CN108739050B - Cordyceps sinensis liquid culture medium and method for efficiently obtaining blastospores for Cordyceps sinensis host insect infection - Google Patents
Cordyceps sinensis liquid culture medium and method for efficiently obtaining blastospores for Cordyceps sinensis host insect infection Download PDFInfo
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- CN108739050B CN108739050B CN201810513326.2A CN201810513326A CN108739050B CN 108739050 B CN108739050 B CN 108739050B CN 201810513326 A CN201810513326 A CN 201810513326A CN 108739050 B CN108739050 B CN 108739050B
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01G18/00—Cultivation of mushrooms
Abstract
The invention discloses a Chinese caterpillar fungusA liquid culture medium of grass fungi and a method for efficiently obtaining budding spores for infecting host insects of cordyceps sinensis. The cordyceps sinensis liquid culture medium contains 150-300 g of potatoes, 10-30 g of peptone, 20-40 g of maltose, 0.5-1.5 g of magnesium sulfate, 1.5-30 g of monopotassium phosphate and vitamin B per liter120-30 g of galleria mellonella larva grinding liquid, 5-10 g of galleria mellonella larva grinding liquid and the balance of water. The cordyceps sinensis liquid culture medium and the method for efficiently obtaining the blastospores for infecting the cordyceps sinensis host insects greatly shorten the culture time for obtaining the blastospores, can obtain a large amount of the blastospores for infecting the cordyceps sinensis host insects, and provide technical support for the industrial development of artificial cordyceps sinensis cultivation.
Description
The technical field is as follows:
the invention belongs to the technical field of microbial culture, and particularly relates to a cordyceps sinensis liquid culture medium and a method for efficiently obtaining blastospores for cordyceps sinensis host insect infection.
Background art:
cordyceps sinensis is a complex of stiff insects and fungal stroma formed by infecting hepialdae larvae with Cordyceps sinensis Ophiococcineps sinensis (Berk), has important medicinal and economic values, and is known as Chinese medicine with ginseng and pilose antler. Because of its special habitat (natural reproduction only in alpine regions), slow growth speed, long period and extremely limited natural resources, relevant departments have classified Cordyceps as a national second-level protective species.
In recent years, due to the rapid increase of the demand for cordyceps sinensis at home and abroad, resources are being exhausted. The artificial cultivation of the cordyceps sinensis can not only meet the national demand and market demand, but also protect biological resources and ecological environment. After more than 30 years of research, the artificial culture of hirsutella sinensis and the large-scale feeding technology of the host hepialus larva both make great progress, but how to quickly obtain the geminispores for infecting the host insects of the cordyceps sinensis, improve the success rate of the hirsutella sinensis infecting the larva to form stiff insects and grow stroma, and shorten the stroma culture time is an important problem facing the artificial culture industry of the cordyceps sinensis at present.
The invention content is as follows:
the invention aims to overcome the defects in the prior art and provides a cordyceps sinensis liquid culture medium and a method for efficiently obtaining blastospores for cordyceps sinensis host insect infection.
The first purpose of the invention is to provide a cordyceps sinensis liquid culture medium, wherein each liter of the cordyceps sinensis liquid culture medium contains 150-300 g of potatoes, 10-30 g of peptone, 20-40 g of maltose, 0.5-1.5 g of magnesium sulfate, 1.5-30 g of monopotassium phosphate and vitamin B120-30 g of galleria mellonella larva grinding liquid, 5-10 g of galleria mellonella larva grinding liquid and the balance of water.
The galleria mellonella larva grinding fluid is preferably galleria mellonella larva grinding fluid of 5-instar galleria mellonella.
The second purpose of the invention is to provide a preparation method of the cordyceps sinensis liquid culture medium, wherein each liter of the culture medium is prepared by the following method: weighing 150-300 g of peeled potatoes, adding water, boiling, filtering to obtain a potato liquid, adding 10-30 g of peptone, 20-40 g of maltose, 0.5-1.5 g of magnesium sulfate, 1.5-30 g of monopotassium phosphate, 120-30 g of vitamin B and 5-10 g of galleria mellonella larva grinding liquid, and adding water to a constant volume of 1000 mL.
