Method for preparing pneumocandin B0 by microbial fermentation

[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...

[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

[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...