Cordyceps sinensis optimized culture medium and culture method thereof

Abstract

The invention relates to the technical field of biology, and discloses a cordyceps sinensis optimized culture medium and a culture method.The cordyceps sinensis optimized culture medium comprises a culture dish and further comprises a solid layer, a nutrition layer and an induction layer; the solid layer comprises a substrate filled in the inner bottom layer of the culture dish, and the substrate comprises 60%-70% of corncob powder, 15%-20% of wheat bran, 10%-15% of hepialus armoricanus larva residue powder, 0.5%-1% of monopotassium phosphate, 0.2%-0.4% of magnesium sulfate heptahydrate, 0.01%-0.05% of ferric sulfate and 5%-10% of calcium carbonate. Through the synergistic effect of the solid layer, the nutrition layer and the induction layer, the solid layer takes corncob powder, wheat bran and sawdust as substrates, provides physical support and water-holding and ventilation functions, and promotes hypha adhesion and expansion; the nutrient layer realizes selective permeation through a microporous filter membrane, so that solid matrix particles are prevented from settling to interfere with liquid flow, small-molecular nutrient substances such as glucose and amino acid are ensured to be efficiently transmitted to the matrix layer, and dynamic balance required by hypha metabolism is maintained.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and in particular to an optimized culture medium for Cordyceps sinensis and a culture method thereof. BACKGROUND

[0002] Cordyceps sinensis is a complex formed by the infection of bat moth larvae with Cordyceps fungus, and has wide medicinal value. Its main active ingredients include cordycepin, adenosine, cordycepic acid, etc., and are widely used in immune regulation, anti-fatigue, anti-tumor and other fields. However, due to the scarcity of natural resources and the limitations of ecological environment and climate conditions, wild collection has been unable to meet the market demand. Therefore, artificial cultivation of Cordyceps sinensis has become a key way to solve the resource shortage.

[0003] Although certain progress has been made in the artificial cultivation of Cordyceps sinensis in recent years, there are still many technical difficulties. First, the single nature of the culture medium limits the efficient growth of mycelium and stroma. Traditional culture media rely on a single carbon source (such as rice, wheat bran), which is difficult to meet the nutritional needs of different growth stages. In addition, the differentiation of mycelium and stroma requires different nutrient supplies, and a single culture medium cannot achieve precise control, resulting in slow mycelial growth or incomplete stroma differentiation. Second, insufficient environmental control is an important factor affecting the cultivation effect. The mycelial stage and stroma stage of Cordyceps sinensis have different requirements for environmental conditions such as temperature, humidity, and light. The existing cultivation equipment lacks dynamic adjustment capability and is difficult to maintain the best cultivation environment. For example, the mycelial stage requires lower temperature (15-18℃) and higher humidity (85%-90%), while the stroma stage requires higher temperature (20-25℃) and lower humidity (60%-70%). If the environmental parameters are not adjusted in time, it will directly affect the survival rate of mycelium and the morphological integrity of stroma.

[0004] In addition, bacterial contamination is a common problem in artificial cultivation. If the culture medium and culture environment are not strictly sterilized, they are easy to be infected by other microorganisms, thereby reducing the survival rate of mycelium and even leading to the failure of the entire cultivation. The existing cultivation device lacks effective antibacterial mechanism and is difficult to effectively inhibit the breeding of bacteria. For example, if appropriate antibacterial measures are not taken during the cultivation process, bacteria, molds and other bacteria may multiply in large quantities in the culture medium, compete with the target strain for nutrients, and even secrete harmful metabolites to inhibit the growth of mycelium.

[0005] Finally, the efficiency of the inducer also restricts the artificial cultivation of Cordyceps sinensis. The existing inducers (such as ecdysterone and flavonoids) have certain effects on promoting the differentiation of mycelium into stroma, but their concentration and release mode still need to be optimized. High concentration of inducer may cause excessive differentiation of mycelium or inhibit its growth, while low concentration cannot effectively trigger the formation of stroma. In addition, if the release mode of the inducer is not uniform, it may cause inconsistent growth of mycelium, affecting the quality of the final product.

