CN108040752B - Artificial cultivation method of isaria cicadae and traditional Chinese medicine cordyceps sobolifera - Google Patents
Artificial cultivation method of isaria cicadae and traditional Chinese medicine cordyceps sobolifera Download PDFInfo
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Abstract
The invention provides an artificial cultivation method of isaria cicadae and traditional Chinese medicinal material isaria cicadae, the collection number of the isaria cicadae is CGMCC No 13684, and a large amount of traditional Chinese medicinal material isaria cicadae can be obtained by performing slant activated culture, seed culture and solid cultivation culture on the strain. The isaria cicadae produced spore has extremely strong capability, the provided artificial cultivation method is simple and easy to implement, and the isaria cicadae which is the same as the traditional Chinese medicine material isaria cicadae collected in natural mountain forests can be obtained, so that the problem of shortage of wild isaria cicadae medicinal materials is effectively solved.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine material cultivation and bioengineering, and relates to an artificially separated wild traditional Chinese medicine cordyceps sobolifera strain isaria cicadae and an artificial cultivation method for traditional Chinese medicine cordyceps sobolifera by adopting the strain.
Background
The Chinese medicinal cicada fungus is a complex of fungus Paecilomyces cicadae Miquel nymph of Clavicipitaceae, and is similar to Cordyceps; the infected insect body native to the transected mountain range and Tianmu mountain range is the wild cicada fungus of bamboo cicada, called as cicada fungus or big cicada grass, and mainly produced in Jiangsu and Zhejiang provinces. China has long-term understanding and utilization of cordyceps sobolifera, and the record of processing cordyceps sobolifera is recorded in Lei Gong Pao Zhi treatise of Lei 25989 from north and south, which is 800 years earlier than that of cordyceps sinensis. The "materia medica" (the materia medica of the picture) of Song Chaosu song, the "xi luo" (the width of Song Yao), the "syndrome type materia medica" (the cautious word of Song Dynasty Tang), the "compendium of materia medica" (the compendium of materia medica) of Ming Dynasty Li Zhen and the later pharmacopoeia have recorded effects. After the establishment of new China, the medicinal use of cordyceps sobolifera is recorded in numerous book notes such as Chinese medicine universities, Chinese medicine seas, Chinese medicine dictionaries, Chinese materia medica and the like in numerous materia medica compiled in China or pharmacopoeias. Only two cordyceps sinensis are recorded in the traditional Chinese medicine book, one cordyceps sinensis is cordyceps sinensis, the other cordyceps sinensis is cordyceps sobolifera, the cordyceps sinensis is commonly called as small cordyceps due to large flowers and the cordyceps sobolifera is called as big cordyceps due to small flowers, the cordyceps militaris which is sold in a market more hot at present is cordyceps sinensis, and the cordyceps militaris is not recorded in the traditional Chinese medicine book at all and is only eaten as common food. In recent years, with the exhaustion of cordyceps sinensis resources, artificial cultivation is not successful until now because of difficulty in simulating growth conditions, and the price rises sharply, so that the use of cordyceps sinensis is limited.
In the classification of traditional Chinese medicine properties, cordyceps sobolifera and liquorice; cold; non-toxic, it enters lung and liver meridians, and is cool in nature, and cordyceps sinensis is sweet and neutral. It enters lung and kidney meridians and is warm in nature. At present, common diseases such as hyperlipidemia, hypertension, diabetes, tumor and the like are all febrile diseases, and comparatively speaking, cicada fungus is superior to cordyceps sinensis in the treatment of the diseases. Therefore, the cordyceps sobolifera as the only cordyceps sobolifera recorded in ancient books can replace cordyceps sinensis, has positive clinical significance and wide market value, and becomes a hot spot of domestic and foreign research. The demand of cordyceps sobolifera in the current market is more than 400 tons every year. However, as the cordyceps sobolifera needs specific ecological environment and host insects for growth, the picking period can only be about 20 days in the plum rain season in one year, the natural cordyceps sobolifera is very rare, and the total annual output of wild cordyceps sobolifera in China is only about 25 tons, which limits the large-scale use of cordyceps sobolifera.
