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

Inactive Publication Date: 2010-04-07
CHANGCHUN SHENGJINNUO BIOLOGICAL PHARMA
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Problems solved by technology

Cordyceps sinensis commonly used in traditional Chinese medicine is a precious medicinal fungus, but its natural resources ...

Method used

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  • Mutagenic strain of cordyceps militaris and breeding method
  • Mutagenic strain of cordyceps militaris and breeding method
  • Mutagenic strain of cordyceps militaris and breeding method

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Effect test

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...

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 yi...

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Abstract

The invention provides a mutagenic strain of cordyceps militaris and a breeding method. A new high-yield strain of cordyceps militaris is obtained by chemical mutagenesis, the preservation number thereof is CGMCC NO. 2909, and compared with original starting strain, the dry weight of mycelium is increased by 103%, the content of adenosine is increased by 536%, the content of polysaccharide is increased by 22.2%, the content of protein is increased by 55.5%, and the content of mannitol is increased by 37.5 %. The new strain of cordyceps militaris does not degenerate after more than 20 times of subculturing, has genetic stability, and utilizes the research of random amplified polymorphic DNA (RAPD) to verify the difference on DNA level between the strain and the original starting strain. The invention also discloses the optimum process condition for extracting polysaccharide and adenosine of cordyceps militaris.

Description

technical field [0001] The invention discloses a mutagenic strain of Cordyceps militaris, which is a new type of strain. The invention also provides a cultivation method of the strain, which belongs to the technical field of microorganisms. Background technique [0002] Cordyceps militaris (L.) Link, also known as Cordyceps militaris, Cordyceps militaris or Cordyceps wu-style, and Cordyceps sinensis belong to the genus Ascomycetes, Sclerotia, Sclerotia, Sclerotia, Ergotaceae, Cordyceps, and are mainly distributed in Shanxi, my country. Shaanxi, Jilin, Hebei, Guangdong and other places. Cordyceps militaris is a complex formed by fungi parasitizing insect pupae or larval corpses. Cordyceps sinensis commonly used in traditional Chinese medicine is a precious medicinal fungus, but its natural resources are scarce, expensive, and artificial cultivation is very difficult, so far it has not been able to achieve large-scale production. [0003] At present, the artificial cultivatio...

Claims

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Application Information

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IPC IPC(8): A01H15/00A01H1/06
Inventor 滕利荣高陆孟庆繁逯家辉王迪金璐张嫒莉杜研王彦峰李珊珊沈畏刘海雕白冰朱明光翟景波高朝辉姜丽艳王贞佐林凤任晓冬郭伟良张凯明
Owner CHANGCHUN SHENGJINNUO BIOLOGICAL PHARMA
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