CN113348965B - Proliferation method of Cordyceps sinensis for infecting hepialus larva - Google Patents
Proliferation method of Cordyceps sinensis for infecting hepialus larva Download PDFInfo
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- 241001248610 Ophiocordyceps sinensis Species 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 title claims abstract description 22
- 241000330899 Hepialus Species 0.000 title claims abstract description 13
- 230000035755 proliferation Effects 0.000 title abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 34
- 244000060011 Cocos nucifera Species 0.000 claims abstract description 32
- 235000013162 Cocos nucifera Nutrition 0.000 claims abstract description 32
- 238000012258 culturing Methods 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 13
- 238000004321 preservation Methods 0.000 claims description 12
- 241000196324 Embryophyta Species 0.000 claims description 8
- 230000001580 bacterial effect Effects 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 7
- 238000004140 cleaning Methods 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 239000002689 soil Substances 0.000 claims description 7
- 241000238631 Hexapoda Species 0.000 claims description 6
- 241000607479 Yersinia pestis Species 0.000 claims description 6
- 238000005520 cutting process Methods 0.000 claims description 6
- 201000010099 disease Diseases 0.000 claims description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 6
- 239000012535 impurity Substances 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 235000015097 nutrients Nutrition 0.000 claims description 6
- 238000006213 oxygenation reaction Methods 0.000 claims description 6
- 238000003860 storage Methods 0.000 claims description 6
- 238000009423 ventilation Methods 0.000 claims description 6
- 238000005286 illumination Methods 0.000 claims description 5
- 230000002062 proliferating effect Effects 0.000 claims 2
- 238000003306 harvesting Methods 0.000 abstract description 3
- 238000005406 washing Methods 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 16
- 230000035784 germination Effects 0.000 description 11
- KRALOLGXHLZTCW-UHFFFAOYSA-L calcium;2-acetyloxybenzoate Chemical compound [Ca+2].CC(=O)OC1=CC=CC=C1C([O-])=O.CC(=O)OC1=CC=CC=C1C([O-])=O KRALOLGXHLZTCW-UHFFFAOYSA-L 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- 238000004659 sterilization and disinfection Methods 0.000 description 5
- 238000011160 research Methods 0.000 description 4
- 230000002411 adverse Effects 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 238000012136 culture method Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000001902 propagating effect Effects 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000130660 Hepialidae Species 0.000 description 1
- 241001558929 Sclerotium <basidiomycota> Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
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- 238000010790 dilution Methods 0.000 description 1
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- 238000001125 extrusion Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000004763 spore germination Effects 0.000 description 1
- 230000006019 sporocarp development involved in sexual reproduction Effects 0.000 description 1
- 230000028070 sporulation Effects 0.000 description 1
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- A01G18/00—Cultivation of mushrooms
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Abstract
The application belongs to the field of cordyceps sinensis cultivation, and particularly relates to a cordyceps sinensis proliferation method for infecting hepialus larvae, which comprises the following steps: 1) Collecting natural strains; 2) Pretreatment: washing the strain acquired in the step 1) with water, and airing the surface moisture of the sub-seat for later use; 3) And (3) temporary planting: planting the pretreated strain in a planting box containing sterile coconut coir with the water content of 60%, and placing the planting box in a clean room with the temperature of 18-20 ℃ and the RH of 85-90% for cultivation; 4) Ascus shell collection: harvesting the ascus when the ascus is full in growth and the ascus protrudes; 5) Ascus shell planting: planting the ascus shells acquired in the step 4) in a collecting box containing pure water with the depth of 1-2cm, and placing the collecting box in a clean room with the temperature of 18-20 ℃ and the RH of 65-70% for culture; 6) And (3) strain collection: collecting the culture solution in a concentrated manner; the proliferation method effectively reduces the collection and dependence on natural strains, reduces the input cost of enterprises, and protects the natural strains.
Description
Technical Field
The application relates to the field of cordyceps sinensis, in particular to a proliferation method of cordyceps sinensis for infecting hepialus larvae.