[0006] In summary, the existing artificial cultivation technology of Cordyceps sinensis has problems such as single culture medium, insufficient environmental control, contamination of miscellaneous bacteria, and low efficiency of inducer, and an optimized culture medium and its cultivation method are urgently needed to improve the survival rate of mycelium, promote the differentiation of stroma, and improve the overall cultivation efficiency and product quality.

[0007] In the patent with the patent number CN1775943A, a semi-synthetic culture medium for Cordyceps sinensis is disclosed. The semi-synthetic culture medium for Cordyceps sinensis provided by the present application comprises 12.0-20.0 g of carbon, 0.70-1.49 g of nitrogen, 1.0-1.5 g of potassium dihydrogen phosphate, and 0.5-1.0 g of magnesium sulfate, and is diluted with water to 1000 ml. The carbon source is sucrose or glucose or maltose or lactose, and the nitrogen source is yeast extract and peptone or yeast extract and beef extract. The semi-synthetic culture medium for Cordyceps sinensis has the advantages of simple process, no heavy metal pollution, fast growth of mycelium, high yield of mycelium (more than 23 g / l), stable product quality, etc., and is suitable for physiological and biochemical research of Cordyceps sinensis and industrialized fermentation production of Cordyceps sinensis. The yield and cultivation efficiency of Cordyceps sinensis in the prior art can still be further improved. SUMMARY

[0008] In view of the problems existing in the prior art, an optimized culture medium for Cordyceps sinensis and its cultivation method are proposed.

[0009] In one aspect of the present application, an optimized culture medium for Cordyceps sinensis is provided, which comprises a culture dish, a solid layer, a nutrient layer, and an induction layer.

[0010] The solid layer comprises a substrate filled in the inner bottom layer of the culture dish, and the composition of the substrate is 60%-70% corn cob powder, 15%-20% wheat bran, 10%-15% bat moth larva residue powder, 0.5%-1% potassium dihydrogen phosphate, 0.2%-0.4% magnesium sulfate heptahydrate, 0.01%-0.05% ferric sulfate, and 5%-10% calcium carbonate.

[0011] Further, the nutrient layer comprises a total spray pipe fixedly connected to the culture dish, a spray head fixedly connected to the total spray pipe, a microporous filter membrane arranged on the solid layer, and a nutrient solution filled in the microporous filter membrane.

[0012] Further, the nutrient solution components are glucose 50%-60%, proteose peptone 40%-50%, yeast extract 2%-3%, potassium dihydrogen phosphate 2%-3%, magnesium sulfate heptahydrate 1%-2%, and vitamin B group 0.01%-0.05%.

[0013] Further, the induction layer comprises a water-absorbing expansion film covering the surface of the nutrient solution and a thin film active layer filled on the water-absorbing expansion film.

[0014] Further, the components of the thin film active layer are ecdysterone 16%-18% and flavonoids 80%-90%.

[0015] Further, the plurality of nozzles are circumferentially equidistantly arranged on the inner wall of the culture dish, and the microporous filter membrane is located between the nutrient solution and the substrate.

[0016] Further, the application also provides a culture method of the optimized cordyceps sinensis culture medium, comprising the following steps,

[0017] The solid layer raw materials are prepared by weighing and mixing corn cob powder 60%-70%, wheat bran 15%-20%, bat moth larva residue powder 10%-15%, potassium dihydrogen phosphate 0.5%-1%, magnesium sulfate heptahydrate 0.2%-0.4%, ferric sulfate 0.01%-0.05%, and calcium carbonate 5%-10%; the corn cob powder is crushed to a particle size of less than or equal to 2 mm, the wheat bran is selected from defatted products, and the bat moth larva residue powder is a low-temperature dried and crushed product; the inorganic salts are dissolved in a small amount of water to prepare a mother liquor, and the water content of the mixture is controlled to be 35%-40%.

[0018] Further, the corn cob powder, the wheat bran, and the bat moth larva residue powder are stirred at a low speed for 10 minutes until uniform, the calcium carbonate is continuously stirred for 5 minutes, the inorganic salt mother liquor is slowly injected, and the mixture is stirred until the water content reaches the standard.

[0019] Further, the mixture is loaded into a fermentation tank and sealed, the temperature is maintained at 30-35 DEG C, the humidity is maintained at 70%-75%, the pile is turned over every 5 days, and the fermentation period is 25-30 days.