The third purpose of the invention is to provide a method for efficiently obtaining budding spores for infecting host insects of cordyceps sinensis, which comprises the following steps: inoculating the hirsutella sinensis mycelia into the cordyceps sinensis liquid culture medium, culturing for 30 days at 12 +/-3 ℃ and the rotating speed of 80-120 rpm in the dark, and obtaining the budding spores for infecting the cordyceps sinensis host insects.
The hypha blocks are preferably hypha blocks with mucus-shaped surfaces.
The culture medium and the method greatly shorten the culture time for obtaining the blastospores, can obtain a large amount of the blastospores for infecting the host insects of the cordyceps sinensis, and provide technical support for the industrialized development of artificial cultivation of the cordyceps sinensis.
Description of the drawings:
FIG. 1 shows the colony morphology of hirsutella sinensis (surface slime state of colony) used for inoculating a liquid medium according to the present invention.
FIG. 2 shows the colony morphology of hirsutella sinensis (surface of colony grown with aerial hyphae).
FIG. 3 is a microscopic examination (400X) of blastospores of Cordyceps sinensis (hirsutella sinensis) cultured according to the present invention.
FIG. 4 is a microscopic examination (400X) of spores of Cordyceps sinensis (hirsutella sinensis) prepared by the present invention after fluorescence-28 staining.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1:
the liquid culture medium of the cordyceps sinensis strain of the embodiment is prepared by the following method per liter of culture medium: weighing 300g peeled potato, shredding, adding appropriate amount of water, boiling, decocting with slow fire for 20min, cooling, and filtering to obtain potato liquid; 30g of peptone, 40g of maltose, 1.5g of magnesium sulfate, 30g of monopotassium phosphate and vitamin B are weighed1(grinding into powder is needed) 30g, adding water and stirring evenly to obtain solution A; mixing the solution A and the potato liquid to obtain a mixed solution, weighing 10g of 5-instar larvae of the greater wax moth, grinding the 5-instar larvae of the greater wax moth to obtain mucus, adding the 5-instar larvae grinding liquid of the greater wax moth to the mixed solution, adding water to the mixed solution to a constant volume of 1000mL, carrying out autoclaving at 121 ℃ for 30min, and placing the mixed solution in a culture chamber at 12 +/-3 ℃ for precooling for later use.
Inoculating hirsutella sinensis (Ophiococcus sinensis) to PPDA solid culture medium, culturing at 12 + -3 deg.C in dark for 30d (FIG. 1), selecting mycelium block with viscous surface (size of 0.3cm × 0.5cm) and inoculating to 250mL triangular flask containing 100mL Cordyceps liquid culture medium, placing the triangular flask in sterile constant temperature dark room on a shaking table with rotation speed of 120 rpm, culturing at 12 + -3 deg.C in dark, after 30 days, taking out 1mL culture medium, counting with blood count plate, and microscopic examining blastospores (FIG. 3-4) to obtain 3.44 × 107one/mL.
Example 2:
the liquid culture medium of the cordyceps sinensis strain of the embodiment is prepared by the following method per liter of culture medium: weighing 150g peeled potato, shredding, adding appropriate amount of water, boiling, decocting with slow fire for 20min, cooling, and filtering to obtain potato liquid; weighing 10g of peptone, 20g of maltose, 0.5g of magnesium sulfate, 1.5g of potassium dihydrogen phosphate and vitamin B120g (needing to be ground into powder), adding water and stirring uniformly to obtain a solution A; mixing the solution A and the potato liquid to obtain a mixed solution, weighing 5g of 5-instar larvae of the greater wax moth, grinding the 5-instar larvae of the greater wax moth to obtain mucus, adding the 5-instar larvae grinding liquid of the greater wax moth to the mixed solution, adding water to the mixed solution to a constant volume of 1000mL, carrying out autoclaving at 121 ℃ for 30min, and placing the mixed solution in a culture chamber at 12 +/-3 ℃ for precooling for later use.