Background
Cordyceps sinensis is a complex of a stroma of Cordyceps sinensis (Cordycepssienesis (Berkeley) Saccaro) fungus of Clavicepitaceae (Clavicepitaceae) on a larva of Hepialidae (Hepialus) and a cadaver of Hepialus larva, the Hepialus larva is infected by Cordyceps sinensis (Cordyceps sinensis), the mycelia are filled in the whole tissue of the worm body to form hard pseudosclerotium, the worm shape is kept unchanged for several months in winter low-temperature dry soil, and stick-shaped fruiting bodies (stroma) are grown from the sclerotium and the ground (grass) is exposed when the temperature and humidity are proper in summer, so that Cordyceps sinensis is named, is one of special rare medicinal materials in China, has a flat taste and has the effects of tonifying lung and tonifying kidney. Wild Cordyceps sinensis only grows in alpine vegetation such as alpine meadow and plateau meadow with the elevation of Qinghai-Tibet plateau of 3000-5000 meters, and the yield is reduced due to over-digging in recent years.
At present, in the research of culturing cordyceps sinensis, the research of conidium is crucial, the main difficulty in the research of conidium of cordyceps sinensis is that the yield of conidium is low, the period is long, the quantity of conidium obtained by artificial culture is small, the collection and dependence on natural strains in the culturing process of cordyceps sinensis are aggravated, and how to obtain a large quantity of conidium by culture becomes a hot spot in the research of culturing cordyceps sinensis.
Disclosure of Invention
The technical problem to be solved by the application is to provide a proliferation method of Cordyceps sinensis bacteria for infecting hepialus larvae, so that collection and dependence on natural bacteria are reduced, the input cost of enterprises is reduced, and the natural bacteria are protected.
The technical scheme for solving the technical problems is as follows: a method for propagating Cordyceps sinensis bacteria infecting hepialus larvae, comprising the following steps:
1) Collecting natural strains;
2) Pretreatment: washing the strain acquired in the step 1) with water, and airing the surface moisture of the sub-seat for later use;
3) And (3) temporary planting: planting the pretreated strain in a planting box containing sterile coconut coir with the water content of 60%, and placing the planting box in a clean room with the temperature of 18-20 ℃ and the RH of 85-90% for cultivation;
4) Ascus shell collection: harvesting the ascus when the ascus is full in growth and the ascus protrudes;
5) Ascus shell planting: planting the ascus shells acquired in the step 4) in a collecting box containing pure water with the depth of 1-2cm, and placing the collecting box in a clean room with the temperature of 18-20 ℃ and the RH of 65-70% for culture;
6) And (3) strain collection: collecting the culture solution after planting and culturing in the step 5) in a concentrated manner every afternoon, placing the culture solution in a strain temporary storage barrel of 10kg, starting an oxygenation pump, and placing the culture solution in a clean room at 18-20 ℃ for 3 days;
7) And (3) strain preservation: mixing the bacterial liquid after being placed for 3 days in the step 6) with nutrient soil with the humidity of 65%, and then placing the mixture in a refrigerator with the temperature of about 4 ℃ for preservation.
Further, in step 1), the standard of collection of the natural strain is that the length of the ascus is not less than 3cm, the diameter of the ascus is not less than 3mm, and the stroma is not damaged by diseases and insect pests, is not sunken, and is not broken or folded.
Further, in step 2), the temperature of the water is controlled to be less than or equal to 20 ℃, and the temperature of the clean room is less than or equal to 20 ℃.
Further, in the step 3), the preparation method of the sterile coconut coir comprises the step of placing the coconut coir in 50kw for microwave sterilization for 30min to obtain the sterile coconut coir.
Further, in step 3), the specific requirements of the temporary planting are: the row spacing of the strain is 3 x 3cm, the sub-seats are not mutually overlapped and extruded, ventilation is carried out 2 times a day, and the illumination intensity is 800-1000lux.
Further, in step 4), the specific method for collecting the ascal shell is as follows: cutting the surface along the position 3cm below the base of the ascus shell by using sterilized scissors, and then cleaning impurities such as coconut chaff stained on the ascus shell by using pure water, wherein the temperature is less than or equal to 20 ℃, and airing the surface moisture.
