Method and culture medium for producing riprestatin by microbial fermentation
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A technology of microbial fermentation and fermentation medium, which is applied in the field of microbial fermentation to produce riprestatin and medium, which can solve the problems of low fermentation level and achieve the effects of increasing potency, increasing production and reducing costs
Inactive Publication Date: 2016-01-20
ZHEJIANG UNIV OF TECH +1
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European patent EP0803576 describes the process of improving liprestatin by adding linoleic acid, octanoic acid and N-formyl-L-leucine or leucine during the fermentation process, but the fermentation level is relatively low, lower than 1g / L
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Embodiment 1
[0020] 1. Strains:
[0021] Stretomycestoxytricini mutant strain XC-lp-69 (provided by Xinchang Pharmaceutical Factory of Zhejiang Pharmaceutical Co., Ltd.).
[0022] 2. Medium:
[0023] Slant medium preparation: soluble starch 10g, wheat flour 5g, yeast extract 1g, casein 1g, potassium dihydrogen phosphate 0.5g, magnesium sulfate 0.5g, agar 20g, add water to dilute to 1L, and adjust pH to 7.1, 121~ Sterilize at 125°C for 20 minutes. This medium is used for strain activation.
[0024] Seed medium preparation: 20g soybean meal powder, 20g glycerin, 5g yeast extract, add water to make the volume to 1L, adjust the pH to 7.1, 121~125℃, and sterilize for 20min. This medium is used for seed liquid preparation.
[0025] Initial fermentation medium: 32.5g soybean meal powder, 8g corn gluten meal, 100g sunflower oil, 25g soybean soft phospholipid, 22.5g glycerin, Tween-800.05g, 0.8g calcium carbonate, 0.025g vitamin E, 0.125g vitamin C, Add water to make the volume to 1L, adjust the pH to 7....
Embodiment 2
[0053] Slant medium preparation: soluble starch 10g, wheat flour 5g, yeast extract 1g, casein 1g, potassium dihydrogen phosphate 0.5g, magnesium sulfate 0.5g, agar 20g, add water to dilute to 1L, and adjust pH to 7.1, 121~ Sterilize at 125°C for 20 minutes. This medium is used for strain activation.
[0054] Seed medium preparation: 20g soybean meal powder, 20g glycerin, 5g yeast extract, add water to make the volume to 1L, adjust the pH to 7.1, 121~125℃, and sterilize for 20min. This medium is used for seed liquid preparation.
[0055] Fermentation medium preparation: 32.5g soybean meal powder, 8g corn gluten meal, 100g sunflower oil, 25g soybean soft phospholipid, 22.5g glycerin, Tween-800.05g, 0.8g calcium carbonate, 0.025g vitamin E, 0.125g vitamin C, MgSO 4 12mmol, CoSO 4 ·7H 2 O1mmol, ZnSO 4 ·7H 2 O0.25mmol, add water to make the volume to 1L, adjust the pH to 7.1, 121~125℃, sterilize for 20min. The medium is used for fermentation production of lipstatin.
[0056] Slant cul...
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Abstract
The invention provides a method for producing lipstatin through microbial fermentation, and a used fermentation culture medium. The method comprises the following steps: inoculating streptomyces toxytricini into the fermentation culture medium, adding Mg<2+>, Co<2+> and Zn<2+> with the final concentrations of 10-15 mmol / L, 0.5-2.0 mmol / L and 0.1-0.5 mmol / L respectively in the culture medium, conducting fermental cultivation at 220 r / min and at the temperature of 28 DEG C, adding aminocaproic acid on the fourth day of fermental cultivation to enable the final concentration of the aminocaproic acid to be 40-50 mmol / L, adding n-caprylic acid on the sixth day of fermental cultivation to enable the final concentration of the n-caprylic acid to be 30-40 mmol / L, and keeping the total fermental cultivation time to be 150-180 hours, so as to obtain the lipstatin from the fermentation liquid after the fermentation is completed. According to the invention, the addition combination of a precursor and metal ions affecting the fermentation and synthesis of the lipstatin is optimized, and the best combination of the precursor and the metal ions is found, so that the yield of the lipstatin is remarkably increased, the potency is remarkably improved after the method and the culture medium are applied to the industrial production of the lipstatin, and the cost is lowered.
Description
(1) Technical field [0001] The invention relates to a method for producing lipstatin by microbial fermentation, and a fermentation medium used. (2) Background technology [0002] Lipstatin (Lipstatin) is a metabolite produced by the fermentation of Streptomycestoxytricini, and its tetrahydro derivative is orlistat (olistat), a weight-loss drug successfully developed by Roche. Nico), Orlistat is the first gastrointestinal lipase inhibitor. It forms a covalent bond with the active serine sites of gastric lipase and pancreatic lipase in the stomach and small intestine to inactivate the enzyme and reduce fat. Decomposition and absorption, so as to achieve the effect of weight control. Olista is currently the only weight loss drug that does not enter the blood and does not act on the central nervous system. [0003] Orlistat can be synthesized by hydrogenation after lipstatin is produced by a full chemical synthesis method or by a fermentation synthesis method. Due to the relatively ...
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