Cordyceps militaris culture medium and cultivation method of cordyceps militaris

Abstract

The invention relates to the technical field of cordyceps militaris cultivation, in particular to a cordyceps militaris cultivation medium and a cordyceps militaris cultivation method.The cordyceps militaris cultivation medium is prepared from, by weight, 80-90 parts of sustained release microspheres, 6-10 parts of silkworm chrysalis meal, 1-3 parts of yeast extract, 4-6 parts of bran, 0.5-2 parts of rice hull powder and 0.1-0.2 part of mineral elements. By optimizing the component compatibility of the culture medium, a microenvironment suitable for cordyceps militaris hypha growth and sporocarp differentiation is jointly created, the nutrition utilization rate is increased, and the risk of infectious microbe pollution is reduced through physical barriers and chemical inhibition.

Description

TECHNICAL FIELD

[0001] The present application relates to the cultivation technology field of Cordyceps militaris, and particularly relates to a Cordyceps militaris cultivation substrate and a cultivation method of Cordyceps militaris. BACKGROUND

[0002] Cordyceps militaris, also known as North Cordyceps or Cordyceps flower, belongs to Ascomycota and Cordyceps. Under natural conditions, Cordyceps militaris usually grows in the litter layer of forest land, and parasitizes on the pupae or larvae of Lepidoptera insects to complete its unique life cycle. The life cycle of Cordyceps militaris starts from ascospores produced by its fruiting body. The ascospores are spread by wind and infect suitable host insects, germinate into mycelium in the insect body, and gradually grow by utilizing the nutrients in the insect body. When the nutrients are almost exhausted, the mycelium further develops into sclerotia, and under suitable environmental conditions, the fruiting body with obvious head and stem grows from the sclerotia. The mature fruiting body can release ascospores again to achieve cyclic infection. In artificial cultivation, Cordyceps militaris can obtain fruiting bodies by solid cultivation, or obtain mycelium and its fermentation products by liquid fermentation. These products contain polysaccharides, cordycepic acid, nucleoside components, and other bioactive substances such as pentostatin, which endow them with immunomodulatory, antioxidant, antitumor, and neuroprotective functions, and thus have important application value in the fields of health food and medical research and development.

[0003] In the prior art, the traditional substrate mainly based on grains has fixed nutrient components and rapid release, which cannot meet the growth needs of Cordyceps militaris for several weeks. Excessive nutrients in the early stage easily cause pollution, and insufficient nutrients in the later stage lead to early senescence and insufficient accumulation of active components. In addition, the substrate rich in carbon and nitrogen is easily contaminated by miscellaneous bacteria after high-temperature and high-pressure sterilization if the inoculation and culture environment are not properly controlled. At the same time, the rapidly released sugar provides convenience for the growth of miscellaneous bacteria. SUMMARY

[0004] In order to solve the problems mentioned in the background, the present application provides a Cordyceps militaris cultivation substrate and a cultivation method of Cordyceps militaris.

[0005] In order to achieve the above-mentioned purposes, the present application adopts the following technical solutions: A Cordyceps militaris cultivation substrate, comprising the following components in parts by weight: slow-release microspheres 80-90 parts, silkworm pupa powder 6-10 parts, yeast extract 1-3 parts, bran 4-6 parts, rice husk powder 0.5-2 parts, and mineral elements 0.1-0.2 parts. The slow-release microspheres are core-shell structure microspheres with silica particles loaded with Cordyceps militaris nutrients as the core and temperature-sensitive copolymer as the shell.

[0006] Further, the slow-release microspheres are prepared by the following steps: S1, adding the dried silica powder into the sterile nutrient solution, stirring for 20-40 min, and drying at 60℃ until constant weight to obtain a uniform slurry; S2, dispersing the slurry in deionized water to form a suspension, adding monomers N-isopropyl acrylamide and acrylic acid into the suspension, and adding a crosslinking agent methylene bisacrylamide, under the protection of inert atmosphere, warming the reaction system, then adding an initiator, stirring for 4-8 h, after the reaction is completed, centrifuging and washing the obtained product, and drying at 60℃ until constant weight to obtain the slow-release microspheres.

[0007] Further, the mineral elements are composed of the following components: 0.05-0.15 parts of potassium dihydrogen phosphate, 0.03-0.08 parts of magnesium sulfate heptahydrate, and 0.01-0.03 parts of trace element compounds, wherein the trace element compounds are selected from at least one of zinc sulfate, ferrous sulfate, or calcium chloride.

