Cordyceps sinensis cultivation soil and control method thereof

Abstract

The invention relates to the technical field of cordyceps sinensis cultivation, in particular to cordyceps sinensis cultivation soil and a control method thereof, and the cordyceps sinensis cultivation soil comprises first-stage cultivation soil and second-stage cultivation soil; the first-stage culture soil is prepared from the following components in parts by weight: 60 to 80 parts of coco coir, 1 to 10 parts of perlite and 0.5 to 3 parts of nutrient solution; the second-stage culture soil comprises a basal layer, a water storage layer, a nutrition layer and a turf layer on the uppermost layer; the nutrient layer is prepared from the following components in parts by weight: 40 to 50 parts of coco coir, 1 to 5 parts of perlite, 10 to 15 parts of humus, 0.5 to 3 parts of nutrient solution and 1 to 5 parts of plant ash. By preparing soil in different culture periods and artificially regulating the growth soil of cordyceps sinensis, the quality of the artificially cultured cordyceps sinensis is improved to be equivalent to that of wild cordyceps sinensis, and the content of effective components in the cordyceps sinensis is increased.

Description

Technical Field

[0001] The present invention relates to the technical field of Cordyceps sinensis cultivation, and in particular to a Cordyceps sinensis cultivation soil and a control method thereof. Background Art

[0002] Cordyceps sinensis, also known as Chinese caterpillar fungus, is composed of a caterpillar body and a stroma connected together. The host of wild Cordyceps sinensis fungus, Hepialus armoricanus Oberthur, generally grows in alpine meadow areas at an altitude of 3,650 - 4,250 meters, with a slope of 15 - 30 degrees, an air temperature of 0 - 23°C, a ground temperature of 3 - 7.5°C at a depth of 20 cm, a relative air humidity of 50% - 70%, and a soil humidity of 40% - 60%. The life cycle of Hepialus armoricanus Oberthur: The larval stage is the longest, and it takes 2 years to pupate. Generally, it is distributed more than 10 cm deep in the soil. In late May, part of the larvae form pupae, and most of them emerge as moths in early August. Eggs are laid on the soil surface and hatch into the soil about 2 months later. Artificial cultivation of Cordyceps sinensis cannot be separated from the natural environment for its growth.

[0003] Currently, artificially bred Cordyceps sinensis has weak stress resistance and is easily infected by microorganisms, which is an important limiting factor affecting the development of Cordyceps sinensis resources. In addition, previous research by the research group found that the protein content and types of wild Cordyceps sinensis are higher than those of artificially cultivated Cordyceps sinensis, and there are also differences in the contents of components such as polysaccharides, cordycepin, and adenosine, suggesting that differences in the growth environment may have an important impact on Cordyceps sinensis. Comparing the microecological environments of wild Cordyceps sinensis and artificially bred Cordyceps sinensis, it is not difficult to find that in the natural environment, both the host of Cordyceps sinensis - Hepialus armoricanus Oberthur larvae and the main place for the development of Cordyceps sinensis - the soil contain a more abundant microbial community. For artificially bred Cordyceps sinensis, larvae are generally raised in a sterile environment, and the feed and soil need to be sterilized in advance, while in the wild environment, the larvae feed on complex substances and the surrounding soil microorganisms are rich.

[0004] There are almost no relevant reports on the study of endophytic fungi of artificially cultivated Cordyceps sinensis and soil microbial communities under artificial conditions, and there are also few relevant reports on the impact of the soil environment of wild and artificially bred Cordyceps sinensis on the growth and quality of Cordyceps sinensis. Therefore, we have conducted in - depth research in this direction in order to provide a cultivation soil for the artificial cultivation technology of Cordyceps sinensis that can effectively improve the survival rate and the quality of Cordyceps sinensis, and ensure the nutritional value of artificially cultivated Cordyceps sinensis. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a Cordyceps sinensis cultivation soil and a control method thereof. By preparing soils for different cultivation periods and artificially regulating the growth soil of Cordyceps sinensis, the quality of artificially cultivated Cordyceps sinensis is improved to be equivalent to that of wild Cordyceps sinensis, and the content of effective components in Cordyceps sinensis is increased.

[0006] To solve the above - mentioned technical problems, the present invention adopts the following technical solutions: An Ophiocordyceps sinensis cultivation soil, including a first-stage cultivation soil and a second-stage cultivation soil; the first-stage cultivation soil consists of the following components in parts by weight: 60 - 80 parts of coconut coir, 1 - 10 parts of perlite, and 0.5 - 3 parts of nutrient solution; the second-stage cultivation soil includes a base layer, a water storage layer, a nutrient layer, and a grass skin layer on the top; the nutrient layer consists of the following components in parts by weight: 40 - 50 parts of coconut coir, 1 - 5 parts of perlite, 10 - 15 parts of humus, 0.5 - 3 parts of nutrient solution, and 1 - 5 parts of plant ash; the humus is premixed with spores of ectomycorrhizal fungi of the genus Lactarius, wherein the dosage of the spores of ectomycorrhizal fungi of the genus Lactarius is 1 - 3% of the total weight of the humus.

