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Method for breeding endo-inulinase strains by using protoplast fusion technology

A protoplast fusion and protoplast technology, applied in the field of genetics, can solve problems such as the enzyme activity of no strains, and achieve highly reproducible results

Inactive Publication Date: 2013-11-27
XUZHOU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to literature reports, cell mutagenesis and optimization of process conditions are used to improve the enzyme activity of endo-inulinase produced by microorganisms, but there is no report on the enzyme activity of strains using protoplast fusion technology

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] The two starting strains of Aspergillus niger were incubated and activated at 30°C for 7 days, and the activated bacterial mass was taken in a 250ml shaker flask, and cultured on a shaker at 28°C for 5 days; filtered, collected mycelium balls, and rinsed with sterile water; Put 1g of mycelia into a 100ml Erlenmeyer flask filled with 20ml of 1% enzymatic hydrolysis solution (1:20), vibrate at 32°C for 2 hours, stop the enzymatic hydrolysis with an ice bath, and double-layer aseptic Filter through lens paper, wash the precipitate with PBA solution for 3 times, add PBA reagent solution to the precipitate, this is the protoplast solution; add PEG6000 and CaCl to each of the two protoplast solutions 2 Centrifuge the solution at 2000r / min for 10min, filter it with sterile lens paper, suspend 20ml of the precipitate with PBA solution, absorb 0.1ml and apply it to the regeneration medium respectively, and cultivate it at 30°C for 2 days; select the strain with a large transparen...

Embodiment 2

[0020] Take the two starting strains of Aspergillus niger that were activated at 28°C and put them into a 250ml shaker flask, and culture them on a shaking table at 30°C for 4 days; filter, collect mycelial balls, and rinse with sterile water; take 1g each Put the mycelia into a 100ml Erlenmeyer flask filled with 20ml and 1% enzymatic hydrolysis solution (1:20), shake the enzymatic hydrolysis at 30°C for 3 hours, stop the enzymatic hydrolysis in an ice bath, pass through a double-layer sterile lens Filter through paper, wash the precipitate with PBA solution for 3 times, add PBA reagent solution to the precipitate to prepare protoplast solution; add 1ml of each of the two protoplast solutions and then add PEG6000 and CaCl 2 Centrifuge the solution at 2000r / min for 10min, filter it with sterile lens paper, suspend 20ml of the precipitate with PBA solution, suck 0.1ml and apply it to the regeneration medium respectively, and cultivate it at 28°C for 4 days; select the strain with...

Embodiment 3

[0022] Take the two starting strains of Aspergillus niger that were activated at 29°C and put them into a 250ml shaker flask, and culture them on a shaking table at 29°C for 4 days; filter, collect mycelial balls, and rinse with sterile water; take 1g each Put the mycelium into a 100ml Erlenmeyer flask filled with 20ml, 1% enzymatic hydrolysis solution (1:20), shake the enzymatic hydrolysis at 28°C for 4 hours, stop the enzymatic hydrolysis in an ice bath, pass through a double-layer sterile lens Filter through paper, wash the precipitate with PBA solution for 3 times, add PBA reagent solution to the precipitate to prepare protoplast solution; add 1ml of each of the two protoplast solutions and then add PEG6000 and CaCl 2 Centrifuge the solution at 2000r / min for 10min, filter it with sterile lens paper, suspend 20ml of the precipitate with PBA solution, absorb 0.1ml and apply it to the regeneration medium respectively, and cultivate it at 29°C for 3 days; select the strain with...

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Abstract

The invention relates to a method for breeding microbes by using the protoplast fusion technology, in particular to a method for breeding endo-inulinase strains by using the protoplast fusion technology, and belongs to the technical field of heredity. According to the method, two aspergillus nigers are used as starting strains and subjected to activation by a culture medium containing inulin as well as shake culture by burdock juice, cellulase and helicase are utilized to hydrolyze cell walls of aspergillus niger hypha cells, and the oscillation is performed at constant temperature to prepare protoplasts; through the fusion of the protoplasts, the enzyme activities of endo-inulinase are respectively improved by 35.08 percent and 48.26 percent compared with the two aspergillus nigers. The protoplasts can be regenerated after the fusion, after ten generations passage experiments, the deviation of the enzyme activity of the endo-inulinase is smaller compared with regenerated strains, and inheritable characters are stable; the strains can be used for industrial production of endo-inulinase.

Description

technical field [0001] The invention relates to a preparation method for protoplast fusion and breeding microorganisms, in particular to a preparation method for using protoplast fusion technology to select and breed endo-inulinase strains, which belongs to the field of genetic technology. Background technique [0002] Fructose-oligosaccharide is a good bifidus factor and water-soluble dietary fiber, which can be used as raw material or additive of functional food or feed. Fructose-oligosaccharides have the functions of anti-tumor, stimulating the growth of bifidobacteria, preventing and treating constipation, inhibiting the formation of spoilage substances in the intestine, improving the body's immunity, improving lipid metabolism, and lowering cholesterol. It can also be used to improve gastrointestinal function, lower blood lipids and prevent hypercholesterolemia. Therefore, it has important health and medicinal development value. [0003] Traditional industrial large-s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/04C12R1/685
Inventor 曹泽虹高兆建董玉玮
Owner XUZHOU UNIV OF TECH
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