Cordyceps sinensis cultivation soil and control method thereof

Abstract

The present application relates to the field of Ophiocordyceps sinensis cultivation technology, and in particular to an Ophiocordyceps sinensis cultivation soil and a control method thereof, comprising a first-stage cultivation soil and a second-stage cultivation soil; the first-stage cultivation soil is composed of the following components in parts by weight: 60-80 parts of coconut husk, 1-10 parts of perlite, and 0.5-3 parts of nutrient solution; the second-stage cultivation soil comprises a base layer, a water storage layer, a nutrient layer, and a grass layer on the topmost layer; the nutrient layer is composed of the following components in parts by weight: 40-50 parts of coconut husk, 1-5 parts of perlite, 10-15 parts of humus, 0.5-3 parts of nutrient solution, and 1-5 parts of wood ash. The present application prepares soils for different cultivation stages, artificially controls the growth soil of Ophiocordyceps sinensis, and improves the quality of artificially cultivated Ophiocordyceps sinensis to be equivalent to that of wild Ophiocordyceps sinensis, and enhances the content of effective ingredients in Ophiocordyceps sinensis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of Ophiocordyceps sinensis cultivation technology, in particular to an Ophiocordyceps sinensis cultivation soil and a control method thereof. BACKGROUND

[0002] Ophiocordyceps sinensis is also known as Xiaocaodongchong and Chongcao, which is connected by a worm body and a fruiting body. The host of wild Ophiocordyceps sinensis is bat moth, which generally grows in the alpine meadow zone at an altitude of 3650-4250 meters, with a slope of 15-30 degrees, an air temperature of 0-23℃, a ground temperature of 3-7.5℃ at a depth of 20 cm, an air relative humidity of 50%-70%, and a soil humidity of 40%-60%. The life history of bat moth: the larval stage is the longest, which needs to undergo pupation for 2 years, and generally distributes below 10 cm deep in the soil. In late May, the larvae partially form pupae, and most of them become moths in early August. The eggs are laid on the soil surface, and hatch into the soil after about 2 months. Artificial planting of Ophiocordyceps sinensis cannot be separated from the natural environment of the growth of Ophiocordyceps sinensis.

[0003] At present, artificially bred Ophiocordyceps sinensis has weak resistance and is easily infected by microorganisms, which is an important factor restricting the development of Ophiocordyceps sinensis resources. In addition, the previous research of the research group found that the protein content and species of wild Ophiocordyceps sinensis are higher than those of artificially bred Ophiocordyceps sinensis, and the contents of polysaccharide, cordycepin and adenosine also differ, which indicates that the difference in growth environment may have an important impact on Ophiocordyceps sinensis. By comparing the microecological environment of wild Ophiocordyceps sinensis and artificially bred Ophiocordyceps sinensis, it is not difficult to find that in the natural environment, whether it is the host of Ophiocordyceps sinensis, bat moth larvae, or the main place for the development of Ophiocordyceps sinensis, soil, all contain more abundant microbial communities. Artificially bred Ophiocordyceps sinensis generally feeds larvae in a sterile environment, and the feed and soil need to be sterilized in advance, while in the field environment, the larvae feed on complex food, and the soil microorganisms are rich.

[0004] There is almost no related report on the endophytic bacteria of artificial Ophiocordyceps sinensis and the soil microbial community under artificial conditions, and there are few related reports on the influence of the soil environment of wild and artificially bred Ophiocordyceps sinensis on the growth and quality of Ophiocordyceps sinensis. Therefore, we have carried out in-depth research in this direction in order to provide an artificial breeding technology for Ophiocordyceps sinensis, which can effectively improve the survival rate and the quality of Ophiocordyceps sinensis and ensure the nutritional value of artificially bred Ophiocordyceps sinensis. SUMMARY

[0005] The technical problem to be solved by the present application is to provide an Ophiocordyceps sinensis cultivation soil and a control method thereof, which can prepare soil for different cultivation periods, artificially control the growth soil of Ophiocordyceps sinensis, improve the quality of artificially bred Ophiocordyceps sinensis to be equivalent to that of wild Ophiocordyceps sinensis, and increase the content of effective ingredients in Ophiocordyceps sinensis.

[0006] In order to solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0007] A cordyceps cultivation soil, comprising a first-stage cultivation soil and a second-stage cultivation soil; the first-stage cultivation soil is composed of the following components in parts by weight: 60-80 parts of coconut husk, 1-10 parts of perlite and 0.5-3 parts of nutrient solution; the second-stage cultivation soil comprises a base layer, a water storage layer, a nutrient layer and a topmost turf layer; the nutrient layer is composed of the following components in parts by weight: 40-50 parts of coconut husk, 1-5 parts of perlite, 10-15 parts of humus, 0.5-3 parts of nutrient solution and 1-5 parts of wood ash; the humus is premixed with lactarius ectomycorrhizal fungal spores, wherein the amount of lactarius ectomycorrhizal fungal spores is 1-3% of the total weight of the humus.

