-Candida albicans activitywas obtained from a number of bacterial strains which could inhibit many fungi andbacteria. By conventional biochemical identification, we found that anti-CA strain hadsimilar characteristics to those of Bacillus amyloliquefaciens. By using molecularbiology method, including determination of16S rDNA sequence and alignment, theanti-CA strain was identified be one strain of B. amyloliquefaciens. This strain caninduce the death of C. albicans which is a species of opportunistic pathogens.Furthermore, the optimal medium compositions for the anti-Candida albicansactivity substance production by the anti-CA strain were that2.0%（w/v） of soybeancake powder,1.0%（w/v） of wheat flour. The optimal cultivation conditions were28℃, pH6.5and cultivation time27h. Fermentation was carried out via2-Lfermenter and antifungal activity, amylase activity and protease activity produced bythe anti-CA strain during the fermentation was confirmed. The results revealed thatwithin21h of the fermentation, cell growth reached the stationary phase with a celldensity of about3.3×108cells/mL, the highest anti-Candida albicans activity of thesupernatant was reached at the27th hour of cultivation with a Φ31.33mm inhibitionzone. A high amylase activity of110.75U/mL and proteinase activity of3.82U/mLwere detected. All the results would lay the foundation for the future industrialfermentation production. The cultivation reached to the death phase from the33rd hour. An antifungal lipopeptide in the supernatant was purified by acid precipitation,thin layer chromatography and high performance liquid chromatography （HPLC）. Themass spectrometry result showed that its molecular weight was about1021and itsmolecular formula was C₅₂H₉₁N₇O₁₃. The anti-Candida albicans activity wasconducted under the condition of28℃, pH7.0, and the purified lipopeptide showedthe best antimicrobial activity under these conditions.The purified lipopeptide was able to kill the whole cells of C. albicans cells, andcould also kill the protoplast of the C. albicans cells. After the whole sensitive cellswere treated by the lipopeptide, the cell walls were invaginated and wrinkled, and thematrix leaked from the cells. The treated cells also can be stained by PI. However, thenormal cells can not be affected by the lipopeptide. This confirmed that thelipopeptide produced by anti-CA strain acted on the cell wall of the sensitive yeastcells, causing defective integrity of the cell wall, damage of cell membrane and celldeath.