[0020] Further, after the fermentation is completed, the mixture is sterilized by high-pressure steam at 121 DEG C for 30 minutes, and then cooled, divided, and sealed for storage.

[0021] The application has the following beneficial effects:

[0022] 1、Through the synergistic effect of the solid layer, the nutrient layer and the induction layer, the medium can realize precise regulation and efficient management of the mycelium growth environment, the solid layer takes corn cob powder, wheat bran and sawdust as the matrix, provides physical support and water holding and aeration function, and slowly releases part of the nutrient components to promote mycelium adhesion and expansion; the nutrient layer realizes selective permeation through the microporous filter membrane, which not only prevents the interference of solid substrate particles with liquid flow, but also ensures that small molecular nutrients such as glucose and amino acids are efficiently transported to the substrate layer, maintaining the dynamic balance required for mycelium metabolism; the induction layer activates the mycelium secondary metabolic pathway in a targeted manner through the complex ratio of ecdysterone and flavonoids, significantly improving the synthesis efficiency of target products, and the slow-release characteristics of the water-absorbing swelling film prolong the action time of active ingredients.

[0023] 2、Through the combination of the solid layer, the nutrient layer and the induction layer, the advantages are to optimize the nutrient supply path, strengthen the metabolic regulation ability, and reduce the pollution risk, finally realize the high-density growth of mycelium and the efficient accumulation of target products, and provide stable and controllable technical support for the industrialized production of high-value biological products such as Cordyceps sinensis. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a schematic diagram of the three-dimensional structure of the present application;

[0025] Figure 2 is a partial sectional view of the present application;

[0026] Figure 3 is Figure 2 is a schematic diagram of each structure separation.

[0027] In the figure: 1, culture dish; 2, solid layer; 21, matrix; 3, nutrient layer; 31, total spray pipe; 32, spray head; 33, microporous filter membrane; 34, nutrient solution; 4, induction layer; 41, water-absorbing swelling film; 42, thin film active layer. DETAILED DESCRIPTION

[0028] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below with reference to the drawings.

[0029] Example 1, refer to Figures 1-3 , the first embodiment of the present application provides an optimized medium for Cordyceps sinensis, which includes a culture dish 1, and further includes a solid layer 2, a nutrient layer 3 and an induction layer 4;

[0030] The solid layer 2 includes a substrate 21 filled in the inner bottom layer of the culture dish 1, and the substrate 21 is composed of 60-70% of corn cob powder, 15-20% of wheat bran, 10-15% of bat moth larva residue powder, 0.5-1% of potassium dihydrogen phosphate, 0.2-0.4% of magnesium sulfate heptahydrate, 0.01-0.05% of ferric sulfate and 5-10% of calcium carbonate.

[0031] Specifically, the solid layer 2 is one of the core structures of artificial cultivation of Cordyceps sinensis, and the design needs to consider the functions of physical support, nutrient supply and environmental regulation. The main raw materials of this layer include corn cob powder, wheat bran, bat moth larva residue powder, inorganic salt and regulator, and the total mass is 908.1 grams. Each component is mixed according to the accurate percentage ratio to ensure the functional synergy and stability.

[0032] The specific formula is: corn cob powder accounts for 66.07%, wheat bran accounts for 16.52%, bat moth larva residue powder accounts for 10.99%, potassium dihydrogen phosphate accounts for 0.55%, magnesium sulfate heptahydrate accounts for 0.33%, ferric sulfate accounts for 0.01%, and calcium carbonate accounts for 5.51%.

[0033] The functions and requirements of each component are as follows:

[0034] The corn cob powder, as the main carbon source and structural support material, needs to be crushed (particle size ≤2mm) to provide a loose and porous physical structure to promote mycelium attachment and expansion. The wheat bran, rich in cellulose and trace elements, needs to be selected from uncontaminated defatted wheat bran, accounting for 16.52% to supplement organic matter and nitrogen source. The bat moth larva residue powder, as a high-protein source, needs to be low-temperature dried and crushed into finished products, accounting for 10.99% to enhance mycelial metabolic activity. Potassium dihydrogen phosphate and magnesium sulfate heptahydrate provide essential elements such as phosphorus, potassium and magnesium, and the purity needs to be strictly controlled. Ferric sulfate, as an iron source, accounts for 0.01% and needs to be accurately weighed to avoid toxicity. Calcium carbonate is used to adjust the pH value to 6.5-7.0, accounting for 5.51% and needs to be an industrial-grade powder to ensure solubility and stability.