[0008] Preferably, the nutrient solution is specifically prepared from the following components: 2% of glucose, 0.5% of proteose peptone, 3% of soluble starch, 0.2% of potassium dihydrogen phosphate, 0.2% of magnesium sulfate, 10 mg of vitamin B1 and water per 100 mL of the nutrient solution.

[0009] Preferably, the base layer is composed of the following components in parts by weight: 5-10 parts of bamboo charcoal particles, 5-15 parts of perlite, 20-30 parts of rock sand, 10-15 parts of coconut husk and 20-30 parts of straw fiber and polylactic acid degradation compound; wherein the weight ratio of straw fiber to polylactic acid is 15:1.

[0010] Preferably, the water storage layer is composed of the following components in parts by weight: 20-30 parts of pottery clay, 3-7 parts of chitosan / polyacrylic acid composite hydrogel and 5-8 parts of rock sand.

[0011] Preferably, the turf layer is planted by cross-distribution of the seedlings of pinus taeda and pennisetum clandestinum, and the distance between the seedlings of pinus taeda and pennisetum clandestinum is not less than 15 cm.

[0012] Preferably, the humus is mixed from animal humus and plant humus in a mass ratio of 1:7-9, the animal humus is mixed from cow dung and shell powder in a mass ratio of 20:1, and the plant humus is made by adding a fermentation agent to wood chips, corn cobs, starch, pinus taeda and pennisetum clandestinum in a mass ratio of 3:5:2:7:2 for composting fermentation.

[0013] A control method of a cordyceps cultivation soil, specifically comprising the following steps:

[0014] S1, placing the first-stage cultivation soil in a dry and ventilated container after sterilization, and placing the larvae infected with mycelium by artificial injection into the first-stage cultivation soil, controlling the soil moisture to be 70-80%, the environmental temperature to be 15-25℃ and the environmental humidity to be 75-85%, until the strobilus of the larvae germinates;

[0015] S2, the second stage of breeding soil layers are prepared for disinfection treatment, and then laid in a dry and ventilated container in the order of the basement layer, the water storage layer, the nutrient layer and the turf layer from bottom to top, the step S1 sub-germination of the insect body is transplanted to the second stage of breeding soil, the lower end of the insect body is located in the nutrient layer, the water content of the nutrient layer and the turf layer is controlled at 70~80%, the environmental temperature is 15~30℃, the environmental humidity is 75~85%, until the insect grass is grown;

[0016] S3, when the insect grass in step S2 grows to the ascospore maturation stage, the uninfected bat moth larvae are laid in the second stage of breeding soil for natural infection, forming an ecological self-cycle;

[0017] S4, the larvae that are naturally infected in step S3 are transplanted to the first stage of breeding soil, the cultivation steps of steps S1 and S2 are repeated, and the nutrient solution is supplemented in time.

[0018] Preferably, the preparation thickness of the second stage of breeding soil layers is specifically: 1~2cm of the basement layer, 2~3cm of the water storage layer, 10~20cm of the nutrient layer, and the height of the pine and the centipede grass seedlings in the turf layer is controlled within 3~15cm.

[0019] Preferably, the height of the pine and the centipede grass in the turf layer in step S2 is controlled within 3~10cm during the cultivation of the insect grass, so as to avoid blocking the insect grass.

[0020] The beneficial effects of the present application are:

[0021] 1、The present application studies the nutrients and growth environment required by insect grass in different growth periods, attempts to design breeding soil in stages, divides the life cycle of insect grass into mycelial infection period and stroma growth period, and matches different soil environments, which meets the biological characteristics, improves the survival rate of larvae as much as possible, simulates the wild ecological environment as much as possible, improves the protein content of insect grass, and also improves other effective components in insect grass, such as polysaccharide, cordycepin and adenosine, greatly improves the quality of artificially cultivated insect grass.

