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A method for cultivating actinomycetes to obtain rapamycin fermentation broth

A technology of actinomycetes and rapamycin, which is applied in the field of fermentation engineering, can solve the problems of unfavorable extraction and purification, can not fully meet the needs of industrial production, and the unit of rapamycin biological fermentation is not high, and achieve the benefit of extraction work Effect

Active Publication Date: 2015-09-09
天津北洋百川生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the shortcomings of rapamycin bio-fermentation production mainly lie in the low bio-fermentation unit of rapamycin, which is not conducive to extraction and purification, and cannot fully meet the requirements of industrial production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The Actinoplanes sp. mutagenic strain BCStr-096 was deposited in the General Microbiology Center of China Committee for the Collection of Microbial Cultures on November 05, 2013, with a preservation number of CGMCC NO.8430; the strain The rapamycin-producing actinomycete hn-016 (Actinoplanes sp.) screened from the soil samples of Hainan Island was used as the starting strain, and it was screened by multiple groups of compound mutagenesis.

[0024] 1. Taking Actinomycetes hn-016 as the starting strain, the strain BCStr-096 was screened by plasma mutagenesis at room temperature at atmospheric pressure.

[0025] (1) Select the well-growing rapamycin-producing actinomycete hn-016 slant strain, wash the spores with saline, filter through gauze, and make a spore suspension of 106 / mL;

[0026] (2) Pipette 10 μL of the spore suspension in (1) onto a circular iron sheet with a diameter of 1 cm, and place it under normal pressure with helium as the working gas, power 110W, and wo...

Embodiment 2

[0038] The fermentation culture of 30L fermentor comprises the following steps:

[0039] (1) Cultivation of slant strains

[0040] Transfer the motile actinomycetes preserved in glycerol to the preservation isolation medium with a 1mL pipette, and inoculate them with an inoculation needle, and culture them in a constant temperature incubator at 28°C for 8 days. The slope changes from orange to yellowish brown.

[0041] (2) Shake flask seed culture

[0042] Select a well-growth activated slant, take 1 cm2 strains and transfer them to fresh seed culture medium, and culture them on a vertical shaker at 28°C and 220r / min for 48h.

[0043] (3) Seed tank seed culture

[0044]Put 6L of seed tank culture medium in a 10L primary seed tank, sterilize, inoculate the shake flask seed culture solution with 8% inoculum, tank pressure 0.05Mpa, aeration ratio 0.7vvm, DO control at 35-38% , 28°C, cultivated for 46h to obtain seed solution.

[0045] (4) Fermentation culture

[0046] Put 18...

Embodiment 3

[0054] The fermentation culture of 200L fermentor comprises the following steps:

[0055] (1) Cultivation of slant strains

[0056] Use a 1mL pipette to transfer the motile actinomycetes preserved in glycerol to the preservation isolation medium, and use an inoculation needle to inoculate densely. Culture in a constant temperature incubator at 28°C for 10 days until the slope changes from orange to yellowish brown.

[0057] (2) Shake flask seed culture

[0058] Select a well-growth activated slant, take 1 cm2 strains and transfer them to fresh seed culture medium, and culture them on a vertical shaker at 28°C and 220r / min for 48h.

[0059] (3) Primary seed tank seed cultivation

[0060] Put 6L of seed tank medium into a 10L first-level seed tank, sterilize, inoculate the shake flask seed culture solution with 10% inoculum, tank pressure 0.05Mpa, aeration ratio 1.0vvm, DO control at 35-38%, Cultivate for 42 hours at 28°C to obtain a first-grade seed solution.

[0061] (4) S...

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Abstract

The invention discloses a method for obtaining rapamycin fermentation liquor by utilizing actinoplanes, belonging to the technical field of fermentation engineering. The method comprises the following steps of culturing actinoplanes CGMCC No.8430 on a slant culture medium, inoculating the cultured slant strain onto a shake flask seed culture medium, then inoculating the cultured seed liquor into a seeding tank, and then inoculating the seed liquor into a fermentation culture medium to undergo fermentation culture, thus obtaining the rapamycin fermentation liquor. The method has the beneficial effects that rapamycin with relatively high concentration can be generated from actinoplanes by optimizing the culture medium and controlling the culture conditions; the yield of rapamycin can be about 750mg / L in fermentation experiments in a 30L fermentation tank, and the yield of rapamycin can be about 840mg / L in fermentation experiments in a 200L fermentation tank, so that rapamycin more easily meets the requirement of industrial production; moreover, high concentration is more beneficial for subsequent extraction operation.

Description

technical field [0001] The invention belongs to the technical field of fermentation engineering, and in particular relates to a method for obtaining rapamycin fermentation liquid by using actinomycetes. Background technique [0002] Rapamycin (RPM), also known as sirolimus, is a nitrogen-containing triene macrolide antibiotic with a unique mechanism of action. It has antifungal, antiproliferative and antitumor effects. Rapamycin is a triene macrolide compound composed of a 31-membered ring, containing a hemiketal covered by a special α, β-diketopidine amide molecule, with a molecular formula of C51H79NO13 and a molecular weight of 991, which is White solid crystal, melting point 183-185℃, lipophilic, slightly soluble in water, almost insoluble in ether, but soluble in methanol, ethanol, acetone, chloroform and other organic solvents. [0003] Rapamycin is currently the most promising new powerful immunosuppressant in the world. Its molecular structure is similar to FK506, b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P17/18C12R1/045
Inventor 乔长晟石漫漫李雪朱明
Owner 天津北洋百川生物技术有限公司
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