[0035] Production mode and deployment method:

[0036] 1. Raw material pretreatment: corn cob is crushed and sieved, then mixed with wheat bran, and bat moth larva residue powder is prepared separately. Inorganic salt needs to be weighed according to the proportion and dissolved in a small amount of water to prepare a mother liquor.

[0037] 2. Mixing process: add corn cob powder, wheat bran and bat moth larva residue powder into a mixer according to the proportion, mix at low speed for 10 minutes to ensure uniformity, then add calcium carbonate powder and continue mixing for 5 minutes, finally slowly inject the inorganic salt mother liquor into the mixed material, stirring while adding, and the mother liquor addition amount is 35-40% of the moisture content;

[0038] 3. Fermentation treatment: the mixture is loaded into a fermentation tank, covered with a film and sealed, and kept at a temperature of 30-35℃ and a humidity of 70%-75% for a fermentation period of 25-30 days; during this period, the heap is turned over every 5 days to ensure sufficient degradation and inactivation of potential pathogens;

[0039] 4. Sterilization and shaping: after the fermentation is complete, the substrate 21 needs to be sterilized at 121℃ for 30 minutes, and then cooled to room temperature and divided into blocks or granules for subsequent use.

[0040] Notes:

[0041] (1) The raw materials need to be strictly selected to avoid contamination with weed seeds or heavy metals;

[0042] (2) The moisture content needs to be controlled during mixing, as too high a moisture content can cause clumping, and too low a moisture content can affect mycelial adhesion;

[0043] (3) The fermentation temperature needs to be stabilized at 30-35℃, as too high a temperature can damage the active ingredients, and too low a temperature can prolong the cycle;

[0044] (4) After sterilization, the substrate 21 should be stored in a sealed manner to avoid secondary contamination;

[0045] (5) Before use, the pH value (6.5-7.0) and moisture content (35%-40%) need to be tested to ensure that they meet the growth requirements of the Cordyceps mycelium.

[0046] Through the above formulation and process, the solid layer 2 can provide a stable physical structure and balanced nutritional environment for Cordyceps, and through precise control of the physicochemical parameters, the growth efficiency of the mycelium and the success rate of the stroma differentiation can be significantly improved.

[0047] Reference Figures 2-3 , the nutrient layer 3 includes a total spray pipe 31 fixedly connected to the culture dish 1, a spray head 32 fixedly connected to the total spray pipe 31, a microporous filter membrane 33 arranged on the solid layer 2, and a nutrient solution 34 filled in the microporous filter membrane 33; the nutrient solution 34 is composed of 50%-60% glucose, 40%-50% protein peptone, 2%-3% yeast extract, 2%-3% potassium dihydrogen phosphate, 1%-2% magnesium sulfate heptahydrate, and 0.01%-0.05% vitamin B group.

[0048] Specifically, the nutrient layer 3 is a key functional layer in the artificial culture system of Cordyceps sinensis, and its core role is to provide readily available carbon source, nitrogen source and trace elements for mycelium, promote the rapid growth of mycelium and maintain metabolic activity. The layer exists in the form of liquid culture solution, and based on 1 liter of culture solution, the total dry matter mass is 37.51 grams, and each component is accurately proportioned to ensure the balance and stability of nutrient supply; the nutrient solution 34 is connected to the total spray pipe 31 through a peristaltic pump, and slowly supplements the liquid to the nutrient layer 3 through 12 circumferentially equidistantly arranged spray heads 32, the nutrient solution 34 is separated from the substrate 21 by a microporous filter membrane 33, and the independence of the two is ensured by the pore size screening effect of the microporous filter membrane 33, and the synergistic regulation of nutrition and environment is realized through selective permeation.