At present, artificial culture of cordyceps sobolifera is mainly divided into two forms: mycelium obtained by liquid submerged fermentation and solid cultured Cordyceps cicadae fruiting body. The cordyceps sobolifera mycelium is finished in a liquid fermentation mode, generally only about 3-4 days, and the method is independent of natural conditions, short in growth period, less in pollution, high in yield and capable of realizing large-scale production. However, the application of mycelium instead of sporophore has natural defects due to the obvious difference between the mycelium and sporophore in nutrient and medicinal components. The cordyceps sobolifera used as the medicinal material must have the same external form and tissue structure as the wild cordyceps sobolifera, and the cordyceps sobolifera is mainly cultivated by adopting a biosolid fermentation technical method, but the existing strains are not easy to form the sporophyte bundles on an artificial culture medium, and have the defects of no branching of the sporophyte, low content of spore powder and longer cultivation period, so the cultivation is not easy to succeed and the medicinal use cannot be realized.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide an artificially separated wild traditional Chinese medicine cordyceps sobolifera strain isaria cicadae and an artificial cultivation method of a traditional Chinese medicine cordyceps sobolifera. The method can form the coremium bundles consistent with the external form and the tissue structure of the wild cordyceps sobolifera, and effectively solves the problem of shortage of wild medicinal materials.
In order to realize the purpose of the invention, the inventor finally collects the wild cordyceps sobolifera traditional Chinese medicinal material from the millstone mountain area of Tianwang town of Tianwang of sentence capacity in Jiangsu province through a large number of experimental studies and explorations, and obtains a strain of Isaria cicadae with extremely strong spore production capability through strain separation and identification, and the inventor names the Isaria cicadae (Isaria cicada) Y.R.L-3 and simultaneously reserves the Isaria cicadae in the China general microbiological culture collection management center in 2017 for 20 days 2 and 20 days, and the address is the microbial research institute of China academy of sciences No. 3 of North West Lu No. 1 of the Yangyo of Beijing city, with the preservation number of CGMCC No. 13684.
It should be noted that the high-yield Isaria cicadae (Isaria cicadae) Y.R.L-3 related by the invention has the following characteristics and characteristics:
morphological characteristics: the fruiting body is yellow brown to brown, and is covered by a large amount of white powdery conidia, and the texture is hard and crisp.
Physiological properties: the hyphae are thin-walled, transparent, smooth, have separation and branching, and have a width of 1.6-3.7 mu m. Conidiophores are simply branched, 2-4 conidiophores are grown oppositely and laterally or are grown at the top end in a rotation mode, the base parts are woven and shrunk, the middle parts are expanded, and the upper ends are suddenly thinned. Conidium cylinder of 5.2-10.5 × 2.8-4.2 μm, partial slightly curved, slightly pointed at two ends, thin wall, colorless, 4.4-11.5 × 1.8-3.9 μm.
Metabolic characteristics: the strain grows fast on a potato glucose culture medium, is cultured for 5 days at 28 ℃ in the dark, and has a colony diameter of 13-15 mm, and is white, velvet and raised; the back of the colony is beige, and no water-soluble pigment exists.
In addition, the invention also provides an artificial cultivation method of the traditional Chinese medicine cordyceps sobolifera, which comprises the steps of slant activated cultivation, seed cultivation and solid cultivation of the Isaria cicadae (Isaria cicadae) Y.R.L-3 to obtain the traditional Chinese medicine cordyceps sobolifera, wherein a culture medium used for the solid cultivation preferably takes rice as a substrate.
Further preferably, the artificial cultivation method of the traditional Chinese medicine cordyceps sobolifera as described above comprises the slant activating culture step: transferring Isaria cicadae (Isaria cicadae) Y.R.L-3 strain to PDA culture medium slant test tube under aseptic condition, culturing at 22-28 deg.C for 5-7 days, selecting strain with good growth vigor, and storing.