Inoculating hirsutella sinensis (Ophiococcus sinensis) to PPDA solid culture medium, culturing at 12 + -3 deg.C in dark for 30d (FIG. 1), selecting mycelium block with viscous surface (size of mycelium block is 0.3cm × 0.5cm), inoculating into 250mL triangular flask containing 100mL Cordyceps liquid culture medium, placing the triangular flask into a shaking table with rotation speed of 80 rpm in sterile constant-temperature dark room, culturing at 12 + -3 deg.C in dark, after 30 days, taking out 1mL culture medium, counting with blood counting plate, and microscopically detecting blastospores (FIG. 3-4) to 2.98 × 107one/mL.
Example 3:
the liquid culture medium of the cordyceps sinensis strain of the embodiment is prepared by the following method per liter of culture medium: weighing 300g peeled potato, shredding, adding appropriate amount of water, boiling, decocting with slow fire for 20min, cooling, and filtering to obtain potato liquid; 30g of peptone, 40g of maltose, 1.5g of magnesium sulfate, 30g of monopotassium phosphate and vitamin B are weighed1(grinding into powder is needed) 30g, adding water and stirring evenly to obtain solution A; mixing the solution A and the potato liquid to obtain a mixed solution, weighing 10g of 5-instar larvae of the greater wax moth, grinding the 5-instar larvae of the greater wax moth to obtain mucus, adding the 5-instar larvae grinding liquid of the greater wax moth to the mixed solution, adding water to the mixed solution to a constant volume of 1000mL, carrying out autoclaving at 121 ℃ for 30min, and placing the mixed solution in a culture chamber at 12 +/-3 ℃ for precooling for later use.
Inoculating hirsutella sinensis (Ophiococcus sinensis) to PPDA solid culture medium, culturing at 12 + -3 deg.C in dark for 90d (FIG. 2), selecting hard mycelium block (mycelium block size 0.3cm × 0.5cm) with aerial mycelium spread on surface, inoculating to 250mL triangular flask containing 100mL Cordyceps liquid culture medium, placing the triangular flask in sterile constant temperature dark room, culturing at rotation speed of 120 rpm, culturing at 12 + -3 deg.C in dark, after 30 days, taking out 1mL culture medium, counting with blood counting plate, and microscopic examining blastospores (FIG. 3-4) to obtain 8.6 × 105one/mL.
Example 4:
the liquid culture medium of the cordyceps sinensis strain of the embodiment is prepared by the following method per liter of culture medium: weighing 300g peeled potato, shredding, adding appropriate amount of water, boiling, decocting with slow fire for 20min, cooling, and filtering to obtain potato liquid; 30g of peptone, 40g of maltose, 1.5g of magnesium sulfate, 30g of monopotassium phosphate and vitamin B are weighed130g (ground into powder), adding water, stirring uniformly, adding into the potato liquid, adding water to a constant volume of 1000mL, autoclaving at 121 ℃ for 30min, and placing in a 12 +/-3 ℃ culture room for precooling for later use.
Inoculating hirsutella sinensis (Ophiococcus sinensis) to PPDA solid culture medium, culturing at 12 + -3 deg.C in dark for 30d (FIG. 1), selecting mycelium block with viscous surface (size of mycelium block is 0.3cm × 0.5cm), inoculating into 250mL triangular flask containing 100mL Cordyceps liquid culture medium, placing the triangular flask into a shaking table with sterile constant temperature dark room at rotation speed of 120 r/min, culturing at 12 + -3 deg.C in dark, after 30 days, taking out 1mL culture medium, counting with blood counting plate, and microscopic examination of blastospores (FIG. 3-4) to 4 × 104one/mL.
Example 5:
the liquid culture medium of the cordyceps sinensis strain of the embodiment is prepared by the following method per liter of culture medium: weighing 150g peeled potato, shredding, adding appropriate amount of water, boiling, decocting with slow fire for 20min, cooling, and filtering to obtain potato liquid; weighing 10g of peptone, 20g of maltose, 0.5g of magnesium sulfate, 1.5g of potassium dihydrogen phosphate and vitaminB120g (ground into powder), adding water, stirring uniformly, adding into the potato liquid, adding water to a constant volume of 1000mL, autoclaving at 121 ℃ for 30min, and placing in a 12 +/-3 ℃ culture room for precooling for later use.
