Culture method of functional cordyceps militaris sporocarp

Abstract

The invention discloses a method for culturing functional cordyceps militaris sporocarp, and belongs to the technical field of biology. The culture method comprises the following steps: step 1, inoculating a cordyceps militaris preservation strain into a mother strain slant culture medium for culture to obtain a first-grade cordyceps militaris seed; step 2, inoculating the first-stage cordyceps militaris seeds into a second-stage liquid strain culture medium for culturing to obtain second-stage cordyceps militaris seeds; step 3, inoculating the second-level cordyceps militaris seeds into a third-level liquid strain culture medium for culturing to obtain third-level cordyceps militaris seeds; and step 4, inoculating the third-level cordyceps militaris seeds into a grass-growing experimental culture medium, and culturing until cordyceps militaris grass-growing seeds are obtained. The process is simple, culture substitute raw materials are rich and easy to obtain, waste is recycled, environmental pollution is reduced, and a scientific basis is provided for large-scale production of functional cordyceps militaris.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for culturing a functional Cordyceps militaris fruiting body, a functional Cordyceps militaris fruiting body prepared by the culturing method, and an application of the functional Cordyceps militaris fruiting body in extracting cordycepin and / or cordycepic acid. Background Art

[0002] Cordyceps militaris, also known as northern cordyceps and northern caterpillar fungus, is a traditional medicinal and edible fungus that is loved by people for its nutritional and economic value. Since the discovery and isolation of Cordyceps militaris in the 1980s, my country's cultivation technology has developed rapidly and has initially achieved large-scale cultivation without seasonal restrictions.

[0003] Cordyceps militaris belongs to the genus Cordyceps, class Pyrheomycetes, order Glomerulates, family Claviceps, and is a bipolar heterothallic fungus. Cordycepin is a secondary metabolite of Cordyceps militaris, and its synthesis ability is also closely related to the species, nutritional requirements, environmental conditions and other factors. At the same time, Cordyceps militaris strains are prone to changes in genetic traits under artificial culture conditions, especially in agronomic traits and fruiting ability, and are very prone to sterility, yield reduction or fruiting body deformities.

[0004] Pueraria root is a native plant of my country, and it is widely distributed in my country. From the North China Plain to the land of Jingchu, from the shores of the East China Sea to the Northwest Plateau, it can be found almost everywhere. Pueraria root is a plant that can be used as both medicine and food, and is usually divided into medicinal Pueraria root and edible Pueraria root. Medicinal Pueraria root includes wild Pueraria root and bitter Pueraria root, which taste bitter and difficult to swallow, but have high medicinal value. Edible Pueraria root includes sweet Pueraria root and powder Pueraria root, which taste sweet and refreshing and have the effect of beautifying and nourishing the skin. Its roots, stems, leaves, and flowers can all be used as medicine. Modern medicine shows that the active ingredients in Pueraria root are isoflavones, among which puerarin has the highest content. These isoflavone compounds can treat diseases such as acute myocardial infarction, unstable angina, hyperviscosity, acute cerebral infarction, cervical spondylosis, etc., and have an antagonistic effect on memory impairment caused by scopolamine and ethanol; its edible part is mainly the root tuber, people extract its starch and process it into a series of products such as kudzu vermicelli, kudzu cake, kudzu beverage, kudzu jelly, kudzu wine, kudzu biscuits, etc., and there is a certain amount of puerarin in the wastewater and waste residue produced by the processing. At the same time, the stems and leaves of kudzu root are good animal feeds, and because of its tenacious adaptability and vitality, it is also a soil and water conservation plant that countries around the world attach great importance to, and is known as the "good doctor of the earth". Therefore, the cultivation of kudzu root has developed rapidly in recent years. Since the research field of kudzu root and Cordyceps militaris has the same efficacy, this also provides feasibility for using kudzu root in the cultivation of Cordyceps militaris. Summary of the invention

[0005] The present application aims to provide a method for cultivating high-quality Cordyceps militaris fruiting bodies using kudzu root and puerarin powder as culture matrix, mainly using kudzu root rhizomes and leaves rich in functional ingredients to replace grain for Cordyceps militaris cultivation, reprocessing kudzu root processing wastewater and waste residue to obtain puerarin, and utilizing it in culture medium, thereby improving the biological conversion rate and preparing high-quality Cordyceps militaris fruiting bodies. The process of the present application is simple, the cultivation alternative raw materials are abundant and easy to obtain, and the recycling of waste reduces environmental pollution, providing a scientific basis for the large-scale production of functional Cordyceps militaris.