Further preferably, the artificial cultivation method of the traditional Chinese medicine cordyceps sobolifera as described above comprises the following seed cultivation steps: inoculating the strain preserved in the slant activation culture step into a liquid PDA culture medium in an aseptic manner, and culturing for 3-7 days in a conical flask or a seeding tank for a shaking table at the temperature of 20-25 ℃ and the oscillation frequency of 110-150r/min until a large number of mycelium pellets appear in the conical flask to be used as seed liquid for later use.
Further preferably, the above artificial cultivation method for cordyceps sobolifera as a traditional Chinese medicinal material comprises the following solid cultivation and culture steps: inoculating the seed solution obtained in the seed culture step into a culture medium for solid cultivation, and culturing for 30-50 days at 20-25 ℃ under the condition that the relative humidity is 60% -80% to obtain the traditional Chinese medicine cordyceps sobolifera sporostalk bunch. When the height of the culture of the coremium is more than 3cm, a large number of conidia appear on the surface, and the coremium is bent, the culture can be harvested.
The invention discovers through experiments that the culture medium used for solid cultivation is easy to form the bundle of the sporophores by taking rice as a substrate, and on the basis, the inventor carries out improvement and optimization and obtains the solid cultivation culture medium formula of the Isaria cicadae (Isaria cicadae) Y.R.L-3, wherein the culture medium comprises the following components in the following dosage:
further preferably, the solid cultivation medium of Isaria cicadae (Isaria cicadae) Y.R.L-3 as described above comprises the following components in the following amounts:
still further preferably, the solid culture medium for Isaria cicadae (Isaria cicadae) Y.R.L-3 as described above is prepared from the following components in parts by weight:
in tests, the Isaria cicadae (Isaria cicadae) Y.R.L-3 provided by the invention is found to be easy to form an encystment bundle on an artificial culture medium and has high yield, so the invention also provides the application of the Isaria cicadae (Isaria cicadae) Y.R.L-3 in preparing the cicada fungus bundle. In addition, the rice is used as the main substrate of the artificial culture medium, and the spore-forming capability of Isaria cicadae (Isaria cicadae) Y.R.L-3 is higher. Therefore, the invention also provides the application of the rice in preparing the solid or liquid culture medium of Isaria cicadae (Isaria cicadae) Y.R.L-3; and application of rice, silkworm pupa powder and rice bran or/and wheat bran in preparing solid or liquid culture medium of Isaria cicadae (Isaria cicadae) Y.R.L-3.
Compared with the prior art, the isaria cicadae strain provided by the invention has the advantage of strong spore production capacity, and compared with artificial culture mediums such as insects, barley (rye) and the like, the provided artificial culture medium has higher spore production capacity, simpler culture and collection conditions, more convenient raw material obtaining approaches, no bacteria in the whole process and difficult infectious microbes, so that pure isaria cicadae can be obtained. In addition, the method is simple and easy to implement, can obtain the coremium just like the Chinese medicinal material cicada fungus collected in natural mountain forest, and has great market popularization value.
Drawings
FIG. 1 is a flow chart of an artificial cordyceps sobolifera cultivation process;
FIG. 2 is a drawing of wild (left) and artificial (right) cordyceps sobolifera bundles.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.
Example 1
① preparation of PDA solid inclined plane comprises cutting peeled potato into small pieces, placing in a container, adding 1000mL of water, boiling for 20min, filtering, adding glucose and agar (liquid culture medium without agar), adding 1000mL of water, heating to dissolve, packaging into test tube (1/4) with long test tube, plugging in a cotton plug, sterilizing under 0.1MPa for 30min, and cooling on inclined plane plate.
② slant strain preparation, which comprises activating lyophilized patent preserved strain in PDA liquid culture medium under aseptic condition, rotating tube, culturing at 22 deg.C for 3 days, and selecting the strain with good growth condition;
③ preparation method of PDA conical flask seed liquid comprises cutting peeled potato into small pieces, placing in a container, adding 1000mL water, boiling for 30min, filtering, adding glucose, adding water to 1000mL, heating to dissolve, subpackaging in conical flask with a content of 60% of conical flask volume, plugging with cotton plug, wrapping with kraft paper, sterilizing under 0.15MPa for 30min, taking out, and cooling to below 20 deg.C.