[0022] 2、The application divides the second soil layer into a hierarchical structure: in the base layer, bamboo charcoal particles are introduced to enhance air permeability through their porous structure, while adsorbing heavy metals and pathogenic bacteria to reduce the risk of soil pollution. Meanwhile, the composite degradation material of straw fiber and polylactic acid (PLA) is used to improve drainage while slowly degrading, continuously releasing organic acids to promote microbial activity in the lower layer. In the water storage layer, chitosan / polyacrylic acid composite hydrogel is applied to significantly improve the soil water holding capacity by using the high water absorption of polyacrylic acid (can absorb 500 times its weight of water) and the viscous network of chitosan, maintaining the substrate humidity at 65-80%, meeting the high humidity requirements of the cordyceps mycelium expansion period, effectively reducing the irrigation frequency by 30-50%, and reducing the risk of mycelial growth stagnation caused by humidity fluctuations. In addition, chitosan improves soil aggregate structure through hydrogen bonding, and the swelling properties of composite hydrogel increase soil porosity, combined with pottery clay and rock sand, which can effectively promote the penetration of cordyceps mycelium and oxygen exchange, and inhibit the substrate from hardening. The humus in the nutrient layer is premixed with lactarius ectomycorrhizal fungal spores, which can form a symbiotic network with cordyceps mycelium to promote the efficiency of mycelial colonization in host larvae. The turf layer optimizes lactarius fungal spores for better results. Moreover, the combination of humus and wood ash not only provides organic matter but also adjusts the pH value to the suitable range for cordyceps fungus (slightly acidic to neutral). The specific plants in the turf layer may secrete substances that promote bacteria (such as terpenoids from pinus), indirectly increasing the success rate of natural infection of larvae.

[0023] 3、The application controls the growth environment of cordyceps and uses the first batch of ascospores naturally infected larvae to form a natural cycle of growth. Through experiments, it is found that the larvae that successfully naturally infected grow into cordyceps with higher nutritional content and quality. At the same time, the application can cultivate both artificially injected and naturally infected larvae using the same set of cultivation soil system to cultivate cordyceps of different qualities, maximizing the utilization rate of the cultivation system. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Effect of coconut fiber dosage on the germination of stipe;

[0025] Figure 2 Effect of perlite dosage on the germination of stipe;

[0026] Figure 3 Effect of nutrient solution dosage on the germination of stipe;

[0027] Figure 4 State of stipe bud germination of larvae; DETAILED DESCRIPTION

[0028] For the convenience of those skilled in the art to understand, the present application is further illustrated below in conjunction with the embodiments and drawings, and the content mentioned in the embodiments is not a limitation on the present application.

[0029] Example 1

[0030] The nutrient solution was prepared by containing 2% glucose, 0.5% proteose peptone, 3% soluble starch, 0.2% potassium dihydrogen phosphate, 0.2% magnesium sulfate, 10 mg vitamin B1 and water per 100 mL.

[0031] Preparation of the first-stage cultivation soil:

[0032] Take compressed coconut coir, soak in water, add perlite and nutrient solution, mix evenly, sterilize and reserve.

[0033] Test Example 1

[0034] 1.1 Test the effect of the amount of coconut coir on the germination of strobilus

[0035] Take 600g, 650g, 700g, 750g, 800g compressed coconut coir, soak to 70% moisture content, mix with 50g perlite and 1mL nutrient solution respectively to prepare the first-stage cultivation soil, sterilize and put into dry containers numbered Y1-Y5 respectively, take 20 injected and infected larvae respectively and put into containers numbered Y1-Y5 for cultivation, control the soil moisture at 70-80%, the environmental temperature at 15-25℃ and the environmental humidity at 75-85%, observe the strobilus bud germination and the required time, as shown in Table 1 and Figure 1 .

[0036] Table 1 Effect of the amount of coconut coir on the germination of larvae

[0037]

[0038] 1.2 Test the effect of the amount of perlite on the germination of strobilus

[0039] Take 700g compressed coconut coir, soak to 70% moisture content, take 1mL nutrient solution, take 10g, 20g, 40g, 60g, 80g and 100g perlite respectively, mix to prepare the first-stage cultivation soil, sterilize and put into dry containers numbered Z1-Z6 respectively, take 20 injected and infected larvae respectively and put into containers numbered Y1-Y5 for cultivation, control the soil moisture at 70-80%, the environmental temperature at 15-25℃ and the environmental humidity at 75-85%, observe the strobilus bud germination and the required time, as shown in Table 2 and Figure 2 .

[0040] Table 2 Effect of the amount of perlite on the germination of larvae

[0041]

[0042] 1.3 Test the effect of nutrient solution dosage on the germination of stolon

[0043] Take 700g compressed coconut shell and soak it until the water content is 70%. Take 50 pieces of perlite and mix 0.5mL, 1mL, 1.5mL, 2mL, 2.5mL, and 3mL of perlite to prepare the first-stage breeding soil. After sterilization, put it into a dry container and number it X1-X6. Take 20 infected larvae and put them into containers numbered Y1-Y5 for breeding. Control the soil moisture at 70-80%, the environmental temperature at 15-25℃, and the environmental humidity at 75-85%. Observe the stolon germination of the larvae and the time required, as shown in Table 3 and Figure 3 .