[0049] The specific formula is: glucose accounts for 53.33% (20 grams), protein peptone 40% (15 grams), yeast extract 2.67% (1 gram), potassium dihydrogen phosphate 2.67% (1 gram), magnesium sulfate heptahydrate 1.33% (0.5 gram), vitamin B group 0.03% (0.01 gram).

[0050] The functions and requirements of each component are:

[0051] Glucose as a readily available carbon source, food grade analytical pure, accounts for 53.33% to provide the energy required for rapid metabolism of mycelium; protein peptone as a nitrogen source and amino acid source, industrial grade product meeting the standard of microbial culture, accounts for 40% to support protein synthesis of mycelium; yeast extract rich in B vitamins and trace elements, high-activity defatted yeast extract, accounts for 2.67% to supplement cofactors; potassium dihydrogen phosphate and magnesium sulfate heptahydrate provide essential elements such as phosphorus, potassium and magnesium respectively, and the purity needs to be strictly controlled, accounts for 2.67% and 1.33% to maintain osmotic pressure and enzyme activity; vitamin B group needs to use a composite preparation (containing B1, B2, B6, B12, etc.), accounts for 0.03% to participate in coenzyme synthesis.

[0052] Production mode and deployment method:

[0053] 1. Raw material pretreatment: glucose and protein peptone need to be weighed and dissolved in a small amount of reverse osmosis water (pH 5.8-6.2) respectively, and stirred until completely dissolved; yeast extract needs to be preheated to 40℃ to soften before adding to the solution;

[0054] 2. Mixing process: mix the dissolved glucose solution and protein peptone solution according to the proportion, then add the yeast extract solution, continue to stir for 30 minutes; potassium dihydrogen phosphate and magnesium sulfate heptahydrate need to be dissolved separately and added slowly, stirring while adding until homogeneous; finally add the vitamin B group solution and mix evenly;

[0055] 3. Sterilization: the mixed solution needs to be sterilized at 121℃ for 30 minutes, and then cooled to room temperature before being divided into sterile containers. After sterilization, the pH value (6.0-6.5) and electrical conductivity (EC value ≤1.5 mS / cm) need to be tested to ensure that the mycelium growth requirements are met.

[0056] Notes:

[0057] (1) The raw materials need to be strictly selected to avoid containing heavy metals or organic pollutants;

[0058] (2) Glucose and proteose peptone need to be prepared and used immediately, as long-term storage can lead to degradation;

[0059] (3) The temperature needs to be controlled during mixing (≤40℃), as high temperature can damage the activity of vitamin B;

[0060] (4) After sterilization, the culture solution should be stored in the dark at 4℃ in the refrigerator, and restored to room temperature before use;

[0061] (5) Before use, the pH value and EC value need to be tested, and if necessary, adjust the pH to 6.0-6.5 with dilute hydrochloric acid or sodium hydroxide.

[0062] Through the above formula and process, the nutrient layer 3 can provide precise and readily available nutrients for the Cordyceps mycelium, and through strict control of the physicochemical parameters, the growth rate of the mycelium and the accumulation efficiency of the metabolites are significantly improved.

[0063] Reference Figures 2-3 , the induction layer 4 includes a water-absorbing and expanding film 41 covering the surface of the nutrient solution 34, and a thin film-shaped active layer 42 filled on the water-absorbing and expanding film 41, the composition of the thin film-shaped active layer 42 is ecdysterone 16%-18% and flavonoids 80%-90%.

[0064] Specifically, the induction layer 4 is a key functional layer in the artificial culture system of Cordyceps, its core function is to regulate the metabolic pathway of the mycelium and promote the directional synthesis of the target products: β-ecdysterone and flavonoids through the synergistic effect of specific active ingredients. This layer exists in the form of solid powder, based on the case of 1 gram of culture system, the total mass is 0.06 grams, the formula strictly follows the ratio of ecdysterone 16.67% (0.01 grams) and flavonoids 83.33% (0.05 grams), and the flavonoids include a water-absorbing and expanding film 41 covering the surface of the nutrient solution 34.

[0065] The specific formula is: ecdysterone accounts for 16.67% (0.01 grams), flavonoids accounts for 83.33% (0.05 grams), and flavonoids are composed of plant extracts and water-absorbing and expanding film 41 in a ratio of 7:3.