④ collecting mycelium, inoculating the PDA liquid culture medium into a conical flask under aseptic condition, culturing at 22 deg.C and oscillation frequency of 100r/min until a large amount of mycelium balls appear in the conical flask;
when the density of mycelium pellet of culture reaches 104Centrifuging at 10000rpm above per mL, collecting mycelium pellet, oven drying at 60 deg.C overnight, cooling, vacuum packaging with film bag, and storing at-20 deg.C.
⑤ yield and property testing:
the culture product obtained in this example was mycelia (pellets), and the yield was 82.1g (water content 8% or less)/L of the liquid medium in terms of dry weight, wherein the adenosine content was 29.1mg/g dry weight, i.e., 2.91%.
Example 2
① preparation of PDA solid inclined plane comprises cutting peeled potato into small pieces, placing in a container, adding 1000mL of water, boiling for 20min, filtering, adding glucose and agar (liquid culture medium without agar), adding 1000mL of water, heating to dissolve, packaging into test tube (1/4) with long test tube, plugging in a cotton plug, sterilizing under 0.1MPa for 30min, and cooling on inclined plane plate.
② slant strain preparation, which comprises activating lyophilized patent preserved strain in PDA liquid culture medium under aseptic condition, rotating tube, culturing at 22 deg.C for 3 days, and selecting the strain with good growth condition;
③ preparation method of PDA conical flask seed liquid comprises cutting peeled potato into small pieces, placing in a container, adding 1000mL water, boiling for 30min, filtering, adding glucose, adding water to 1000mL, heating to dissolve, subpackaging in conical flask with a content of 60% of conical flask volume, plugging with cotton plug, wrapping with kraft paper, sterilizing under 0.15MPa for 30min, taking out, and cooling to below 20 deg.C.
Inoculating the PDA liquid culture medium into a conical flask under aseptic condition, culturing at 22 deg.C and oscillation frequency of 100r/min for 5 days until a large amount of mycelium pellets appear in the conical flask;
④ cultivation:
(1) preparing a rice solid culture medium: filling the prepared culture medium into a culture box (the specification of the culture box is that the diameter of the circle is 9-10 cm, and the height is 6-10 cm), and sealing the culture box by using a breathable sealing film; the mixture ratio is that 10 weight portions of rice matrix, 2 weight portions of rice bran, 3 weight portions of silkworm chrysalis meal, 0.05 weight portion of potassium dihydrogen phosphate, 0.3 weight portion of peptone, 0 weight portion of yeast powder and 12 weight portions of water are mixed evenly according to the proportion.
(2) And (3) sterilization: placing the culture box into a sterilizing pot, and sterilizing at 0.15MPa for 30 min;
(3) inoculation: after the solid culture medium is cooled to below 20 ℃, inoculating under aseptic condition, and inoculating 15 culture boxes per 300mL of seed liquid;
(4) cultivation: transferring the inoculated culture box to a culture room, culturing at 20-25 deg.C and relative humidity of 60-80% for 30d-50d, and maintaining fresh air in the culture room, wherein the environmental temperature is 2-3 deg.C lower than that in the early mycelium growth stage, and ventilation is required at proper time every day;
⑤ harvesting when the height of the culture reaches above 3cm, there is a great amount of conidium on the surface, and the sporophore bunch bends, removing the sealing film, taking out the culture with a tool, separating the sporophore bunch, oven drying, grading, cooling, vacuum packaging with film bag, and storing the mycoplasm at-20 deg.C.
⑥ yield and property testing:
the culture product obtained in this example consisted of a bundle of sporophores, conidia, mycelia and a solid culture medium, and the yield was 110-. Pharmacological experiments prove that the culture product of isaria cicadae in the embodiment has the function of improving the renal function.
Example 3
① preparation of PDA solid inclined plane comprises cutting peeled potato into pieces, placing in a container, adding 1000mL water, boiling for 50min, filtering, adding glucose and agar, adding 1000mL water, heating to dissolve, packaging into test tube (1/4) with long test tube, plugging a cotton plug, sterilizing under 0.15MPa for 30min, and cooling.
