Mutagenic strain of cordyceps militaris and breeding method
A technology of Cordyceps militaris and mutagenic strains, applied in the field of microorganisms, can solve the problems of failure to achieve large-scale production, scarce natural resources, difficulty in artificial cultivation, etc., and achieves optimization of the ultrasonic extraction process of polysaccharides, yield and effective ingredient content improvement, protection The effect of wild resources
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Embodiment 1
[0044] Embodiment 1: Mutagenesis and breeding method of Cordyceps militaris mutant strain
[0045] (1) Mutation of Cordyceps militaris
[0046] Inoculate the strain of Cordyceps militaris with the preservation number CGMCC 5.699 on a PDA slant, cultivate it in a 26°C incubator for 5 days, inject 1.5mL of sterile normal saline into the slant test tube, filter it with sterile absorbent cotton after shaking, and dilute it to a concentration of 3× 10 7 individual / mL spore suspension. After adding 10 mg of nitrosoguanidine and 0.5 mL of acetone to a petri dish, use 9 mL of sterile phosphate buffer (pH=6.0), and after the nitrosoguanidine is fully dissolved, add 1 mL of spore suspension for mutagenesis. After 10 min of mutagenesis, the bacterial suspension was diluted step by step in sterile distilled water to a concentration of 3×10 4 cells / mL, after 4 days of PDA medium plate culture, the strains were stored in 96-well plates. Use a sterilized toothpick to pick the strains in ...
Embodiment 2
[0056] Optimization of extraction conditions of polysaccharides from the mycelia of Cordyceps militaris mutagenic strains
[0057] (1) Ultrasonic extraction method of mycelium polysaccharide
[0058] The mycelium of the mutagenic strain of Cordyceps militaris was obtained by liquid submerged fermentation, freeze-dried to constant weight, crushed, and passed through a 60-mesh sieve. Weigh 0.1 g of mycelium dry powder, add a certain amount of deionized water according to a certain water-to-material ratio, and ultrasonically extract in a water bath for a certain period of time. The extract was centrifuged at 12000 rpm at 4°C for 10 min, and the content of total sugar and reducing sugar in the supernatant was determined. The total sugar content was determined by anthracene copper-sulfuric acid method; the reducing sugar content was determined by DNS method. The polysaccharide content was obtained by subtracting the reducing sugar content from the total sugar content.
[0059] (...
Embodiment 3
[0067] Optimization of extraction conditions of adenosine from the mycelium of the mutagenic strain of Cordyceps militaris
[0068] (1) Vacuum freeze-dry the mycelia of Cordyceps militaris obtained by liquid submerged fermentation, pulverize, and pass through a 60-mesh sieve for experimentation. Accurately weigh 0.15 g of the pretreated mycelium, add distilled water and extract in a constant temperature water bath for a certain period of time. Centrifuge at 8000rpm for 10min, and separate the supernatant for determination of adenosine content. According to the method introduced in the 2005 edition of "Chinese Pharmacopoeia", the content of adenosine was determined by high performance liquid chromatography. The mobile phase was phosphate buffer at pH 6.5:methanol (volume ratio)=85:15. The determination conditions of HPLC are chromatographic column: C-18, flow rate: 1 mL / min, detection wavelength: 260 nm, column temperature: 35° C., and injection volume: 10 μL. Adenosine yiel...
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