Study On Pristinamycins Fermentation And The Integrated Fermentation-separation Process By Streptomyces Pristinaespiralis

Pristinamycins, produced by Streptomyces pristinaespiralis, is a member of streptogramin family of antibiotics. It not only has strong antibacterial activity against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus, but exhibits a prolonged post-antibiotics effect. So pristinamycins is considered as the specially selected medicament against the stubborn gram-positive bacterium infection. Up to now, the production level of pristinamycins is still so low that it could not meet the clinical requirement. In this paper, the fermentation, separation and purification techniques for pristinamycins production were all systematically studied. In addition, a new technique, the integrated fermentation and separation technology based on the in situ product removal technique by adsorbent resin, was developed to produce pristinamycins.The fermentation condition in shake flasks by Streptomyces pristinaespiralis XC416 was studied. The production medium was optimized both by 'one-variable-at-a-time' approach and the statistical methods based on Response Surface Method. The optimized medium composition was as follows: soluble starch 65.45 g/L, glucose 9.30 g/L, soybean flour 10 g/L, peptone 5 g/L, fish flour 10 g/L, yeast extract 3 g/L, (NH₄)SO₄ 1.5 g/L, MgSO₄·7H₂O 4.93 g/L, KH₂PO₄ 0.2 g/L, NaNO₃ 0.75 g/L, CaCO₃ 4 g/L. And the optimized cultural condition in shake flasks was as follows: inoculum age of 48 h, inoculum level of 8%, medium volume of 25 mL in a 250 mL shake flask, initial pH value of 6.5-7.0, temperature of 23℃, shaking speed of 240 rpm. Under the above conditions, S. pristinaespiralis XC416 could produce 490.7 mg/L pristinamycins in the shake flask.Taking the integrated bioreaction and separation process as guide, a new technique, viz. the technique of in situ product removal based on the adsorption of the product by macroreticular resin JD-1 directly from the fermentation broth, was developed for pristinamycins production. When 8% (w/v) resin JD-1 was added into the broth at 45 h during the fermentation process, the yield of pristinamycins could reach 1353 mg/L and the adsorption capacity of resin JD-1 on pristinamycins was about 13.5 mg/g.The batch fermentation of Streptomyces pristinaespiralis XC416 in 5 L bioreactor was studied, and the yield of pristinamycins reached 604 mg/L by controlling the different dissolved oxygen levels during the fermentation process. On the other hand, a morphologically structured model was developed, well simulating the batch fermentation process for pristinamycins production. Also, a fuzzy growth model of Streptomyces pristinaespiralis XC416 was developed based on the fuzzy theory. From the model, the coupled degree of the cell growth and the pristinamycins production was 1.64%, indicating that it is a non growth-associated process.The separation and purification of pristinamycins from the fermentation broth by the method of silica gel column chromatography was developed. And the operational condition was as follows: 100-200 mesh silica gel to be the stationary phase, solvent system of methlene chloride: ethyl acetate: ethanol = 80:35:5(V:V) to be the eluent, the flow rate of the eluent to be 2.0 mL/min, the feeding concentration of pristinamycins to be 2.26 g/L, the total loading amount of the silica gel to be 0.81 mg/g and the temperature to be 25℃. Under the above condition, the product with the purity of about 96% and the recovery of about 97% was finally obtained. After then, by the preparative HPLC method to purify and refine the product obtained above further, the final product, viz. pristinamycinâ…¡_A with the purity of more than 99% was obtained. And the chemical structure of pristinamycinâ…¡_A was elucidated by UV, IR, NMR and MS methods.The exploring of a new technique to produce pristinamycins by using the integrated bioreaction and separation process based on expanded bed adsorption technique was conducted. When using the integrated mode of free cell, the high viscosity of the broth could easily cause some negative factors, such as the liquid channels, hampering the successful operation. And when using the integrated mode of immobilized cell, although the viscosity of the broth was effectively reduced and the expanded bed was stabilized, the broth was contaminated by some bacteria in 24 h after the onset of the integrated operation, also hampering the successful operation.