② slant strain preparation, which comprises activating lyophilized patent preserved strain in PDA liquid culture medium under aseptic condition, rotating tube, culturing at 22 deg.C for 5 days, and selecting the strain with good growth condition;
③ preparation method of PDA conical flask seed liquid comprises cutting peeled potato into small pieces, placing in a container, adding 1000mL water, boiling for 50min, filtering, adding glucose, adding water to 1000mL, heating to dissolve, subpackaging in conical flask with a content of 60% of conical flask volume, plugging with cotton plug, wrapping with kraft paper, sterilizing under 0.20MPa for 30min, taking out, and cooling to below 20 deg.C.
Inoculating the PDA liquid culture medium into a conical flask under aseptic condition, culturing at 22 deg.C and oscillation frequency of 130r/min for 7 days until a large amount of mycelium pellets appear in the conical flask;
④ cultivation:
(1) preparing a solid culture medium for rice cultivation, filling the prepared culture medium into a culture box (the specification of the culture box is that the diameter of a circle is 9-10 cm, and the height is 6-10 cm), and sealing the culture box by using a breathable sealing film; the mixture ratio is that 20 weight portions of rice substrate, 4 weight portions of bran, 1 weight portion of silkworm chrysalis meal, 0 weight portion of monopotassium phosphate, 1 weight portion of peptone, 2 weight portions of yeast powder and 10 weight portions of water are mixed evenly according to the proportion.
(2) And (3) sterilization: placing the culture box into a sterilizing pot, and sterilizing at 0.15MPa for 30 min;
(3) inoculation: after the solid culture medium is cooled to below 20 ℃, inoculating 20 culture boxes per 300mL under a sterile condition;
(4) cultivation: transferring the inoculated culture box to a culture room, culturing at 20-25 deg.C and relative humidity of 60-80% for 30d-50d, and maintaining fresh air in the culture room, wherein the environmental temperature is 2-3 deg.C lower than that in the early mycelium growth stage, and ventilation is required at proper time every day;
⑤ harvesting when the height of the culture reaches above 3cm, there is a great amount of conidium on the surface, and the sporophore bunch bends, removing the sealing film, taking out the culture with a tool, separating the sporophore bunch, oven drying, grading, cooling, vacuum packaging with film bag, and storing the mycoplasm at-20 deg.C.
⑥ yield and property testing:
the culture product obtained in this example consisted of a bundle of sporophores, conidia, mycelia and a solid culture medium, and the yield was 90-100g (water content below 8%) of the bundle of sporophores per kg of solid culture medium.
Example 4
② preparation of PDA solid inclined plane comprises cutting peeled potato into pieces, placing in a container, adding 1000mL water, boiling for 350min, filtering, adding glucose and agar, adding 1000mL water, heating to dissolve, packaging into test tube (1/4) with long test tube, plugging a cotton plug, sterilizing under 0.15MPa for 30min, and cooling.
② slant strain preparation, which comprises activating lyophilized patent preserved strain in PDA liquid culture medium under aseptic condition, rotating tube, culturing at 22 deg.C for 5 days, and selecting the strain with good growth condition;
③ preparation method of PDA conical flask seed liquid comprises cutting peeled potato into small pieces, placing in a container, adding 1000mL water, boiling for 30min, filtering, adding glucose, adding water to 1000mL, heating to dissolve, subpackaging in conical flask with a content of 60% of conical flask volume, plugging with cotton plug, wrapping with kraft paper, sterilizing under 0.15MPa for 30min, taking out, and cooling to below 20 deg.C.
Inoculating the PDA liquid culture medium into a conical flask under aseptic condition, culturing at 22 deg.C and oscillation frequency of 150r/min for 5 days until a large amount of mycelium pellets appear in the conical flask;
④ 10L seed tank culture
Placing 7L PDA culture medium into an lOL fermenter (70% volume), sterilizing at 123 deg.C for 30min, cooling to 24 deg.C, inoculating 10% of conical flask seed, controlling fermentation condition at 24 deg.C, pot pressure of 0.01-0.05Mpa, and air flow rate of 0.5m3Fermenting for 72 hr.