[0066] Functions and requirements of each component:

[0067] Ecdysterone needs to choose high purity (HPLC≥98%) β-ecdysterone, its role is to simulate insect molting hormone signal pathway, activate the secondary metabolic pathway of mycelium, promote the synthesis and accumulation of β-ecdysterone, flavonoids need to choose natural extracts with antioxidant and anti-inflammatory activity, its function is to remove free radicals, regulate oxidative stress environment, and assist mycelium to maintain metabolic balance; The water-swellable film 41 as a physical carrier needs to have the characteristics of rapid water absorption and swelling (80-100 times), and after swelling, a dense film layer is formed to cover the surface of the nutrient solution 34, preventing the volatilization of active ingredients and delaying the release rate.

[0068] Production method and deployment method:

[0069] 1. Raw material pretreatment: Ecdysterone needs to be dissolved in anhydrous ethanol under light-proof conditions to a concentration of 2 mg / mL, and ultrasonic assisted dissolution for 30 minutes, flavonoids need to be ground to 200 mesh or less, and the impurities are removed after mixing and sieving;

[0070] 2. Mixing process: slowly add the dissolved ecdysterone solution to the flavonoid powder, stir while adding until completely absorbed and uniform, the mixture needs to be dried in a 40°C vacuum drying oven to constant weight, ensuring that the moisture content is ≤2%;

[0071] 3. Forming and packaging: the dried mixed powder is packaged into aluminum foil bags, 0.06 grams per bag, and the water-swellable film 41 needs to be prepared separately: mix polyacrylic acid sodium and crosslinking agent in a ratio of 100:1, cast into a 1cm×1cm sheet, and place in a dryer for standby.

[0072] Notes:

[0073] (1) Ecdysterone needs to be stored in a -20°C refrigerator in the dark to avoid decomposition caused by high temperature or light;

[0074] (2) The environmental humidity during the mixing process of flavonoids needs to be controlled (≤40%) to prevent the water-swellable film 41 from absorbing moisture in advance;

[0075] (3) The mixing process needs to be carried out in a sterile operation table to avoid bacterial contamination;

[0076] (4) The swelling performance of the water-swellable film 41 needs to be regularly tested to ensure that its water absorption ratio is stable at 80-100 times;

[0077] (5) The proportion of each component needs to be strictly controlled when deploying the solid layer 2, and the particle size of the corn cob powder that is too large or too small will affect the mycelium attachment efficiency.

[0078] Through the above formula and process, the induction layer 4 can precisely regulate the metabolic direction of the mycelium, combined with the antioxidant effect of flavonoids and the slow-release function of the water-absorbing and swelling film 41, significantly improving the yield and activity of β-ecdysterone in Cordyceps sinensis.

[0079] During use, through the synergistic effect of the solid layer 2, the nutrient layer 3 and the induction layer 4, the culture medium can realize precise regulation and efficient management of the mycelium growth environment. The solid layer 2 takes corn cob powder, wheat bran and sawdust as the matrix 21, providing physical support and water-holding and aeration functions, while slowly releasing part of the nutrient components to promote the attachment and expansion of the mycelium. The nutrient layer 3 realizes selective permeation through the microporous filter membrane 33, preventing the solid matrix 21 particles from interfering with the liquid flow, while ensuring efficient transmission of small-molecule nutrients such as glucose and amino acids to the matrix 21 layer, maintaining the dynamic balance required for mycelial metabolism. The induction layer 4 activates the secondary metabolic pathway of the mycelium by complexing ecdysterone and flavonoids, significantly improving the synthesis efficiency of the target product, while the slow-release characteristics of the water-absorbing and swelling film 41 extend the action time of the active ingredients. The advantages of the combination of the three are to optimize the nutrient supply path, strengthen the metabolic regulation ability and reduce the pollution risk, ultimately realizing high-density growth of the mycelium and efficient accumulation of the target product, providing stable and controllable technical support for the industrialized production of high-value biological products such as Cordyceps sinensis.

