Fermentation Process On A New Antifungal Antibiotic

In recent years, the deep fungal infection increases gradually, which threatens sufferers’ health and lives. A strain of Bacillus sp. N-50 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 amyloliquefaciens based on sequencing 16S ribosomal RNA 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 4%, soybean meal 0.825%, (NH4)2SO4 1.375%, K2HPO4 0.24%, NaCl 0.2%, MgSO4 0.05%, CaCO30.072%, CuSO4 0.002%, FeSO4 0.002%, MnSO4 0.002%. Furthermore, the culture condition was also 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 6364.343 U/mL, which was increased 8.37-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 influenced 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 2.77, soluble starch 27.2, (NH4)2SO43.73. Under the RSM-optimized conditions, the potency activity of culture broth was up to 16866.29 U/mL, which was in good agreement with the model-predicted value and was increased 2.65-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 1.0 vvmandfermentation period 36 h. Under the above-mentioned fermentation conditions, the antifungal potency of broth reached its peak value as 8305.841 U/mL. 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.