⑤ cultivation:
(1) preparing a solid culture medium for rice cultivation, putting the prepared culture medium into a culture box (the specification of the culture box is 33cm square and 6-10 cm high), and sealing by using a breathable sealing film; the mixture ratio is that 20 weight portions of rice matrix, 3 weight portions of rice bran, 2 weight portions of silkworm chrysalis meal, 0.01 weight portion of potassium dihydrogen phosphate, 0 weight portion of peptone, 1 weight portion of yeast powder and 16 weight portions of water are mixed evenly according to the proportion.
(2) And (3) sterilization: placing the culture box into a sterilizing pot, and sterilizing at 0.15MPa for 30 min;
(3) inoculation: after the solid culture medium is cooled to below 20 ℃, inoculating 2 culture boxes per 300mL under aseptic conditions;
(4) cultivation: transferring the inoculated culture box to a culture room, culturing for 50d at 25 ℃ and 80% relative humidity, wherein the environmental temperature is 2-3 ℃ lower than that of the early mycelium growth stage, ventilating at proper time every day, and keeping the air in the culture room fresh;
⑥ harvesting when the height of the sporophore bundle of the culture is above 3cm, a large number of conidia appear on the surface and the sporophore bundle is bent, removing the sealing film during harvesting, taking out the culture with a tool to separate the sporophore bundle, drying and grading, cooling, vacuum packaging with a film bag, storing the mycoplasm at-20 deg.C, and storing the sporophore bundle at-45 deg.C.
⑦ yield and property testing:
the culture product obtained in this example consisted of a bundle of sporophores, conidia, mycelia and a solid culture medium, and the yield was 150g (water content below 8%) of the bundle of sporophores per kg of solid culture medium.
Example 5
① preparation of PDA solid inclined plane comprises cutting peeled potato into small pieces, placing in a container, adding 1000mL of water, boiling for 20min, filtering, adding glucose and agar (liquid culture medium without agar), adding 1000mL of water, heating to dissolve, packaging into test tube (1/4) with long test tube, plugging in a cotton plug, sterilizing under 0.1MPa for 30min, and cooling on inclined plane plate.
② slant strain preparation, which comprises activating lyophilized patent preserved strain in PDA liquid culture medium under aseptic condition, rotating tube, culturing at 22 deg.C for 3 days, and selecting the strain with good growth condition;
③ preparation method of PDA conical flask seed liquid comprises cutting peeled potato into small pieces, placing in a container, adding 1000mL water, boiling for 30min, filtering, adding glucose, adding water to 1000mL, heating to dissolve, subpackaging in conical flask with a content of 60% of conical flask volume, plugging with cotton plug, wrapping with kraft paper, sterilizing under 0.15MPa for 30min, taking out, and cooling to below 20 deg.C.
Inoculating the PDA liquid culture medium into a conical flask under aseptic condition, culturing at 22 deg.C and oscillation frequency of 100r/min for 5 days until a large amount of mycelium pellets appear in the conical flask;
④ cultivation:
(1) preparing a rice solid culture medium: filling the prepared culture medium into a culture box (the specification of the culture box is that the diameter of the circle is 9-10 cm, and the height is 6-10 cm), and sealing the culture box by using a breathable sealing film; the mixture ratio is 10 parts of wheat substrate, 2 parts of rice bran, 3 parts of silkworm chrysalis meal, 0.05 part of monopotassium phosphate, 0.3 part of peptone, 0 part of yeast powder and 12 parts of water, and all the raw materials are uniformly mixed according to the proportion.
(2) And (3) sterilization: placing the culture box into a sterilizing pot, and sterilizing at 0.15MPa for 30 min;
(3) inoculation: after the solid culture medium is cooled to below 20 ℃, inoculating under aseptic condition, and inoculating 15 culture boxes per 300mL of seed liquid;
(4) cultivation: transferring the inoculated culture box to a culture room, culturing at 20-25 deg.C and relative humidity of 60-80% for 30d-50d, and maintaining fresh air in the culture room, wherein the environmental temperature is 2-3 deg.C lower than that in the early mycelium growth stage, and ventilation is required at proper time every day;
⑤ harvesting when the height of the culture reaches above 3cm, there is a great amount of conidium on the surface, and the sporophore bunch bends, removing the sealing film, taking out the culture with a tool, separating the sporophore bunch, oven drying, grading, cooling, vacuum packaging with film bag, and storing the mycoplasm at-20 deg.C.
