深海独岛枝芽孢杆菌A493产活性物质特性与发酵条件优化研究

以一株抗菌谱较窄并且稳定拮抗水稻黄单胞菌的深海独岛枝芽孢杆菌(V.dokdonensis)A493为研究对象,研究了其产活性物质特性及发酵条件的优化。A493产抗菌活性物质可以耐受100℃的高温,在pH值4～10和紫外照射环境中活性稳定,可以通过3kDa的超滤管,对蛋白酶不敏感,只能溶解于水和甲醇。酸沉淀和硫酸铵沉淀实验结果排除了活性物质是脂肽类物质的可能性,初步研究显示该活性物质可能是极性较强的水溶性活性小分子新化合物。确定A493最佳发酵条件为:接种菌龄12h、接种量1%、发酵时间48h、发酵温度28℃、培养转速180r·min-1、250mL锥形瓶中的装液量为70mL。优化后,其单位体积发酵液抑菌效果提高了18.7%,活性物质对水稻黄单胞菌的抑菌效价相当于15.6μg·mL-1的标准链霉素。

Active Substance Characteristics of Virgibacillus Dokdonensis A493 from Deep Sea and Optimization of Its Fermentation Conditions

Deep sea V.dokdonensis A493 was taken as the research object,which had specific antimicrobial spectrum and stable resistance against Xanthomonas oryzae pv.oryzae pxo99.The research of characteristics of active substance produced by A493 and the optimization of its fermentation conditions were carried out.The active substance from A493 could withstand temperatures up to 100 ℃ and UV irradiation.Furthermore,it kept strong activity over a wide pH range from 4 to 10.It could permeated through <3 kDa ultrafiltration membrane and was protease-insensitive.The active substance could only be dissolved in water and methanol.Acid precipitation and ammonium sulfate precipitation indicated that the active substance was not lipopeptide antibiotic.The preliminary research results showed the active substance may be a stable,polar,water-soluble small molecule new compound.Preliminary optimization of fermentation conditions of A493 was conducted.The results showed that activity of A493 fermentation broth against X.oryzae pv.oryzae after optimization was increased by 18.7% under the optimum fermentation conditions as follows:inoculated cell age 12 h,inoculation amount 1%,fermentation time 48 h,fermentation temperature 28 ℃,rotation speed 180 r·min-1,liquid volume 70 mL/250 mL.The relative inhibitive potency of the active substance of sterile fermentation supernatant produced by A493 against X.oryzae pv.oryzae was equivalent to that of 15.6 μg·mL-1 of streptomycin.