A method for artificially cultivating Cordyceps sinensis fruiting bodies

Abstract

The present invention relates to the field of biotechnology, and in particular to a method for artificially cultivating Cordyceps sinensis fruiting bodies, comprising: placing Cordyceps sinensis fungi in a liquid fermentation medium for fermentation to obtain a conidia solution; injecting the conidia solution onto the surface of a bat moth larva to infect the larvae; placing the injected bat moth larvae in a medium simulating natural ecological conditions, and raising them in a first artificial climate chamber to obtain dead larvae; placing the dead larvae in a substrate simulating natural ecological conditions, and covering the dead larvae with a layer of substrate; and culturing them in a second artificial climate chamber to obtain dead larvae covered with a fungus coat; and planting the dead larvae covered with a fungus coat in soil, and culturing them in a third artificial climate chamber to obtain Cordyceps sinensis fruiting bodies. The Cordyceps sinensis cultivated by this method has a short production cycle, low heavy metal content, and can be produced on a large scale.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology cultivation, and in particular to a method for artificially cultivating Cordyceps sinensis fruiting bodies. Background Art

[0002] Cordyceps sinensis is a fungus-like complex formed when the Cordyceps sinensis fungus infects the larvae of the bat moth (Hepialus spp.), a parasitic insect that has become inert after being infected by the fungus. Its growth process involves the following steps: Cordyceps sinensis eggs are laid in autumn and hatch into larvae. These larvae then parasitize specific moth larvae. With the arrival of spring, the larvae absorb nutrients from the moth larvae, allowing the fungus' hyphae to take over the larvae's body, eventually completely replacing the larvae's organs. The moth larvae are no longer viable, and the larvae themselves become mycelium. The mycelium gradually gathers energy and begins to develop into a fruiting body, which emerges from the moth larva's head. When the spores on the fruiting body mature, they are dispersed by wind into the surrounding environment, seeking new hosts.

[0003] Because Cordyceps sinensis is a precious medicinal herb with high economic value, it is in high demand. However, due to the ecological fragility of the Qinghai-Tibet Plateau where it grows and its stringent requirements for both the environment and the host, its production is limited. Therefore, artificial cultivation of Cordyceps sinensis has become an inevitable option.

[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 artificial and efficient 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 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 of Cordyceps sinensis are injected into the surface of the bat moth larvae to infect the larvae, and the injected bat moth larvae are placed in a culture medium simulating natural ecological conditions, and then the bat moth larvae are raised in a first artificial climate chamber to obtain dead insects;

[0009] The dead insects are placed in a substrate simulating natural ecological conditions, and a layer of substrate is covered on the dead insects, and dead insects covered with fungus coats are obtained after being cultured in a second artificial climate chamber;

[0010] The dead insects covered with fungus coat are planted in soil and cultured in a third artificial climate chamber to obtain Cordyceps sinensis fruiting bodies.

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

[0012] 1. Highly efficient induction and cultivation of Cordyceps sinensis conidia. The special conidia fermentation medium designed in the present invention has high efficiency and low cost in inducing conidia of Cordyceps sinensis and can be applied in large-scale industrialization.

[0013] 2. Cordyceps sinensis uses conidia to infect bat moth larvae in an ecologically simulated manner. This invention simulates the natural habitat conditions and conidia of Cordyceps sinensis to infect bat moth larvae, effectively inducing the production of infected dead insects and has excellent potential for commercial development.

[0014] 3. Producing Cordyceps sinensis fruiting bodies using an artificial ecological induction technology system. This method simulates the native growth environment and ecological conditions of Cordyceps sinensis fruiting bodies, such as culture medium, temperature, humidity, and light conditions, enabling large-scale production of high-quality Cordyceps sinensis, a valuable traditional Chinese medicine 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 obvious 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 is a brief introduction to 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 any 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 Cordyceps sinensis conidia;

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

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

[0021] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts 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 understanding and reading by those familiar with this technology, and are not used to limit the conditions for implementation of the present invention. Therefore, they have no substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the efficacy and purpose that can be achieved by the present invention. At the same time, 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 implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of 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. Figure 1 As shown, the method includes:

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

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

[0026] The conditions for 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 rotary fermentation condition parameters 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 solution is obtained, such as Figure 2 shown.

[0028] The fermentation medium formula 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 bodies (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 dissolution, the volume is adjusted to 1L with double distilled water, and sterilized to obtain a liquid fermentation medium.

