高产洛伐他汀棒曲霉菌株的筛选、鉴定及发酵条件优化

Screening and identification of Aspergillus clavatus strain with high-yielding lovastatin and its optimization of fermentation conditions

从不同生境(食品、土壤、空气、有机质等)收集到的自然发酵样品中分离得到150株曲霉属菌株。用高效液相色谱(HPLC)法检测发酵液中洛伐他汀(Lovastatin)含量,筛选获得1株稳定高产Lovastatin的曲霉菌株(编号:Ac-32)。根据菌落形态特征并结合18S r DNA测序,鉴定其为棒曲霉(Aspergillus clavatus)。通过摇瓶发酵单因素实验优化了碳氮源种类、碳氮源含量、碳氮比(C/N)、发酵温度、初始pH、转速、种龄和接种量,确定了棒曲霉菌株Ac-32摇瓶发酵产Lovastatin的适宜条件为:乳糖为碳源、蛋白胨为氮源、碳源含量为100 g/L、氮源含量为12 g/L、碳氮比(C/N)为15∶1.8、温度28℃、转速180 r/min、初始pH 5.2、种龄4 d、接种量6%。采用Minitab 17软件的P-B实验设计法,筛选对Lovastatin产量有显著影响的因素为:温度、pH、碳源含量和氮源含量。根据P-B实验结果,运用响应面法分析,确定棒曲霉菌株Ac-32产Lovastatin的最优条件为:碳源含量100 g/L,氮源含量11.8 g/L,温度28℃,pH 5.2。在此条件下,Lovastatin最高产量为236.221μg/m L。

150 Aspergillus strains were selected from different natural fermentation samples of food, soil, air and so on, and their products of lovastatin were measured by high performance liquid chromatography （HPLC） method. Among them, the Aspergillus strain Ac-32 producing higher-level lovastatin was screened. Based on 18S rDNA sequence and morphology characteristics, the strain Ac-32 was identified as AspergiUus clavatus. Through analyzing the influence of C source, N source, C content, N content, C/N, temperature, initial pH and so on, the results indicated that optimal fermentation conditions were as follows： C source lactose, N source peptone, C content 100 g/L, N content 12 g/L, C/N 15： 1.8, temperature 28 ℃, shaking speed 180 r/min, initial pH 5.2, inoculating age 4 d and inoculum size 6%. Using Plackett- Burman （P-B） in Minitab 17, 4 significant effects （C content, N content, temperature and pH） on lovastatin yields were screened out. According to the P-B experiment results and analysis of response surface methodology in Design Expert, the optimum conditions were as follows： C content 100 g/L, N content 11.8 g/L, temperature 28 ℃ , pH 5.2. Under the optimized conditions, the maximum lovastatin yield was 236. 221 μg/mL.