摘要
从北极王湾地区湖泊沉积物中分离出的菌株AFN2001,其代谢产物对金黄色葡萄球菌有明显的抑制作用,抑菌圈直径达23 mm,对大肠杆菌和绿脓杆菌没有抑制作用。为了确定AFN2001菌株发酵获得最多的活性物质,对单因素确定的营养因素淀粉、黄豆浸出物、蔗糖和NaNO3及另外的MgSO4、K2HPO3和水质进行7因素3水平正交优化实验。对结果进行直观分析及方差分析,发现NaNO3对发酵液活性的影响最大,其次依次为淀粉、K2HPO3、MgSO4、黄豆浸出物、蔗糖和水质对发酵液活性的影响;并找出产生活性物质的最佳组合。确定优化培养基(淀粉为10 g、蔗糖为10 g、黄豆浸出物为6 g、MgSO4为0.5 g和自来水为1 000 mL)和优化发酵条件(发酵温度为28℃、起始pH值为6.0和装液量为50%)。利用优化培养基和优化发酵条件发酵的菌株AFN2001,其发酵液的抑菌活性是基础培养基发酵液的1.6倍。
There are unique geographical and climatological characteristics in the Arctic and Antarctic areas. Only during short summer time ice and snow melt in some region, both areas are covered by ice and snow all the year round. Highly variable illumination radiation, seasonal sunshine time and extremely low temperature result in unique biological characteristics of the polar microorganism. Since the Ekelof＇s report on the first isolation of microorganism from the Antarctic in 1908, many microbiologists from different countries did numerous work successively in the polar areas and confirmed that polar microorganisms have broad prospects in fundamental research, exploitation and application. Therefore the polar region is regarded as a potential and important microorganism resource base, and may become a potential source place of new types of active substance and forerunner compounds.
The strain AFN2001 is an actinomycete isolated from the lake sediment of the Arctic area. Fermenting in the basal fermentation broth FI（KNO3 1.0 g, Soluble starch 20.0 g, K2HPO4 0.5 g,MgSO4 0. 5 g,NaCl 0.5 g, H20 1 000 mL, pH 7.2-7.4）, the metabolites of AFN2001 possesses evident inhibition on Staphylococcus aureus （ATCC 25923, the diameter of the inhibiying circle was 23 mm）, but no inhibition on Pseudomonas aeruginosa （ATCC 25916） and Escherichia coli （ATCC 25922）.
In order to find out suitable nutrients required for generating antibacterial activated substance against Staphylococcus aureus, various carbon sources （glucose, sucrose, starch, 2%, w/v） or nitrogen sources （yeast extract, casin, peptone, NaNO3, KNO3, NH4NO3, NH4Cl, 20%, w/v） were respectively used in the basal medium with the one-factor-at-a-time method. The results show that sucrose and starch is the suitable carbon source for generating antibacterial activated substance with AFN2001, while soybean extract,yeast extract, NaNO3 and KNO3 is suitable nitrogen source. In which soybean extra has evident superiority. Thus, 4 nutrient material （starch, sucrose, soybean extract, NaNO3） were selected for the orthogonal matrix.
Orthogonal optimization tests at tree levels with seven nutrients （starch, soybean extract, sucrose, NaNO3, MgSO4, K2 HPO3 and water quality） were made to get the maximum antibacterial activated substance, and visual and variance analyses were carried out on the results, which show that they could be ranked in importance as NaNO3〉starch〉K2 HPO3〉 MgSO4 〉soybean extract〉sucrose〉water quality. The optimized nutrient medium FII is starch 10 g, sucrose 10 g, soybean extract 6 g, MgSO4 0. 5 g, tap water 1 L.
To determine the optimum fermentation condition, the optimized medium FII was used as the nutrient medium and the orthogonal design L9 （3^4） was used to optimize the fermentation temperature（A）, starting pH value（B） and volume of broth（C）. Visual and variance analyses of the results show that the factors could be ranked in importance as temperature〉 pH value〉volume, the optimal combination is A2B1C1 or A2B1C3 and the optimal condition of fermentation is 28 ℃, pH 6, and 250/500 mL flask. The optimized medium is starch 10 g, sucrose 10 g, soybean extract 6 g, MgSO4 0. 5 g and tap water 1 L.
AFN2001 strain cultivation with the basal medium and conditions and optimized medium and conditions shows that the maximum bacteria inhibition circle was 22 mm on the third day when FI basal medium was used, while it was 36 mm when FII optimized medium was used, which is 1.64 times higher than that with the basal medium. This shows that the optimized medium and cultivation conditions from the orthogonal experiment can significantly raise the activity of AFN2001 fermentation fluid and thus make it possible to isolate antibacterial activated substance from Arctic actinomycete.
出处
《海洋学研究》
北大核心
2008年第4期66-71,共6页
Journal of Marine Sciences
基金
极地战略基金2006资助项目(200605)
关键词
极地放线菌
抗菌活性
发酵优化
正交实验
arctic actinomycete
antibacterial activity
fermentation optimization
orthogonal matrix method