[0044] Table 3 Effect of perlite dosage on larval germination

[0045]

[0046] Based on the above test results, it can be seen that when the coconut shell dosage is between 650-750g, the germination rate and germination period of the larvae are optimal. When the perlite dosage is between 40-80g, the germination rate and germination period of the larvae are optimal. When the nutrient solution dosage is between 1.5-2.5mL, the germination rate and germination period of the larvae are optimal. On this basis, continue to carry out L9(3 4 ) orthogonal test experiment, as shown in Table 4.

[0047] Table 4 Effect of three factors on larval germination

[0048]

[0049] The software calculates that the optimal ratio of the first-stage breeding soil is 685g of coconut shell, 59g of perlite, and 2.2mL of nutrient solution.

[0050] The state of stolon germination of the larvae is shown in Figure 4 .

[0051] Example 2

[0052] Prepare the base layer: 5-10 parts of bamboo charcoal particles, 5-15 parts of perlite, 20-30 parts of rock sand, 10-15 parts of coconut shell, and 20-30 parts of straw fiber and polylactic acid degradation compound; preferably 9 parts of perlite, 24 parts of rock sand, 13 parts of coconut shell, and 27 parts of straw fiber and polylactic acid degradation compound are mixed to prepare, wherein the weight ratio of straw fiber and polylactic acid is 15:1.

[0053] Prepare the water storage layer:

[0054] Preparation of chitosan / polyacrylic acid composite hydrogel: chitosan is dissolved in 1% acetic acid solution, and acrylic acid monomer (neutralization degree 70-80%), crosslinking agent (MBA, amount 0.5% of the mass of acrylic acid) and initiator (APS, amount 1.2% of the mass of monomer) are added, and the reaction is carried out at 50-60℃ for 4-6 hours. Due to the high concentration of crosslinking agent, the network will be too dense, which will reduce the swelling rate. It is found through tests that the mass ratio of chitosan to acrylic acid is 1:7.8, and the performance is the best.

[0055] 20-30 parts of clay, 3-7 parts of chitosan / polyacrylic acid composite hydrogel, 5-8 parts of gravel, preferably 26 parts of clay, 5.7 parts of chitosan / polyacrylic acid composite hydrogel, and 7.2 parts of gravel are mixed to obtain.

[0056] Preparation of humus:

[0057] Wood chips, corn cobs, starch, pine and centipede grass are mixed in a mass ratio of 3:5:2:7:2 to produce plant humus by fermentation with a fermentation agent.

[0058] Animal humus is prepared by mixing cow dung and shell powder in a mass ratio of 20:1.

[0059] Animal humus and plant humus are mixed in a mass ratio of 1:7-9, preferably 1:7.68, to obtain humus.

[0060] The spores of the Lactarius deliciosus fungus are premixed with the humus, and the amount of the ectomycorrhizal fungal spores is 1-3% of the total weight of the humus, and the preferred ratio by weight is 2:95.

[0061] Preparation of nutrient layer:

[0062] 40-50 parts of compressed coconut husk, 1-5 parts of perlite, 10-15 parts of humus, 0.5-3 parts of nutrient solution, and 1-5 parts of wood ash are mixed to obtain.

[0063] Select pine and centipede grass with a height of about 5 cm as seedlings for sod planting.

[0064] Example 3

[0065] S1, the first stage of breeding soil is treated by disinfection and placed in a dry and ventilated container, and the larvae infected with artificial injection of mycelium are placed in the first stage of breeding soil, the soil moisture is controlled at 70-80%, the environmental temperature is 15-25℃, and the environmental humidity is 75-85%, until the bud of the insect body stroma germinates;

[0066] S2, the second stage of breeding soil is prepared and disinfected, and then is laid in a dry and ventilated container in the order of bottom layer, water storage layer, nutrient layer and turf layer from bottom to top. The thickness of each layer is specifically: 1-2 cm of bottom layer, 2-3 cm of water storage layer, 10-20 cm of nutrient layer, and the height of the seedlings of Pinus massoniana and Paspalum notatum in the turf layer is controlled within 3-15 cm. Pinus massoniana and Paspalum notatum are planted in the nutrient layer, and the seedlings of Pinus massoniana and Paspalum notatum are planted in cross distribution, and the distance between the seedlings is not less than 15 cm. The insect body of the sub-ascidial bud germination in step S1 is transplanted into the second stage of breeding soil, and the lowermost end of the insect body is located in the nutrient layer. The water content of the nutrient layer and the turf layer is continuously controlled at 70-80%, the environmental temperature is 15-30°C, and the environmental humidity is 75-85%, until the insect grass 1 is grown;

[0067] S3, when the insect grass in step S2 grows to the ascospore maturation stage, uninfected bat moth larvae are placed in the second stage of breeding soil to naturally infect and form an ecological self-cycle;

[0068] S4, the larvae that successfully naturally infect in step S3 are transplanted into the first stage of breeding soil, and the cultivation steps of steps S1 and S2 are repeated, and the nutrient solution is supplemented in time, and the insect grass 2 is grown.

