Isolation and Purification of a Novel Antifungal Antibiotic and Optimization of Its Fermentation Conditions
|School||Zhejiang University of Technology|
|Keywords||antifungal-antibiotics 16S rDNA response surface method fermentation kinetics model|
In recent years, the infection of deep fungus increases gradually, which threatens sufferers’health and lives. A strain of Bacillus sp. N-23 which can produce a kind of macrolide antifungal-antibiotic—called Zhexinmycin was screened. This kind of antibiotic has high antifungal activity against Canidia albicans.The strain was identified as Bacillus subtilis based on sequencing 16S ribosomal DNA gene and blasting the sequence in NCBI database.Both the culture condition and medium composition for producing the antibiotic were optimized by single-factor methodology in 250 mL shaking flask. The optimum composition of culture medium was determined as follows: soluble starch 40, soybean meal 8.25,（NH4）2SO4 13.75,K2HPO4 2.4, NaCl 2, MgSO4 0.5, CaCO3 0.72, CuSO4 0.02, FeSO4 0.02, MnSO4 0.02. The culture condition was optimized as follows: pH 6.5, temperature 28℃, inoculate amount 8%（v/v）, shaking speed 200 r/min, liquid volume in flask 40 mL/250 mL, fermentation period 36 h. Under the aforementioned culture conditions with the optimal composition of fermentation medium, the potency activity of culture broth reached 7026.788 U/mL, which was increased 3.49-fold in comparsion with the results under the initial culture conditions.Statistically based experimental designs were applied to further optimize the medium composition. First, a two-level Plackett–Burman design was constructed to determine which factors in the fermentation medium influence the potency activity of metabolites produced by the strain. Then, the concentrations of the soybean bran, starch soluble and （NH4）2SO4 were optimized using the response surface methodology （RSM）. A quadratic model was constructed to fit the potency activity and the variables. The main composition of the fermentation medium was optimized as follows （g/L）: Soybean flour 2.72, soluble starch 26.67, （NH4）2SO4 3.95. Under the RSM-optimized conditions, the potency activity of culture broth was up to 16806.48U/mL, which was in good agreement with the model-predicted value and was increased 3.49-fold compared with the results under the culture conditions optimized by single-factor methodology.Fermentation scale-up was carried out in 5 L continuous stirred tank reactor （CSTR） with the following culture conditions: rotation speed 200 r/min, broth content 60% （v /v）, pH 6.5, inoculum 10% （v/v）, aeration rate 200L/h, fermentation period 36 h. Under the above-mentioned fermentation conditions, the antifungal potency of broth reached its peak value as 7137.45 U/mL. Fermentation scale-up was carried out in 5 L continuous stirred tank reactor （CSTR） with the following culture conditions: rotation speed 500 r/min, broth content 60% （v /v）, pH 6.0, inoculum 8% （v/v）, aeration rate 500L/h, and fermentation period 28 h. Under the above-mentioned fermentation conditions, the antifungal potency of broth was 1.2-fold compared with which in 250 mL shaking flask. Meanwhile, a mathematical model was developed to describe the dynamic kinetics of Zhexinmycin production and the model parameters were simulated from experimental data by Origin 8.0 software using Logistic equation, Luedeking- Piret equation and Luedeking-Piret-like equation. The results indicated the model fitted the experimental data well.The antifungal compound was identified as a kind of Macrolactins, which not only had inhibitory effect against Aspergillus niger ATCC 20611, Escherichia coli ATCC 8739, Klebsiella pneumoniae ATCC 10031, but also had inhibitory effect against C. albicans ATCC 64548. It possessed great potential of being developed as a new antifungal drug.