新型抗真菌代谢产物发酵条件的响应面优化

从浙江东海土壤中筛选获得1株菌株(ZS09-33)能产生抑制白色念珠菌的代谢产物,经16S rRNA序列测定和Genbank数据库搜索,确定其属于Bacillus amyloliquefaciens. Plackett Burman实验发现,影响抗真菌效价的主要因素为：黄豆饼粉、可溶性淀粉以及(NH4)2SO4.最陡爬坡实验和Box-Behnken实验得到响应面（RSM）优化的最佳培养基质量浓度为：黄豆饼粉2.72 g/L,可溶性淀粉 26.67 g/L,(NH4)2SO4 3.95 g/L,K2HPO4 1.8 g/L,NaCl 2 g/L,MgSO4 0.5 g/L,CaCO3 0.72 g/L,CuSO4 0.02 g/L,FeSO4 0.02 g/L,MnSO4 0.02 g/L,初始pH 6.5.在此条件下发酵液抗真菌效价达16 806.48 U/mL,与模型值相符.与单因素优化结果相比,效价提高了239%.产物经ESI/MS、¹H-NMR及¹³C-NMR确定为相对分子质量为402的大环内脂类物质.

Optimization of fermentation conditions for production of a novel antifungal metabolite by response surface methodology

A strain ZS09-33, antagonising human pathogenic fungi—Candida albicans, was screened from soil samples of East Sea, Zhejiang Province, China. The strain was identified as Bacillus amyloliquefaciens based on sequencing 16S rRNA gene and blasting in Genbank. Then a two-level Plackett-Burman design was established to determine which factors in the fermentation medium influence the potency activity of metabolites. The concentrations of soybean bran, starch soluble and (NH4)2SO4 were optimized using the response surface methodology (RSM). The optimum composition of medium was as follows (per liter): soybean flour 2.72, soluble starch 26.67, (NH4)2SO4 3.95, K2HPO4 1.8, NaCl 2, MgSO4 0.5, CaCO3 0.72, CuSO4 0.02, FeSO4 0.02, MnSO4 0.02, initial pH 6.5. Under the RSM-optimized conditions, the potency activity of culture broth was 16 806.48 U/mL, which accorded well with the model-predicted value and resulted in a 2.39 fold increase in antifungal activity compared to the original culture conditions obtained by single factor methodology. After analyzed by ESI/MS, ¹H-NMR and ¹³C-NMR, the substance was determined as a macrolides antibiotic with relative molecular weight 402.