[0069] Comparative Example 1

[0070] (Difference from Example 3: only the first stage of breeding soil is used)

[0071] The first stage of breeding soil is placed in a dry and ventilated container after being disinfected, and the larvae artificially injected with mycelium infection are placed in the first stage of breeding soil. The water content of the soil is controlled at 70-80%, the environmental temperature is 15-25°C, and the environmental humidity is 75-85%, until the insect grass 3 is grown.

[0072] Comparative Example 2

[0073] (Difference from Example 3: only the second stage of breeding soil is used)

[0074] The second stage of breeding soil is prepared and disinfected, and then is laid in a dry and ventilated container in the order of bottom layer, water storage layer, nutrient layer and turf layer from bottom to top. The thickness of each layer is specifically: 1-2 cm of bottom layer, 2-3 cm of water storage layer, 10-20 cm of nutrient layer, and the height of the seedlings of Pinus massoniana and Paspalum notatum in the turf layer is controlled within 3-15 cm. Pinus massoniana and Paspalum notatum are planted in the nutrient layer, and the seedlings of Pinus massoniana and Paspalum notatum are planted in cross distribution, and the distance between the seedlings is not less than 15 cm. The insect body of the sub-ascidial bud germination in step S1 is transplanted into the second stage of breeding soil, and the lowermost end of the insect body is located in the nutrient layer. The water content of the nutrient layer and the turf layer is continuously controlled at 70-80%, the environmental temperature is 15-30°C, and the environmental humidity is 75-85%, until the insect grass 4 is grown.

[0075] Comparative Example 3

[0076] (Difference from Example 3: no water storage layer is placed)

[0077] S1, the first-stage cultivation soil is placed in a dry and ventilated container after sterilization treatment, and the larvae artificially injected with mycelium are placed in the first-stage cultivation soil, the soil moisture is controlled at 70-80%, the environmental temperature is 15-25°C, and the environmental humidity is 75-85%, until the insect body stroma bud germination;

[0078] S2, each layer of the second-stage cultivation soil is prepared and sterilized, and then is laid in a dry and ventilated container in the order of bottom layer, nutrient layer and turf layer from bottom to top, the thickness of each layer is prepared as follows: 10-20 cm of the nutrient layer, the height of the seedlings of Pinus elliottii and Paspalum notatum in the turf layer is controlled within 3-15 cm, Pinus elliottii and Paspalum notatum are planted in the nutrient layer, the seedlings of Pinus elliottii and Paspalum notatum are cross-distributed and planted, and the distance between the seedlings is not less than 15 cm, the insect body stroma bud germination in step S1 is transplanted into the second-stage cultivation soil, the lowermost end of the insect body is located in the nutrient layer, and the soil moisture in the nutrient layer and the turf layer is continued to be controlled at 70-80%, the environmental temperature is 15-30°C, and the environmental humidity is 75-85%, until the insect grass grows to 5.

[0079] In this example, the bottom layer and the water storage layer are not placed, and the irrigation frequency is obviously more than that in Example 3, which is actually close to 1.5 times of the irrigation frequency in Example 3.

[0080] The above situation shows that the application of chitosan / polyacrylic acid composite hydrogel in the water storage layer can significantly improve the soil water holding capacity by using the high water absorption of polyacrylic acid (which can absorb 500 times of water of its own weight) and the viscous network of chitosan, maintain the substrate humidity at 65-80%, meet the high humidity requirement of the mycelium expansion period, effectively reduce the irrigation frequency by 30-50%, and reduce the risk of mycelium growth stagnation caused by humidity fluctuation; in addition, chitosan improves the soil aggregate structure through hydrogen bonding, the swelling property of the composite hydrogel increases the soil porosity, and the combination of pottery clay and rock sand can effectively promote the penetration of mycelium and oxygen exchange, and inhibit the substrate compaction.

