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
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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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