[0080] Embodiment 2, refer to Figures 1-3 As a second embodiment of the present application, a culture method of an optimized culture medium for Cordyceps sinensis is provided, comprising the following steps,

[0081] S1: Prepare the solid layer 2 raw materials, mix 66.07% (600g) of corn cob powder, 16.52% (150g) of wheat bran, 10.99% (100g) of bat moth larva residue powder, 0.55% (5g) of potassium dihydrogen phosphate, 0.33% (3g) of magnesium sulfate heptahydrate, 0.01% (0.09g) of ferric sulfate and 5.51% (50g) of calcium carbonate, with a total mass of 908.1g. The corn cob powder needs to be crushed to a particle size of ≤2mm, the wheat bran is a defatted product, and the bat moth larva residue powder is a low-temperature dried and crushed product. The inorganic salts are dissolved in a small amount of water to form a mother liquor, and the water content is controlled at 35%-40% after mixing;

[0082] S2: Stir the corn cob powder, wheat bran and bat moth larva residue powder at low speed for 10 minutes until uniform, continue to stir for 5 minutes after adding calcium carbonate, slowly inject the inorganic salt mother liquor and stir until the water content of the mixture reaches the standard;

[0083] S3: Put the mixture into the fermentation tank and seal it, keep the temperature at 30-35℃ and the humidity at 70%-75%, turn the pile every 5 days, and the fermentation period is 25-30 days;

[0084] S4: After fermentation is completed, high-pressure steam sterilization is performed at 121℃ for 30 minutes, and after cooling, the product is packed into blocks or granules and sealed for storage;

[0085] S5: A nutrient layer 3 liquid culture solution is prepared by weighing 53.33% (20 g) of glucose, 40% (15 g) of proteose peptone, 2.67% (1 g) of yeast extract, 2.67% (1 g) of potassium dihydrogen phosphate, 1.33% (0.5 g) of magnesium sulfate heptahydrate, and 0.03% (0.01 g) of vitamin B group, with a total dry matter mass of 37.51 g; the glucose and proteose peptone are dissolved in reverse osmosis water, and the yeast extract is added after being softened by heating to 40℃;

[0086] S6: The glucose solution and the proteose peptone solution are mixed for 30 minutes, the yeast extract solution is then added and stirred, followed by the slow addition of the potassium dihydrogen phosphate and magnesium sulfate heptahydrate solutions until homogenization, and finally the vitamin B group solution is added and mixed evenly;

[0087] S7: The mixed solution is sterilized at 121℃ for 30 minutes, and after cooling, it is packed into sterile containers, and the pH value is detected to be 6.0-6.5 and the conductivity EC value is ≤1.5 mS / cm, and it is stored in a 4℃ refrigerator in the dark;

[0088] S8: An induction layer 4 is prepared by weighing 16.67% (0.01 g) of ecdysterone and 83.33% (0.05 g) of flavonoids, and the flavonoids are mixed with the water-absorbing and swelling film 41 at a ratio of 7:3;

[0089] S9: The ecdysterone is dissolved in anhydrous ethanol to a concentration of 2 mg / mL, and ultrasonic assistance is performed for 30 minutes; the flavonoid component is ground to 200 mesh or less and sieved, and is mixed with the ecdysterone solution and stirred until adsorption is uniform;

[0090] S10: The mixture is dried in a vacuum drying oven at 40℃ until the moisture content is ≤2%, and is packed into aluminum foil bags at 0.06 g per bag; the water-absorbing and swelling film 41 is prepared separately: sodium polyacrylate is mixed with a crosslinking agent at a ratio of 100:1, cast into a mold, and cut into 1 cm x 1 cm sheets;

[0091] S11: A culture system is built, with the solid layer 2 placed at the bottom, covered with a microporous filter membrane 33 (pore size 0.22 μm) to separate the nutrient layer 3; the nutrient permeate solution is slowly replenished through a peristaltic pump connected to 12 circumferential spray heads 32;

[0092] S12: The water-absorbing and swelling film 41 of the induction layer 4 is laid on the surface of the nutrient solution 34, ensuring uniform coverage, and the inducer powder is layered and spread on the surface of the substrate 21 layer;

[0093] S13: control the culture environment temperature 18-22℃, humidity 70%~75%, light intensity ≤500lux, monitor pH value (substrate 21 layer 6.5-7.0, nutrient solution 34 6.0-6.5) and water content every day;

[0094] S14: check the growth every 3 days after mycelium inoculation, adjust the nutrient solution 34 supply frequency to twice a week, and supplement the induction layer 4 every 10 days;

[0095] S15: the culture period is 90-120 days, and the sclerotia are harvested after formation, inactivated active ingredients are obtained by soaking in hot water at 60-80℃ for 10-15 minutes, and drying to ≤10% water content is completed.