[0006] On the one hand, the present application provides a method for culturing a functional Cordyceps militaris fruiting body, comprising the following steps:

[0007] Step 1: inoculating the preserved strain of Cordyceps militaris into the mother strain slant culture medium for cultivation to obtain the first-grade seeds of Cordyceps militaris;

[0008] Step 2: inoculating the first-level seeds of Cordyceps militaris into a second-level liquid culture medium for cultivation to obtain second-level seeds of Cordyceps militaris;

[0009] Step 3: inoculating the second-level seeds of Cordyceps militaris into a third-level liquid culture medium for cultivation to obtain third-level seeds of Cordyceps militaris;

[0010] Step 4: inoculating the Cordyceps militaris grade 3 seeds into a grass-growing experimental medium, and culturing until Cordyceps militaris grass-growing seeds are obtained;

[0011] The mother species slant culture medium, the secondary liquid strain culture medium, the tertiary liquid strain culture medium and the grass growing test medium all contain pueraria root and puerarin powder, the mass ratio of puerarin powder to pueraria root in the mother species slant culture medium, the secondary liquid strain culture medium and the tertiary liquid strain culture medium is 1:(5-10), and the mass ratio of puerarin powder to pueraria root in the grass growing test medium is 1:(7-10);

[0012] The kudzu root is kudzu root particles processed by the following method: drying fresh kudzu root in an environment of 50-60° C., crushing and sieving after drying, and crushing to 5-50 meshes to obtain the kudzu root particles.

[0013] Optionally, the puerarin powder extraction method comprises: subjecting pueraria processing waste residue and pueraria processing wastewater to subcritical water extraction at a mass ratio of 1:(15-17) to obtain an extract, and then subjecting the extract to solid-liquid separation, concentration, and drying to obtain puerarin powder.

[0014] Optionally, the culture method of step one comprises: aseptically inoculating the preserved strain of Cordyceps militaris onto a mother strain slant culture medium under light-proof conditions, and culturing for 7 days at 18-22° C. in the dark for activation.

[0015] Optionally, under sterile conditions, the first-level Cordyceps militaris seeds are inoculated into a second-level liquid culture medium, and cultured at a constant temperature and shaken at 18-22° C. in the dark for 84-96 hours.

[0016] Optionally, the culturing method of step three includes: the charging volume accounts for 50-60% of the tank volume, and after actual disinfection, the temperature is lowered to 22-24°C, and Cordyceps militaris secondary seeds are added according to the charging volume in the tank, the inoculation amount is 4-6%, the tank pressure is 0.02-0.06Mpa, 20-22°C, the rotation speed is 130-150rpm, and the light-proof fermentation time is 2-3 days. When the mycelium is strong and the volume of the ball is rich and accounts for 50-70%, the cultivation is stopped.

[0017] Optionally, the culturing method of step four includes: the culturing method of step four includes: aseptically inoculating 6% to 8% of Cordyceps militaris grade 3 seeds into a grass-growing experimental culture medium, first culturing in the dark at 18-22°C, then entering the color change stage, controlling the light intensity to 200-400Lx, the air humidity to 65-75%, waiting for the mycelium color to turn orange-yellow, puncturing holes for ventilation to form Cordyceps militaris fruiting body primordia, controlling the air humidity to 85%-95%, and culturing until it can be harvested.

[0018] Optionally, the mother culture slant culture medium, the secondary liquid culture medium, the tertiary liquid culture medium and the grass growing experiment culture medium also contain crop nutrients, and the crop nutrients are selected from any one or more of potatoes, wheat grains, rice, corn and buckwheat.

[0019] Optionally, the mother culture slant culture medium, secondary liquid culture medium, tertiary liquid culture medium, and weeding experiment culture medium also contain nutrient supplements, and the nutrient supplements are selected from any several of glucose, peptone, magnesium sulfate, potassium dihydrogen phosphate, vitamin B1, agar, glycine, zinc sulfate, and calcium chloride.

[0020] On the other hand, the present application provides a functional Cordyceps militaris fruiting body prepared by the above method.

[0021] In another aspect, the present application provides a use of a functional Cordyceps militaris fruiting body in extracting cordycepin and / or cordycepic acid.

[0022] The beneficial effects of the present invention include at least one of the following:

[0023] 1. The cultivation method provided in this application and the method for producing Cordyceps militaris using pueraria root and puerarin powder can not only reduce resource waste and environmental pollution, but also save production raw materials and grain, and reduce production costs;

[0024] 2. The cultivation method provided by the present application has improved fruiting body yield and cordycepin content to varying degrees, has good cordycepic acid synthesis ability, a 100% grass yield rate in liquid strains, excellent agronomic traits of strain fruiting bodies, and an average biological efficiency of 90.80%;

[0025] 3. The cultivation method provided in this application and the liquid strain production technology are stable and reliable and can be used for large-scale production and application;

[0026] 4. The cultivation method provided in the present application uses pueraria root and puerarin powder to replace part of the nutrients in the culture medium during the culture process, thereby increasing the content of isoflavones in the fruiting bodies of Cordyceps militaris, reducing costs, taming the strains, and improving vitality;

