Optimization Of Fermentation Process For Wuyiencin And Determination Of Wuyiencin In Fermentation Broth

Wuyiencin is an agricultural antibiotic with extensive inhibition effect, which was obtained from the culture filtrate of Streptomyces ahygroscopicus. var. wuyiensis. Wuyiencin exhibits strong effects on many pathogenic fungi of crops. To improve the proficiency of wuyiencin, investigations were conducted to optimize the components of the fermentation medium and cultural conditions in the laboratory. Also, a technique for measuring the wuyiencin yield in the GSCP liquid medium of 5. ahygroscopicus was developed with reverse phase high performance liquid chromatography (HPLC).Temperature and pH were shown key factors affecting wuyiencin productions by S. ahygroscopicus. It was found that the best temperature and pH value for growth of the microbe were 25℃to 28℃and pH8.0. Experiment also indicated that the best inoculum was obtained when the microbe was cultured at the best temperature and pH value conditions for 18 hours to 24hours, and the best incubation time of highest wuyiencin yield was 64 hours at 28℃and pH8.0. This cultural conditions were tested in cultivating the 'high yielding' S-40 isolate of S. ahygroscopicus var. wuyiensis and wuyiencin production was 4443μg/mL, a yield increase of about 26.4% based on the yield proficiency (3515μg/mL) of wuyiencin of this isolate on the original cultural conditions.Fractional factorial design was employed to evaluate effects of the 10 components of GSCP medium, i.e. glucose, soybean cake powder, corn flour, potato starch, MgSO₄, FeSO₄, NaCl, compounds A, B, and C. Regression analysis was performed to determine the significant factors affecting wuyiencin production; glucose and soybean cake powder were found the most important two components. Therefore, a five-level two-factor central composite design (CCD) was used to optimize the dosages of glucose(x₂) and soybean cake powder(x₄).The relation of wuyiencin production(y) to the two significant factors was fitted with data from the CCD experiment and the fitted model was: y = 6643.78 + 687.42 x₂ + 275.13 x₄ - 824.69 x₂² - 122.45 x₂x₄ -620.94x₄²(R=0.8999, P<0.01).The possible maximum yield of wuyiencin could be obtained when the concentrations of glucose and soybean cake powder were 27.2 g/L and 31.8 g/L, respectively, calculated from the fitted model. An optimized medium was thus formulated based on these results. This medium was tested in cultivating the 'high yielding' S-40 isolate of S. ahygroscopicus var. wuyiensis and wuyiencin production was 6643μg/mL, a yield increase of about 50% based on the yield proficiency (4443μg/mL) of wuyiencin of this isolate on the original medium.A reverse phase high performance liquid chromatography (HPLC) method for the detennination of wuyiencin of an agricultural antibiotic in fermentation broth was developed. Analytical conditions involved use of a SB-AQ column (4.6mm×250mm, 5μm) with temperature of 25℃. The mobile phase was 1.4g/L trichloroacetic acid, flow rate was 1ml/min and detection wavelength was 254nm. In linear range of 0.2mg/mL-1.6mg/mL, there was a regressive equation y =0.272+0.345x (R=0.9994, P<0.01), the correlation coefficient was 0.9997, the recovery rate was 96.59%-100.57% and the relative standard deviation was 0.43%-2.08%.According to cylinder-plate method, standard curve was obtained with inhibiting diameter as abscissa and the logarithmic concentration of wuyiencin as ordinate. In linear range of 20μg/mL-60μg/mL, there was a regressive equation: y =-1.5097+0.1199x (R=0.9948, P<0.01).Besides, empirical relationship between cylinder-plate method and HPLC method was established: y = 1025.041 -0.375A+3.997B- 1.072C+0.220D(R=0.9958, P<0.01).