⑥ yield and property testing:
the culture product obtained in this example consisted of a bundle of sporophores, conidia, mycelia and a solid culture medium, and the yield was 60-70g (water content below 8%) of the bundle of sporophores per kg of solid culture medium. Pharmacological experiments prove that the culture product of isaria cicadae in the embodiment has the function of improving the renal function.
Example 6
① preparation of PDA solid inclined plane comprises cutting peeled potato into small pieces, placing in a container, adding 1000mL of water, boiling for 20min, filtering, adding glucose and agar (liquid culture medium without agar), adding 1000mL of water, heating to dissolve, packaging into test tube (1/4) with long test tube, plugging in a cotton plug, sterilizing under 0.1MPa for 30min, and cooling on inclined plane plate.
② slant strain preparation, which comprises activating lyophilized patent preserved strain in PDA liquid culture medium under aseptic condition, rotating tube, culturing at 22 deg.C for 3 days, and selecting the strain with good growth condition;
③ preparation method of PDA conical flask seed liquid comprises cutting peeled potato into small pieces, placing in a container, adding 1000mL water, boiling for 30min, filtering, adding glucose, adding water to 1000mL, heating to dissolve, subpackaging in conical flask with a content of 60% of conical flask volume, plugging with cotton plug, wrapping with kraft paper, sterilizing under 0.15MPa for 30min, taking out, and cooling to below 20 deg.C.
Inoculating the PDA liquid culture medium into a conical flask under aseptic condition, culturing at 22 deg.C and oscillation frequency of 100r/min for 5 days until a large amount of mycelium pellets appear in the conical flask;
④ cultivation:
(1) preparing a barley solid cultivation medium: filling the prepared culture medium into a culture box (the specification of the culture box is that the diameter of the circle is 9-10 cm, and the height is 6-10 cm), and sealing the culture box by using a breathable sealing film; the mixture ratio is 10 parts of barley matrix, 2 parts of rice bran, 3 parts of silkworm chrysalis meal, 0.05 part of monopotassium phosphate, 0.3 part of peptone, 0 part of yeast powder and 12 parts of water, and all the raw materials are uniformly mixed according to the proportion.
(2) And (3) sterilization: placing the culture box into a sterilizing pot, and sterilizing at 0.15MPa for 30 min;
(3) inoculation: after the solid culture medium is cooled to below 20 ℃, inoculating under aseptic condition, and inoculating 15 culture boxes per 300mL of seed liquid;
(4) cultivation: transferring the inoculated culture box to a culture room, culturing at 20-25 deg.C and relative humidity of 60-80% for 30d-50d, and maintaining fresh air in the culture room, wherein the environmental temperature is 2-3 deg.C lower than that in the early mycelium growth stage, and ventilation is required at proper time every day;
⑤ harvesting when the height of the culture reaches above 3cm, there is a great amount of conidium on the surface, and the sporophore bunch bends, removing the sealing film, taking out the culture with a tool, separating the sporophore bunch, oven drying, grading, cooling, vacuum packaging with film bag, and storing the mycoplasm at-20 deg.C.
⑥ yield and property testing:
the culture product obtained in this example consisted of a bundle of sporophores, conidia, mycelia and a solid culture medium, and the yield was 40-50g (water content below 8%) of the bundle of sporophores per kg of solid culture medium.
Claims (8)
1. Isaria cicadae (Isaria cicadae) Y.R.L-3 is separated from wild cicada fungus in Zhengsu province of Tianwang Zhenshanshan area of sentence capacity, and the preservation number of the Isaria cicadae (Isaria cicadae) Y.R.L-3 is CGMCC No 13684.
2. A solid culture medium of Isaria cicadae (Isaria cicadae) Y.R.L-3 according to claim 1, wherein the culture medium comprises rice as a substrate.