[0007] Preferably, the nutrient solution is specifically prepared from the following components: every 100 mL of the nutrient solution contains 2% glucose, 0.5% peptone, 3% soluble starch, 0.2% potassium dihydrogen phosphate, 0.2% magnesium sulfate, 10 mg of vitamin B1, and water. Preferably, the base layer consists 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 coir, and 20 - 30 parts of a compound degradant of straw fiber and polylactic acid; wherein, the weight ratio of straw fiber to polylactic acid is 15:1.

[0008] Preferably, the water storage layer consists of the following components in parts by weight: 20 - 30 parts of clay, 3 - 7 parts of chitosan / polyacrylic acid composite hydrogel, and 5 - 8 parts of rock sand.

[0009] Preferably, the grass skin layer is formed by cross-distributing and planting seedlings of Lycopodium cernuum and Paspalum notatum, and the distance between the seedlings of Lycopodium cernuum and Paspalum notatum is not less than 15 cm.

[0010] Preferably, the humus is mixed by animal humus and plant humus in a mass ratio of 1:7 - 9, the animal humus is made by mixing cow dung and shell powder in a mass ratio of 20:1, and the plant humus is composted and fermented by adding fermentation agents according to a mass ratio of 3:5:2:7:2 of sawdust, corn cob, starch, Lycopodium cernuum, and Paspalum notatum.

[0011] A control method for an Ophiocordyceps sinensis cultivation soil specifically includes the following steps: S1, after disinfecting the first-stage cultivation soil, place it in a dry and ventilated container, place the larvae infected with artificial injection of mycelium into the first-stage cultivation soil, control the soil moisture at 70 - 80%, the environmental temperature at 15 - 25 °C, and the environmental humidity at 75 - 85%, until the stroma buds of the insect body germinate; S2, prepare and disinfect each layer of the second-phase breeding soil, and then lay them in a dry and ventilated container in the order of a base layer, a water storage layer, a nutrient layer and a turf layer from bottom to top, transplant the insect bodies that have germinated from the fruiting buds in step S1 into the second-phase breeding soil, with the lowest end of the insect body located in the nutrient layer, continue to control the moisture content of the nutrient layer and the turf layer at 70-80%, the ambient temperature at 15-30° C., and the ambient humidity at 75-85%, until the Cordyceps grows; S3, when the Cordyceps in step S2 grows to the ascospore maturity stage, uninfected bat moth larvae are placed in the second-stage breeding soil for natural infection, forming an ecological self-circulation; S4, transplanting the larvae that are successfully naturally infected in step S3 into the first-stage breeding soil, repeating the breeding steps of steps S1 and S2, and replenishing nutrient solution at any time.

[0012] Preferably, the thickness of each layer of the second-phase breeding soil is as follows: 1-2 cm base layer, 2-3 cm water storage layer, 10-20 cm nutrient layer, and the height of the ground pine and bahia grass seedlings in the turf layer is controlled within 3-15 cm.

[0013] Preferably, the height of the ground pine and bahia grass in the turf layer in step S2 is controlled within 3 to 10 cm during the Cordyceps cultivation process to avoid blocking the Cordyceps.

[0014] Beneficial effects of the present invention: 1. The present invention studies the nutrients and growth environment required for different growth stages of Cordyceps, attempts to design the cultivation soil in stages, divides the life cycle of Cordyceps into the mycelium infection stage and the stroma growth stage, and matches different soil environments in accordance with its biological characteristics, while maximizing the infection survival rate of the larvae and imitating the wild ecological environment as much as possible, so as to increase the protein content of Cordyceps, and also increase the content of other effective ingredients in Cordyceps, such as polysaccharides, cordycepin and adenosine, thereby greatly improving the quality of artificially cultivated Cordyceps.

