Study On High Producing Strain Breeding, Fermentation Condition Optimization, Fermentation Kinetics And Solubility Of Natamycin

Natamycin (Pimaricin ) is one of the kind of macrolide antibiotics with 26-membered ring polyene structure, which is produced by several strains of actinomycetes such as Streptomyces natalensis, Streptomyces chatanoogensis and Streptomyces gilvosporeus. Natamycin is predominantly a strong, broad-spectrum antifungal agent. Because of low toxicity of natamycin to the mammalian cells compared to other antifungal compounds, it is widely used in food industries and medicial treatment. However, the yield of natamycin at present is very low, so the application of natamycin is limited severely due to the high cost of production. Therefore, it is of great importance to enhance the research and development of natamycin production process. In this work, the high productivity strain was selected and bred for natamycin production, the medium composition and fermentation conditions were optimized, the solubility of natamycin in various solution were studied and the separation technology of natamycin from fermentation broth was developed. The major research contents and results are as follows:A RP-HPLC method for the analysis of natamycin in fermentation broth was developed. The method is simple, rapid, accurate and reliable for the determination of natamycin concentration.5. gilvosporeus SG 1-4 was used as a starting strain. After treated with diethyl sulfate and ultraviolet light in series, a mutant stain SG 9-16 with high natamycin productivity was screened out by using the medium with selective pressure of 2-DOG, sodium acetate, sodium propionate as well as streptomycin sulfate. Under the fermentation condition without being optimized, the production of natamycin was 0.79 g/L, which was 1.5 times higher than the parental strain SG 1-4. The strain is genetically stable.The batch fermentation of natamycin production with the strain SG 9-16 in shake flask was studied. The optimal medium for seed culture, the culture conditions, and the fermentation medium for natamycin production were examined. Also, the effects of cultivation conditions such as seed age, inoculum, medium volume, initial pH value, temperature and precursors on natamycin production by Streptomyces gilvosporeus were evaluated. Under the optimum conditions, strain SG 9-16 could produce natamycin 3.0 g/L by shaking-flask batch fermentation, which was 280% higher than that of initial conditions.A 5 L bioreactor was used to study the kinetics of natamycin fermentation, The productivity of natamycin reached 2.86 g/L in the optimized operating conditions, which was 170% higher than that in shaking-flask fermentation (without addition of precursors). A morphological-structured model was adopted to simulate the experiment data from 5 L bioreactor batch fermentation. The model simulations for mycelium growth and natamycin formation were in good agreement with the experiment data. Also, based on the fussy model, the coupled degree of the cell growth and the production of natamycin by Streptomycesgilvosporeus was 18.3%, therefore, it is a typical growth-prodution separated process.According to the preliminary study of shaking-flask fermentation, statistically-based experimental designs were empolyed to optimize the natamycin production by Streptomyces gilvosporeus. In the first optimization step, Plackett-Burman (P-B) design was used to evaluate the effects of many internal and external related factors on natamycin fermentation. Based on the results of the P-B experiment, the path of steepest ascent was undertaken to approach the optimal region of these factors. By regression analysis, initial pH, temperature, KH2PO4, CaCC>3, yeast extract, peptone and age of inoculum were found to be important for the natamycin production. Subsequently, a central composite rotatable design and response surface analysis were used to optimize the above critical seven factors, to find out the optimal concentration levels and to elucidate the relationships among these factors. Then a response surface function was fit, the results indicated that the stationary point was a saddle point with neither global maximum nor minimum. Finally, the optimal fermentation parameters were calculated by the nonlinear constrained optimization methods.Based on the known biosynthesis pathway and the metabolic regulation of natamycin, the optimization strategy was further considered to increase the yield of natamycin. The addition of prussiate and magnesium phosphate into fermentation medium could promote the natamycin biosynthesis. The optimized cultivation conditions were as follows: glucose 40 g/L, beef extract 10 g/L, MgSO4 ? 7H2O 1.0 g/L, NaCl 2 g/L, soybean meal 15 g/L, soluble starch 30 g/L, KH2PO4 0 g/L, CaCO3 2.4 g/L, yeast extract 0.34 g/L, peptone 6.34 g/L, IQFe (CN)6 ? 3H2O 3xl0'5 mol/L, Mg3(PO4)2 5 g/L;initial pH 7.4, age of inoculum 35.7 h, inoculum level 15%, working volume 25 mL/250 mL, temperature 22 °C, cultivation time 120 h, adding 0.6% sodium propionate into the broth at 24 h of cultivation. This optimization strategy rendered the natamycin production to be increased from 3.0 g/L to 5.67 g/L.The solubility of natamycin in water, isopropanol and methanol at different temperature were measured using balance method, and the results were regressed with different correlation equations. It was found that the empirical equation was the best to correlate the solubility data of natamycin in water and isopropanol, while the solubility data of natamycin in methanol was well correlated with the empirical equation, apelblat equation, simplified state equation and ^ h equation, respectively. In addtion, the backpropagation (BP) artificial neural network(ANN) was also used to correlate these experimental data of three systems, the prediction of interpolation and extrapolation of the data were satisfactory.The solubility data of natamycin in water-methanol mixture at 0~35 °C were experimentally determined by balance method. The solubility of natamycin increases slowly with the increase of temperature and decreases sharply as water concentration increases. The experimental data was correlated in the form of A h equation by the nonlinear least-square technique. The parameter X and h were expressed by the function of water concentration. The solubility data by interpolation calculation were satisfactory to some degree.The influence of pH value on natamycin dissolution was studied. It was found that the minimum solubility was obtained at pH 6.5-7.5, and the dissolution is remarkably enhancedwith the increase or decrease of solution pH, especially at pH above 9 or below 4.5. Furthermore, the solubility of natamycin is considerably increased when CaCl2 was added.Natamycin was isolated and purified from broth by methanol extraction method. Its molecular weight and chemical structure were elucidated by UV, IR, NMR and MS methods.