[0008] Further, the particle size of the silkworm chrysalis powder is 80-200 mesh, the particle size of the rice husk powder is 40-100 mesh, and the total nitrogen content of the yeast extract is ≥8%.

[0009] Further, the sterile nutrient solution in step S1 includes the following components by weight / volume concentration (w / v): 15-25% of carbon source, 2-4% of nitrogen source, 0.5-1% of potassium dihydrogen phosphate, 0.2-0.5% of magnesium sulfate heptahydrate, and 0.01-0.05% of vitamin B1, and the balance is deionized water, wherein the carbon source is selected from at least one of glucose or sucrose, and the nitrogen source is selected from at least one of peptone or yeast powder.

[0010] Further, the ratio of silica and sterile nutrient solution in step S1 is 1g: (0.8-1.5) mL, and the particle size of the silica powder is 800-1500 mesh.

[0011] Further, the solid content of the suspension in step S2 is 0.5-2.0% (w / v), and the mass ratio of N-isopropyl acrylamide and acrylic acid is 1: (0.32-0.51).

[0012] Further, the addition amount of methylene bisacrylamide in step S2 is 0.5-1% of the total mass of N-isopropyl acrylamide and acrylic acid, and the addition amount of the initiator is 1-3% of the total mass of N-isopropyl acrylamide and acrylic acid, and the initiator is selected from at least one of ammonium persulfate, potassium persulfate, or azobisdimethylaminoformamide hydrochloride.

[0013] Further, the target temperature of warming in step S2 is 65-75℃, and the stirring speed is 100-300 rpm.

[0014] According to another aspect of the present application, a cultivation method of Cordyceps militaris is provided, comprising the following steps: A1, the components of the Cordyceps militaris cultivation substrate are weighed according to the proportion, mixed uniformly, then water is added to adjust the water content to 55-65%, the material is stirred after being mixed, and then the cultivation containers are filled with the material, and inoculation holes are prepared in the center of the material; A2, the cultivation containers after being filled are sterilized by high-pressure steam at 121-123 DEG C, 0.11-0.12 MPa for 30-40 min; A3, after the culture medium is cooled to 25-28 DEG C, 10-15 mL of Cordyceps militaris liquid strain or silkworm bean size solid strain is inoculated in a sterile environment; A4, the inoculated container is placed in a dark environment at 18-20 DEG C and a relative humidity of 60-70% for cultivation until the mycelium grows full of the culture medium and turns orange yellow; A5, the cultivation temperature is reduced to 15-17 DEG C, and 200-500 lx scattered light is given for stimulation, after primordium formation, the temperature is maintained at 18-22 DEG C and the humidity is 85-90%, and the cultivation is continued until the fruiting body matures; A6, when the height of the fruiting body reaches 8-10 cm, the top end swells and orange yellow spore powder appears, the whole cluster is harvested.

[0015] The beneficial effects of the present application are: 1, in the technical scheme of the present application, the slow-release microspheres construct a core-shell structure nutrient release system, in which the silicon dioxide particles loaded with Cordyceps militaris nutrients are used as the core to provide high specific surface area and adsorption capacity, which can effectively carry and protect key nutrients such as carbon source and nitrogen source; the copolymer of N-isopropyl acrylamide and acrylic acid is used as the shell, and through the swelling-shrinking behavior at different temperatures, the intelligent controllable release of nutrients is realized. In the mycelial growth stage, the shell structure is relatively loose, allowing slow release of nutrients, matching the slow nutrient demand of Cordyceps militaris, and avoiding the pollution of miscellaneous bacteria caused by excessive nutrients in the early stage. In the fruiting body formation stage, the shell shrinks to accelerate the release of nutrients, meeting the demand of Cordyceps militaris for rapid growth, thereby solving the problem of mismatching between the release of nutrients in the traditional substrate and the demand of Cordyceps militaris at different stages, and effectively preventing premature aging and insufficient accumulation of active ingredients.