Further, in step 5), the ascal shell planting method is to ensure that the base can contact the cultivation liquid, and the ascal shell remains dry.
The application at least comprises the following beneficial effects: the collection and dependence on natural strains are reduced, and the purposes of reducing the input cost of enterprises and protecting the natural strains are achieved; the culture method ensures that the ascus of the strain has better development, the ascus is thicker and longer, the number of ascus shells is further increased, and finally, the number of ascospores is greatly increased. The germination rate of the ascospores is improved by processing and preserving the bacterial liquid, and the number of the germination tubes of the unit ascospores is increased, so that the number of the conidiophores is further increased. The method mainly increases the yield per unit, and reduces the collection quantity of the strain under the requirement of collecting the conidium with the same purpose.
Detailed Description
The principles and features of the present application are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the application.
A method for propagating Cordyceps sinensis bacteria infecting hepialus larvae, comprising the following steps:
1) Collecting natural strains;
2) Pretreatment: washing the strain acquired in the step 1) with water, and airing the surface moisture of the sub-seat for later use;
3) And (3) temporary planting: planting the pretreated strain in a planting box containing sterile coconut coir with the water content of 60%, and placing the planting box in a clean room with the temperature of 18-20 ℃ and the RH of 85-90% for cultivation;
4) Ascus shell collection: harvesting the ascus when the ascus is full in growth and the ascus protrudes;
5) Ascus shell planting: planting the ascus shells acquired in the step 4) in a collecting box containing pure water with the depth of 1-2cm, and placing the collecting box in a clean room with the temperature of 18-20 ℃ and the RH of 65-70% for culture;
6) And (3) strain collection: collecting the culture solution after planting and culturing in the step 5) in a concentrated manner every afternoon, placing the culture solution in a strain temporary storage barrel of 10kg, starting an oxygenation pump, and placing the culture solution in a clean room at 18-20 ℃ for 3 days;
7) And (3) strain preservation: mixing the bacterial liquid after being placed for 3 days in the step 6) with nutrient soil with the humidity of 65%, and then placing the mixture in a refrigerator with the temperature of about 4 ℃ for preservation.
RH-relative humidity; the reason for controlling the water content of the sterile coconut coir to 60% is that the ascus is properly supplemented with water, the adverse effect of the water content exceeding 60% is easy to cause the decomposition and deterioration of thalli, and the adverse effect of the water content below 60% is that the ascus is not properly supplemented with water to cause wilting and death;
the reason why the culture temperature of the planting box in the step 3) is controlled at 18-20 ℃ and the relative humidity is controlled at 85-90% is to improve the survival rate of the strain, so that the ascus development is thicker and longer,
the reason why the temperature of the collection box culture in the step 5) is controlled at 18-20 ℃ and RH65-70% is that the ascus is in a proper temperature and humidity environment, which is beneficial to the ascus ejection of ascospores and the extension of the ejection time; the reason for placing the cultivation liquid in a clean room at 18-20 ℃ for 3 days is that ascospores germinate to emit a large number of bud tubes and generate conidia.
Further, in step 1), the standard of collection of the natural strain is that the length of the ascus is not less than 3cm, the diameter of the ascus is not less than 3mm, and the stroma is not damaged by diseases and insect pests, is not sunken, and is not broken or folded. The reason for adopting the natural strain collection standard is that on one hand, the mixed bacterial infection in the culture process is reduced, and on the other hand, the method is beneficial to culturing the robust ascus shell.
Further, in step 2), the temperature of the water is controlled to be less than or equal to 20 ℃, and the temperature of the clean room is less than or equal to 20 ℃. The reason for controlling the clean room temperature to be less than or equal to 20 ℃ is to provide the ascus with a proper survival temperature, and the adverse effect of being higher than 20 ℃ is to cause the death of the strain or the ascus.
Further, in the step 3), the preparation method of the sterile coconut coir comprises the step of placing the coconut coir in 50kw for microwave sterilization for 30min to obtain the sterile coconut coir.
