| Sixty bacterial strains with the activity against Candida albicans, a main pathogenic strain of system fungal infection, were screened out from 2427 bacteria, which were separated form ten soil samples of China. One strain LDT917 was detected to have significant bioactivity against Candida albicans and determined as the objected strain. The strain LDT917, which is a gram-positive bacterium, has a spore in the center of cell. The physiological and biochemical studies have shown that strain LDT917 has the characteristics of Bacillus family. Based on 16S rDNA sequence analysis, the strain LDT917 has been identified to be most close to the strain Bacillus subtilis BFSA.The antimicrobial spectrum of Bacillus sp.LDT917 was assayed through cup-plate method. Results showed that antibiotic LDT917 had a wide and remarkable inhibitory effect against most of the tested strains including a broad range of pathogenic yeasts and gram-positive bacteria, as follows: Candida albicans, Candida glabrata, Candida tropicalsis, Cryptococcus neoformans, Rhodotorula glutinis, Staphylococcus aureus and Bacillus subtilis. Noticeably, antibiotic LDT917 displayed a significant antimicrobial activity against clinical isolates fluconazole-resistant Candida albicans, fluconazole-resistant Candida glabrata and methicillin-resistant Staphylococcus aureus.The production of the antibiotic by strain LDT917 was promoted by natural screening and UV mutational screening. A stable and high-yielding strain LDT917-383 was obtained. The flask fermentation conditions were optimized by single-factor experiment and orthogonal design experiment. The optimum fermentation medium was glucose monohydrate 2.0%, beef extract 1.0%, peptone 0.5%. The optimum culture conditions were as follows: fermentation temperature 30℃, seed culture time 12h, inoculum percentage 10%, medium content 20ml/250ml, fermentation time 24h.The antibiotic LDT917 was existed mainly in the fermentation liquid. The physical and chemical properties of the antibiotic were studied. The results showed the antibiotic was stable in hot, acid, neutral and weak alkaline conditions but not in strong alkaline condition. The experiment of pH paper chromatography showed the antibiotic might be weak acid and water-soluble. By UV scanning, its absorption peak was observed at 256nm. Antifungal antibiotic LDT917 was purified by pretreatment, silica thin-layer chromatography, macropore absorption resin and ion exchange chromatography. Finally, the white crystallized antibiotic LDT917 was obtained by concentrated and dried, it could be used for further refinement. |