[0015] 2. The present invention designs a layered structure for the secondary soil layer: introducing bamboo charcoal particles into the base layer, using its porous structure to enhance air permeability, adsorb heavy metals and pathogenic bacteria at the same time, reduce the risk of soil pollution, and using a composite degradation material of straw fiber and polylactic acid (PLA) to achieve slow degradation while improving drainage, continuously release organic acids to promote the activity of microorganisms in the lower layer. Applying a chitosan / polyacrylic acid composite hydrogel in the water storage layer, using the high water absorption of polyacrylic acid (able to absorb 500 times its own weight of water) and the viscous network of chitosan to significantly improve the water holding capacity of the soil, maintain the substrate humidity stable at 65 - 80%, meet the high humidity requirements during the mycelium expansion period of Cordyceps sinensis, effectively reduce the irrigation frequency by 30 - 50%, and reduce the risk of mycelium growth stagnation caused by humidity fluctuations; in addition, chitosan improves the soil aggregate structure through hydrogen bonding, and the swelling characteristics of the composite hydrogel increase the soil porosity. Combining with clay and rock sand can effectively promote the penetration of Cordyceps sinensis mycelium and oxygen exchange, and inhibit substrate compaction. Premix the spores of ectomycorrhizal fungi of the genus Lactarius in the humus of the nutrient layer, which can form a symbiotic network with the Cordyceps sinensis mycelium, promote the colonization efficiency of mycelium in the host larvae, and cooperate with the fungal spores of Lactarius deliciosus selected in the grass layer for better results; moreover, the combination of humus and plant ash not only provides organic matter but also adjusts the pH value to the suitable range for Cordyceps sinensis (slightly acidic to neutral). Specific plants in the grass layer may secrete bacteria-promoting substances (such as terpenoids of Lycopodium clavatum), indirectly increasing the success rate of natural infection of larvae. 3. The present invention controls the growth environment of Cordyceps sinensis, and uses the ascospores shed from the first batch of mature Cordyceps to naturally infect larvae to form a natural cycle of growth. Through experiments, it is found that the nutritional components and quality of the larvae that have successfully undergone natural infection are higher after growing into Cordyceps sinensis. At the same time, the present invention can cultivate larvae with natural infection while cultivating larvae with artificial injection infection, and cultivate Cordyceps sinensis with different qualities using the same set of cultivation soil system, maximizing the utilization rate of the breeding system. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Effect of the coconut coir dosage of the present invention on the germination of stromata Figure 2 Effect of the perlite dosage of the present invention on the germination of stromata Figure 3 Effect of the nutrient solution dosage of the present invention on the germination of stromata Figure 4 Germination state of the stromata buds of the insect body of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] For the convenience of those skilled in the art to understand, the present invention will be further described below in conjunction with the embodiments and the drawings. The content mentioned in the embodiments does not limit the present invention.

[0018] Example 1

[0019] Prepare the nutrient solution for standby according to the composition that every 100 mL of the nutrient solution contains 2% glucose, 0.5% peptone, 3% soluble starch, 0.2% potassium dihydrogen phosphate, 0.2% magnesium sulfate, 10 mg of vitamin B1 and water.

[0020] Prepare the first-stage cultivation soil: Take the compressed coconut coir, soak it in water until it swells, add perlite and the nutrient solution, mix them evenly, and sterilize it for standby.

[0021] Test Example 1 1.1 Test the effect of the amount of coconut coir on the germination of stroma Take 600 g, 650 g, 700 g, 750 g, and 800 g of compressed coconut coir respectively, soak them until the water content reaches 70%, mix them with 50 g of perlite and 1 mL of nutrient solution respectively to prepare the first-stage cultivation soil. After disinfection, put them into dry containers respectively, numbered Y1 - Y5. Take 20 infected larvae by injection and put them into the containers numbered Y1 - Y5 respectively for cultivation. Control the soil moisture at 70 - 80%, the environmental temperature at 15 - 25 °C, and the environmental humidity at 75 - 85%. Observe the germination situation and the required time of the stroma buds of the larvae, as shown in Table 1 and Figure 1 .

[0022] Table 1 Effect of the amount of coconut coir on the germination of larvae

[0023] 1.2 Test the effect of the amount of perlite on the germination of stroma Take 700 g of compressed coconut coir and soak it until the water content reaches 70%. Take 1 mL of nutrient solution. Take 10 g, 20 g, 40 g, 60 g, 80 g, and 100 g of perlite respectively, mix them to prepare the first-stage cultivation soil. After disinfection, put them into dry containers respectively, numbered Z1 - Z6. Take 20 infected larvae by injection and put them into the containers numbered Y1 - Y5 respectively for cultivation. Control the soil moisture at 70 - 80%, the environmental temperature at 15 - 25 °C, and the environmental humidity at 75 - 85%. Observe the germination situation and the required time of the stroma buds of the larvae, as shown in Table 2 and Figure 2 .