[0081] Comparative Example 4

[0082] (Difference from Example 3: no turf layer is placed)

[0083] S1, the first-stage cultivation soil is placed in a dry and ventilated container after sterilization treatment, and the larvae artificially injected with mycelium are placed in the first-stage cultivation soil, the soil moisture is controlled at 70-80%, the environmental temperature is 15-25°C, and the environmental humidity is 75-85%, until the insect body stroma bud germination;

[0084] S2, the second stage of soil is prepared and disinfected, and then is laid in a dry and ventilated container according to the order from bottom to top as the substrate layer, the water storage layer and the nutrient layer. The thickness of each layer is specifically: 1-2 cm of the substrate layer, 2-3 cm of the water storage layer, and 10-20 cm of the nutrient layer. The insect body with the subiculum bud germination in step S1 is transplanted into the second stage of soil, and the lowermost end of the insect body is located in the nutrient layer. The water content of the nutrient layer and the turf layer is controlled at 70-80%, the environmental temperature is 15-30°C, and the environmental humidity is 75-85%. Until the insect grass is grown 6.

[0085] In this embodiment, the turf layer is not placed, and the self-circulation cultivation of steps S3 and S4 in Example 3 is continued: S3, when the insect grass in step S2 grows to the ascospore maturation stage, the uninfected bat moth larvae are arranged in the second stage of soil to naturally infect and form an ecological self-circulation;

[0086] S4, the larvae naturally infected in step S3 are transplanted into the first stage of soil, and the cultivation steps of steps S1 and S2 are repeated, and the nutrient solution is supplemented in time. The insect grass is grown 6'.

[0087] The results show that the natural infection success rate of naturally infected larvae decreases by about 8% compared with Example 3. It is indicated that the specific plants in the turf layer in Example 3 may secrete bacterium-promoting substances (such as terpenoids of Pinus strobus), which indirectly increase the natural infection success rate of larvae.

[0088] Comparative Example 5

[0089] (Difference from Example 3: no substrate layer is placed)

[0090] S1, the first stage of soil is placed in a dry and ventilated container after being disinfected, and the larvae artificially injected with mycelium are placed in the first stage of soil. The water content of the soil is controlled at 70-80%, the environmental temperature is 15-25°C, and the environmental humidity is 75-85%. Until the subiculum bud of the insect body germinates;

[0091] S2, the second stage of soil is prepared and disinfected, and then is laid in a dry and ventilated container according to the order from bottom to top as the substrate layer, the water storage layer and the nutrient layer. The thickness of each layer is specifically: 1-2 cm of the substrate layer, 2-3 cm of the water storage layer, and 10-20 cm of the nutrient layer. The thickness of each layer is specifically: 1-2 cm of the substrate layer, 2-3 cm of the water storage layer, and 10-20 cm of the nutrient layer. The insect body with the subiculum bud germination in step S1 is transplanted into the second stage of soil, and the lowermost end of the insect body is located in the nutrient layer. The water content of the nutrient layer and the turf layer is controlled at 70-80%, the environmental temperature is 15-30°C, and the environmental humidity is 75-85%. Until the insect grass is grown 7.

[0092] By observation, the growth process of Cordyceps in this embodiment is prolonged compared to that in Example 3, and the size of the Cordyceps is uneven, and the worm body is not as thick as that in Example 3. Therefore, it is illustrated that by introducing bamboo charcoal particles in the base layer, the porous structure of the bamboo charcoal particles is used to enhance the air permeability, and the heavy metals and pathogenic bacteria are adsorbed to reduce the risk of soil pollution. At the same time, the composite degradable material of straw fiber and polylactic acid (PLA) is used to improve the drainage performance and achieve slow degradation, and the organic acid is continuously released to promote the activity of microorganisms in the lower layer, thereby promoting the growth of Cordyceps.

[0093] Test Example 2

[0094] Cordycepin content determination

[0095] 2.1 Chromatographic analysis conditions

[0096] A Kromasil C18 (4.6 mm x 250 mm, 5 μm) reversed-phase silica gel column was used, the mobile phase was 10 mM KH2PO4 in methanol / double distilled water (15:85), the flow rate of the mobile phase was set to 1 mL / min during HPLC determination; the detection wavelength was 254 nm, the column temperature was 30°C, and the injection volume was 20 μL.

[0097] 2.2 Preparation of cordycepin standard curve

[0098] 5 mg of cordycepin standard was accurately weighed and dissolved in 50 mL of double distilled water to give a solution with a concentration of 100 μg / mL. The solution was then diluted in sequence to obtain cordycepin standard solutions with concentrations of 50, 25, 12.5, 6.25 and 3.125 μg / mL, respectively. Then, the standard solution was determined by HPLC under the following chromatographic conditions: flow rate of mobile phase 1 mL / min, detection wavelength 254 nm, column temperature 30°C, and injection volume 20 μL. The peak area was measured after injection, and the peak area-concentration plot was obtained, and the regression equation C = 3.41192 x 10 -5 A-1.28460, R 2 = 0.9996. Wherein, C is the concentration of the cordycepin standard solution (unit: μg / mL); A is the peak area determined by HPLC; the correlation coefficient R 2 = 0.9996, indicating a good linear relationship.

