A kind of preservation method of actinomycetes teikao

The technology of actinomycetes and ticao is applied in the field of preservation and preparation of microbial strains, which can solve the problems of loss of preservation efficiency, decline, and decrease of cysts and spores.

Active Publication Date: 2022-08-09
NCPC NEW DRUG RES & DEV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims at the problem that the preservation efficiency of Actinoplanes teichomyceticus decreases due to the decrease in cyst spores during cryopreservation, and provides a method for cultivating Actinoplanes teichomyceticus. Bacteria, obtaining abundant zoospores, and preparing a multi-level strain library for the production of strain preservation methods for species

Method used

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  • A kind of preservation method of actinomycetes teikao
  • A kind of preservation method of actinomycetes teikao

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Preparation of improved sporulation medium:

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

[0026] In addition to the agar powder, mix the above components, adjust the pH to 7.0-7.2 with 2.5M sodium hydroxide solution, then add the agar powder, heat to melt, sterilize at 121°C for 30 minutes, and distribute it in a petri dish while still 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, heptahydrate Water zinc sulfate 0.02g, cobalt chloride 0.002g, agar powder 20g, distilled water 1000mL;

[0028] In addition to the aga...

Embodiment 2

[0041] According to the improved sporulation medium formula in Example 1, double-disc petri dish solids, mid-test tube slopes, eggplant bottle production slopes, and ventilation conditions are respectively: 9cm double-disc petri dish loading capacity 20mL; pilot tube slope loading capacity 10mL, rubber stopper ; Eggplant bottle inclined surface loading capacity 50mL, 10g cotton plug mouth.

[0042] Take out the strain storage tube, insert 0.2-0.5mL of seed liquid into the above medium under aseptic conditions, inoculate evenly with a sterile inoculation needle, culture at 29°C for 6 days, and then transfer to 24°C for 2 days; cysts The live counts of spores reached 8.8 × 10, respectively 6 , 6.5×10 6 , 8.1×10 6 .

Embodiment 3

[0044] According to Example 1, the formula of the sporulation medium was improved to prepare a first-generation eggplant bottle strain slant, take out the strain storage tube, and under aseptic conditions, insert 0.2-0.5 mL of seed liquid into the above medium, and evenly inoculate it with a sterile inoculating needle. The cells were incubated at 28°C for 6 days, and then transferred to 25°C for 2 days. Add glycerol and sclerodextran composite formula cryoprotectant (glycerol 200g / L+scleroglucan 10g / L) to the spore culture to make a 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 bank for teicoplanin production.

[0045] The second-generation eggplant bottle was prepared by inoculating the original seed bank of produ...

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Abstract

The invention provides a culturing method for promoting the industrial production strain of Actinomycetes teikao to produce abundant zoosporocysts. Wheat germ powder is selected as the main carbon and nitrogen source for spore germination in the formula of the culture medium. Asparagine and ammonium sulfate are used as supplementary nitrogen sources, and trace elements supplement iron, manganese, cobalt, and zinc ions. The culture temperature was controlled in stages. In addition, glycerol and scleroglucan are used as composite cryoprotectants to prepare a strain library by preservation at -80°C or ultra-low temperature liquid nitrogen, and the preservation stability of the strain library is greatly improved.

Description

technical field [0001] The present invention relates to a method for preserving and preparing microbial strains for antibiotic fermentation production, in particular to a method for culturing and preserving a large number of zoosporocysts from Actinomycetes tektorii. Background technique [0002] Teicoplanin is another antibiotic with important clinical efficacy against drug-resistant bacteria after vancomycin and desmethyl vancomycin. Its mechanism of action produces strong antibacterial activity against Gram-positive bacteria by inhibiting the synthesis of bacterial cell walls. It is able to resist severe infections caused by multi-drug 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, which is obtained by using a rare actinomycete, Actinoplanes teichomyceticus, for pure-bred fermentation production. In fer...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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