Isolation, Purification, Characterization And Producing Conditions Of The Antifungal Peptides Produced By Bacillus Sp. AH-E-1

The diseases caused by fungi are increasingly serious. Because Bacillus sp. canproduce a variety of antifungal agents, it has attracted the attention of people. Bacillussp. AH-E-1was a newly discovered bacteria strain which could strongly inhibitCandida albicans in previous studies. To further study antifungal activities of Bacillussp. AH-E-1and screen novel antifungal substances, we studied Bacillus sp. AH-E-1and the fermentation supernatant from it. Subsequently, the isolation, purification, andcharacterization of the antifungal peptides produced by Bacillus sp. AH-E-1werestudied. Finally, we preliminarily optimized the fermentation conditions of producingthe antifungal peptides. The studies were as follows:To study the antibiotic activities of Bacillus sp. AH-E-1and its fermentationsupernatant, confrontation method, overlay assay, the coverslip insertion method andso on were used. The studies showed that the strain could strongly inhibit against13strains of phytopathogenic fungi and11strains of human pathogenic fungi, and thatthe strain could induce mycelial distortion of filamentous fungi and so on. Theantifungal substances produced by Bacillus sp. AH-E-1were in its fermentationsupernatant which could inhibit spore germination and give suppression to germ tubeand mycelial growth. The inhibitory effect of the fermentation supernatant to Candidaalbicans was stronger than Amphotericin B (1mg/mL).The antifungal substances were crudely extracted by ammonium sulfateprecipitation, solvents extraction, organic solvents precipitates, isoelectric pointprecipitation and methanol diffusion, at last methanol diffusion was used to extractthe antifungal substances. The preliminarily qualitative studies of antifungalsubstances were done by the reactions of triketohydrindene hydrate and so on, then3routes of separation and purification of the antifungal peptides were compared, it wasselected that the antifungal peptides were isolated and purified by ultrafiltration,reversed-phase liquid and HILIC chromatograph. The antifungal peptides had beenpurified to chromatography-class. The study showed that the antifungal substanceswere determined as peptides, of which the molecular weights were about500~1000Da.It was proved that the peptides were strong polar and non-ionizing. Besides the peptides were not sensible to high temperature, X-ray, pH (2~12),5kinds of organicsolvents such as chloroform, and6kinds of enzymes such as Trypsin at1mg/mL.The fermentation conditions of producing the antifungal peptides werepreliminarily optimized by single factor and orthogonal experiments. The studiesshowed that the production of antifungal peptides was affected by many factors, andthat the increasing of the ratio of carbon to nitrogen in culture was in favor ofproducing the antifungal peptides. The effects of5factors (medium, fermentationtime, medium volume, inoculation size and initial pH of the medium) against theproduce of the antifungal peptides were as follows: medium> fermentation time>medium volume=inoculation size> initial pH of the medium. The optimalfermentation condition was: Bacillus sp. AH-E-1was cultured in150ml of PDB (200)culture medium with initial pH7.0in500ml shaking flasks at37℃, inoculation sizeand fermentation time were10%and24h, respectively.