[0016] 2, by optimizing the component compatibility of the cultivation substrate, the silkworm chrysalis powder provides natural insect-derived protein and lipids, the yeast extract supplements vitamins and growth factors, the bran and rice husk powder adjusts the porosity and air permeability of the substrate, and the mineral elements balance the supply of trace elements, which synergistically act with the slow-release microspheres to jointly create a microenvironment suitable for the growth of Cordyceps militaris mycelium and the differentiation of fruiting bodies, improve the nutrient utilization rate, and reduce the risk of miscellaneous bacterial pollution through physical barriers and chemical inhibition, especially after high-temperature high-pressure sterilization, the stability can still be maintained.

[0017] 3、The cultivating method of the present application realizes fine regulation and control of the growth cycle of Cordyceps militaris by precisely controlling environmental parameters such as temperature, humidity and light. In the mycelium culture stage, low temperature and darkness are adopted to promote the robust growth of mycelium, in the primordium formation stage, temperature reduction and scattered light are used to stimulate the differentiation of fruiting bodies, and in the fruiting body maturation stage, high humidity and suitable temperature are maintained to ensure the integrity of fruiting body morphology and the accumulation of active ingredients. The method, in combination with the slow-release substrate, significantly improves the bioconversion rate, fruiting body yield and consistency of Cordyceps militaris, and is suitable for large-scale production. DETAILED DESCRIPTION

[0018] The technical solutions of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0019] Unless otherwise specified, the raw materials used in the present application are all obtained from market-purchased conventional products.

[0020] Preparation Example 1 The slow-release microspheres are prepared by the following steps: 10 g of dried silica powder with a particle size of 1000 mesh is added to 10 mL of sterile nutrient solution (containing 20% glucose, 3% peptone, 0.8% potassium dihydrogen phosphate, 0.3% magnesium sulfate heptahydrate and 0.03% vitamin B1, and the balance is deionized water), stirred at a speed of 200 rpm for 30 min, and then dried at 60°C to constant weight to obtain silica particles loaded with nutrients; S2, the particles obtained in step S1 are dispersed in deionized water to prepare a suspension with a solid content of 1.0% (w / v), 1:0.4 of N-isopropyl acrylamide and acrylic acid (total mass of monomers is 10 g) are added, and 0.7% of methylene bisacrylamide based on the total mass of monomers is added, the reaction system is heated to 70°C under nitrogen protection, then 2% of ammonium persulfate based on the total mass of monomers is added, and the reaction is stirred at a speed of 200 rpm for 6 h. After the reaction is completed, the obtained product is centrifuged, washed with deionized water three times, and dried at 60°C to constant weight to obtain slow-release microspheres.

[0021] Preparation Example 2 The slow-release microspheres are prepared by the following steps: S1, 10 g of dried silica powder with a particle size of 800 mesh was added to 12 mL of sterile nutrient solution (containing 18% sucrose, 3.5% yeast powder, 0.5% potassium dihydrogen phosphate, 0.5% magnesium sulfate heptahydrate and 0.01% vitamin B1, and the balance was deionized water), stirred at 250 rpm for 20 min, and then dried at 60°C to constant weight to obtain silica particles loaded with nutrients; S2, the particles obtained in step S1 were dispersed in deionized water to prepare a suspension with a solid content of 1.5% (w / v), N-isopropyl acrylamide and acrylic acid were added in a mass ratio of 1:0.5 (the total mass of monomers was 10 g), and methylene bisacrylamide was added at 0.5% of the total mass of monomers. Under nitrogen protection, the reaction system was heated to 65°C, and then 1.5% of the total mass of monomers was added. Potassium persulfate was stirred at 150 rpm for 8 h. After the reaction was completed, the product was centrifuged, washed with deionized water three times, and dried at 60°C to constant weight to obtain the sustained-release microspheres.

[0022] Preparation Example 3 The sustained-release microspheres were prepared by the following steps: S1, 10 g of dried silica powder with a particle size of 800 mesh was added to 12 mL of sterile nutrient solution (containing 18% sucrose, 3.5% yeast powder, 0.5% potassium dihydrogen phosphate, 0.5% magnesium sulfate heptahydrate and 0.01% vitamin B1, and the balance was deionized water), stirred at 250 rpm for 20 min, and then dried at 60°C to constant weight to obtain silica particles loaded with nutrients; S2, the particles obtained in step S1 were dispersed in deionized water to prepare a suspension with a solid content of 1.5% (w / v), N-isopropyl acrylamide and acrylic acid were added in a mass ratio of 1:0.5 (the total mass of monomers was 10 g), and methylene bisacrylamide was added at 0.5% of the total mass of monomers. Under nitrogen protection, the reaction system was heated to 65°C, and then 1.5% of the total mass of monomers was added. Potassium persulfate was stirred at 150 rpm for 8 h. After the reaction was completed, the product was centrifuged, washed with deionized water three times, and dried at 60°C to constant weight to obtain the sustained-release microspheres.

