Artificial cultivation method for cordyceps sinensis sporocarp

Abstract

The invention relates to the technical field of biotechnology, in particular to an artificial cultivation method of cordyceps sinensis sporocarp, which comprises the following steps: placing cordyceps sinensis in a liquid fermentation culture medium for fermentation to obtain cordyceps sinensis conidia liquid; the method comprises the following steps: injecting a cordyceps sinensis fungus conidium solution to the surface of a hepialus armoricanus larva body to infect the larva, placing the injected hepialus armoricanus larva in a culture medium simulating natural ecological conditions, and breeding in a first artificial climate box to obtain bombyx batryticatus; putting the bombyx batryticatus in a substrate simulating natural ecological conditions, covering the bombyx batryticatus with a layer of substrate, and culturing in a second artificial climate box to obtain the bombyx batryticatus overgrown with fungus coats; and planting the bombyx batryticatus overgrown with the fungus coats in soil, and culturing in a third artificial climate box to obtain cordyceps sinensis sporocarps. The cordyceps sinensis cultivated by the method is short in production period and low in heavy metal content, and can be produced on a large scale.

Description

Technical Field

[0001] The invention relates to the field of biotechnology cultivation, and in particular to an artificial cultivation method for cordyceps sinensis fruiting bodies. Background Art

[0002] Cordyceps sinensis is a fungus-insect complex formed by the Cordyceps sinensis fungus infecting the larvae of the bat moth (Hepialus spp.), that is, a dead insect body infected by the fungus. Its growth process includes: Cordyceps sinensis eggs are produced in autumn and hatch into larvae; the hatched larvae parasitize the body of a specific moth larva; with the arrival of spring, the larvae absorb nutrients from the moth larvae so that the hyphae occupy the moth larvae and finally completely replace the organs in the moth larvae. At this time, the moth larvae can no longer survive, and the larvae also become mycelium; the mycelium gradually condenses energy and begins to stretch out fruiting bodies, which drill out from the head of the moth larvae; when the spores on the fruiting bodies of Cordyceps sinensis mature, they are spread by the wind to the surrounding environment to find new hosts.

[0003] Since Cordyceps sinensis is a precious medicinal material with high economic value, the demand for Cordyceps sinensis is relatively large. However, due to the ecological fragility of the Qinghai-Tibet Plateau and the strict requirements of the environment and host for the growth of Cordyceps sinensis, the yield of Cordyceps sinensis is not high. Therefore, artificial cultivation of Cordyceps sinensis has become an inevitable choice.

[0004] At present, the artificial cultivation technology of Cordyceps sinensis is affected by factors such as the reproduction of infected host insects, spore cultivation and the efficient artificial induction of Cordyceps sinensis fruiting bodies, resulting in its production cost still being relatively high. It has no obvious advantages over naturally collected Cordyceps sinensis and urgently needs further breakthroughs. Summary of the invention

[0005] The present invention aims to provide a method for artificially cultivating Cordyceps sinensis fruiting bodies, so as to solve the above-mentioned technical problems.

[0006] To achieve the above object, the present invention provides a method for artificially cultivating Cordyceps sinensis fruiting bodies, the method comprising:

[0007] placing the Cordyceps sinensis in a liquid fermentation medium for fermentation to obtain a Cordyceps sinensis conidia liquid;

[0008] The conidia liquid of Cordyceps sinensis is injected into the body surface of bat moth larvae to infect the larvae, and the injected bat moth larvae are placed in a culture medium simulating natural ecological conditions, and dead insects are obtained after being raised in a first artificial climate box;

[0009] Placing the dead insects in a substrate simulating natural ecological conditions, and covering the dead insects with a layer of substrate, and obtaining dead insects covered with fungus coats after culturing in a second artificial climate chamber;

[0010] The dead insects covered with fungus coat are planted in soil, and the Cordyceps sinensis fruiting bodies are obtained after being cultured in a third artificial climate chamber.

[0011] Technical effects and advantages of the present invention:

[0012] 1. Highly efficient induction and cultivation of Cordyceps sinensis conidia liquid. The special conidia fermentation medium designed by the present invention has high efficiency and low cost induction efficiency of Cordyceps sinensis conidia, and can be industrialized and expanded for application on a large scale.