[0024] Table 2 Effect of the amount of perlite on the germination of larvae

[0025] 1.3 Test the effect of the amount of nutrient solution on the germination of stroma Take 700 g of compressed coconut coir and soak it until the water content reaches 70%. Take 50 perlite particles, and take 0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL, and 3 mL of perlite respectively. Mix them to prepare the first-stage cultivation soil. After disinfection, put them into dry containers respectively, numbered X1 - X6. Take 20 larvae after injection infection respectively and put them into containers numbered Y1 - Y5 for cultivation. Control the soil moisture at 70 - 80%, the environmental temperature at 15 - 25 °C, and the environmental humidity at 75 - 85%. Observe the germination situation and the required time of the stroma buds of the larvae, as shown in Table 3 and Figure 3 .

[0026] Table 3 Influence of Perlite Dosage on Larval Germination

[0027] Based on the above test results, it can be seen that when the dosage of coconut coir is between 650 - 750 g, the germination rate and germination cycle of the larvae are both the best. When the dosage of perlite is between 40 - 80 g, the germination rate and germination cycle of the larvae are both the best. When the dosage of nutrient solution is between 1.5 - 2.5 mL, the germination rate and germination cycle of the larvae are both the best. On this basis, continue to conduct the L9(3 4 ) orthogonal test, as shown in Table 4.

[0028] Table 4 Influence Test of Three Factors on Larval Germination

[0029] Using software to calculate, the best ratio of the first-stage cultivation soil is 685 g of coconut coir, 59 g of perlite, and 2.2 mL of nutrient solution.

[0030] The germination state of the stroma buds of the larvae is shown in Figure 4 .

[0031] Example 2

[0032] 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 coir, and 20 - 30 parts of straw fiber and polylactic acid compound degradant; preferably, 9 parts of perlite, 24 parts of rock sand, 13 parts of coconut coir, and 27 parts of straw fiber and polylactic acid compound degradant are mixed and prepared. Among them, the weight ratio of straw fiber to polylactic acid is 15:1.

[0033] Prepare the water storage layer: Preparation of chitosan / polyacrylic acid composite hydrogel: Chitosan was dissolved in 1% acetic acid solution, and acrylic acid monomer (neutralization degree 70 - 80%), cross-linking agent (MBA, dosage 0.5% of the mass of acrylic acid), and initiator (APS, dosage 1.2% of the mass of the monomer) were added. The reaction was carried out at 50 - 60 °C for 4 - 6 hours. Since too high a concentration of the cross-linking agent would lead to an overly dense network and reduce the swelling rate, through testing, it was found that when the mass ratio of chitosan to acrylic acid was 1:7.8, the optimal performance items were obtained.

[0034] It was prepared by mixing 20 - 30 parts of clay, 3 - 7 parts of chitosan / polyacrylic acid composite hydrogel, and 5 - 8 parts of rock sand. Preferably, 26 parts of clay, 5.7 parts of chitosan / polyacrylic acid composite hydrogel, and 7.2 parts of rock sand were mixed.

[0035] Preparation of humus: Wood chips, corn cobs, starch, Lycopodium cernuum, and Paspalum notatum were taken and composted with a fermentation inoculant in a mass ratio of 3:5:2:7:2 to prepare plant humus for standby.

[0036] Cow dung and shell powder were taken and mixed in a mass ratio of 20:1 to prepare animal humus for standby.

[0037] Animal humus and plant humus were taken and mixed in a mass ratio of 1:7 - 9. The preferred ratio was 1:7.68 to prepare humus for standby.

[0038] The spores of Lactarius deliciosus fungi were premixed with humus. The dosage of the spores of ectomycorrhizal fungi of the genus Lactarius was 1 - 3% of the total weight of the humus. The preferred ratio in terms of weight ratio was 2:95.

[0039] Preparation of the nutrient layer: It was prepared by mixing 40 - 50 parts of compressed coconut coir, 1 - 5 parts of perlite, 10 - 15 parts of humus, 0.5 - 3 parts of nutrient solution, and 1 - 5 parts of plant ash. Preferably, 48 parts of coconut coir, 4 parts of perlite, 14 parts of humus, 1.8 parts of nutrient solution, and 3 parts of plant ash were mixed.

[0040] Lycopodium cernuum and Paspalum notatum with a height of about 5 cm and seedlings with thick and healthy roots were selected as the turf for planting and standby.