[0023] Example 1 A method for cultivating Cordyceps militaris, comprising the following steps: The raw materials were weighed by weight fraction: 80 parts of sustained-release microspheres prepared in Preparation Example 1, 10 parts of silkworm chrysalis powder with a particle size of 100 mesh, 1 part of yeast extract with a total nitrogen content of 8%, 6 parts of bran, 0.5 parts of rice husk powder with a particle size of 60 mesh, and 0.2 parts of mineral elements (0.1 parts of potassium dihydrogen phosphate, 0.07 parts of magnesium sulfate heptahydrate, and 0.03 parts of zinc sulfate).

[0024] Subsequently, cultivation is carried out in the following steps: A1, water is added to the above mixed substrate, the moisture content is adjusted to 60%, after mixing, it is divided and packed in a cultivation bottle (500 mL), the wet weight of each bottle is about 300 g, and an inoculation hole is prepared in the center of the material; A2, the cultivation bottle is sterilized by high-pressure steam at 121°C and 0.11 MPa for 35 min; A3, after the medium is cooled to 26°C, 12 mL of Cordyceps militaris liquid strain is inoculated into each inoculation hole on a sterile operation table; A4, the inoculated cultivation bottle is placed in a dark culture room at 19°C and 65% relative humidity for culture until the mycelium grows full of the medium and turns orange; A5, the culture temperature is reduced to 16°C, and 300 lx scattered light stimulation is given, after primordium formation, the temperature is maintained at 20°C and the humidity is 88%, and the culture is continued until the fruiting body matures; A6, when the height of the fruiting body reaches 8 cm, the top is swollen, and orange yellow spore powder appears, the whole cluster is harvested.

[0025] Example 2 A method for cultivating Cordyceps militaris, comprising the following steps: The raw materials are weighed by weight fraction: 85 parts of the slow-release microspheres prepared in Preparation Example 2, 8 parts of silkworm pupa powder with a particle size of 150 mesh, 2 parts of yeast extract with a total nitrogen content of 8%, 5 parts of bran, 1.2 parts of rice husk powder with a particle size of 80 mesh, and 0.15 parts of mineral elements (0.08 parts of potassium dihydrogen phosphate, 0.05 parts of magnesium sulfate heptahydrate, and 0.02 parts of ferrous sulfate), which are mixed uniformly.

[0026] Subsequently, cultivation is carried out in the following steps: A1, water is added to the above mixed substrate, the moisture content is adjusted to 58%, after mixing, it is divided and packed in a cultivation pot, and an inoculation hole is prepared in the center of the material; A2, the cultivation container is sterilized by high-pressure steam at 122°C and 0.115 MPa for 30 min; A3, after the medium is cooled to 25°C, 10 mL of Cordyceps militaris liquid strain is inoculated in a sterile environment; A4, the inoculated container is placed in a dark environment at 18°C and 70% relative humidity for culture until the mycelium grows full of the medium and turns orange; A5, the culture temperature is reduced to 15°C, and 200 lx scattered light stimulation is given, after primordium formation, the temperature is maintained at 18°C and the humidity is 90%, and the culture is continued until the fruiting body matures; A6, when the height of the fruiting body reaches 8 cm, the top is swollen, and orange yellow spore powder appears, the whole cluster is harvested.

[0027] Example 3 A cultivating method of Cordyceps militaris, comprising the following steps: Each raw material is weighed by weight fraction: 90 parts of the slow-release microspheres prepared in Preparation Example 3, 6 parts of silkworm chrysalis powder with a particle size of 200 mesh, 3 parts of yeast extract with a total nitrogen content of 10%, 4 parts of bran, 2 parts of rice husk powder with a particle size of 100 mesh, and 0.1 parts of mineral elements (0.05 parts of potassium dihydrogen phosphate, 0.03 parts of magnesium sulfate heptahydrate, and 0.02 parts of calcium chloride).

