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Preparation method of breeding high-yield extracellular polysaccharide strains by using lithium chloride to mutagenize red ganoderma bioplast

A protoplast, extracellular polysaccharide technology, applied in the field of genetics, can solve the problems of poor mutagenesis effect and low yield of active substances of mutagenized strains

Inactive Publication Date: 2009-11-25
XUZHOU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the mutagenesis of protoplasts basically adopts physical ultraviolet mutagenesis, and the yield of active substances of the mutagenized strains is low, and the mutagenesis effect is poor.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] The red Ganoderma lucidum strain was incubated and activated at 28°C for 7 days, and the activated bacterial mass was inoculated in the starch liquid medium of the Erlenmeyer flask, cultured on a shaker at 28°C for 5 days, and the mycelium was collected by filtration. After washing with mannitol, transfer to 0.3% mercaptoethanol, vibrate for 40 minutes, collect mycelium by filtration, wash and filter with mannitol, and centrifuge at 1200 r / min for 8 minutes. Take 0.5 g of mycelium and add it to the compound enzyme solution at a ratio of 1:15, shake the enzymolyzed mycelium at 28°C for 3 hours, and centrifuge the filtrate at 1000 r / min for 3 minutes, twice. The supernatant was centrifuged at 3000r / min for 10min to obtain protoplasts. 0.02% lithium chloride was added to the regeneration medium, 1 ml of protoplasts were taken and spread on the regeneration medium, and cultured at constant temperature for 7 days. The activated bacterial block was inoculated into the solubl...

Embodiment 2

[0019] Activate the red Ganoderma lucidum strain at 27°C, inoculate the activated bacterial block into a 250ml shaker flask, and culture on a shaker at 27°C for 5 days. Collect the mycelium by filtration, rinse with sterile water and mannitol, transfer to 0.2% mercaptoethanol, oscillate on a shaker for 30 minutes, the temperature is 28°C, collect the mycelium by filtration, wash and filter with mannitol, and use 1500r / min for the mycelium The mycelium was separated by centrifugation for 10 min. Take 1 g of mycelia and add the compound enzyme solution (lysozyme + helicase + cellulase) at a ratio of 1:20, shake the enzymolysis at 30°C for 4 hours, and centrifuge the filtrate twice at 600r / min for 5min. The supernatant was centrifuged at 4000r / min for 5min to obtain protoplasts. Add 0.2% lithium chloride to the regeneration medium, take 1.5 ml of protoplasts, spread on the regeneration medium, and culture at 27° C. for 6 days. The activated bacterial block was inoculated in a 2...

Embodiment 3

[0021] The ganoderma lucidum strain was activated for 7 days at 29°C, and the activated bacterial block was inoculated in the soluble starch liquid medium of a 150 ml shake flask, and cultured on a shaking table at 29°C for 4 days. Collect the mycelium by filtration, rinse with Tris buffer and mannitol solution, transfer to 0.2% mercaptoethanol, shake for 60min, temperature 28°C, collect the mycelium by filtration, wash with Tris buffer, and wash the mycelium with 1000r / centrifuge for 5min. Take 1g of mycelium and add it to the compound enzyme solution at a ratio of 1:10, oscillate at 32°C for 2 hours, filter it with sterile gauze, centrifuge the filtrate at 600r / min for 5min, twice, and centrifuge the supernatant at 3500r / min for 10min Get protoplasts. Add 0.04% lithium chloride to the regeneration medium, take 2ml of protoplasts and spread it on the regeneration medium, cultivate it at 29°C for 6 days, and inoculate small pieces of the activated strain into a 250ml shake f...

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Abstract

The invention relates to a preparation method of ganoderma polysaccharide, particularly relates to a preparation method of breeding high-yield extracellular polysaccharide strains by using lithium chloride to mutagenize red ganoderma bioplast, belongs to genetic technology field. The invention uses reg ganoderma as strains, activation by potato dextrose agar medium, starch liquid shaking cultivation, using lywallzyme, helicase and cellulase to hydrolyze red ganoderma mycelium, shaking at constant temperature to prepare red ganoderma bioplast. After mutagenesis of lithium chloride, content of extracellular polysaccharide is improved 8 to 10 times than original strains. After generation of the mutagenized strains, content of extracellular polysaccharide is improved 6 to 8 times than original strains, and its genetic character is stable, the engineering strain can be used in industrial production of ganoderma polysaccharide.

Description

technical field [0001] The invention relates to a preparation method of ganoderma lucidum polysaccharides, in particular to a preparation method for breeding high-yield exopolysaccharide strains by mutagenizing red ganoderma lucidum protoplasts with lithium chloride, which belongs to the field of genetic technology. Background technique [0002] At present, in the field of cultivation and development of edible fungi, there are many varieties of edible fungi with different qualities. Red Ganoderma lucidum polysaccharide is the most valuable active ingredient in red Ganoderma lucidum. Wide range of pharmacological activities such as immunity and anti-tumor. However, the yield of red Ganoderma lucidum polysaccharide is generally not high and it is difficult to produce industrially. The protoplast mutagenesis technique is an effective new method for microbial breeding. In 2001, Li Gang et al. used ultraviolet mutagenesis to mutate Ganoderma lucidum protoplasts, and bred two hi...

Claims

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Application Information

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IPC IPC(8): C12N15/01C12P19/04C12R1/645
Inventor 董玉玮苗敬芝刘全德吕兆启高明侠曹泽虹
Owner XUZHOU UNIV OF TECH
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