- 學位論文
枯草桿菌發酵生成孢子與生產iturin A之研究
Studies on spore and iturin A production from a Bacillus subtilis
摘要
枯草桿菌為格蘭氏陽性菌，能產內生孢子，被視為一般安全之菌株，報告指出枯草桿菌具有對抗許多植物病原菌的潛力。枯草桿菌之內生孢子在逆境下容易存活因此能以孢子型態長久保存，且在產孢過程中，可產生具強力抗菌活性之抗生物質iturin A，其對多種病原菌具有抑制作用，將其噴施於田間植株上，可防治特定病害的發生。 本研究首先針對枯草桿菌發酵產孢條件進行探討，使用5 L發酵槽，先將枯草桿菌培養至營養細胞濃度約10^9 CFU/ml以上，接著改變溫度或pH值以誘導營養細胞轉換成孢子，結果發現37℃、pH 6.5有最高的轉孢率為48%。 接著探討不同培養基以批次發酵或饋料發酵方式將枯草桿菌培養至高細胞濃度，再各別將細胞轉移至內含有FeCl2、MgSO4、MnCl2等產孢配方之5 L醱酵槽中，進行誘導產生孢子。結果發現利用饋料發酵能得到 4 x 10^9 CFU/ml 的營養細胞，這些營養細胞回收後轉移到含有FeCl2、MgSO4、MnCl2等離子的產孢配方，可以得到超過90%的轉孢率。 而在枯草桿菌產iturin A之培養基的最適化中，使用濃度2%之蔗糖，1% 大豆蛋白做為氮源的條件下，可以得到353.55 mg/l之iturin A。
並列摘要
Bacillus subtilis is a Gram-positive, spore forming bacteria. It is considered as GRAS (generally recognized as safe). Several strains of B. subtilis have been reported that have potential for biological control of several plant diseases. It is able to sporulate under adverse environmental conditions, and can be preserved for a long time. During sporulation, it is able to produce many antimicrobial compounds and has been the subject of much work as a biocontrol agent. It can be sprayed on plants to prevent specific diseases. The study investigated control conditions of sporulation for Bacillus subtilis from vegetable cell. Bacillus subtilis was cultured to a vegetable cell count of about 10^9 CFU/ml in a 5 L bioreactor. The pH or temperature was changed to induce sporulation. The results indicated the highest rate of sporulation was 48% at 37℃, pH 6.5. Batch fermentation under different medium and fed batch fermentation were employed to cultivate the Bacillus subtilis to high cell density. The cells were harvested and transferred to a medium containing FeCl2、MgSO4、MnCl2 to induce sporulation. The best result was obtained by fed-batch fermentation. The vegetable cell density of 4 x 10^9 CFU/ml was achieved and sporulation rate was more than 90%. A Bacillus subtilis YT-1 was employed to investigate the optimum medium to produce inturin A. It was found that 353.55 mg/l of iturin A can be obtained in 2 % sucrose and 1 % soybean protein.