[0028] Subsequently, cultivation is carried out by the following steps: A1, water is added to the above-mentioned mixed substrate, the water content is adjusted to 65%, and after mixing, the material is divided and packed in a cultivation bag, and an inoculation hole is prepared in the center of the material; A2, the cultivation container is sterilized with high-pressure steam at 123℃ and 0.12 MPa for 40 min; A3, after the culture medium is cooled to 28℃, 15 mL of Cordyceps militaris liquid strain is inoculated in a sterile environment; A4, the inoculated container is placed in a dark environment at 20℃ and 60% relative humidity for cultivation until the mycelium fills the culture medium and turns orange; A5, the cultivation temperature is reduced to 17℃, and 500 lx scattered light is given for stimulation, and after the primordium is formed, the temperature is maintained at 22℃ and the humidity is 85%, and the cultivation is continued until the fruiting body matures; A6, when the height of the fruiting body reaches 10 cm, the top is swollen and orange spore powder appears, the whole cluster is harvested.

[0029] Example 4 A cultivating method of Cordyceps militaris, comprising the following steps: Each raw material is weighed by weight fraction: 82 parts of the slow-release microspheres prepared in Preparation Example 1, 9 parts of silkworm chrysalis powder with a particle size of 120 mesh, 1.5 parts of yeast extract with a total nitrogen content of 8%, 5.5 parts of bran, 1.5 parts of rice husk powder with a particle size of 70 mesh, and 0.18 parts of mineral elements (0.12 parts of potassium dihydrogen phosphate, 0.04 parts of magnesium sulfate heptahydrate, and 0.02 parts of zinc sulfate).

[0030] Subsequently, cultivation is carried out by the following steps: A1, water is added to the above-mentioned mixed substrate, the water content is adjusted to 57%, and after mixing, the material is divided and packed in a cultivation bottle (500 mL), with a wet weight of about 300 g per bottle, and an inoculation hole is prepared in the center of the material; A2, the cultivation bottle is sterilized with high-pressure steam at 121℃ and 0.11 MPa for 38 min; A3, after the culture medium is cooled to 27℃, 13 mL of Cordyceps militaris liquid strain is inoculated into each inoculation hole on a sterile operation table; A4. The inoculated cultivation bottle is placed in a dark culture room at 19°C and 68% relative humidity for culture until the mycelium grows over the medium and turns orange yellow; A5. The culture temperature is lowered to 16°C, and 400 lx scattered light is given for stimulation. After primordium formation, the temperature is maintained at 21°C and the humidity is maintained at 87% for further culture until the fruiting bodies mature; A6. When the height of the fruiting bodies reaches 9 cm, the top end swells and orange yellow spore powder appears, the whole cluster is harvested.

[0031] Example 5 A cultivating method of Cordyceps militaris, comprising the following steps: The raw materials are weighed by weight fraction: 88 parts of the slow-release microspheres prepared in Preparation Example 2, 7 parts of silkworm chrysalis powder with a particle size of 180 mesh, 2.5 parts of yeast extract with a total nitrogen content of 12%, 4.5 parts of bran, 0.8 parts of rice husk powder with a particle size of 90 mesh, and 0.12 parts of mineral elements (0.07 parts of potassium dihydrogen phosphate, 0.06 parts of magnesium sulfate heptahydrate, 0.01 parts of ferrous sulfate, and 0.01 parts of calcium chloride), which are uniformly mixed.

[0032] Subsequently, cultivation is carried out according to the following steps: A1. Water is added to the above-mentioned mixed medium to adjust the water content to 62%, and after mixing, the medium is divided into cultivation pots, and an inoculation hole is prepared in the center of the medium; A2. The cultivation container is autoclaved at 122°C and 0.118 MPa for 32 min; A3. After the medium cools to 25.5°C, 11 mL of Cordyceps militaris liquid strain is inoculated in a sterile environment; A4. The inoculated container is placed in a dark environment at 18.5°C and 62% relative humidity for culture until the mycelium grows over the medium and turns orange yellow; A5. The culture temperature is lowered to 15.5°C, and 250 lx scattered light is given for stimulation. After primordium formation, the temperature is maintained at 19°C and the humidity is maintained at 89% for further culture until the fruiting bodies mature; A6. When the height of the fruiting bodies reaches 8.5 cm, the top end swells and orange yellow spore powder appears, the whole cluster is harvested.

