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Method for preparing pneumocandin B0 by microbial fermentation

A technology of neumocontin and fermentation process, applied in the field of fermentation, can solve the problems of lack of large-scale, high-purity and stable production of nemocontin B, affecting the fermentation purity and yield of nemocontin B, etc. pressure, stable production effect

Active Publication Date: 2018-07-10
JIANGSU HENGRUI MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the fermentative production of pneumocidine B 0 In the process, a series of pneumocantine analogues will also be produced, such as pneumocantine A 0 , serine analogs and D 0 , thus affecting the neomerizine B 0 Fermentation purity and yield
Although the prior art has reported a lot of neomocontin B 0 Fermentation methods, but still lack large-scale, high-purity stable production of pneumocidine B 0 Methods

Method used

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  • Method for preparing pneumocandin B0 by microbial fermentation
  • Method for preparing pneumocandin B0 by microbial fermentation
  • Method for preparing pneumocandin B0 by microbial fermentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: Neomocontin B 0 5L fermentation production

[0024] Prepare the primary shake flask seed medium, the medium components are as follows: anhydrous glucose 4%, soybean cake powder 2%, cottonseed cake powder 1%, corn steep liquor dry powder 1%, potassium dihydrogen phosphate 0.1%, trace element source solution (magnesium chloride hexahydrate 0.486g / L, zinc sulfate heptahydrate 0.418g / L, ferric chloride hexahydrate 0.840g / L, copper sulfate pentahydrate 0.078g / L) 0.1%. Prepare drinking water and adjust the pH to 5.5 before sterilization. Use a sterilizer to sterilize at 121°C for 30 minutes.

[0025] First-level shake flask seed culture: Inoculate 1ml of the seed glycerol tube into the first-level seed shake flask, place on a shaker at 25°C, 220rpm, and cultivate for 3 days.

[0026] Prepare the secondary shake flask seed medium, the medium components are as follows: anhydrous glucose 4%, soybean cake powder 2%, cottonseed cake powder 1%, corn steep liquor dr...

Embodiment 2-7

[0030] Embodiment 2-7: Different fermentation media produce pneumocidine B 0

[0031] Using fermentation media composed of different carbon sources, according to the fermentation method of Example 1, Nemocontin B was prepared 0 . The inorganic salt components in the fermentation medium are: 0.4% of dipotassium hydrogen phosphate, 0.05% of ferrous sulfate, 0.03% of manganese sulfate, 0.2% of ammonium sulfate and 0.2% of sodium nitrate. The carbon source and nitrogen source components of the fermentation medium are shown in the table below.

[0032] Example

Embodiment 8

[0034] Sampling during the fermentation process for the determination of pneumocidine B in the fermentation broth 0 and main impurity pneumocontin A 0 , serine analogs, D 0 The processing process is as follows: take a small amount of fermentation liquid, add ethanol, ultrasonic for 30 minutes, centrifuge to get the supernatant, and measure its yield by HPLC.

[0035] HPLC detection conditions: YMC J'sphere ODS-M80 4μm 250×4.6mm chromatographic column; A: 0.1% phosphoric acid aqueous solution B: acetonitrile as mobile phase, gradient elution; detection wavelength is 210nm; flow rate is 1.5mL / min; The column temperature was 30°C. The elution gradient is shown in the table below:

[0036]

[0037] Adopt above-mentioned method to measure the pneumocantine B of the fermented liquid gained in embodiment 1-7 0 And the main impurity content, see the table below.

[0038]

[0039] It can be seen from the above table that when the carbon source of the fermentation medium is a...

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Abstract

The invention relates to a method for preparing pneumocandin B0 by microbial fermentation. The method is concretely characterized in that mold Glarea lozoyensi undergoes liquid deep fermentation, eachof a seed medium and a fermentation medium contains a proper carbon source, a proper nitrogen source, a proper inorganic salt and a proper trace element source, wherein the fermentation medium uses sorbitol as a main carbon source, and glucose is added to form a composite carbon source. The fermentation method can realize the stable production in an industrial fermentation tank, and allows the yield of the pneumocandin B0 to reach 2.5 g / L.

Description

technical field [0001] The invention belongs to the technical field of fermentation, and relates to a method for preparing pneumocidine B by microbial fermentation 0 The method and the fermentation medium used. Background technique [0002] Caspofungin (caspofungin), the first approved echinocandin antifungal drug, was developed by Merck. It interferes with the synthesis of fungal cell walls by inhibiting 1,3-β-D-glucan synthase, forming an antifungal effect. Since mammalian cells do not have cell walls, it is harmless to humans. Caspofungin exhibited in vitro antibacterial activity against both Aspergillus and Candida fungi. It is mainly used for the treatment of candida bacteremia and other candida infections, and esophageal candidiasis. It is suitable for patients infected with invasive aspergillus and who are ineffective or intolerable to previous treatment, as well as empiric treatment of neutropenic fever patients. [0003] caspofungin 0 (Pneumocandin B 0 , PB 0...

Claims

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Application Information

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IPC IPC(8): C12P21/04C12N1/14C12R1/645
CPCC07K7/56C12N1/14C12P21/02
Inventor 任宏杰陈舟舟王宏伟朱小龙
Owner JIANGSU HENGRUI MEDICINE CO LTD
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