[0027] 5. The cultivation method provided in the present application can improve the appearance and biological indicators of Cordyceps militaris, increase the content of isoflavones and the like in the fruiting bodies of Cordyceps militaris, and enhance the antioxidant capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0029] Figure 1 This is a micrograph of the mycelium of the secondary seeds of Cordyceps militaris in Example 1;

[0030] Figure 2 This is a schematic diagram of the germination of the fruiting body primordium of Cordyceps militaris in Example 1;

[0031] Figure 3 This is a schematic diagram of the grass growth of Cordyceps militaris fruiting bodies in Example 1;

[0032] Figure 4 This is a schematic diagram of the growth of the fruiting body of Cordyceps militaris in Example 1. DETAILED DESCRIPTION

[0033] In order to more clearly illustrate the overall concept of the application, the following is described in detail by way of embodiment. In the following description, a large amount of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0034] If no specific conditions are specified in the examples, the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials, instruments and reagents involved in the following examples can be purchased from commercial sources.

[0035] In the following examples, pueraria root and puerarin powder were prepared by the following method:

[0036] 1. Treatment of Pueraria root:

[0037] The fresh, mildew-free and rotten kudzu root is selected after the kudzu root is harvested in autumn or winter, washed, dewormed and peeled, and the kudzu root is placed in a grinder to grind the sweet potato vine into small pieces of 3-4 cm, and then the ground kudzu root is placed in a drying oven at 50-60° C. for drying, and after drying, it is crushed and sieved, and dried until the water content reaches 6-10%, and then bagged for standby use. The dried kudzu root is further crushed by a grinder until it can pass through a 5-10 mesh sieve to obtain kudzu root coarse particles; the kudzu root coarse particles are further crushed by a grinder until it can pass through a 30-50 mesh sieve to obtain kudzu root granules.

[0038] 2. Preparation of puerarin powder:

[0039] (1) placing kudzu root waste residue and kudzu root waste water in a mass ratio of 1:(15-17) into a subcritical water extraction device, adding 10% propane solution and 5% ethanol solution by weight of the kudzu root waste residue and the kudzu root waste water, mixing and performing subcritical water extraction at an extraction pressure of 10 MPa, an extraction temperature of 115° C., and an extraction time of 60 minutes to obtain an extract;

[0040] (2) After the extract is cooled to room temperature, it is centrifuged at 4000 rpm / min for 5 min, then evaporated and concentrated to one twentieth of the original volume, and then freeze-dried at -60°C to obtain puerarin powder.

[0041] In the following examples, a functional Cordyceps militaris fruiting body is prepared by the following method:

[0042] Step 1: Preparation of primary seeds

[0043] After the crop nutrients, kudzu vine root particles and distilled water are mixed and boiled for 20-30 minutes, the filtrate is obtained by filtering with filter cloth, and puerarin powder and nutritional supplements are added to the filtrate, the mass ratio of puerarin powder to kudzu vine root is 1: (5-10), and the mass ratio of crop nutrients to kudzu vine root particles is (15-20): 1. After mixing evenly, put them into a test tube, and then put the test tube into a steam sterilizer for sterilization at 121°C for 25-30 minutes, cool to room temperature, and preserve the Cordyceps militaris strain, which is obtained by culturing CICC14014, CICC14015 or CICC14013 strains. Under sterile conditions, use an inoculation hook to scratch a 4×4mm mycelium sheet away from the original inoculation point, use an inoculation shovel to scoop out the mycelium sheet and place it in the middle and lower part of the fresh mother slope, press it slightly, and activate and culture it in a constant temperature incubator at 18-22°C in the dark for 7 days, stop culturing after the tube is full, and store it at low temperature to obtain the first-class Cordyceps militaris seeds.

[0044] The crop nutrients are selected from any one or more of potatoes, wheat grains, rice, corn, and buckwheat, however, the present invention is not limited thereto.

[0045] Nutritional supplements include glucose, glycine, peptone, potassium dihydrogen phosphate, magnesium sulfate, vitamin B1 and agar.

[0046] Step 2: Preparation of secondary seeds

[0047] After the crop nutrients, kudzu vine root particles and distilled water are mixed and boiled for 20-30 minutes, the filtrate is filtered through a filter cloth to obtain a filtrate, and puerarin powder and nutritional supplements are added to the filtrate, the mass ratio of puerarin powder to kudzu vine root is 1: (5-10), and the mass ratio of crop nutrients to kudzu vine root particles is (15-20): 1. After being evenly mixed, the mixture is put into a triangular bottle, sterilized at 121°C for 30 minutes and cooled to room temperature, and the first-level seeds of Cordyceps militaris are aseptically scratched into 3 to 5 2×2mm mycelium pieces away from the original inoculation point with an inoculation hook, and the mycelium pieces are scooped out with an inoculation shovel and placed in a second-level triangular bottle, and cultured at 18-22°C, 130-150rpm in the dark for 3 to 5 days, and the culture is stopped when the mycelium is strong and the volume of the rich balls reaches 50-70%, and the second-level seeds of Cordyceps militaris are obtained.