Further, in step 3), the specific requirements of the temporary planting are: the row spacing of the strain is 3 x 3cm, the sub-seats are not mutually overlapped and extruded, ventilation is carried out 2 times a day, and the illumination intensity is 800-1000lux. The reason that the row spacing of the strains is controlled to be 3cm is that the strains are not extruded mutually, pollution caused by mixed bacteria generated by overlapped extrusion is avoided, 2 LED lamps with the intensity of 15w can be placed at the position 30cm above the planting box in specific operation with the illumination intensity of 800-1000lux, on one hand, the illumination intensity is provided, and on the other hand, the development degree of ascopes is convenient to observe.
Further, in step 4), the specific method for collecting the ascal shell is as follows: cutting the surface along the position 3cm below the base of the ascus shell by using sterilized scissors, and then cleaning impurities such as coconut chaff stained on the ascus shell by using pure water, wherein the temperature is less than or equal to 20 ℃, and airing the surface moisture. The reason for airing the surface moisture at the temperature of less than or equal to 20 ℃ is to ensure the activity of the ascus while the moisture on the surface of the ascus shell is quickly lost.
Further, in step 5), the ascal shell planting method is to ensure that the base can contact the cultivation liquid, and the ascal shell remains dry.
The following will describe in detail a proliferation method of Cordyceps sinensis infected with hepialus larva according to the present application with reference to examples, comparative examples and experimental data.
Example 1
1) Collecting natural strains: the length of the ascus shell is 3cm, the diameter of the ascus shell is 3mm, and the sub-seat has no plant diseases and insect pests, no dent and no fracture or crease;
2) Pretreatment: cleaning the strain acquired in the step 1) by using water with the temperature of 20 ℃, and airing the surface moisture of the sub-seat in a clean room with the temperature for later use;
3) And (3) temporary planting: planting the pretreated strain in a planting box containing sterile coconut coir with water content of 60%, wherein the plant row spacing of the strain is 3 x 3cm, the sub-bases are not mutually overlapped and extruded, ventilation is carried out for 2 times a day, 2 LED lamps with the weight of 15w are placed at the position 30cm above the planting box, and the planting box is placed in a clean room with the temperature of 18 ℃ and the RH of 85% for cultivation; the preparation method of the sterile coconut coir comprises the steps of placing the coconut coir at 50kw for microwave sterilization for 30min to obtain the sterile coconut coir;
4) Ascus shell collection: when the ascus shell is full in growth and the ascus is protruded, the ascus shell is collected by cutting along the position 3cm below the base of the ascus shell by using a pair of sterilized scissors, impurities such as coconut chaff and the like stained on the ascus shell are cleaned by pure water, and the surface moisture is dried at 20 ℃;
5) Ascus shell planting: planting the ascus shells acquired in the step 4) in a collecting box containing pure water with the depth of 1-2cm, ensuring that a base can be contacted with a cultivation liquid, keeping the ascus shells dry, and culturing in a clean room with the temperature of 18 ℃ and RH 65%;
6) And (3) strain collection: collecting the culture solution after planting and culturing in the step 5) in a concentrated manner every afternoon, placing the culture solution in a strain temporary storage barrel of 10kg, starting an oxygenation pump, and placing the culture solution in a clean room at 18-20 ℃ for 3 days;
7) And (3) strain preservation: mixing the bacterial liquid after being placed for 3 days in the step 6) with nutrient soil with the humidity of 65%, and then placing the mixture in a refrigerator with the temperature of about 4 ℃ for preservation.