[0033] Example 6 A cultivating method of Cordyceps militaris, comprising the following steps: The raw materials are weighed by weight fraction: 84 parts of the slow-release microspheres prepared in Preparation Example 3, 9.5 parts of silkworm chrysalis powder with a particle size of 80 mesh, 1.2 parts of yeast extract with a total nitrogen content of 8%, 5.8 parts of bran, 1.8 parts of rice husk powder with a particle size of 50 mesh, and 0.16 parts of mineral elements (0.14 parts of potassium dihydrogen phosphate, 0.03 parts of magnesium sulfate heptahydrate, and 0.02 parts of calcium chloride), which are uniformly mixed.

[0034] Subsequently, the following steps are taken for cultivation: A1, water is added to the above mixed substrate, and the moisture content is adjusted to 63%, after mixing, the material is divided and packed in cultivation bags, and inoculation holes are prepared in the center of the material; A2, the cultivation container is sterilized by high-pressure steam at 123°C and 0.12 MPa for 36 min; A3, after the medium is cooled to 27.5°C, 14 mL of Cordyceps militaris liquid strain is inoculated in a sterile environment; A4, the inoculated container is placed in a dark environment at 20°C and 66% relative humidity for cultivation, until the mycelium covers the medium and turns orange; A5, the cultivation temperature is reduced to 16.5°C, and 450 lx scattered light is given for stimulation, after the primordium is formed, the temperature is maintained at 22°C and the humidity is 86%, and the cultivation is continued until the fruiting body matures; A6, when the height of the fruiting body reaches 9 cm, the top is swollen and orange spore powder appears, the whole cluster is harvested.

[0035] Comparative Example 1 The difference between this comparative example and Example 1 is that the slow-release microspheres prepared in Example 1 are replaced with silica powder with a particle size of 1000 mesh, and the remaining steps are the same as Example 1.

[0036] Comparative Example 2 The difference between this comparative example and Example 2 is that the slow-release microspheres prepared in Example 2 are replaced with silica powder with a particle size of 800 mesh, and the remaining steps are the same as Example 2.

[0037] Comparative Example 3 The difference between this comparative example and Example 3 is that the slow-release microspheres prepared in Example 3 are replaced with silica powder with a particle size of 1500 mesh, and the remaining steps are the same as Example 3.

[0038] Comparative Example 4 The difference between this comparative example and Example 4 is that the slow-release microspheres prepared in Example 1 are replaced with N-isopropyl acrylamide, and the remaining steps are the same as Example 4.

[0039] Comparative Example 5 The difference between this comparative example and Example 5 is that no slow-release microspheres are added, and the remaining steps are the same as Example 5.

[0040] Examples 1-6 and Comparative Examples 1-5 are respectively arranged in parallel with 30 replicates (i.e. 30 bottles). During the cultivation process, the following indicators are observed and recorded: (Ⅰ) Contamination rate: During the mycelium cultivation stage (step A4), observe and record the contamination of each cultivation bottle regularly (contamination is the appearance of miscellaneous bacterial colonies). When the mycelium covers the culture medium, calculate the contamination rate of each group. Contamination rate (%) = (number of contaminated bottles / total number of bottles) x 100%. The results are shown in Table 1:

[0041]

[0042] (Ⅱ) Mycelial growth rate: From the 3rd day after inoculation, measure the spread diameter of mycelium on the surface of the culture medium at the same time every day, take the average value by cross method, until the mycelium covers the entire surface of the culture medium. Calculate the average daily growth rate of mycelium (mm / day) and the time required for full bottle (days). The results are shown in Table 2:

[0043]

[0044] (Ⅲ) When the fruiting body matures (reaches the harvesting standard), the following tests are performed: Growth cycle: Record the total number of days required from inoculation to the maturity of the fruiting body for harvesting.

[0045] Primordium formation rate: Record the percentage of bottles in each group that can normally form primordia out of the total number of bottles.

[0046] Fruiting body yield: Harvest all the fruiting bodies in each cultivation bottle, dry them in an oven at 60°C to constant weight, weigh and record the dry weight of fruiting bodies in each bottle (g / bottle).

[0047] Fruiting body morphology: Measure the average height of the fruiting bodies (cm).