[0048] The crop nutrients are selected from any one or more of potatoes, wheat grains, rice, corn, and buckwheat, however, the present invention is not limited thereto.

[0049] Nutritional supplements include glucose, peptone, glycine, potassium dihydrogen phosphate, magnesium sulfate, and vitamin B1.

[0050] Step 3: Preparation of tertiary seeds

[0051] The crop nutrients, kudzu vine root particles and distilled water are mixed and boiled for 20-30 minutes, and then filtered with a filter cloth to obtain a filtrate, and puerarin powder and nutritional supplements are added to the filtrate, wherein the mass ratio of puerarin powder to kudzu vine root particles is 1:(5-10), and the mass ratio of crop nutrients to kudzu vine root particles is (15-20):1. After being evenly mixed, the mixture is put into a triangular bottle, and the filling volume accounts for 60% of the tank volume. After being actually sterilized, the temperature is lowered to 22-24° C., and Cordyceps militaris secondary liquid seeds are added according to the filling volume in the tank, the inoculation amount is 4-6%, the tank pressure is 0.02-0.06Mpa, 20-22° C., the rotation speed is 130-150rpm, and the light-proof fermentation time is 2-3 days. When the mycelium is strong and the volume of the rich fungus balls accounts for 50-70%, the cultivation is stopped to obtain Cordyceps militaris tertiary seeds.

[0052] The crop nutrients are selected from any one or more of potatoes, wheat grains, rice, corn, and buckwheat, however, the present invention is not limited thereto.

[0053] Nutritional supplements include glucose, peptone, glycine, potassium dihydrogen phosphate, magnesium sulfate, and vitamin B1.

[0054] The tertiary liquid bacterial culture medium also includes a defoamer, and the defoamer is any one of natural oils and fats, polyethers, higher alcohols and silicone resins. However, the present invention is not limited thereto.

[0055] Step 4: Preparation of grass seeds

[0056] The crop nutrients, kudzu root coarse particles and puerarin powder are placed in a 1000 ml volumetric culture bottle, the mass ratio of puerarin powder to kudzu root is 1: (2-5), the nutritional supplements are added to the volumetric culture bottle at a ratio of 1: (1.4-2.0) g / ml, the bottle mouth is covered with a polyethylene film, and it is tied with a rubber band, and the culture bottle is placed in a steam sterilizer at 121° C. for 60-70 minutes, cooled to room temperature, and the third-grade Cordyceps militaris seeds prepared in step 3 are inoculated, and the mixture is inoculated. The seeding amount is 6-8%, and the culture is carried out in a dark environment at a temperature of 18-22°C; after the white mycelium grows and covers the surface of the culture medium in the bottle, the culture enters the color change stage, the light intensity of the culture is 200-400Lx, and the air humidity is 65-75%; when the mycelium color turns to orange, use a sterile needle to evenly pierce holes at the mouth of the culture bottle for ventilation, and the primordium protrudes and grows out, and the culture is carried out in an environment with an air humidity of 85-95%, and the Cordyceps militaris fruiting body is harvested when the top is slightly swollen and as high as the bottle mouth.

[0057] In one embodiment, in the above-mentioned step 4, during the culture process of Cordyceps militaris fruiting bodies, when the mycelium changes color and the primordium protrusions are just formed, the culture can be stopped according to the production purpose, that is, the primordium of Cordyceps militaris fruiting body is formed. At this time, the culture medium is rich in mycelium and rich in nutrients such as protein, carbohydrates, amino acids, etc., and can be used for the development of downstream products such as health products.

[0058] Example 1

[0059] A method for cultivating a functional Cordyceps militaris fruiting body in this embodiment comprises the following steps:

[0060] Step 1: Preparation of Cordyceps militaris first-grade seeds

[0061] 200 g / L potato, 10 g / L kudzu vine granules and 1000 ml distilled water were mixed and boiled for 20 min, and then filtered through filter cloth to obtain a filtrate. Puerarin powder 2 g / L, glucose 20 g / L, glycine 2 g / L, peptone 10 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate 1.5 g / L, vitamin B1 10 mg / L, and agar 15 g / L were added to the filtrate, mixed evenly and put into a test tube, and then the test tube was placed in a steam sterilizer for sterilization at 121°C for 30 min, cooled to room temperature, and the Cordyceps militaris preserved strain was aseptically scratched with an inoculation hook away from the original inoculation point into 4×4 mm mycelium slices, and the strain was scooped out with an inoculation shovel and placed in the middle and lower part of the slope of a fresh first-level seed, slightly pressed, and activated and cultured in a constant temperature incubator at 18-22°C in the dark for 7 days, and the culture was stopped after the tube was full, and stored at low temperature to obtain the first-level seeds of Cordyceps militaris.