[0099] 2.3 Detection of cordycepin content in fruiting bodies, post-harvest culture medium and industrial liquid fermented Cordyceps sinensis mycelium

[0100] The mature Cordyceps sinensis fruiting bodies were dried at 50℃, and were crushed by a food crusher. 0.2g of the powder was precisely weighed, and was placed in a test tube with a plug. 10mL of double distilled water was added, and was ultrasonically treated for 30min. Then, it was centrifuged at 5000g for 15min. Finally, the solid impurities were removed by filtering through a 0.45μm filter membrane, and the content of cordycepin was detected by high performance liquid chromatography. When the HPLC was determined, the flow rate of the mobile phase was set to 1mL / min, the detection wavelength was 254nm, the column temperature was 30℃, and the injection amount was 20μL. All the results were the average values of three parallel sample values.

[0101] The test results are shown in Table 5:

[0102] Table 5 Cordycepin content of cultivated Cordyceps sinensis in Example 3 and comparative examples

[0103]

[0104] The above test results show that the present application studies the nutrients and growth environment required by Cordyceps sinensis in different growth periods, attempts to design the cultivation soil in stages, divides the life cycle of Cordyceps sinensis into mycelium infection period and stipe growth period, and matches different soil environments, which conforms to the biological characteristics, imitates the wild ecological environment as much as possible under the condition of improving the survival rate of the larvae as much as possible, designs a multi-layer cultivation soil structure, improves the content of cordycepin in Cordyceps sinensis, and greatly improves the quality of artificially cultivated Cordyceps sinensis.

[0105] Test Example 3

[0106] Adenosine content test

[0107] 3.1 Instruments

[0108] Waterse2695-2998 type high performance liquid chromatograph; Photodiode Array Detector detector; EMPOWER chromatography workstation; AE240 type electronic balance; SK-250H type numerical control ultrasonic instrument, KUDOS ultrasonic instrument Co., Ltd.

[0109] 3.2 Experimental materials

[0110] Adenosine control (batch number: 110879-200202) was provided by China Drug and Biological Product Inspection Institute; Cordyceps sinensis was a commercially available sample, which was provided by Chengdu Jinxin Chinese Herbal Medicine Slice Co., Ltd.

[0111] Methanol was a chromatographic pure reagent, and the others were analytical pure reagents; water was ultrapure water.

[0112] 3.3 Methods

[0113] 3.3.1 Chromatographic conditions

[0114] Chromatographic column: Welchrom C18 (4.6 mm x 250 mm, 5 μm); mobile phase: 0.1% phosphoric acid solution (pH 2.8) - methanol (95:5); flow rate: 1.0 mL / min; column temperature: 25 °C; detection wavelength: 260 nm.

[0115] 3.3.2 Preparation of control solution

[0116] Take the adenosine control sample, accurately weigh, place in a 50 mL volumetric flask, add 90% methanol to make a solution containing 200 μg per 1 mL, shake well, as a stock solution; another accurately take 1 mL of the stock solution into a 10 mL volumetric flask, dilute to the mark with 90% methanol, shake well, as a control solution (containing 20 μg of adenosine per 1 mL).

[0117] 3.3.3 Preparation of test solution

[0118] Take about 0.5 g of the powder (passed through a No. 3 sieve) prepared from Cordyceps militaris in Example 3 and Comparative Example, accurately weigh, place in a stoppered conical flask, accurately add 90% methanol 20 mL, tightly stop, shake well, weigh, heat to reflux for 30 min, cool, weigh again, make up the weight loss with 90% methanol, shake well, filter, take the filtrate, and obtain. The test results are shown in Table 6.

[0119] Table 6 Adenosine content determination results of samples

[0120]

[0121] The above test results show that the present application studies the nutrients and growth environment required by Cordyceps militaris in different growth periods, attempts to design breeding soil in stages, divides the Cordyceps militaris life cycle into mycelium infection period and stroma growth period, and matches different soil environments, which meets the biological characteristics, imitates the wild ecological environment as much as possible under the condition of improving the survival rate of larvae as much as possible, designs a multi-layer breeding soil structure, and improves the content of adenosine in Cordyceps militaris, greatly improves the quality of artificially cultivated Cordyceps militaris.

