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Preservation method of actinoplanes teichomyceticus

A technology of actinomycetes and testosteroids, which is applied in the field of preservation and preparation of microbial strains, can solve the problems of decline and less storage efficiency of cyst spores

Active Publication Date: 2020-03-13
NCPC NEW DRUG RES & DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The present invention is aimed at the industrial production strain of teicoplanin ( Actinoplanes teichomyceticus ) in cryopreservation due to the problem of decreased preservation efficiency due to less or no cyst spores, a method for cultivating actinomycetes for the testozoans is provided to obtain abundant zoospores and prepare multi-stage Strain bank is used to produce strain preservation method for supplying species

Method used

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  • Preservation method of actinoplanes teichomyceticus
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Examples

Experimental program
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Embodiment 1

[0024] Preparation of improved sporulation medium:

[0025] Example 1) 20g of wheat germ powder, 0.1g of aspartic acid, 2.0g of ammonium sulfate, 0.5g of potassium dihydrogen phosphate, 2.0g of calcium carbonate, 0.02g of ferrous sulfate heptahydrate, 0.01g of manganese chloride tetrahydrate, seven Zinc sulfate water 0.01g, cobalt chloride 0.005g agar powder 20g, distilled water 1000mL;

[0026] In addition to agar powder, mix the above ingredients, adjust the pH value to 7.0-7.2 with 2.5M sodium hydroxide solution, then add agar powder, heat to dissolve, sterilize at 121°C for 30 minutes, and distribute it in petri dishes while it is hot .

[0027] Example 2) wheat germ powder 10g, aspartic acid 0.2g, ammonium sulfate 3.0g, potassium dihydrogen phosphate 1.0g, calcium carbonate 4.0g, ferrous sulfate heptahydrate 0.03g, manganese chloride tetrahydrate 0.005g, seven Zinc sulfate water 0.02g, cobalt chloride 0.002g, agar powder 20g, distilled water 1000mL;

[0028] In additio...

Embodiment 2

[0041] Improve the spore-producing medium formula according to Example 1 to prepare the solid double-disc petri dish, the slant of the pilot test tube, and the production slant of the eggplant bottle, and the ventilation conditions are respectively: 20 mL of the 9 cm double-disc petri dish; 10 mL of the slant of the pilot test tube, rubber stopper ; Eggplant bottle slant capacity 50mL, 10g cotton plug mouth.

[0042] Take out the strain preservation tube, insert 0.2-0.5mL seed solution into the above-mentioned medium under aseptic conditions, inoculate evenly with a sterile inoculation needle, cultivate at 29°C for 6 days, and then transfer to 24°C for 2 days; Live cyst spore counts reached 8.8×10 6 , 6.5×10 6 , 8.1×10 6 .

Embodiment 3

[0044] Improve the sporulation medium formula according to example 1, prepare the first-generation eggplant bottle strain slope, take out the strain preservation tube, under aseptic conditions, insert 0.2-0.5mL seed liquid into the above-mentioned medium, evenly inoculate with a sterile inoculation needle, Cultured at 28°C for 6 days, then transferred to 25°C for 2 days. Add glycerol and scleroglucan compound formula cryoprotectant (glycerol 200g / L + scleroglucan 10g / L) into the spore culture to make spore suspension, shake at 220r / min for 10min, filter through medium-speed filter paper, After filtration, the spore liquid was transferred into a sterile cryopreservation tube, and the cooling rate was 7°C / min to -35°C, and then quickly placed in ultra-low temperature liquid nitrogen for preservation, as a seed stock for teicoplanin production.

[0045] Take the original seed bank of production seeds and inoculate the improved spore-forming medium formula to prepare the slope of ...

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Abstract

The invention provides a culture method for promoting an actinoplanes teichomyceticus industrial production strain to generate rich zoosporangiophore, in the formula of a strain culture medium, wheatgerm powder is selected as a main carbon and nitrogen source for spore germination, asparagine and ammonium sulfate are used as supplementary nitrogen sources, and trace elements supplement iron, manganese, cobalt and zinc ions. The culture temperature is controlled by stages. Glycerol and scleroglucan are used as composite cryoprotectants for -80 DEG C or ultralow-temperature liquid nitrogen preservation to prepare a strain library, so that the preservation stability of the strain library is greatly improved.

Description

technical field [0001] The invention relates to a method for preserving and preparing microbial strains used for antibiotic fermentation production, in particular to a method for culturing and preserving the strains for producing a large number of zoospores for actinomycetes. Background technique [0002] Teicoplanin is another antibiotic with important clinical efficacy against drug-resistant bacteria following vancomycin and desmethylvancomycin. Its mechanism of action produces strong antibacterial activity against Gram-positive bacteria by inhibiting bacterial cell wall synthesis, especially It can resist severe infections caused by multidrug-resistant bacteria Staphylococcus aureus and Enterococcus, and is another drug barrier for the treatment of antibiotic-resistant infections. The industrial production of teicoplanin is a microbial fermentation method, through the use of rare actinomycetes - Actinomycetes teicoplanans ( Actinoplanes teichomyceticus ) is obtained thro...

Claims

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Application Information

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IPC IPC(8): C12N3/00C12N1/04C12R1/045
CPCC12N3/00C12N1/04Y02P60/85
Inventor 王耀耀张雪霞朱研研郝惠云郄丽萍王亚莉
Owner NCPC NEW DRUG RES & DEV
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