[0062] Step 2: Preparation of Cordyceps militaris secondary seeds

[0063] Mix 200g / L potato, 10g / L kudzu vine granules and 1000ml distilled water and boil for 25min, filter through filter cloth to obtain filtrate, add 2g / L puerarin powder, 20g / L glucose, 2g / L glycine, 10g / L peptone, 3g / L potassium dihydrogen phosphate, 1.5g / L magnesium sulfate and 10mg / L vitamin B1 to the filtrate, mix well and put into a conical flask, sterilize at 121℃ for 30min and cool to room temperature, and use an inoculation hook to aseptically mark 3-5 2×2mm mycelium pieces away from the original inoculation point for the first-level seeds of Cordyceps militaris, use an inoculation shovel to scoop out the mycelium pieces and place them in a second-level seed conical flask, and culture them at 18-22℃, 130-150rpm in the dark for 3 days, and stop culturing when the mycelium is strong and the volume of the rich balls reaches 50-70%, and obtain the second-level seeds of Cordyceps militaris.

[0064] Step 3: Preparation of Cordyceps militaris Grade 3 Seeds

[0065] 200 g / L potato, 10 g / L kudzu vine granules and 1000 ml distilled water were mixed and boiled for 20-30 min, and then filtered through filter cloth to obtain a filtrate. Puerarin powder 2 g / L, glucose 20 g / L, glycine 2 g / L, peptone 10 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate 1.5 g / L, vitamin B1 10 mg / L, and defoamer 0.06 mL / L were added to the filtrate, and the mixture was evenly mixed and put into a triangular flask. The filling volume accounted for 60% of the tank volume. After actual disinfection, the temperature was lowered to 22-24° C., and Cordyceps militaris secondary seeds were inoculated according to the filling volume in the tank. The inoculation amount was 4-6%, the tank pressure was 0.02-0.06 MPa, 20-22° C., the rotation speed was 130-150 rpm, the light-proof fermentation time was 2-3 days, and the culture was stopped when the mycelium was strong and the volume of the rich balls accounted for 50-70%, and the Cordyceps militaris tertiary seeds were obtained.

[0066] Step 4: Cultivation of Cordyceps militaris fruiting bodies

[0067] 35g / L wheat, 8g / L kudzu root coarse grains, and 4g / L puerarin powder were placed in a 650ml volumetric culture bottle. 1g / L magnesium sulfate, 2g / L potassium dihydrogen phosphate, and vitamin B1 were added to the volumetric culture bottle. The method comprises the following steps: mixing 5 mg / L of 1,2-dichlorobenzene and 500 mL of water, covering the bottle mouth with a polyethylene film, and fastening it with a rubber band, placing the culture bottle in a steam sterilizer and sterilizing at 121° C. for 60-70 min, cooling it to room temperature, and inoculating the third-grade Cordyceps militaris seeds prepared in step 3 with an inoculation amount of 6-8%, and culturing it in a dark environment at a temperature of 18-22° C.; culturing it in a color change stage after the white mycelium grows and covers the surface of the culture medium in the bottle, the light intensity of the culture is 200-400 Lx, and the air humidity is 65-75%; when the mycelium color turns to orange-yellow, evenly piercing holes at the mouth of the culture bottle with a sterile needle for ventilation, and the primordium protrudes and grows out, and culturing it in an environment with an air humidity of 85-95%, and harvesting it when the top of the fruiting body of the Cordyceps militaris slightly swells up to the bottle mouth.

[0068] Example 2

[0069] The preparation method of this embodiment is substantially the same as that of embodiment 1, except that, in the preparation of the first-grade seeds of Cordyceps militaris in step 1, 13 g / L of Pueraria lobata granules and 1.3 g / L of puerarin powder are used; in the preparation of the second-grade seeds of Cordyceps militaris in step 2, 13 g / L of Pueraria lobata granules and 2.2 g / L of puerarin powder are used; in the preparation of the third-grade seeds of Cordyceps militaris in step 3, 13 g / L of Pueraria lobata granules and 2.0 g / L of puerarin powder are used; and in the cultivation of the fruiting bodies of Cordyceps militaris in step 4, 50 g / L of wheat, 14 g / L of Pueraria lobata coarse grains and 7 g / L of puerarin powder are used.

[0070] Example 3

[0071] The preparation method of this embodiment is substantially the same as that of Embodiment 1, except that, in the preparation of the first-grade seeds of Cordyceps militaris in step 1, 12 g / L of Pueraria lobata granules and 2 g / L of puerarin powder are used; in the preparation of the second-grade seeds of Cordyceps militaris in step 2, 12 g / L of Pueraria lobata granules and 1.5 g / L of puerarin powder are used; in the preparation of the third-grade seeds of Cordyceps militaris in step 3, 12 g / L of Pueraria lobata granules and 1.3 g / L of puerarin powder are used; and in the cultivation of the fruiting bodies of Cordyceps militaris in step 4, 40 g / L of wheat, 10 g / L of Pueraria lobata coarse grains and 2.5 g / L of puerarin powder are used.