[0122] All the technical features in the present embodiment can be modified in appearance according to actual needs.

[0123] The above embodiments are the preferred implementation of the present application, in addition to this, the present application can be implemented in other ways, without departing from the technical solution concept of the present application, any obvious replacement within the protection scope of the present application.

Claims

1. A soil for cultivating Cordyceps sinensis, characterized by comprising: The first-stage cultivation soil and the second-stage cultivation soil are included. ​ The first-stage cultivation soil is composed of the following components in parts by weight: 60-80 parts of coconut husk, 1-10 parts of perlite, and 0.5-3 parts of nutrient solution; the first-stage cultivation soil is used for culturing the larva infected by mycelium to the stage of the emergence of the insect body stroma bud; The second-stage cultivation soil includes, from bottom to top, a base layer (1), a water storage layer (2), a nutrient layer (3), and a topmost turf layer (4); the second-stage cultivation soil is used for culturing the insect body with the emerged stroma bud into insect grass; The base layer (1) is composed of the following components in parts by weight: 5-10 parts of bamboo charcoal particles, 5-15 parts of perlite, 20-30 parts of rock sand, 10-15 parts of coconut husk, and 20-30 parts of straw fiber and polylactic acid compound degradation products; the weight ratio of the straw fiber to the polylactic acid is 15:1; The water storage layer (2) is composed of the following components in parts by weight: 20-30 parts of pottery clay, 3-7 parts of chitosan / polyacrylic acid composite hydrogel, and 5-8 parts of rock sand; The nutrient layer (3) is composed of the following components in parts by weight: 40-50 parts of coconut husk, 1-5 parts of perlite, 10-15 parts of humus, 0.5-3 parts of nutrient solution, and 1-5 parts of wood ash; the humus is premixed with Lactarius ectomycorrhizal fungal spores; the amount of the Lactarius ectomycorrhizal fungal spores is 1-3% of the total weight of the humus; The humus is mixed from animal humus and plant humus at a mass ratio of 1:7-9; the animal humus is mixed from cow dung and shell powder at a mass ratio of 20:1; the plant humus is fermented from wood chips, corn cobs, starch, pine bark, and centipede grass at a mass ratio of 3:5:2:7:2; The turf layer (4) is planted by cross-distribution of the seedlings of the pine bark and the centipede grass; the distance between the seedlings of the pine bark and the centipede grass is not less than 15 cm.

2. The Ophiocordyceps sinensis cultivation soil according to claim 1, characterized in that: The nutrient solution is specifically prepared from the following components: 100 mL of the nutrient solution contains 2% of glucose, 0.5% of protein peptone, 3% of soluble starch, 0.2% of potassium dihydrogen phosphate, 0.2% of magnesium sulfate, 10 mg of vitamin B1, and water.

3. The method according to any one of claims 1-2, wherein the method comprises: Specifically includes the following steps: ​ S1, after the first-stage cultivation soil is sterilized, the soil is placed in a dry and ventilated container, the larva infected by mycelium is artificially injected, and the larva is placed in the first-stage cultivation soil; the soil moisture is controlled at 70-80%, the environmental temperature is 15-25℃, and the environmental humidity is 75-85%; until the emergence of the insect body stroma bud; S2, after each layer of the second-stage cultivation soil is prepared and sterilized, the layers are sequentially laid in a dry and ventilated container from bottom to top as the base layer (1), the water storage layer (2), the nutrient layer (3), and the turf layer (4); the insect body with the emerged stroma bud in step S1 is transplanted into the second-stage cultivation soil, the lowermost end of the insect body is located in the nutrient layer (3), the moisture of the nutrient layer (3) and the turf layer (4) is controlled at 70-80%, the environmental temperature is 15-30℃, and the environmental humidity is 75-85%; until the insect grass is grown; S3, when the step S2 is grown to ascospore maturity period, the uninfected bat moth larvae are placed in the second stage of cultivation soil to form ecological self-circulation; S4, the step S3 is successfully infected larvae are transplanted to the first stage of cultivation soil, repeat the steps S1 and S2 cultivation steps, and replenish the nutrient solution at any time.

4. The method for controlling the Ophiocordyceps sinensis breeding soil according to claim 3, characterized in that: The preparation thickness of each layer of the second stage of cultivation soil is specifically: 1-2cm of the base layer (1), 2-3cm of the water storage layer (2), 10-20cm of the nutrient layer (3).

5. The method for controlling the Ophiocordyceps sinensis breeding soil according to claim 3, characterized in that: The height of the turf layer (4) in step S2 is controlled within 3-10cm during the cultivation of cordyceps, so as to avoid blocking cordyceps.

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