Inoculating hirsutella sinensis (Ophiococcus sinensis) to PPDA solid culture medium, culturing at 12 + -3 deg.C in dark for 90d (FIG. 2), selecting hard mycelium block (mycelium block size 0.3cm × 0.5cm) with aerial mycelium spread on surface, inoculating into 250mL triangular flask containing 100mL Cordyceps liquid culture medium, placing the triangular flask in sterile constant temperature dark room on shaking table with rotation speed of 80 rpm, culturing at 12 + -3 deg.C in dark, after 30 days, taking out 1mL culture medium, counting with blood counting plate, and microscopic examining blastospores (FIG. 3-4) to obtain 1 × 104one/mL.
Example 6:
the liquid culture medium of the cordyceps sinensis strain of the embodiment is prepared by the following method per liter of culture medium: weighing 300g peeled potato, shredding, adding appropriate amount of water, boiling, decocting with slow fire for 20min, cooling, and filtering to obtain potato liquid; weighing 10g of peptone, 20g of maltose, 1.5g of magnesium sulfate, 30g of potassium dihydrogen phosphate and vitamin B120g (needing to be ground into powder), adding water and stirring uniformly to obtain a solution A; mixing the solution A and the potato liquid to obtain a mixed solution, weighing 5g of 5-instar larvae of the greater wax moth, grinding the 5-instar larvae of the greater wax moth to obtain mucus, adding the 5-instar larvae grinding liquid of the greater wax moth to the mixed solution, adding water to the mixed solution to a constant volume of 1000mL, carrying out autoclaving at 121 ℃ for 30min, and placing the mixed solution in a culture chamber at 12 +/-3 ℃ for precooling for later use.
Inoculating hirsutella sinensis (Ophiococcus sinensis) to a PPDA solid culture medium, culturing at 12 +/-3 ℃ for 30d in the dark (figure 1), selecting a mycelium block (the size of the mycelium block is 0.3cm multiplied by 0.5cm) with a viscous liquid surface, inoculating the mycelium block into a 250mL triangular flask containing 100mL of a cordyceps sinensis liquid culture medium, placing the triangular flask of the inoculated mycelium block in a sterile constant-temperature dark room, culturing at 12 +/-3 ℃ under the condition of illumination (common fluorescent lamp, illumination intensity is about 200lux) on a shaking table, taking out 1mL of culture medium after 30 days, counting by using a counting plate, and observing by using a microscope to find that no blastospore is generated.
Example 7:
the liquid culture medium of the cordyceps sinensis strain of the embodiment is prepared by the following method per liter of culture medium: weighing 300g peeled potato, shredding, adding appropriate amount of water, boiling, decocting with slow fire for 20min, cooling, and filtering to obtain potato liquid; 30g of peptone, 40g of glucose, 1.5g of magnesium sulfate, 30g of monopotassium phosphate and vitamin B are weighed1(grinding into powder is needed) 30g, adding water and stirring evenly to obtain solution A; mixing the solution A and the potato liquid to obtain a mixed solution, weighing 10g of 5-instar larvae of the greater wax moth, grinding the 5-instar larvae of the greater wax moth to obtain mucus, adding the 5-instar larvae grinding liquid of the greater wax moth to the mixed solution, adding water to the mixed solution to a constant volume of 1000mL, carrying out autoclaving at 121 ℃ for 30min, and placing the mixed solution in a culture chamber at 12 +/-3 ℃ for precooling for later use.
Inoculating hirsutella sinensis (Ophiococcus sinensis) to a PPDA solid culture medium, culturing at 12 +/-3 ℃ for 30d in the dark (figure 1), selecting a mycelium block with a viscous liquid surface (the size of the mycelium block is 0.3cm multiplied by 0.5cm), inoculating the mycelium block into a 250mL triangular flask containing 100mL of cordyceps sinensis liquid culture medium, placing the triangular flask inoculated with the mycelium block into a shaking table with a sterile constant-temperature dark room and a rotating speed of 120 revolutions per minute, culturing at 12 +/-3 ℃ in the dark, taking out 1mL of culture solution after 30 days, counting by using a counting plate, and performing microscopic examination, wherein no blastospore is produced.