[0072] Example 4

[0073] The preparation method of this embodiment is substantially the same as that of embodiment 1, except that, in the preparation of the first-grade seeds of Cordyceps militaris in step 1, 11 g / L of Pueraria lobata granules and 1.4 g / L of puerarin powder are used; in the preparation of the second-grade seeds of Cordyceps militaris in step 2, 11 g / L of Pueraria lobata granules and 1.8 g / L of puerarin powder are used; in the preparation of the third-grade seeds of Cordyceps militaris in step 3, 11 g / L of Pueraria lobata granules and 2.2 g / L of puerarin powder are used; and in the cultivation of the fruiting bodies of Cordyceps militaris in step 4, 45 g / L of wheat, 12 g / L of Pueraria lobata coarse grains and 4 g / L of puerarin powder are used.

[0074] Example 5

[0075] The preparation method of this embodiment is substantially the same as that of embodiment 1, except that, in the preparation of the first-grade seeds of Cordyceps militaris in step 1, 10.5 g / L of Pueraria lobata granules and 2.1 g / L of puerarin powder are used; in the preparation of the second-grade seeds of Cordyceps militaris in step 2, 10.5 g / L of Pueraria lobata granules and 1.75 g / L of puerarin powder are used; in the preparation of the third-grade seeds of Cordyceps militaris in step 3, 10.5 g / L of Pueraria lobata granules and 1.3 g / L of puerarin powder are used; and in the cultivation of the fruiting bodies of Cordyceps militaris in step 4, 55 g / L of wheat, 16 g / L of Pueraria lobata coarse grains and 8 g / L of puerarin powder are used.

[0076] Comparative Example 1

[0077] The preparation method of this comparative example is roughly the same as that of Example 1, except that:

[0078] The composition of the mother slant culture medium is as follows: mix 200 g / L potato and 1000 ml distilled water and boil for 20-25 minutes, filter with filter cloth to obtain the filtrate, add 20 g / L glucose, 2 g / L glycine, 10 g / L peptone, 3 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate, 10 mg / L vitamin B1, and 15 g / L agar to the filtrate.

[0079] The composition of the secondary liquid culture medium is as follows: mix 200 g / L potatoes and 1000 ml distilled water and boil for 20-25 minutes, filter with filter cloth to obtain a filtrate, add 20 g / L glucose, 2 g / L glycine, 10 g / L peptone, 3 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate, and 10 mg / L vitamin B1 to the filtrate.

[0080] The composition of the tertiary liquid culture medium is as follows: mix 200g / L potatoes and 1000ml distilled water and boil for 20-30min, filter with filter cloth to obtain the filtrate, add 20g / L glucose, 2g / L glycine, 10g / L peptone, 3g / L potassium dihydrogen phosphate, 1.5g / L magnesium sulfate, 10 mg / L vitamin B1, and 0.06mL / L defoaming agent to the filtrate.

[0081] The composition of the medium for the grass growing experiment was as follows: wheat was 50 g / L, and the parameters of the other steps were the same.

[0082] Depend on Figure 1 It can be seen that the secondary liquid seed strains of Example 1 are all light yellow, with moderate ball density, uniform size, strong vitality, strong hyphae and abundant branches under the microscope, and no foreign bacteria. Figure 2 It can be seen that on the culture medium for the grass growing experiment, the mycelium is white, the color changes quickly, and the primordium density is uniform. Figure 3 It can be seen that in Example 1, the fruiting body grows fast and has a high rate of emergence. Figure 4 It can be seen that the fruiting body in Example 1 is golden in color, uniform and neat, and has a swollen head.

[0083] Test 1: Biological indicator test

[0084] In each example, 12 bottles of Cordyceps militaris fruiting bodies were cultivated at the same time. The biological indicators of Cordyceps militaris in the above examples are shown in Table 1. The data in the following table are all average values ​​of the test data of 12 bottles of Cordyceps militaris fruiting bodies.

[0085] Table 1

[0086]

[0087] As shown in Table 1, the fruit bodies of Examples 1-5 have good shape, golden color, uniform and neat, enlarged heads, moderate density, and the grass yield rate reaches 100%. The fruit bodies have excellent agronomic traits and the biological efficiency reaches an average of 90.80%. The yield is between 37.8% and 40.4%, which is suitable for large-scale cultivation. The biological indicators of the fruit bodies of Examples 1-5 are all higher than those of the comparative example, indicating that the fruit bodies in the examples have good appearance and high commercial value.