[0041] Example 3

[0042] S1, After disinfecting the first-stage breeding soil, it was placed in a dry and ventilated container. The larvae infected with artificial injection of hyphae were placed in the first-stage breeding soil, and the soil moisture was controlled at 70 - 80%, the environmental temperature was 15 - 25 °C, and the environmental humidity was 75 - 85% until the stroma buds of the insect body germinated; S2. Prepare each layer of the second-stage cultivation soil, disinfect it, and then lay it in a dry and well-ventilated container in the order from bottom to top as the base layer, water storage layer, nutrient layer, and turf layer. The specific preparation thickness of each layer is as follows: a base layer of 1 - 2 cm, a water storage layer of 2 - 3 cm, a nutrient layer of 10 - 20 cm. The height of the seedlings of Dichondra repens and Paspalum notatum in the turf layer is controlled within 3 - 15 cm. Dichondra repens and Paspalum notatum are planted in the nutrient layer, and the seedlings of Dichondra repens and Paspalum notatum are cross-distributed for planting, with the seedling spacing not less than 15 cm. Transplant the insects with germinated stromata in step S1 into the second-stage cultivation soil, with the lowest end of the insects located in the nutrient layer. Continue to control the moisture in the nutrient layer and turf layer at 70 - 80%, the environmental temperature at 15 - 30 °C, and the environmental humidity at 75 - 85% until Cordyceps 1 grows. S3. When the Cordyceps in step S2 grows to the ascospores maturity stage, place the uninfected Hepialus larvae into the second-stage cultivation soil for natural infection to form an ecological self-circulation. S4. Transplant the larvae with successful natural infection in step S3 into the first-stage cultivation soil, repeat the cultivation steps of step S1 and S2, and supplement the nutrient solution at any time until Cordyceps 2 grows.

[0043] Comparative Example 1 (The difference from Example 3 is that only the first-stage cultivation soil is used) After disinfecting the first-stage cultivation soil, place it in a dry and well-ventilated container. Place the larvae infected by artificial injection of hyphae into the first-stage cultivation soil, control the soil moisture at 70 - 80%, the environmental temperature at 15 - 25 °C, and the environmental humidity at 75 - 85% until Cordyceps 3 grows.

[0044] Comparative Example 2 (The difference from Example 3 is that only the second-stage cultivation soil is used) Prepare each layer of the second-stage cultivation soil, disinfect it, and then lay it in a dry and well-ventilated container in the order from bottom to top as the base layer, water storage layer, nutrient layer, and turf layer. The specific preparation thickness of each layer is as follows: a base layer of 1 - 2 cm, a water storage layer of 2 - 3 cm, a nutrient layer of 10 - 20 cm. The height of the seedlings of Dichondra repens and Paspalum notatum in the turf layer is controlled within 3 - 15 cm. Dichondra repens and Paspalum notatum are planted in the nutrient layer, and the seedlings of Dichondra repens and Paspalum notatum are cross-distributed for planting, with the seedling spacing not less than 15 cm. Place the larvae infected by artificial injection of hyphae into the second-stage cultivation soil, with the lowest end of the larvae located in the nutrient layer. Continue to control the moisture in the nutrient layer and turf layer at 70 - 80%, the environmental temperature at 15 - 30 °C, and the environmental humidity at 75 - 85% until Cordyceps 4 grows.

[0045] Comparative Example 3 (The difference from Example 3 is that the water storage layer is not placed) S1. After disinfecting the first-stage cultivation soil, place it in a dry and ventilated container. Place the larvae infected with artificially injected mycelia into the first-stage cultivation soil, control the soil moisture at 70 - 80%, the environmental temperature at 15 - 25°C, and the environmental humidity at 75 - 85% until the stroma buds of the insect bodies germinate. S2. Prepare each layer of the second-stage cultivation soil and disinfect it. Then lay it in a dry and ventilated container in the order from bottom to top as the base layer, the nutrient layer, and the turf layer. The specific thickness of each layer is as follows: a 10 - 20 cm thick nutrient layer, and the height of the seedlings of Dichondra repens and Paspalum notatum in the turf layer is controlled within 3 - 15 cm. Dichondra repens and Paspalum notatum are planted in the nutrient layer, and the seedlings are cross-distributed. The spacing between the seedlings is not less than 15 cm. Transplant the insect bodies with germinated stroma buds in step S1 into the second-stage cultivation soil, with the lowermost end of the insect bodies located in the nutrient layer. Continue to control the moisture in the nutrient layer and the turf layer at 70 - 80%, the environmental temperature at 15 - 30°C, and the environmental humidity at 75 - 85% until the Cordyceps sinensis is grown.

[0046] In this example, the base layer and the water storage layer are not placed. During the cultivation process, the irrigation frequency is significantly higher than that in Example 3, and the actual irrigation frequency is close to 1.5 times that in Example 3.