[0013] 2. Cordyceps sinensis infects bat moth larvae with conidia in a simulated ecological manner. The present invention can efficiently induce the production of infected dead insects by simulating the manner and ecological conditions of Cordyceps sinensis infecting bat moth larvae under natural habitat conditions, and has excellent potential for commercial development.

[0014] 3. Obtain Cordyceps sinensis fruiting bodies based on artificial ecological induction technology system. The present invention simulates the native growth environment and ecological conditions of Cordyceps sinensis fruiting bodies, such as culture medium, temperature, humidity and light conditions, and can mass-produce high-quality Cordyceps sinensis precious traditional Chinese medicine products with excellent economic value.

[0015] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 This is a flow chart of the artificial cultivation method of Cordyceps sinensis;

[0018] Figure 2 This is a picture of the conidia of Cordyceps sinensis;

[0019] Figure 3 This is a picture of bat moth larvae infected with Cordyceps sinensis;

[0020] Figure 4 This is a picture of the fruiting body of Cordyceps sinensis. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention, so they have no substantial technical significance. Any modification of the structure, change of the proportion relationship or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the effects and purposes that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the present invention. The change or adjustment of their relative relationship should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0023] In order to solve the deficiencies of the prior art, the present invention discloses a method for artificially cultivating Cordyceps sinensis, such as Figure 1 As shown, the method includes:

[0024] 1. Place the Cordyceps sinensis in a liquid fermentation medium for fermentation to obtain a Cordyceps sinensis conidia liquid.

[0025] Specifically, the method includes: inoculating the Cordyceps sinensis revived on the PDA plate into the liquid fermentation medium to obtain the Cordyceps sinensis conidia liquid, measuring the Cordyceps sinensis conidia liquid and cultivating it in a 500 ml glass triangular flask for shake flask fermentation, and finally obtaining the Cordyceps sinensis conidia liquid.

[0026] The conditions and parameters of the rotary fermentation include: a rotation rate of 100-200 rpm and a constant temperature of 10-16°C for 35-50 days.

[0027] Preferably, the parameters of the rotary fermentation conditions are: a rotation rate of 150 rpm, a constant temperature of 14°C for 40 days, during which samples are taken regularly to observe the spore production, and finally a Cordyceps sinensis conidia liquid is obtained, such as Figure 2 shown.

[0028] The formula of the fermentation medium is as follows: 10-20g of carbohydrate carbon source, 100-200g of filtered juice after boiling potato juice, 5-20g of nitrogen source, 5-10g of insect body (for stimulating spore production), 3-6g of potassium source, 1-5g of magnesium source, 0.02-0.1g of vitamins, and 0.2-1g of amino acids. After dissolving, the volume is adjusted to 1L with double distilled water, and the liquid fermentation medium is obtained after sterilization.

[0029] Preferably, the specific formula of the fermentation medium is: 15g glucose (or maltose), 150g filtered juice after boiling potato juice, 15g peptone, 6g moth larvae, 5g potassium dihydrogen phosphate, 4g magnesium sulfate heptahydrate, 0.05g vitamin B1, and 0.6g proline.

[0030] 2. Injecting the conidia of Cordyceps sinensis onto the surface of bat moth larvae to infect the larvae, placing the injected bat moth larvae in a culture medium simulating natural ecological conditions, and raising them in a first artificial climate chamber to obtain dead insects.

[0031] Specifically, this step is a key step related to the successful artificial cultivation of Cordyceps sinensis. This study is based on the ecological conditions of the Cordyceps sinensis grassland habitat and formulates the following production process. The cultured Cordyceps sinensis conidia liquid is filtered through three layers of sterile lens paper, the filtrate is centrifuged (centrifugation conditions are 8000 rpm, 10°C, 15min), the supernatant is discarded, the solid is resuspended with a buffer (preferably, a sterile phosphate buffer at pH 7.0), and the collected conidia liquid is diluted to 1-8×10 6 / mL (preferably, 2×10 6 / mL).

[0032] Select 5th instar bat moth larvae (preferably, Hepialus xiaojinensis larvae), inject the diluted conidia solution onto the surface of the Hepialus xiaojinensis larvae to infect the larvae, and spray the diluted conidia solution onto the surface of the Hepialus xiaojinensis larvae. Preferably, a micro-syringe is used to inject the larvae onto the surface of the insect body, and the injection amount is determined according to the size of the insect body. Then, the diluted conidia solution is sprayed onto the surface of the insect body. When the diluted conidia solution is slightly dry on the insect body, the larvae are returned to the culture medium simulating natural ecological conditions for continued feeding, and regular observation is performed until dead insects ( Figure 3 ). The present invention adopts the above-mentioned technical method to make the dead insect production rate of bat moth larvae infected by Cordyceps sinensis reach more than 80%, and has good potential for large-scale commercial application.