[0088] Test 2: Functional index test

[0089] 1. Determination of cordycepic acid content in fruiting bodies

[0090] (1) Reagents and instruments

[0091] Reagents: cordycepin (Solebao Biotechnology Company, purity ≥98%), chromatographic acetonitrile, chromatographic methanol, deionized water, ethanol, glucose, peptone, magnesium sulfate, potassium dihydrogen phosphate, vitamin B1, corn flour, glycine, etc.

[0092] Main instruments and equipment: HFsafe-1200 biological safety cabinet (Shanghai Lishen Scientific Instrument Co., Ltd.), SpH-2102 vertical double-layer large-capacity constant temperature culture shaker (Shanghai Shiping Experimental Equipment Co., Ltd.), SPX-450 biochemical incubator (Zhongyi Guoke (Beijing) Technology Co., Ltd.), LDZX-75KBS vertical high-pressure steam sterilizer (Shanghai Shen'an Medical Equipment Factory), Waters2695 high-performance liquid chromatograph (Waters Company), chromatographic column Luna 5u C18(2)100A (250x4.6mm) (Agilent), KQ-50DA CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0093] (2) Analysis methods

[0094] ① Determination of cordycepin content in mycelium: The determination and analysis were carried out in accordance with the provisions of the Ministry of Agriculture and Rural Affairs standard NY / T2116-2012. The standard curve equation of cordycepin was y=33477x+13493 (R2=0.9999).

[0095] ② Biomass determination: The weight method was used, and the calculation formula was: X = W / V × 100, where: X is the mycelium biomass (g / L), W is the mycelium dry weight (g); V is the sampling volume (mL).

[0096] 2. Determination of cordycepic acid content in fruiting bodies

[0097] (1) Preparation of solvent

[0098] Nash reagent: Weigh 150 g of acetylamine, dissolve it in distilled water, add 2 mL of glacial acetic acid and 2 mL of acetylacetone, and make up to 1000 mL.

[0099] 0.015mol / L sodium periodate: weigh 3.2g of sodium periodate and dissolve it in 0.12mol HCL, and make up to 1000mL.

[0100] 0.1% L-rhamnose: weigh 0.1 g of L-rhamnose, dissolve it in distilled water, and make up to 100 mL.

[0101] Mannitol standard solution: weigh 100 mg of mannitol standard, dissolve in distilled water, dilute to 100 mL, which is 1 mg / mL mannitol standard stock solution, and store at 20°C for later use. Before measurement, accurately pipette 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of 1 mg / mL mannitol standard stock solution into a 100 mL volumetric flask, dilute to 100 mL with distilled water, and set aside.

[0102] (2) Drawing of standard curve

[0103] Measure 1 mL of mannitol standard solution with a mass concentration of 0 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, and 60 mg / L, add 1 mL of potassium periodate solution, mix, and react at room temperature for 10 min; then add 2 mL of 0.1% L-rhamnose solution to remove excess potassium periodate, shake and mix, add 4 mL of freshly prepared Nash reagent, bathe in 53°C water for 15 min to color it, quickly cool to room temperature, and operate with a spectrophotometer at a wavelength of 412 nm, with the standard concentration as the abscissa and the corresponding absorbance as the ordinate. The linear regression equation of the cordycepic acid standard is y=0.0157x-0.0056 (R2=0.9998). The cordycepic acid standard curve has good linearity and the method is stable.

[0104] (3) Determination of cordycepic acid content in samples

[0105] Accurately weigh 0.50g of each of the cultured Cordyceps militaris fruiting body powders in the above examples, place in a 100mL volumetric flask, add 80mL of deionized water, place in an ultrasonic instrument for ultrasonic extraction for 2h, take out and dilute to volume with deionized water, shake well. The extract is filtered through a 0.22μm microporous filter membrane, 1mL of the sample solution to be tested is taken into a 10mL graduated test tube, and the absorbance value is determined according to the cordycepic acid standard curve determination method, and distilled water is used as a control, and the cordycepic acid content is calculated by the regression equation.

[0106] The functional indicators of Cordyceps militaris fruiting bodies in the above example are shown in Table 2:

[0107] Table 2

[0108] Example Cordycepin content (mg / g) Cordycepic acid content (mg / g) Example 1 3.79 36.27 Example 2 3.68 32.45 Example 3 3.50 33.95 Example 4 3.59 34.54 Example 5 3.71 35.78 Comparative Example 1 2.84 26.23

[0109] As shown in Table 2, the cordycepin and cordycepic acid contents of the fruiting bodies of Examples 1-5 are not much different, with the cordycepin content reaching 3.50mg / g-3.79mg / g and the cordycepic acid content being 36.27mg / g-32.45mg / g. Among them, the cordycepin and cordycepic acid contents of the fruiting bodies in Example 1 are the highest, with the cordycepic acid content reaching 3.79mg / g and the cordycepic acid content reaching 36.27mg / g. Overall, the functional indicators of the fruiting bodies of Cordyceps militaris cultivated in Examples 1 to 5 are all higher than those of the comparative example, indicating that the use of Pueraria lobata and puerarin powder as substitutes improves the quality of the fruiting bodies of Cordyceps militaris.