[0047] The above situation shows that the application of chitosan / polyacrylic acid composite hydrogel in the water storage layer, utilizing the high water absorbency of polyacrylic acid (able to absorb 500 times its own weight of water) and the viscous network of chitosan, significantly improves the water holding capacity of the soil, maintains the matrix humidity stably at 65 - 80%, meets the high humidity requirements during the mycelial expansion period of Cordyceps sinensis, can effectively reduce the irrigation frequency by 30 - 50%, and reduces the risk of mycelial growth stagnation caused by humidity fluctuations; in addition, chitosan improves the soil aggregate structure through hydrogen bonding, and the swelling property of the composite hydrogel increases the soil porosity. Combining with clay and rock sand can effectively promote the penetration of Cordyceps sinensis mycelia and oxygen exchange, and inhibit matrix compaction.

[0048] Comparative Example 4 (The difference from Example 3 is that the turf layer is not placed) S1. After disinfecting the first-stage cultivation soil, place it in a dry and ventilated container. Place the larvae infected with artificially injected mycelia into the first-stage cultivation soil, control the soil moisture at 70 - 80%, the environmental temperature at 15 - 25°C, and the environmental humidity at 75 - 85% until the stroma buds of the insect bodies germinate. S2. Prepare each layer of the secondary cultivation soil, disinfect it, and then lay it in a dry and well-ventilated container in the order from bottom to top as the base layer, water storage layer, and nutrient layer. The specific thickness of each layer is as follows: a base layer of 1 - 2 cm, a water storage layer of 2 - 3 cm, and a nutrient layer of 10 - 20 cm. Transplant the insects with germinated stroma buds in step S1 into the secondary cultivation soil, with the lowest end of the insects located in the nutrient layer. Continue to control the moisture in the nutrient layer and the turf layer at 70 - 80%, the environmental temperature at 15 - 30 °C, and the environmental humidity at 75 - 85% until the cordyceps is grown into Cordyceps sinensis 6.

[0049] In this example, the turf layer was not placed, and the self-circulation cultivation in steps S3 and S4 of Example 3 was continued: S3. When the Cordyceps sinensis in step S2 grows to the ascospores maturity stage, place uninfected Hepialus larvae into the secondary cultivation soil for natural infection to form an ecological self-circulation. S4. Transplant the larvae successfully naturally infected in step S3 into the primary cultivation soil, repeat the cultivation steps of steps S1 and S2, and supplement the nutrient solution at any time until Cordyceps sinensis 6' is grown.

[0050] The results show that the natural infection success rate of the naturally infected larvae decreased by about 8% compared with that in Example 3. It shows that the specific plants in the turf layer in Example 3 may secrete bacteria-promoting substances (such as terpenoids of Lycopodium clavatum), indirectly increasing the natural infection success rate of the larvae.

[0051] Comparative Example 5 (The difference from Example 3 is that the base layer is not placed) S1. After disinfecting the primary cultivation soil, place it in a dry and well-ventilated container, place the larvae infected by artificial injection of mycelium into the primary cultivation soil, control the soil moisture at 70 - 80%, the environmental temperature at 15 - 25 °C, and the environmental humidity at 75 - 85% until the stroma buds of the insects germinate. S2. Prepare each layer of the secondary cultivation soil, disinfect it, and then lay it in a dry and well-ventilated container in the order from bottom to top as the water storage layer, nutrient layer, and turf layer. The specific thickness of each layer is as follows: a nutrient layer of 10 - 20 cm, and the height of the seedlings of Lycopodium clavatum and Paspalum notatum in the turf layer is controlled within 3 - 15 cm. Lycopodium clavatum and Paspalum notatum are planted in the nutrient layer, and the seedlings of Lycopodium clavatum and Paspalum notatum are cross-distributed for planting, with the seedling spacing not less than 15 cm. Transplant the insects with germinated stroma buds in step S1 into the secondary cultivation soil, with the lowest end of the insects located in the nutrient layer. Continue to control the moisture in the nutrient layer and the turf layer at 70 - 80%, the environmental temperature at 15 - 30 °C, and the environmental humidity at 75 - 85% until Cordyceps sinensis 7 is grown.

[0052] It was found through observation that the growth process of Cordyceps in this example was extended compared to Example 3, and the size of the mature Cordyceps was uneven, and the worm bodies were not as thick as those in Example 3. Therefore, it shows that introducing bamboo charcoal particles into the base layer, using its porous structure to enhance air permeability, adsorb heavy metals and pathogenic bacteria at the same time, reduce the risk of soil pollution, and using the composite degradation material of straw fiber and polylactic acid (PLA) to achieve slow degradation while improving drainage, continuously release organic acids to promote the activity of microorganisms in the lower layer, thereby promoting the growth of Cordyceps.

