棉花黄萎病拮抗细菌LC-04菌株的抗菌蛋白产生条件研究

Fermentation Conditions of Antagonistic Strain LC-04 Against Verticillium dahliae

LC-04菌株是从棉花叶部组织中分离出的一株类芽孢杆菌,其发酵液上清液的硫酸铵沉淀物对大丽轮枝菌V-190菌株的生长具有明显的拮抗作用,实验证明发酵液中含有蛋白质类抗菌物质。LC-04菌株的适宜发酵条件为:在pH7的LBG培养基中于30℃,180 r.min-1摇床振荡培养48 h,用70%硫酸铵对LC-04菌株发酵液上清液进行盐析,可以沉淀最多的抗菌蛋白。

An antagonistic bacterium LC-04 strain was isolated from cotton leaves, which showed obvious antagonistic effect against Verticilliurn dahliae. It was identified to be a kind of Paenibacillus. By centrifugation it＇s fermentation fluid and removing the bacteria, （NH4）2SO4 which end concentration achieves 70 % degree of saturation was joined into the upper liquid. Over night, collecting precipitates and dissolving with the buffer solution, it is proved to have the antifungal activity; when the precipitate was heated up to 120℃ for 10 minutes, it lost the activity; meanwhile, when the fermentation fluid was extracted with the same volume chloroform, the extract has not the activity. Thus, the antifungal substance produced by strain LC-04 was identified to be a kind of protein; The result indicated, when （NH4）2 SO4 concentration was below 40% degree of saturation, the fermentation fluid precipitate had not displayed the antifungal activity; when the （NH4）2SO4 concentration was 70% degree of saturation, the antifungal activity was the highest; The （NH4）2SO4 saturation increased to 80% degree of saturation, deposit activity had not increased; The 70% （NH4）2SO4 degree of saturation was the precipitation antifungal protein best salinity. And its optimum fermentation conditions of strain LC-04 were studied for the production of the antifungal protein： at the beginning, with the prolonging of the fermentation time, the quantity of the antifungal protein increased, when fermentation time reached to 48 hours, the antifungal protein quantity was the most. If continueing prolonging the fermentation time, the activity droped instead, this was possibly because the exeessivel long fermentation time had caused the antifungal protein digestion. Therefore 48 hour is the best fermentation time to LC-04 strain. Along with fermentation temperature, the fermentation fluid precipitate＇s antifungal activity changed, it was the highest at 30℃ When temperature was higher than 30℃, the activity droped, therefore 30℃ was the optimum fermentation temperature. The antagonistic bacteria strain LC-04 can produce antifungal protein in different media, but when it was cultured in LBG, the amount of the antifungal protein reached maximum, So the optimum medium is LBG. In the fermentation of strain LC-04, it is necessary to provide enough oxygen in the fermentor by changing the rotational speed of the shaker. Along with the rotational speed enhancement, the amount of the antifungal protein is also changing, when the rotational speed is low, oxygen does not favor the bacteria growth; when the shaker rotational speed is 180 r · min^-1, the activity of antifungal protein is the highest. But if the rotational speed is too quick, it would cause the bacteria premature autolysis. As the biomass reduced, antifungal active substance correspondingly reduced, therefore 180 r · min^-1 is the best rotational speed. It indicated that, the LC-04 strain secretes the antifungal protein in the different pH value culture media, when the initial pH of the medium is 7.0, the quantity of the antifungal protein reach the maximum.