[0033] The formula of the culture medium simulating natural ecological conditions includes: 30-50% by mass of a breathable matrix, 40-60% by mass of soil, 5-20% by mass of food, and sterilization treatment to prevent contamination.

[0034] Preferably, the formula of the culture medium under simulated natural ecological conditions is: 40% by mass of sawdust matrix, 50% by mass of humus soil, 10% by mass of carrot or Polygonum aviculare or Polygonum villosa, or a mixture of any two or more thereof (e.g., 10% by mass of carrot, Polygonum aviculare and Polygonum villosa mixed in equal proportions).

[0035] The parameters for raising the fish in the first artificial climate box include: temperature of 12-15°C, time of 10-20 days, and relative air humidity of 80-90%. Preferably, the parameters for raising the fish in the first artificial climate box are: temperature of 14°C, time of 15 days, and relative air humidity of 88%.

[0036] 3. Place the dead insects in a substrate simulating natural ecological conditions, and cover the dead insects with a layer of substrate, and cultivate them in a second artificial climate chamber to obtain dead insects covered with fungus coats.

[0037] Specifically, the present invention is based on the ecological environment conditions of Cordyceps sinensis and specially formulates the following simulated ecological cultivation means: Put a suitable special sterilized matrix in a special container, lay the dead insects flat in the matrix, cover it with a layer of matrix, and cultivate it in a second artificial climate box to obtain dead insects covered with fungus.

[0038] The formula of the matrix simulating natural ecological conditions includes: 40-60% by mass of a breathable matrix, 40-60% by mass of soil, and sterilization treatment to prevent contamination.

[0039] Preferably, the formula of the matrix simulating natural ecological conditions is: 50% by mass of sawdust matrix and 50% by mass of humus soil.

[0040] The culture parameters in the second artificial climate box include: temperature of 12-15° C., culture time of 30-40 days, relative air humidity of 80-90%, and carbon dioxide concentration of 2-5%.

[0041] Preferably, the culture parameters in the second artificial climate box include: temperature of 14° C., relative air humidity of 85%, carbon dioxide concentration of 4%, and dim light irradiation for 35 days to remove the dead insects covered with fungus coat.

[0042] The thickness of the substrate covering the dead insects is 2-3 cm, preferably 2.3 cm.

[0043] 4. Plant the dead insects covered with fungus coat in the soil, and obtain Cordyceps sinensis fruiting bodies by culturing in the third artificial climate chamber.

[0044] Specifically, the method includes: placing the dead insects covered with fungus coat horizontally in the soil, with the soil slightly covering the dead insects' heads, and obtaining the Cordyceps sinensis fruiting bodies by culturing in a third artificial climate chamber.

[0045] The height of the soil above the dead insect heads covered with fungus coat is 0.2-1 cm, preferably 0.5 cm.

[0046] The culture parameters in the third artificial climate box include: temperature of 12-15° C., culture time of 60-80 days, and relative air humidity of 80-90%.

[0047] Preferably, the culture parameters in the third artificial climate box include: temperature of 14°C, relative air humidity of 85%, dim light irradiation, and culture for 70 days to obtain good-quality Cordyceps sinensis fruiting bodies ( Figure 4 ).

[0048] Through the ecologically efficient induction method of Cordyceps sinensis of the present invention, the production cycle of artificially cultivated Cordyceps sinensis products under optimal condition parameters can be shortened to 12 months (at least 2-3 years under natural conditions). In addition, the Cordyceps sinensis has good appearance, and the appearance, weight (about 0.9 grams) and the content of effective ingredients such as cordyceps adenosine, cordycepic acid, and cordyceps polysaccharide meet national standards. In addition, its heavy metal content (mercury, lead, copper and cadmium) is also far lower than the national standard, as shown in Table 1. Therefore, the technical system of the present invention can stably produce high-quality Cordyceps sinensis precious Chinese medicine products on a large scale, with excellent economic value.