3. The solid culture medium of Isaria cicadae (Isaria cicadae) y.r.l-3 according to claim 2, characterized in that it comprises the following components in amounts:
10-25 parts of rice
1-5 parts of rice bran or/and wheat bran
Silkworm chrysalis powder 1-6 weight portions
0 to 0.2 weight portion of monopotassium phosphate
0-1 part by weight of peptone
0-2 parts of yeast powder
10-30 parts of water.
4. A solid culture medium of Isaria cicadae (Isaria cicadae) Y.R.L-3 according to claim 3, characterized in that it comprises the following components in the amounts:
10-20 parts of rice
2-4 parts of rice bran or/and wheat bran
Silkworm chrysalis powder 1-3 weight portions
0.01-0.08 part by weight of monopotassium phosphate
0-1 part by weight of peptone
0-2 parts of yeast powder
10-20 parts of water.
5. The solid culture medium of Isaria cicadae (Isaria cicadae) Y.R.L-3 as claimed in claim 3, which is prepared from the following components in parts by weight:
10-20 parts of rice
2-4 parts of rice bran or/and wheat bran
Silkworm chrysalis powder 1-4 weight portions
0.01-0.08 part by weight of monopotassium phosphate
0-1 part by weight of peptone
0-2 parts of yeast powder
10-20 parts of water.
6. An artificial cultivation method of a traditional Chinese medicine material cordyceps sobolifera is characterized by comprising the steps of carrying out activated culture, seed culture and solid cultivation culture on Isaria cicadae (Isaria cicadae) Y.R.L-3 according to claim 1 to obtain the traditional Chinese medicine cordyceps sobolifera, wherein a culture medium adopted in the solid cultivation culture step comprises the following components in dosage:
10-25 parts of rice
1-5 parts of rice bran or/and wheat bran
Silkworm chrysalis powder 1-6 weight portions
0 to 0.2 weight portion of monopotassium phosphate
0-1 part by weight of peptone
0-2 parts of yeast powder
10-30 parts of water.
7. The artificial cultivation method of traditional Chinese medicinal material cordyceps sobolifera as claimed in claim 6, wherein the solid cultivation and cultivation step comprises the following steps: inoculating the seed solution obtained in the seed culture step into a culture medium for solid cultivation, and culturing for 30-50 days at 20-25 ℃ and relative humidity of 60-80% to obtain the traditional Chinese medicine cordyceps sobolifera sporostalk bunch.
8. Use of Isaria cicadae (Isaria cicadae) y.r.l-3 according to claim 1 for preparing cordyceps sobolifera and/or mycelium.
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| CN102174416A (en) * | 2011-02-16 | 2011-09-07 | 南京中医药大学 | Angelica sinensis endophytic fungi Fusella sp DG09 with plant pathogenic bacteria resistant activity and fermentation solution and application thereof |
| CN102250777A (en) * | 2011-06-20 | 2011-11-23 | 贵州大学 | Isariacicadae strain and use thereof |
| CN103571756A (en) * | 2012-07-30 | 2014-02-12 | 浙江泛亚生物医药股份有限公司 | Test tube screening method of isaria cicadae strain and culture medium |
| CN106167779A (en) * | 2016-08-24 | 2016-11-30 | 南京中医药大学 | A kind of bacillus amyloliquefaciens and the method preparing succinyl ononin in nonaqueous phase |
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| CN102174416A (en) * | 2011-02-16 | 2011-09-07 | 南京中医药大学 | Angelica sinensis endophytic fungi Fusella sp DG09 with plant pathogenic bacteria resistant activity and fermentation solution and application thereof |
| CN102250777A (en) * | 2011-06-20 | 2011-11-23 | 贵州大学 | Isariacicadae strain and use thereof |
| CN103571756A (en) * | 2012-07-30 | 2014-02-12 | 浙江泛亚生物医药股份有限公司 | Test tube screening method of isaria cicadae strain and culture medium |
| CN106167779A (en) * | 2016-08-24 | 2016-11-30 | 南京中医药大学 | A kind of bacillus amyloliquefaciens and the method preparing succinyl ononin in nonaqueous phase |
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