Example 2
1) Collecting natural strains: the length of the ascus shell is 4cm, the diameter of the ascus shell is 3mm, and the sub-seat has no plant diseases and insect pests, no dent and no fracture or crease;
2) Pretreatment: cleaning the strain acquired in the step 1) by using water with the temperature of 16 ℃, and airing the surface moisture of the sub-seat in a clean room with the temperature for later use;
3) And (3) temporary planting: planting the pretreated strain in a planting box containing sterile coconut coir with water content of 60%, wherein the plant row spacing of the strain is 3 x 3cm, the sub-bases are not mutually overlapped and extruded, ventilation is carried out for 2 times a day, 2 LED lamps with the weight of 15w are placed at the position 30cm above the planting box, and the planting box is placed in a clean room with the temperature of 19 ℃ and the RH of 87% for cultivation; the preparation method of the sterile coconut coir comprises the steps of placing the coconut coir at 50kw for microwave sterilization for 30min to obtain the sterile coconut coir;
4) Ascus shell collection: when the ascus shell is full in growth and the ascus is protruded, the ascus shell is collected by cutting along the position 3cm below the base of the ascus shell by using a pair of sterilized scissors, impurities such as coconut chaff and the like stained on the ascus shell are cleaned by pure water, and the surface moisture is dried at the temperature of 16 ℃;
5) Ascus shell planting: planting the ascus shells acquired in the step 4) in a collecting box containing pure water with the depth of 1-2cm, ensuring that a base can be contacted with a culture solution, keeping the ascus shells dry, and culturing in a clean room with the temperature of 19 ℃ and the RH of 68%;
6) And (3) strain collection: collecting the culture solution after planting and culturing in the step 5) in a concentrated manner every afternoon, placing the culture solution in a strain temporary storage barrel of 10kg, starting an oxygenation pump, and placing the culture solution in a clean room at 19 ℃ for 3 days;
7) And (3) strain preservation: mixing the bacterial liquid after being placed for 3 days in the step 6) with nutrient soil with the humidity of 65%, and then placing the mixture in a refrigerator with the temperature of about 4 ℃ for preservation.
Example 3
1) Collecting natural strains: the length of the ascus shell is 5cm, the diameter of the ascus shell is 4mm, and the sub-seat has no plant diseases and insect pests, no dent and no fracture or crease;
2) Pretreatment: cleaning the strain acquired in the step 1) by using water with the temperature of 15 ℃, and airing the surface moisture of the sub-seat in a clean room with the temperature for later use;
3) And (3) temporary planting: planting the pretreated strain in a planting box containing sterile coconut coir with water content of 60%, wherein the plant row spacing of the strain is 3 x 3cm, the sub-bases are not mutually overlapped and extruded, ventilation is carried out for 2 times a day, 2 LED lamps with the weight of 15w are placed at the position 30cm above the planting box, and the planting box is placed in a clean room with the temperature of 20 ℃ and the RH of 90% for cultivation; the preparation method of the sterile coconut coir comprises the steps of placing the coconut coir at 50kw for microwave sterilization for 30min to obtain the sterile coconut coir;
4) Ascus shell collection: when the ascus shell is full in growth and the ascus is protruded, the ascus shell is collected by cutting along the position 3cm below the base of the ascus shell by using a pair of sterilized scissors, impurities such as coconut chaff and the like stained on the ascus shell are cleaned by pure water, and the surface moisture is dried at the temperature of 18 ℃;
5) Ascus shell planting: planting the ascus shells acquired in the step 4) in a collecting box containing pure water with the depth of 1-2cm, ensuring that a base can be contacted with a cultivation liquid, keeping the ascus shells dry, and culturing in a clean room with the temperature of 20 ℃ and the RH of 70%;
6) And (3) strain collection: collecting the culture solution after planting and culturing in the step 5) in a concentrated manner every afternoon, placing the culture solution in a strain temporary storage barrel of 10kg, starting an oxygenation pump, and placing the culture solution in a clean room at 20 ℃ for 3 days;
7) And (3) strain preservation: mixing the bacterial liquid after being placed for 3 days in the step 6) with nutrient soil with the humidity of 65%, and then placing the mixture in a refrigerator with the temperature of about 4 ℃ for preservation.
