| Guanosine is the precursor of the flavor enhancer guanosine monophosphate(GMP) and antiviral drugs(such as ribavirin, et al). So it has broad market prospects. Since microbiological fermentation method has many advantages such as low cost, environmental friendly and high economic benefits, it has become the main production method of guanosine. Yield of guanosine is affected by many factors, thus how to obtain high yield of guanosine with low production cost is the research hotspot in guanosine industry. This article is aimed at improving the yield of guanosine and reducing production cost through analysis of fermentation characteristics of the strain, determination of major influence factors in guanosine fermentation, and optimization of these factors based on the producing strain Bacillus subtilis G3.The main results are as follows:(1) Established high performance liquid chromatography method for the detection of guanosine, hypoxanthine and inosine. The mobile phase was acetonitrile and water, UV 248 nm, flow rate of 1 m L/min, on a C18 column. Theoretical plates number of guanosine hypoxanthine, and inosine under the optimized condition were respectively 50867.37, 45515.64 and 50488.8, the degree of separation among the three peaks was bigger than 1.5. The adaptability of system was good and the linear range were 0.65 mg/L~65 mg/L, 0.35 mg/L~35 mg/L, 0.85 mg/L~85 mg/L.The correlation coefficient was above 0.999 and the average recovery of guanosine, hypoxanthine and inosine were 102.15%, 99.16%, 97.31%. The RSD values were 1.50%, 1.02%, and 1.25%. The detection method established in this research can be used to analyze guanosine fermentation process.(2) The characteristics of guanosine producing strain Bacillus subtilis G3 were studied. It was found that the guanosine synthesis was related to bacterial growth and the fermentation period was determined as 60 h. Meanwhile, the strain has the ability to use xylose, glucose, glycerol, sucrose and soluble starch. Xylose has more contribution on the growth of bacteria, glucose has more contribution on guanosine synthesis. Soy peptone, corn steep liquor and yeast extract can significantly promote cell growth, while the strain could be obviously inhibited by monosodium glutamate, peptone and soybean cake hydrolysate. The strain can make good use of ammonium, while nitrate would significantly inhibit the growth and guanosine production. It was found that manganese sulfate, magnesium sulfate and potassium dihydrogen phosphate can dramatically enhance guanosine productivity. What’s more, addition of guanosine precursors hypoxanthine can also improve guanosine productivity. Fermentation medium was determined by single factor optimization and orthogonal experiment: glucose 140 g/L, corn steep liquor 30 g/L, yeast extract 15 g/L, ammonium sulfate 9 g/L, manganese sulfate 0.005 g/L, magnesium sulfate 4 g/L, potassium dihydrogen phosphate 4 g/L, time xanthine 2 g/L.(3) The fermentation medium were comprehensively optimized by response surface analysis. The optimal fermentation medium were: glucose 152.43 g/L, corn syrup 33.22 g/L, yeast powder 15.16 g/L, ammonium sulfate 11 g/L, manganese sulfate 6.89 mg/L, magnesium sulfate 4 g/L, potassium dihydrogen phosphate 4 g/L, hypoxanthine 2 g/L. Six experiments under the optimized conditions were performed to verify the model, the mean value of guanosine productivity was 5.16 g/L, which was very close to the predicted value and improved by 241.72% than before optimization.(4) The optimal cultivation conditions of Bacillus subtilis G3 were determined based on the optimum culture medium. The results were as follows: the optimal inoculating time 12 h, pH value 7.2, temperature 36°Cand dissolved oxygen concentration 12%. The yield of guanosine 7.33 g/L was obtained under these conditions, enhancing by 38.75% compared with the yield before optimization of conditions. |
PDF Full Text Request