[0053] Test Example 2 Determination of cordycepin content 2.1 Chromatographic analysis conditions The chromatographic column used was a Kromasil C18 (4.6 mm × 250 mm, 5 μm) reverse silica gel column, mobile phase: 10 mM KH 2 PO 4 dissolved in methanol / distilled water (15∶85), the flow rate of the mobile phase was set at 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.

[0054] 2.2 Preparation of cordycepin standard curve Accurately weigh 5 mg of cordycepin standard product, and make up the volume to 50 mL with distilled water, then the concentration of this solution is 100 μg / mL. Dilute it successively to obtain cordycepin standard solutions with concentrations of 50, 25, 12.5, 6.25, and 3.125 μg / mL respectively. Then use HPLC to determine the standard solution. Under the chromatographic conditions of a mobile phase flow rate of 1 mL / min, a detection wavelength of 254 nm, and a column temperature of 30 °C, after injecting 20 μL, measure the peak area, and plot the peak area - concentration to obtain the regression equation C = 3.41192×10 -5 A - 1.28460, R 2 = 0.9996. Among them, C is the concentration of the cordycepin standard solution (unit: μg / mL); A is the peak area measured by HPLC; the correlation coefficient R 2 = 0.9996, indicating a good linear relationship.

[0055] 2.3 Detection of cordycepin content in fruiting bodies, post-harvest culture media and mycelia of Cordyceps sinensis in industrial liquid fermentation The mature Cordyceps sinensis fruiting bodies were dried at 50°C, crushed with a food grinder, and 0.2g of each powder was accurately weighed and placed in a stoppered test tube. 10mL of double distilled water was added, and ultrasonic treatment was performed for 30min. Then, the mixture was centrifuged at 5000g for 15min. Finally, the solid impurities were removed by filtration with a 0.45μm filter membrane, and the cordycepin content was detected by high performance liquid chromatography. During HPLC determination, the mobile phase flow rate was set to 1mL / min, the detection wavelength was 254nm, the column temperature was 30°C, and the injection volume was 20μL. All results are the average values ​​of three parallel samples.

[0056] The test results are shown in Table 5: Table 5 Cordycepin content of Cordyceps cultured in Example 3 and Comparative Example

[0057] The above test results show that the present invention studies the nutrients and growth environment required for different growth stages of Cordyceps, attempts to design the cultivation soil in stages, divides the life cycle of Cordyceps into the mycelium infection stage and the stroma growth stage, and matches different soil environments in accordance with its biological characteristics. While maximizing the infection survival rate of the larvae, the wild ecological environment is imitated as much as possible, a multi-layer cultivation soil structure is designed, the content of cordycepin in Cordyceps is increased, and the quality of artificially cultivated Cordyceps is greatly improved.

[0058] Test Example 3 Adenosine content test 3.1 Instruments Waterse 2695-2998 high performance liquid chromatograph; Photodiode Array Detector; EMPOWER chromatography workstation; AE240 electronic balance; SK-250H CNC ultrasonic instrument, KUDOS Ultrasonic Instrument Co., Ltd.

[0059] 3.2 Experimental Materials Adenosine reference substance (batch number: 110879-200202) was provided by the National Institute for the Control of Pharmaceutical and Biological Products; Cordyceps sinensis was a commercially available sample provided by Chengdu Jinxin Chinese Medicine Pieces Co., Ltd.

[0060] Methanol was a chromatographic-grade reagent, and the rest were analytical-grade reagents; water was ultrapure water.

[0061] 3.3 Methods 3.3.1 Chromatographic conditions Chromatographic column: Welchrom C18 (4.6 mm × 250 mm, 5 μm); mobile phase: 0.1% phosphoric acid solution (adjusted to pH 2.8) - methanol (95:5); flow rate: 1.0 mL / min; column temperature: 25 ° C; detection wavelength: 260 nm.

[0062] 3.3.2 Preparation of reference solution Take adenosine reference substance, weigh it accurately, place it in a 50mL volumetric flask, add 90% methanol to make a solution containing 200μg per 1mL, shake well, and use it as the stock solution; accurately take 1mL of the stock solution and place it in a 10mL volumetric flask, add 90% methanol to dilute to the scale, shake well, and use it as the reference substance solution (containing 20μg adenosine per 1mL).

[0063] 3.3.3 Preparation of test solution Take about 0.5g of the powder (passed through a No. 3 sieve) prepared from Cordyceps in Example 3 and Comparative Example, accurately weigh, place in a stoppered conical flask, accurately add 20mL of 90% methanol, seal, shake well, weigh, heat and reflux for 30min, cool, weigh again, make up the lost weight with 90% methanol, shake well, filter, and take the filtrate to obtain. The test results are shown in Table 6.