[0049] Table 1 Components test data of artificially cultivated Cordyceps sinensis

[0050]

[0051]

[0052] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for artificially cultivating Cordyceps sinensis fruiting bodies, characterized in that: The method comprises: placing the Cordyceps sinensis in a liquid fermentation medium for fermentation to obtain a Cordyceps sinensis conidia liquid; The conidia liquid of Cordyceps sinensis is injected into the body surface of bat moth larvae to infect the larvae, and the injected bat moth larvae are placed in a culture medium simulating natural ecological conditions, and dead insects are obtained after being raised in a first artificial climate box; Placing the dead insects in a substrate simulating natural ecological conditions, and covering the dead insects with a layer of substrate, and obtaining dead insects covered with fungus coats after culturing in a second artificial climate chamber; The dead insects covered with fungus coat are planted in soil, and the Cordyceps sinensis fruiting bodies are obtained after being cultured in a third artificial climate chamber.

2. The method according to claim 1, characterized in that The Cordyceps sinensis is placed in a liquid fermentation medium for fermentation to obtain a Cordyceps sinensis conidia liquid, including: preparing raw materials according to the formula of the fermentation medium, and obtaining a liquid fermentation medium by dissolving; The Cordyceps sinensis is placed in a liquid fermentation medium for rotational fermentation to obtain a Cordyceps sinensis conidia liquid.

3. The method according to claim 2, characterized in that The formula of the fermentation medium includes: 10-20g of carbohydrate carbon source, 100-200g of filtered juice after boiling potato juice, 5-20g of nitrogen source, 5-10g of insect body, 3-6g of potassium source, 1-5g of magnesium source, 0.02-0.1g of vitamins and 0.2-1g of amino acids.

4. The method according to claim 3, characterized in that The formula of the fermentation medium includes: 15g of glucose or maltose, 150g of filtered juice after boiling potato juice, 15g of peptone, 6g of moth larvae, 5g of potassium dihydrogen phosphate, 4g of magnesium sulfate heptahydrate, 0.05g of vitamin B1, and 0.6g of proline.

5. The method according to claim 2, characterized in that: The conditions and parameters of the rotary fermentation include: a rotation rate of 100-200 rpm and a constant temperature of 10-16°C for 35-50 days.

6. The method according to claim 1, characterized in that Before placing the injected bat moth larvae in a culture medium simulating natural ecological conditions, the following steps are also included: filtering the Cordyceps sinensis conidia liquid to obtain a solid; The solid was resuspended with buffer and the collected conidia were diluted; The diluted conidia solution is injected onto the body surface of bat moth larvae to infect the larvae, and the diluted conidia solution is sprayed onto the body surface of bat moth larvae.

7. The method according to claim 1, characterized in that The formula of the culture medium under the simulated natural ecological conditions includes: 30-50% by mass of a breathable matrix, 40-60% by mass of soil, and 5-20% by mass of food.

8. The method according to claim 7, characterized in that The formula of the culture medium under the simulated natural ecological conditions includes: 40% by mass of sawdust matrix, 50% by mass of humus soil, 10% by mass of carrot or Polygonum foetida or Polygonum bulbilum or a mixture of any two or more thereof.

9. The method according to claim 1, characterized in that: The parameters of the conditions for raising the chickens in the first artificial climate box include: a temperature of 12-15° C., a time of 10-20 days, and an air relative humidity of 80-90%.

10. The method according to claim 1, characterized in that The formula of the substrate simulating natural ecological conditions includes: 40-60% by mass of an air-permeable substrate and 40-60% by mass of soil.

11. The method according to claim 10, characterized in that The formula of the substrate simulating natural ecological conditions includes: 50% by mass of sawdust substrate and 50% by mass of humus soil.

12. The method according to claim 1, characterized in that The culture parameters in the second artificial climate box include: temperature of 12-15° C., culture time of 30-40 days, relative air humidity of 80-90%, and carbon dioxide concentration of 2-5%.

13. The method according to claim 1, characterized in that The thickness of the substrate covering the dead insects is 2-3 cm; the height of the soil covering the dead insect heads covered with fungus coat is 0.2-1 cm.

14. The method according to claim 1, characterized in that The culture parameters in the third artificial climate box include: temperature of 12-15°C, culture time of 60-80 days, and relative air humidity of 80-90%.

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