试验设计法优化肺炎支原体培养基

Optimization of medium for Mycoplasma pneumoniae by design of experiment

目的试验设计(design of experiment,DOE)法优化肺炎支原体(Mycoplasma pneumoniae,MP)培养基,以期提高MP产量。方法以MP(FH株)为试验菌株,PPLO培养基为基础,菌体蛋白含量为响应值,采用Plackett-Burman试验、最陡爬坡试验及Box-Behnken试验对脑心浸液、水解乳蛋白、胨3号、精氨酸、小牛胸腺DNA、葡萄糖和胰蛋白胨7种培养基添加成分进行筛选及优化。以菌体蛋白含量及颜色变化单位(color change unit,CCU)为指标,验证改良培养基培养MP的效果。结果培养基各添加成分对MP生长影响作用大小依次为葡萄糖、胰蛋白胨、脑心浸液、精氨酸、月胨3号、水解乳蛋白、小牛胸腺DNA,最终筛选出脑心浸液、葡萄糖及胰蛋白胨3种添加物,最适浓度分别为5. 68、1. 34及2. 64 g/L。采用改良培养基培养MP 96 h时,菌体蛋白含量及CCU达最大,分别为851. 29μg/mL及10~9 ccu/mL。结论 DOE法实现了对MP培养基的优化,获得了影响MP生长的主要参数,提高了MP菌体产量。

Objective To optimize the medium for Mycoplasma pneumoniae(MP)by design of experiment(DOE)so as to increase the yield of MP.Methods Seven addition ingredients,i.e.extraction of brain and heart,lactoalbumin hydrolysate,peptone 3,arginine,calf thymus DNA,glucose and tryptone,were screened and optimized by PlackettBurman test,steepest ascent test and Box-Behnken design,using MP FH strain as test strain,PPLO as basic medium,and bacterial protein content as response value.The effect of culture of MP by using the modified medium was verified serving bacterial protein content and color change unit(CCU)as indicators.Results In the turn of effect on the growth of MP,the addition ingredients were glucose,tryptone,extraction of brain and heart,arginine,peptone 3,lactoalbumin hydrolysate and calf thymus DNA.Three additives,extract of brain and heart,glucose and peptone 3,were screened,of which the concentrations were 5.68,1.34 and 2.64 g/L respectively.The bacterial protein content and CCU of MP cultured in the modified medium for 96 h reached the maximums of 851.29 μg/mL and 10~9 respectively.Conclusion The medium for MP was optimized by DOE,while the significant factors influencing MP growth as well as their optimal concentrations were obtained,and the yield of MP increased.