[0064] Table 6 Adenosine content determination results of samples

[0065] The above test results show that the present invention studies the nutrients and growth environment required for different growth stages of Cordyceps, attempts to design the cultivation soil in stages, divides the life cycle of Cordyceps into the mycelium infection stage and the stroma growth stage, and matches different soil environments in accordance with its biological characteristics. While maximizing the infection survival rate of the larvae, the wild ecological environment is imitated as much as possible, a multi-layer cultivation soil structure is designed, the content of adenosine in Cordyceps is increased, and the quality of artificially cultivated Cordyceps is greatly improved.

[0066] All technical features in this embodiment can be modified in appearance according to actual needs.

[0067] The above embodiments are preferred implementation schemes of the present invention. In addition, the present invention may also be implemented in other ways. Any obvious replacement without departing from the concept of the present technical solution is within the protection scope of the present invention.

Claims

1. A Cordyceps sinensis cultivation soil, characterized by: It includes the first-phase breeding soil and the second-phase breeding soil; The first-stage breeding soil is composed of the following components in parts by weight: 60-80 parts of coconut bran, 1-10 parts of perlite and 0.5-3 parts of nutrient solution; The second-stage breeding soil comprises a base layer (1), an aquifer (2), a nutrient layer (3) and an uppermost turf layer (4); The nutrient layer (3) is composed of the following components in parts by weight: 40-50 parts of coconut bran, 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 ectomycorrhizal fungus spores of the genus Lactarius; wherein the amount of ectomycorrhizal fungus spores of the genus Lactarius is 1-3% of the total weight of the humus.

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

3. The Cordyceps sinensis cultivation soil according to claim 1, characterized in that: 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 bran and 20-30 parts of a composite degradation product of straw fiber and polylactic acid; wherein the weight ratio of straw fiber to polylactic acid is 15:

1.

4. The Cordyceps sinensis cultivation soil according to claim 3, characterized in that: The water storage layer (2) is composed of the following components in parts by weight: 20 to 30 parts of clay, 3 to 7 parts of chitosan / polyacrylic acid composite hydrogel, and 5 to 8 parts of rock sand.

5. The Cordyceps sinensis cultivation soil according to claim 1, characterized in that: The turf layer (4) is formed by cross-planting of pine and bahia grass seedlings, and the spacing between the pine and bahia grass seedlings is not less than 15 cm.

6. The Cordyceps sinensis cultivation soil according to claim 1, characterized in that: The humus is made by mixing animal humus and plant humus in a mass ratio of 1:7-9, the animal humus is made by mixing cow dung and shell powder in a mass ratio of 20:1, and the plant humus is made by adding fermentation agents to sawdust, corn cobs, starch, ground pine and bahia grass in a mass ratio of 3:5:2:7:2 and composting and fermenting.

7. A method for controlling Cordyceps sinensis cultivation soil according to any one of claims 1 to 6, characterized in that: The specific steps include: S1, place the first-stage breeding soil in a dry ventilated container after disinfection, place the larvae infected by artificial injection of hyphae into the first-stage breeding soil, control the soil moisture at 70-80%, the ambient temperature at 15-25℃, and the ambient humidity at 75-85%, until the insect body fruiting buds germinate; S2, preparing and disinfecting each layer of the second-stage cultivation soil, and then laying them in a dry and ventilated container in the order of a base layer (1), a water storage layer (2), a nutrient layer (3) and a turf layer (4) from bottom to top, transplanting the insect bodies that have germinated from the fruiting buds in step S1 into the second-stage cultivation soil, with the lower end of the insect bodies located in the nutrient layer (3), and continuing to control the moisture content of the nutrient layer (3) and the turf layer (4) to 70-80%, the ambient temperature to 15-30°C, and the ambient humidity to 75-85%, until the Cordyceps grows; S3, when the Cordyceps in step S2 grows to the ascospore maturity stage, uninfected bat moth larvae are placed in the second-stage breeding soil for natural infection, forming an ecological self-circulation; S4, transplanting the larvae that are successfully naturally infected in step S3 into the first-stage breeding soil, repeating the breeding steps of steps S1 and S2, and replenishing nutrient solution at any time.

8. The method for controlling Cordyceps sinensis cultivation soil according to claim 7, characterized in that: The thickness of each layer of the second-phase aquaculture soil is specifically: a base layer (1) of 1 to 2 cm, a water storage layer (2) of 2 to 3 cm, and a nutrient layer (3) of 10 to 20 cm.

9. The method for controlling Cordyceps sinensis cultivation soil according to claim 7, characterized in that: In step S2, the height of the ground pine and bahia grass in the turf layer (4) is controlled within 3-10 cm during the cultivation of cordyceps to avoid blocking the cordyceps.

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