Example 8:
the liquid culture medium of the cordyceps sinensis strain of the embodiment is prepared by the following method per liter of culture medium: weighing 150g peeled potato, shredding, adding appropriate amount of water, boiling, decocting with slow fire for 20min, cooling, and filtering to obtain potato liquid; weighing 10g of peptone, 20g of glucose, 0.5g of magnesium sulfate, 1.5g of potassium dihydrogen phosphate and vitamin B1Grinding into powder, adding water 20g, stirring, adding into potato liquid, adding water to a constant volume of 1000mL, autoclaving at 121 deg.C for 30min, and pre-cooling in culture room at 12 + -3 deg.C.
Inoculating hirsutella sinensis (Ophiococcus sinensis) to a PPDA solid culture medium, culturing at 12 + -3 deg.C in the dark for 30d (figure 1), selecting a mycelium block (the size of the mycelium block is 0.3cm multiplied by 0.5cm) with a viscous liquid on the surface, inoculating into a 250mL triangular flask containing 100mL of cordyceps sinensis liquid culture medium, placing the triangular flask of the inoculated mycelium block in a shaking table with a rotating speed of 80 rpm in a sterile constant-temperature dark room, culturing at 12 + -3 deg.C in the dark, after 30 days, taking out 1mL of culture medium, counting by using a blood counting plate, and observing by using a microscope to find that no blastospore is produced.
Example 9:
the liquid culture medium of the cordyceps sinensis strain of the embodiment is prepared by the following method per liter of culture medium: weighing 150g peeled potato, shredding, adding appropriate amount of water, boiling, decocting with slow fire for 20min, cooling, and filtering to obtain potato liquid; weighing 10g of peptone, 20g of glucose, 0.5g of magnesium sulfate, 1.5g of potassium dihydrogen phosphate and vitamin B120g (needing to be ground into powder), adding water and stirring uniformly to obtain a solution A; mixing the solution A and the potato liquid to obtain a mixed solution, weighing 5g of 5-instar larvae of the greater wax moth, grinding the 5-instar larvae of the greater wax moth to obtain mucus, adding the 5-instar larvae grinding liquid of the greater wax moth to the mixed solution, adding water to the mixed solution to a constant volume of 1000mL, carrying out autoclaving at 121 ℃ for 30min, and placing the mixed solution in a culture chamber at 12 +/-3 ℃ for precooling for later use.
Inoculating hirsutella sinensis (Ophiococcus sinensis) to a PPDA solid culture medium, culturing for 90d under the dark condition at 12 +/-3 ℃ and collecting hard mycelium blocks (the size of the mycelium blocks is 0.3cm multiplied by 0.5cm) with aerial hyphae distributed on the surfaces, inoculating the hard mycelium blocks into a 250mL triangular flask filled with 100mL cordyceps sinensis liquid culture medium, placing the triangular flask with the inoculated mycelium blocks into a sterile constant-temperature dark room, culturing at the rotating speed of 80 rpm on a shaking table, culturing under the dark condition at 12 +/-3 ℃, taking out 1mL culture medium after 30 days, counting by using a blood counting plate, and observing under a microscope to find that no blastospores are produced.
Example 10:
the liquid culture medium of the cordyceps sinensis strain of the embodiment is prepared by the following method per liter of culture medium: weighing 150g peeled potato, shredding, adding appropriate amount of water, boiling, decocting with slow fire for 20min, cooling, and filtering to obtain potato liquid; weighing 10g of peptone, 20g of maltose, 0.5g of magnesium sulfate, 1.5g of potassium dihydrogen phosphate and vitamin B1(it is necessary to grind into powderEnd) 20g of water is added and stirred evenly to obtain a solution A; mixing the solution A and the potato liquid to obtain a mixed solution, weighing 5g of 5-instar larvae of the greater wax moth, grinding the 5-instar larvae of the greater wax moth to obtain mucus, adding the 5-instar larvae grinding liquid of the greater wax moth to the mixed solution, adding water to the mixed solution to a constant volume of 1000mL, carrying out autoclaving at 121 ℃ for 30min, and placing the mixed solution in a culture chamber at 12 +/-3 ℃ for precooling for later use.