[0048] The results are shown in Table 3:

[0049]

[0050] (Ⅳ) Randomly sample the dried fruiting bodies harvested from each group, refer to "GB / T 21121-2024 Determination of the oxidation stability of animal and vegetable oils (accelerated oxidation test)", determine the cordycepin content. Refer to "GB / T 15672-2009 Determination of total sugar content in edible fungi", determine the polysaccharide content. The results are shown in Table 4:

[0051]

[0052] From Tables 1-4, the contamination rates of Examples 1-6 are significantly lower than those of the comparative examples, indicating that the prepared sustained-release microspheres can effectively inhibit the growth of miscellaneous bacteria in the cultivation of Cordyceps militaris. The contamination rates of Comparative Examples 1-3, which use ordinary silicon dioxide powder instead of sustained-release microspheres, increase significantly, indicating that ordinary silicon dioxide powder has no antibacterial effect. The contamination rate of Comparative Example 4, which uses N-isopropyl acrylamide instead of sustained-release microspheres, is as high as 53.3%, and the contamination rate of Comparative Example 5, which does not add sustained-release microspheres, is as high as 60.0%, further proving the key role of the overall structure of the sustained-release microspheres in inhibiting miscellaneous bacteria. During the preparation of the sustained-release microspheres, the nutrient-loaded silicon dioxide particles form a specific structure through polymerization, which may change the physical and chemical properties of the culture medium, such as pore structure and surface charge, making it difficult for miscellaneous bacteria to adhere and grow; and the sustained-release microspheres may slowly release substances with antibacterial effects, continuously inhibiting miscellaneous bacteria.

[0053] The average daily growth rate of mycelium (3.61-4.10 mm / day) of Examples 1-6 is faster than that of the comparative examples, and the time required to fill the bottle (17-19 days) is shorter than that of the comparative examples, indicating that the sustained-release microspheres can promote mycelial growth. Comparative Examples 1-3 use ordinary silicon dioxide powder, and the mycelial growth rate and filling time are not as good as Examples; Comparative Examples 4 and 5 perform worse, with slower mycelial growth and longer filling time. The nutrient components in the sustained-release microspheres can be slowly released, providing a continuous and stable source of nutrients for mycelial growth, and their special structure may improve the air permeability and water retention of the culture medium, benefiting mycelial respiration and water and nutrient absorption.

[0054] The growth period (52-53 days) of Examples 1-6 is shorter than that of the comparative examples, the primordium formation rate (93.3-96.7%) is higher than that of the comparative examples, and the dry weight of fruiting bodies (2.06-2.22 g / bottle) and average height (8.4-8.9 cm) are better than those of the comparative examples, indicating that the sustained-release microspheres can promote the growth and development of fruiting bodies, improve yield and quality. Comparative Examples 1-3 use ordinary silicon dioxide powder, the growth period is prolonged, and the primordium formation rate, dry weight of fruiting bodies, and average height are all reduced, and Comparative Examples 4 and 5 perform worse, with lower indicators. The sustained-release microspheres continuously release nutrients to meet the needs of fruiting bodies at different growth stages; their structure may provide a suitable microenvironment for fruiting body growth, such as stable moisture and gas environment; and they may also contain signal substances that promote fruiting body differentiation and growth.

[0055] The cordycepin content (5.36-6.01 mg / g) and polysaccharide content (8.18-8.92%) of examples 1-6 are higher than those of the comparative examples, indicating that the slow-release microspheres can increase the content of active ingredients in the fruiting bodies. The active ingredient content is reduced in comparative examples 1-3 using ordinary silicon dioxide powder; the content is even lower in comparative examples 4 and 5. The nutrient components and special structure in the slow-release microspheres may regulate the metabolic pathways of Cordyceps militaris, promote the synthesis and accumulation of active ingredients such as cordycepin and polysaccharides, and the slow release of nutrients may enable Cordyceps militaris to be in a more suitable physiological state, which is conducive to the synthesis of active ingredients.

[0056] In summary, the present application realizes the intelligent controlled release of nutrients required for the growth of Cordyceps militaris by constructing slow-release microspheres with nutrient-loaded silicon dioxide as the core and temperature-sensitive copolymer as the shell. The core component is optimally combined with silkworm chrysalis powder, yeast extract and other auxiliary materials to form a high-efficiency cultivation substrate with synergistic effects of physical structure regulation and chemical nutrient supply.

[0057] In the description of the specification, the description referring to the terms "embodiment", "each embodiment" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or preparation example are included in at least one embodiment of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments in a suitable manner.