[0110] Each embodiment in this specification is described in a progressive manner, and the same or similar parts between the embodiments can be referred to each other, and each embodiment focuses on the differences from other embodiments. In particular, for the system embodiment, since it is basically similar to the method embodiment, the description is relatively simple, and the relevant parts can be referred to the partial description of the method embodiment.

[0111] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A method for cultivating a functional Cordyceps militaris fruiting body, characterized in that: The culture method comprises the following steps: Step 1: inoculating the preserved strain of Cordyceps militaris into the mother strain slant culture medium for cultivation to obtain the first-grade seeds of Cordyceps militaris; Step 2: inoculating the first-level seeds of Cordyceps militaris into a second-level liquid culture medium for cultivation to obtain second-level seeds of Cordyceps militaris; Step 3: inoculating the second-level seeds of Cordyceps militaris into a third-level liquid culture medium for cultivation to obtain third-level seeds of Cordyceps militaris; Step 4: inoculating the Cordyceps militaris grade 3 seeds into a grass-growing experimental medium, and culturing until Cordyceps militaris grass-growing seeds are obtained; The mother species slant culture medium, the secondary liquid strain culture medium, the tertiary liquid strain culture medium and the grass growing test medium all contain pueraria root and puerarin powder, the mass ratio of puerarin powder to pueraria root in the mother species slant culture medium, the secondary liquid strain culture medium and the tertiary liquid strain culture medium is 1:(5-10), and the mass ratio of puerarin powder to pueraria root in the grass growing test medium is 1:(7-10); The kudzu root is kudzu root particles processed by the following method: drying fresh kudzu root in an environment of 50-60° C., crushing and sieving after drying, and crushing to 5-50 meshes to obtain the kudzu root particles.

2. The culture method according to claim 1, characterized in that The puerarin powder extraction method comprises: performing subcritical water extraction on pueraria processing waste residue and pueraria processing waste water in a mass ratio of 1:(15-17) to obtain an extract, and then performing solid-liquid separation, concentration and drying on the extract to obtain puerarin powder.

3. The culture method according to claim 1, characterized in that The culture method of step 1 comprises: inoculating the preserved strain of Cordyceps militaris aseptically on the mother strain slant culture medium under light-proof conditions, and culturing it for 7 days at 18-22° C. in the dark for activation.

4. The culture method according to claim 1, characterized in that The culture method of step 2 comprises: inoculating the first-level seeds of Cordyceps militaris into a second-level liquid culture medium under sterile conditions, and carrying out constant temperature shaking culture at 18-22° C. in the dark for 84-96 hours.

5. The culture method according to claim 1, characterized in that The cultivation method of step three includes: the charging volume accounts for 50-60% of the tank volume, and after actual disinfection, the temperature is lowered to 22-24°C, and Cordyceps militaris secondary seeds are added according to the charging volume in the tank, the inoculation amount is 4-6%, the tank pressure is 0.02-0.06Mpa, 20-22°C, the rotation speed is 130-150rpm, and the light-proof fermentation time is 2-3 days. When the mycelium is strong and the volume of the ball is rich and accounts for 50-70%, the cultivation is stopped.

6. The culture method according to claim 1, characterized in that The culture method of step 4 comprises: aseptically inoculating 6% to 8% of the third-grade Cordyceps militaris seeds into the grass-growing experimental culture medium, first culturing at 18-22° C. in the dark, then entering the color change stage, controlling the light intensity to 200-400Lx, the air humidity to 65-75%, waiting for the mycelium color to turn to orange-yellow, puncturing holes for ventilation to form the Cordyceps militaris fruiting body primordium, controlling the air humidity to 85%-95%, and culturing until it can be harvested.

7. The culture method according to claim 1, characterized in that The mother species slant culture medium, the secondary liquid strain culture medium, the tertiary liquid strain culture medium and the grass growing experiment culture medium also contain crop nutrients, and the crop nutrients are selected from any one or more of potatoes, wheat grains, rice, corn and bitter buckwheat.

8. The culture method according to claim 1, characterized in that The mother culture slant culture medium, secondary liquid culture medium, tertiary liquid culture medium, and weeding experiment culture medium also contain nutritional supplements, which are selected from any one or more of glucose, peptone, magnesium sulfate, potassium dihydrogen phosphate, vitamin B1, agar, glycine, zinc sulfate, and calcium chloride.

9. A functional Cordyceps militaris fruiting body prepared by the cultivation method according to any one of claims 1 to 8.

10. Use of the functional Cordyceps militaris fruiting body as claimed in claim 9 in extracting cordycepin and / or cordycepic acid.

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