Inoculating hirsutella sinensis (Ophiococcus sinensis) to a PPDA solid culture medium, culturing for 90d under the dark condition at 12 +/-3 ℃ and selecting a hard mycelium block (the size of the mycelium block is 0.3cm multiplied by 0.5cm) with aerial hyphae distributed on the surface, inoculating the hard mycelium block into a 250mL triangular flask filled with 100mL cordyceps sinensis liquid culture medium, placing the triangular flask with the inoculated mycelium block into a sterile constant-temperature dark room, culturing under the conditions of 12 +/-3 ℃ and illumination (common fluorescent lamp and illumination intensity of about 200lux) by using a shaking table at the rotating speed of 120 revolutions per minute, taking out 1mL bacterial liquid after 30 days, counting by using a blood counting plate, and detecting no blastospore generation under a microscope.
Claims (2)
1. A method for efficiently obtaining budding spores for Cordyceps sinensis host insect infection is characterized by comprising the following steps: inoculating hirsutella sinensis mycelia into a cordyceps sinensis liquid culture medium, culturing for 30 days at 12 +/-3 ℃ and the rotating speed of 80-120 rpm in the dark to obtain budding spores for infecting cordyceps sinensis host insects; the mycelium blocks are mycelium blocks with viscous liquid on the surface; the liquid culture medium of the cordyceps sinensis comprises 150-300 g of potatoes, 10-30 g of peptone, 20-40 g of maltose, 0.5-1.5 g of magnesium sulfate, 1.5-30 g of monopotassium phosphate and vitamin B per liter120-30 g of galleria mellonella larva grinding liquid, 5-10 g of galleria mellonella larva grinding liquid and the balance of water; each liter of the culture medium is prepared by the following method: weighing 150-300 g of peeled potatoes, adding water, boiling, filtering to obtain a potato liquid, adding 10-30 g of peptone, 20-40 g of maltose, 0.5-1.5 g of magnesium sulfate, 1.5-30 g of monopotassium phosphate and vitamin B120-30 g of the galleria mellonella larva grinding liquid and 5-10 g of the galleria mellonella larva grinding liquid, and adding water to a constant volume of 1000 mL.
2. The method according to claim 1, wherein the galleria mellonella larva abrasive solution is galleria mellonella larva abrasive solution of 5-instar galleria mellonella larva.
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| CN109952912B (en) * | 2019-03-08 | 2021-06-15 | 广东省科学院动物研究所 | Method for obtaining cordyceps sinensis by infecting host insect hepialus larva with hirsutella sinensis |
| CN109810934B (en) * | 2019-03-13 | 2021-08-13 | 广东省科学院动物研究所 | Application of N-acetylglucosamine in promoting cordyceps sinensis bud spore development to form hypha |
| CN110305795B (en) * | 2019-03-15 | 2021-10-01 | 广东省生物资源应用研究所 | Hirsutella sinensis and application thereof |
| CN110024623B (en) * | 2019-04-30 | 2021-10-15 | 广东省科学院动物研究所 | Application of L-proline in improving cordyceps sinensis bud spore number and hypha biomass |
| CN111172095B (en) * | 2020-02-24 | 2021-01-19 | 广东省科学院动物研究所 | Application of methyl farnesyl ester in promoting cordyceps sinensis to produce budding spores |
| CN112772294B (en) * | 2021-01-05 | 2022-04-01 | 中国科学院动物研究所 | Separation method and application of cordyceps sinensis strain |
| CN115067152B (en) * | 2022-06-28 | 2023-07-25 | 广东省科学院动物研究所 | Culture medium and culture method for artificially culturing cordyceps sinensis fruiting bodies |
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