Sporulation of Cordyceps sinensis strains obtained in examples 1-3
The culture broth collected daily in step 6) of examples 1-3 was sampled after thoroughly mixing, sampled 3 times diagonally with a pipette, 0.033g each time, counted under a microscope for the number of spores, and the current subspore concentration was calculated as dilution factor. Germination monitoring was performed together with the number detection, the number of the sprout tubes per spore germination was counted by observation, and the germination rate was calculated, germination rate (%) = number of germination/number of spores, and the results are shown in the following table:
TABLE 1 detection of spore yield and germination rate
| Conidium number (x 10) 4 Personal computer | Germination rate (%) | |
| Example 1 | 140 | 55.04 |
| Example 2 | 155 | 62.86 |
| Example 3 | 150 | 49.67 |
Under a microscope, the germination of about 9 bud tubes of each spore can be observed;
as is clear from the above table, the spore yield of the individual strain obtained by the proliferation culture method of the present application was increased to about 150 ten thousand. The proliferation culture method of the application ensures that the ascus of the strain has better development, the ascus is thicker and longer, the number of ascus shells is further increased, and finally, the number of ascospores is greatly increased. By processing and preserving the bacterial liquid, the germination rate of the ascospores is improved, and meanwhile, the number of the germination tubes for the unit ascospores is increased, and the number of the conidiophores is further increased. The method mainly increases the yield per unit, and reduces the collection quantity of the strain under the requirement of collecting the conidium with the same purpose.
Although the embodiments of the present application have been disclosed above, it is not limited to the use as set forth in the specification and embodiments, it is fully applicable to the various fields of adaptation of the present application, and further modifications may be readily made by those skilled in the art without departing from the general concept defined by the appended claims and their equivalents, and the application is therefore not to be limited to the specific details and modifications of the application as may be made by those skilled in the art without departing from the spirit and scope of the application.
Claims (3)
1. A method of proliferating Cordyceps sinensis bacteria that infest hepialus larvae, comprising the steps of:
1) Collecting natural strains;
2) Pretreatment: cleaning the strain acquired in the step 1) by using water with the temperature less than or equal to 20 ℃, and airing the surface moisture of the sub-seat in a clean room with the temperature less than or equal to 20 ℃ for later use;
3) And (3) temporary planting: planting the pretreated strain in a planting box containing sterile coconut coir with the water content of 60%, and placing the planting box in a clean room with the temperature of 18-20 ℃ and the RH of 85-90% for cultivation; the specific requirements of the temporary planting are as follows: the plant row spacing of the strain is 3 x 3cm, the sub-seats are not mutually overlapped and extruded, ventilation is carried out for 2 times a day, and the illumination intensity is 800-1000lux;
4) Ascus shell collection: when the ascus shell is full in growth and the ascus is protruded, the disinfected scissors are used for cutting along the position 3cm below the base part of the ascus shell, then pure water is used for cleaning impurities such as coconut chaff and the like stained on the ascus shell, and the surface moisture is dried at the temperature of less than or equal to 20 ℃;
5) Ascus shell planting: planting the ascus shells acquired in the step 4) in a collecting box containing pure water with the depth of 1-2cm, ensuring that a base can be contacted with a culture solution, keeping the ascus shells dry, and placing the collecting box in a clean room with the temperature of 18-20 ℃ and the RH of 65-70% for culturing;
6) And (3) strain collection: collecting the culture solution after planting and culturing in the step 5) in a concentrated manner every afternoon, placing the culture solution in a strain temporary storage barrel of 10kg, starting an oxygenation pump, and placing the culture solution in a clean room at 18-20 ℃ for 3 days;
7) And (3) strain preservation: mixing the bacterial liquid after being placed for 3 days in the step 6) with nutrient soil with the humidity of 65%, and then placing the mixture in a refrigerator with the temperature of about 4 ℃ for preservation.
2. The method according to claim 1, wherein in step 1), the natural strain is collected according to the standard of not less than 3cm in length of ascus shell, not less than 3mm in diameter of ascus shell, and the stroma is free from plant diseases and insect pests, dent, break or crease.
3. The method for proliferating Cordyceps sinensis infected with hepialus larva according to claim 1, wherein in step 3), the method for preparing the sterilized coconut coir is to sterilize the coconut coir with 50kw microwave for 30min to obtain the sterilized coconut coir.
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| CN105941340A (en) * | 2016-05-25 | 2016-09-21 | 浙江农林大学 | An artificial culture method for ghost moth larvae, the hosts of ophiocordyceps sinensis |
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