[0058] The above only describes the preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can make equivalent replacements or changes to the technical solutions and inventive concepts of the present application within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A cultivation substrate for Cordyceps militaris, characterized in that, The product comprises the following components by weight: 80-90 parts of slow-release microspheres, 6-10 parts of silkworm pupa powder, 1-3 parts of yeast extract, 4-6 parts of wheat bran, 0.5-2 parts of rice husk powder, and 0.1-0.2 parts of mineral elements.

2. The Cordyceps militaris cultivation substrate according to claim 1, characterized in that, The sustained-release microspheres are prepared by the following steps: S1. Add the dried silica powder to the sterile nutrient solution, stir for 20-40 minutes, and dry to obtain a slurry; S2. Disperse the slurry in deionized water to form a suspension. Add N-isopropylacrylamide and acrylic acid to the suspension, and add methylenebisacrylamide. Heat the reaction system, add an initiator, and stir the reaction for 4-8 hours. After the reaction is completed, centrifuge, wash, and dry to obtain sustained-release microspheres.

3. The Cordyceps militaris cultivation substrate according to claim 1, characterized in that, The mineral elements are composed of the following components: 0.05-0.15 parts of potassium dihydrogen phosphate, 0.03-0.08 parts of magnesium sulfate heptahydrate, and 0.01-0.03 parts of trace element compounds, wherein the trace element compounds are selected from at least one of zinc sulfate, ferrous sulfate, or calcium chloride.

4. The Cordyceps militaris cultivation substrate according to claim 1, characterized in that, The particle size of silkworm pupa powder is 80-200 mesh, the particle size of rice husk powder is 40-100 mesh, and the total nitrogen content of yeast extract is ≥8%.

5. The Cordyceps militaris cultivation substrate according to claim 2, characterized in that, The sterile nutrient solution in step S1, by weight-volume concentration (w / v), comprises the following components: 15-25% carbon source, 2-4% nitrogen source, 0.5-1% potassium dihydrogen phosphate, 0.2-0.5% magnesium sulfate heptahydrate, and 0.01-0.05% vitamin B1, with the balance being deionized water. The carbon source is selected from at least one of glucose or sucrose, and the nitrogen source is selected from at least one of peptone or yeast extract.

6. The Cordyceps militaris cultivation substrate according to claim 2, characterized in that, In step S1, the ratio of silica to sterile nutrient solution is 1g:(0.8-1.5)mL, and the particle size of silica powder is 800-1500 mesh.

7. The Cordyceps militaris cultivation substrate according to claim 2, characterized in that, In step S2, the solid content of the suspension is 0.5-2.0% (w / v), and the mass ratio of N-isopropylacrylamide to acrylic acid is 1:(0.32-0.51).

8. The Cordyceps militaris cultivation substrate according to claim 2, characterized in that, In step S2, the amount of methylenebisacrylamide added is 0.5-1% of the total mass of N-isopropylacrylamide and acrylic acid, and the amount of initiator added is 1-3% of the total mass of N-isopropylacrylamide and acrylic acid. The initiator is selected from at least one of ammonium persulfate, potassium persulfate, or azobisisobutyramidine hydrochloride.

9. The Cordyceps militaris cultivation substrate according to claim 2, characterized in that, In step S2, the target temperature for heating is 65-75℃, and the stirring speed is 100-300 rpm.

10. A method for cultivating Cordyceps militaris, characterized in that, Includes the following steps: A1. Weigh each component of the Cordyceps militaris cultivation substrate according to the proportions of any one of claims 1-9, mix them evenly, add water to adjust the moisture content to 55-65%, mix the materials and then divide them into cultivation containers, and prepare an inoculation hole in the center of the material. A2. Sterilize the dispensed cultivation containers with high-pressure steam at 121-123℃ and 0.11-0.12 MPa for 30-40 minutes; A3. After the culture medium has cooled to 25-28℃, inoculate 10-15mL of Cordyceps militaris liquid inoculum under aseptic conditions. A4. Place the inoculated container in a dark environment at 18-20℃ and 60-70% relative humidity until the mycelium covers the culture medium and turns orange-yellow. A5. Lower the culture temperature to 15-17℃ and provide 200-500 lx of diffused light stimulation. After the primordia are formed, maintain the temperature at 18-22℃ and the humidity at 85-90% and continue to culture until the fruiting bodies mature. A6. Harvest the whole clump when the fruiting body reaches a height of 8-10cm, the top is swollen and orange-yellow spore powder appears.

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