Extraction and purification method of pneumocandins B0

A technology of pneumocidine and purification method, which is applied in the field of biopharmaceuticals, can solve the problems of cumbersome process and difficulty in removing the pigment of fermentation liquid, etc., and achieve the effect of easy industrial production

Active Publication Date: 2018-03-09
LUNAN PHARMA GROUP CORPORATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0032] In order to solve the technical problem that it is difficult to remove the pigment in the fermentation liquid and the whole process is complicated in the process of preparing pneumocidine B0 i

Method used

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  • Extraction and purification method of pneumocandins B0

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Comparison scheme
Effect test

specific Embodiment approach

[0050] Neomocontin B0HPLC detection condition of the present invention is as follows: chromatographic column: ODS-C18 (5um);

[0051] Detection wavelength: 210nm;

[0052] Mobile phase: water: acetonitrile (55:45); flow rate: 1.0ml / min

[0053] Column temperature: 40°C.

[0054] The B0 fermentation condition of pneumocidine of the present invention is as follows:

[0055] Strain: ATCC 20957

[0056] Incline culture

[0057] Medium: PDA

[0058] Conditions: 25°C, 12 days

[0059] seed culture

[0060] Medium: 1.0% glucose, 0.5% mannitol, 1.0% glycerin, 0.5% peptone, 2.5% cottonseed powder, 0.5% KH2P0, pH before elimination is 6.8-7.0

[0061] Condition: 25℃, 4 days, shaker speed: 250rpm

[0062] Fermentation culture

[0063] Medium: 8.0% mannitol, 2.0% glucose, 3.0% cottonseed powder, 0.5% yeast powder, 0.5% KHZPO4, adjust pH7.0 before elimination.

[0064] Sterilize at 121°C for 30 minutes, inoculum size is 10%, shake bottle volume is 200ml / 1000ml, place in a constan...

Embodiment 1

[0066] 1) With 1000ml of fermented liquid containing pneumocidine B0, purity 2.6%, fermentation titer is 675mg / L, under the state of stirring, adjust pH value 4.2 with phosphoric acid, add 2% filter aid diatomite, mix Evenly, let stand for 0.5 hour, filter to obtain solid scum;

[0067] 2) The bacterial residue was extracted with 2 times of 95% ethanol, filtered to remove mycelium, and the extract was dark red in color; the alcohol concentration of the extract was adjusted to 20 (V / V), and the yield of this step was 81%;

[0068] 3) Install a D101 column with a height of 25 cm and a diameter of about 4 cm, flow the sample solution through the D101 column at a speed of 5 ml / min, rinse the resin column with purified water and 30% (V / V) ethanol-water solution successively, and then Use 80% (V / V) ethanol-water solution to elute the resin column, collect the eluent rich in pneumocantine B0, the purity is 46% through high-pressure liquid phase detection, the color becomes lighter ob...

Embodiment 2

[0074] 1) Take 1000ml of fermented liquid containing pneumocidine B0, with a purity of 2.7%, and a fermentative titer of 701mg / L. While stirring, adjust the pH value to 6 with phosphoric acid, add 5% filter aid perlite, and mix well , stand still for 0.5 hour, filter to obtain solid bacterial residue, and the color of the bacterial residue is lighter;

[0075] 2) The bacterial residue was extracted with 5 times of 95% ethanol, and the mycelia were removed by filtration to obtain the extract, which was dark red in color; the alcohol concentration of the extract was adjusted to 30% (V / V), and the yield of this step was 87%;

[0076] 3) Install a HPD300 column with a height of 25cm and a diameter of about 4cm, flow the sample solution through the HPD300 column at a speed of 5ml / min, rinse the resin column with purified water and 40% (V / V) ethanol-water solution successively, and then Use 90% (V / V) ethanol-water solution to elute the resin column, collect the eluent rich in pneumo...

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Abstract

The invention discloses an extraction and purification method of pneumocandins B0. The method comprises the steps of: 1) adjusting a pH (potential of hydrogen) value of fermentation liquid containingpneumocandins B0, adding a filter aid and performing filtration to form bacterial residue, 2) extracting the bacterial residue with a high-concentration aqueous solution of methanol or ethanol, 3) adsorbing extraction liquid with adsorbent resin, eluting with the 80-90(V/V) aqueous solution of methanol (ethanol), and collecting an eluant rich in pneumocandins B0, 4) adding active carbon into the eluant containing pneumocandins B0 for decoloration treatment, 5) adsorbing with the adsorbent resin, eluting the resin with the 80-100% (V/V) aqueous solution of methanol (ethanol) and collecting an eluant rich in pneumocandins B0, and 6) performing vacuum concentration to form a crude product of pneumocandins B0. Due to improvement of the extraction and purification method, a decoloration and purification effect is obvious, the purity of the product is improved obviously, the production cost is low, and the method is simple in technical step and suitable for industrial production.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a method for extracting and purifying pneumocidine B0. Background technique [0002] Caspofungin is the first echinocandin listed in the world. Its mechanism of action is unique. It targets the fungal cell wall, specifically inhibits the synthesis of β(1,3)-D-glucan in the cell wall, and destroys the fungal cell wall. The integrity of the fungal cells destabilizes the osmotic pressure in the fungal cells, eventually leading to the lysis of the fungal cells. Mammalian cells do not contain β-(1,3)-glucan, and there is no similar cell wall synthesis process. Therefore, while caspofungin is antifungal, it will not damage or affect human cells. In vitro pharmacological studies have shown that caspofungin is effective against a variety of Candida, endemic fungi, Aspergillus and Pneumocystis carinii, so it has attracted more and more attention. [0003] Pneumocandin B0 (P...

Claims

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Application Information

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IPC IPC(8): C07K7/56C07K1/22
CPCC07K7/56
Inventor 张贵民王钦超
Owner LUNAN PHARMA GROUP CORPORATION
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