Optimization Of L-Proline Fermentation Conditions

L-proline is a neutral amino acid with imono. It is one of 20 types of essential amino acids which constitute the protein. It is a non-essential amino acid for people. L-proline has certain physiological activities. It is widely used in medical, agricultural, chemical industrial and food areas, especially in medical area since it is the intermediate of synthesis for many new medicines. Along with the study of it, the application area will enlarge.The dissertation focuses on the shake-flask culture conditions and 5-Liter fermentor of L–proline fermentation.(1) L-proline concentration in fermented broth was qualitative determined with methods of thin layer chromatography and quantitative determined with methods of acidity triketohydrindene hydrate colorimetry. The quantitative determination condition and computing method were obtained.(2) The batch fermentation condition of strain AP117 in shake flask was studied in this dissertation. The seed medium was optimized by orthogonal experiment, and the seed culture conditions were also studied. The optimization seed conditions for Brevibacterium flavum AP117 as follows (%): sucrose 3.0; (NH4)2SO₄ 4.0; L-Ile 300mg/L; biotin 500μg/L; thiamin 500μg/L; MgSO₄·7H₂O 1.0; KH₂PO₄ 0.1; CaCO₃ 3.0; ZnSO₄·7H₂O 0.001; FeSO₄·7H₂O 0.001; MnSO₄·5H₂O 0.001.(3) Based on the single factor experiment of fermentation medium, the fermentation medium was optimized by orthogonal experiment, and the fermentation condition was studied. The optimization fermentation conditions for Brevibacterium flavum AP117 are as follows (%): sucrose 16.0; ammonium acetate 4.0; L- Ile 300mg/L; biotin 400μg/L; thiamin 600μg/L; MgSO₄·7H₂O 2.0; KH₂PO₄ 0.2; CaCO₃ 6.0; ZnSO₄·7H₂O 0.001; FeSO₄·7H₂O 0.001; MnSO₄·5H₂O 0.001; and the optimal culture conditions such as medium initial pH is 7.0-7.2, 30mL medium in 500mL shake-flask, 16-18h for seed growth. Under the optimum condition, the strain AP117 could produce L-proline 52.17g/L after fermentation for 72 hours by flask-shaking batch fermentation.(4) Results of 5-Liter fermentor experiment show that the strain AP117 could produce L-proline 55.08g/L after fermentation for 72 hours, and the optimal culture conditions such as medium initial pH, amount of inoculation, rotation rate are 8.0, 10%, 240r/min, respectively.(5) Fed batch fermentation was studied with AP117 according to the optimum condition of batch fermentation. The optimum initial sucrose concentration was 80g/L in shake flask fed batch fermentation. The intermittent feeding was performed at the 24, 36, 48, 60 hour respectively. The production of L-proline reached 55.28g/L after fermentation for 72 hours. Because of the technology of low glucose, low amino and fed batch, production of L-proline reached 55.38g/L after fermentation for 72 hours. The production was improved by 6.15% than that of fermentation in shake flask fed batch. The production was improved by 49.51% than that of initial condition.