[0096] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A Cordyceps sinensis optimized culture medium, comprising a culture dish (1), characterized in that: It also includes a solid layer (2), a nutrient layer (3) and an induction layer (4); The solid layer (2) includes a matrix (21) filled in the bottom layer inside the culture dish (1), and the matrix (21) comprises 60% to 70% corn cob powder, 15% to 20% wheat bran, 10% to 15% bat moth larvae residue powder, 0.5% to 1% potassium dihydrogen phosphate, 0.2% to 0.4% magnesium sulfate heptahydrate, 0.01% to 0.05% iron sulfate, and 5% to 10% calcium carbonate.

2. The optimized culture medium for Cordyceps sinensis according to claim 1, wherein: The nutrient layer (3) comprises a main nozzle (31) fixedly connected to the culture dish (1), a nozzle (32) fixedly connected to the main nozzle (31), a microporous filter membrane (33) arranged on the solid layer (2), and a nutrient solution (34) filled on the microporous filter membrane (33).

3. The optimized culture medium for Cordyceps sinensis according to claim 2, characterized in that: The nutrient solution (34) comprises 50% to 60% glucose, 40% to 50% peptone, 2% to 3% yeast extract, 2% to 3% potassium dihydrogen phosphate, 1% to 2% magnesium sulfate heptahydrate, and 0.01% to 0.05% B vitamins.

4. The optimized culture medium for Cordyceps sinensis according to claim 3, characterized in that: The induction layer (4) comprises a water-absorbing and expanding membrane (41) covering the surface of the nutrient solution (34), and a thin film active layer (42) filled on the water-absorbing and expanding membrane (41).

5. The optimized culture medium for Cordyceps sinensis according to claim 4, characterized in that: The composition of the thin film active layer (42) is 16% to 18% of ecdysterone and 80% to 90% of flavonoids.

6. The optimized culture medium for Cordyceps sinensis according to claim 5, characterized in that: A plurality of nozzles (32) are circumferentially and equidistantly arranged on the inner wall of the culture dish (1), and a microporous filter membrane (33) is located between the nutrient solution (34) and the matrix (21).

7. The method for cultivating the optimized culture medium of Cordyceps sinensis according to claim 6, characterized in that: Prepare the raw materials for the solid layer (2) by weighing and mixing 60% to 70% corn cob powder, 15% to 20% wheat bran, 10% to 15% bat moth larvae residue powder, 0.5% to 1% potassium dihydrogen phosphate, 0.2% to 0.4% magnesium sulfate heptahydrate, 0.01% to 0.05% iron sulfate, and 5% to 10% calcium carbonate; the corn cob powder needs to be crushed to a particle size of ≤ 2 mm, the wheat bran should be defatted, and the bat moth larvae residue powder should be low-temperature dried and crushed into a finished product; the inorganic salt is dissolved in a small amount of water in proportion to prepare a mother liquor, and the moisture content after mixing is controlled at 35% to 40%.

8. The method for cultivating the optimized culture medium of Cordyceps sinensis according to claim 7, characterized in that: Stir corn cob powder, wheat bran, and bat moth larvae residue powder at low speed for 10 minutes until uniform, add calcium carbonate and continue stirring for 5 minutes, slowly inject inorganic salt mother liquor and stir until the moisture content of the mixture meets the standard.

9. The method for cultivating the optimized culture medium of Cordyceps sinensis according to claim 8, characterized in that: Place the mixture into the fermentation tank and seal it, maintain the temperature at 30-35℃ and the humidity at 70%-75%, turn the pile over every 5 days, and the fermentation cycle is 25-30 days.

10. The method for cultivating the optimized culture medium of Cordyceps sinensis according to claim 9, characterized in that: After fermentation is completed, sterilize with high-pressure steam at 121°C for 30 minutes, cool and package into blocks or granules, and seal and store for later use.

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