[0029] Preferably, the specific formula of the fermentation medium is: 15g glucose (or maltose), 150g filtered juice from boiled 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 the 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 in the successful artificial cultivation of Cordyceps sinensis. This study is based on the ecological conditions of the Cordyceps sinensis grassland habitat and developed 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, 15 minutes), and the supernatant is discarded. The solid is resuspended in a buffer solution (preferably, a sterile phosphate buffer solution 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, use a microsyringe to inject the larvae onto the surface of the insect body to infect the larvae, and determine the injection amount according to the size of the insect body. Then spray the diluted conidia solution onto the surface of the insect body. Wait until the diluted conidia solution is slightly dry on the insect body, then put the larvae back into the culture medium simulating natural ecological conditions and continue to feed them. Regular observation is conducted until dead insects ( Figure 3 The present invention adopts the above-mentioned technical method to enable the dead insect production rate of bat moth larvae infected with Cordyceps sinensis to 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, and 5-20% by mass of food, which is sterilized 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 truncatum or Polygonum umbellatum, or a mixture of any two or more thereof (e.g., 10% by mass of an equal proportion of carrot, Polygonum truncatum, and Polygonum truncatum).

[0035] The parameters for the breeding conditions in the first artificial climate box include: a temperature of 12-15°C, a duration of 10-20 days, and a relative humidity of 80-90%. Preferably, the parameters for the breeding conditions in the first artificial climate box are: a temperature of 14°C, a duration of 15 days, and a relative 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. Cultivate the dead insects covered with fungus coats in a second artificial climate chamber.

[0037] Specifically, the present invention utilizes the following ecologically simulated cultivation methods based on the natural environment of Cordyceps sinensis. A suitable sterilized substrate is placed in a specially designed container, and the dead insects are placed flat on the substrate. A layer of substrate is then added to the substrate, and the dead insects are cultured in a second artificial climate chamber to obtain dead insects covered with a fungus coat.

[0038] The formula of the matrix simulating natural ecological conditions includes: a breathable matrix with a mass fraction of 40-60%, a soil with a mass fraction of 40-60%, and is sterilized 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 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 humidity of 85%, carbon dioxide concentration of 4%, and dim light irradiation for 35 days before removing 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 a third artificial climate chamber.

[0044] Specifically, the method includes: placing the dead insects covered with fungus coats horizontally and planting them in the soil, with the soil slightly covering the dead insect heads; and obtaining the Cordyceps sinensis fruiting bodies by culturing them 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 chamber include: temperature of 12-15° C., culture time of 60-80 days, and relative humidity of 80-90%.

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

[0048] Through the present invention's eco-friendly and efficient induction method for Cordyceps sinensis, the production cycle of artificially cultivated Cordyceps sinensis products can be shortened to 12 months (at least 2-3 years under natural conditions) under optimal conditions and parameters. Furthermore, the Cordyceps sinensis has excellent appearance, with its appearance, individual weight (approximately 0.9 grams), and the content of active ingredients such as cordyceps adenosine, cordycepic acid, and cordyceps polysaccharides meeting national standards. Furthermore, its heavy metal content (mercury, lead, copper, and cadmium) is also well below national standards, as shown in Table 1. Therefore, the present invention's technical system enables large-scale, stable production of high-quality Cordyceps sinensis, a valuable traditional Chinese medicine, with excellent economic value.

[0049] Table 1 Component detection 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, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or 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 scope of protection 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; Injecting a conidia solution 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 larvae. The culture medium simulating natural ecological conditions comprises: a breathable substrate (30-50% by weight), soil (40-60% by weight), and food (5-20% by weight). The raising conditions in the first artificial climate chamber include: a temperature of 12-15°C, a duration of 10-20 days, and a relative humidity of 80-90%. The dead insects are placed in a substrate simulating natural ecological conditions and covered with a layer of substrate. After culturing in a second artificial climate chamber, dead insects covered with fungus coats are obtained. The formulation of the substrate simulating natural ecological conditions includes: a breathable substrate with a mass fraction of 40-60% and soil with a mass fraction of 40-60%. The culturing parameters in the second artificial climate chamber include: a temperature of 12-15°C, a culture time of 30-40 days, a relative humidity of 80-90%, and a carbon dioxide concentration of 2-5%. The dead insects covered with fungus coat are planted in the soil and cultured in a third artificial climate chamber to obtain Cordyceps sinensis fruiting bodies; the culture parameters in the third artificial climate chamber include: temperature of 12-15°C, culture time of 60-80 days, and relative air humidity of 80-90%.

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 raw materials; 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: 10-20 g of carbohydrate carbon source, 100-200 g of filtered juice after boiling potato juice, 5-20 g of nitrogen source, 5-10 g of insect body, 3-6 g of potassium source, 1-5 g of magnesium source, 0.02-0.1 g of vitamins, and 0.2-1 g of amino acids.

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

5. The method according to claim 2, characterized in that The fermentation conditions 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 in buffer and the collected conidia were diluted; The diluted conidia solution is injected onto the body surface of the bat moth larvae to infect the larvae, and the diluted conidia solution is sprayed onto the body surface of the 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: 40% by mass of sawdust matrix, 50% by mass of humus soil, 10% by mass of carrot or Polygonum rotundum or Polygonum bulbilum or a mixture of any two or more thereof.

8. The method according to claim 1, characterized in that The formula of the matrix simulating natural ecological conditions includes: 50% by mass of sawdust matrix and 50% by mass of humus soil.

9. 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.

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