Culture method and application of cordyceps militaris mycelium with high selenium content and high cordycepin content

Abstract

The invention relates to the technical field of mycelium culture, and provides a culture method and application of cordyceps militaris mycelium with high selenium content and high cordycepin content. The culture method comprises the following steps: inoculating cordyceps militaris mycelia into a liquid culture medium, and carrying out shake culture for 4-7 days to obtain cordyceps militaris liquid seeds; adding a plant selenium source into the solid culture medium, sterilizing to obtain a cordyceps militaris selenium-rich solid culture medium, adding the cordyceps militaris liquid seeds into the cordyceps militaris selenium-rich solid culture medium, and continuously culturing for 30-40 days to obtain the high-selenium and high-cordycepin cordyceps militaris mycelium. According to the culture method, the steps are simple and convenient, the raw materials are cheap and safe, the total selenium content of the produced mycelium is 0.76-191.45 mg / kg, the organic selenium content is more than 99%, the cordycepin content is 17707.3-24769 mg / kg, the content of various effective components such as the total selenium, the organic selenium and the cordycepin is outstanding, the yield is high, standardized popularization and application of the selenium industry in multiple fields are facilitated, and the wide market prospect is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of mycelium culture, and particularly relates to a culture method of high-selenium and high-cordycepin content Cordyceps militaris mycelium and application thereof. BACKGROUND

[0002] Cordyceps militaris belongs to Fungi, Ascomycota, Hypocreales, Clavicipitaceae and Cordyceps, and is a medicinal and edible fungus with both nourishing and medicinal values. The core value of Cordyceps militaris is derived from active ingredients such as cordycepin, cordycepic acid, adenosine and polysaccharides. Modern research shows that these ingredients endow Cordyceps militaris with effects such as anti-inflammatory, anti-viral and immune enhancement, and also show potential application prospects in the field of anti-tumor. With excellent nutritional and medicinal properties, Cordyceps militaris gradually gains popularity among consumers, and the market demand is increasing year by year.

[0003] Selenium is an essential trace element for the human body, and is crucial in maintaining the health homeostasis of the body. It is also known as the "king of anti-cancer elements" and "human immune activator". It is the key active component of various selenium-containing enzymes and selenium-containing proteins. Through participating in the regulation of redox balance and the activation of immune cells, it plays biological effects such as anti-oxidative damage and enhanced immune response. For healthy people and people at risk of selenium deficiency, scientific supplementation of selenium within a reasonable range helps to maintain physiological functions and has a positive significance in reducing the risk of some selenium deficiency-related diseases. However, the selenium in the soil and water in nature is mostly inorganic selenium, which has low bioavailability and is easy to cause selenium poisoning in excess. Therefore, it needs to be converted into organic selenium by plants, animals or microorganisms to achieve safe absorption with high bioavailability and low toxicity, which is an effective way for the human body to obtain selenium elements.

[0004] Cordyceps militaris has a unique advantage as a selenium enrichment carrier. Its mycelium has strong selenium tolerance and selenium accumulation capacity. During the growth and metabolism process, Cordyceps militaris can efficiently convert inorganic selenium in the external environment into organic selenium such as protein selenium and polysaccharide selenium through intracellular material metabolism mechanisms, which not only solves the safety problems of inorganic selenium, but also improves the bioavailability of selenium elements, further expanding the application value of Cordyceps militaris. However, the existing culture methods of Cordyceps militaris mycelium are mostly liquid culture, which generally has low yield, low selenium content, low cordycepin content and safety problems of selenium source, and needs to be solved urgently. Therefore, it is of great significance to study the organic combination of Cordyceps militaris and safe selenium source. However, there is no related technical solution in the existing technology, so there is an urgent need for a technical solution for the organic combination of Cordyceps militaris and safe selenium source in the field. SUMMARY

[0005] The application aims to provide a culture method of cordyceps militaris mycelium with high selenium and cordyceps militaris content and application thereof, the cordyceps militaris mycelium cultivated by the technical scheme has high total selenium, organic selenium and cordyceps militaris content, high yield and excellent safety; meanwhile, the operation process is simple, the raw material cost is low and safe and reliable, the culture period is short, and the year-round standardized stable production can be realized. These advantages make it easy to demonstrate and popularize, and have extremely broad application prospects in the related fields.

[0006] In order to achieve the above application purposes, the application provides the following technical scheme. The application provides a culture method of cordyceps militaris mycelium with high selenium and cordyceps militaris content, comprising the following steps: (1) inoculating cordyceps militaris mycelium into a liquid culture medium and oscillating culture for 4-7 days to obtain cordyceps militaris liquid seed; (2) adding a plant selenium source into a solid culture medium, sterilizing to obtain a cordyceps militaris selenium-rich solid culture medium, adding the cordyceps militaris liquid seed into the cordyceps militaris selenium-rich solid culture medium, and continuing to culture for 30-40 days to obtain cordyceps militaris mycelium with high selenium and cordyceps militaris content.

[0007] As preferred, the liquid culture medium in step (1) contains the following final concentrations of raw materials with water as a solvent: glucose 15-25 g / L, peptone 2-3 g / L, yeast extract 0.5-1.5 g / L, potassium dihydrogen phosphate 1-3 g / L, magnesium sulfate 0.5-1.5 g / L, and vitamin B1 0.2-1 g / L; the oscillating culture temperature is 22-25°C, and the oscillating culture speed is 130-150 r / min.

[0008] As preferred, the plant selenium source in step (2) is selenium-rich pyrrosia herb and pellaea viridis extract; the selenium content of the plant selenium source is 1000-2000 mg / kg, the organic selenium content of the plant selenium source is ≥80%, the plant selenium source is sieved before use, the mesh number of the sieving is 80-100 mesh, and the selenium content of the cordyceps militaris selenium-rich solid culture medium is 0-120 mg / kg.

[0009] Preferably, the solid culture medium in step (2) is composed of mixed dry matter and water, with a weight ratio of 1:1.2~1.4; the mixed dry matter is composed of the following raw materials in the following weight ratio: corn flour: wheat bran: soybean meal: corn cob: light calcium carbonate = 40~60:20~30:5~10:5~10:1~3; the ratio of Cordyceps militaris liquid seeds to Cordyceps militaris selenium-enriched solid culture medium is 8~12mL:400g; the pH of the solid culture medium is 7.5~8.5; the continued culture is aseptic culture, the temperature of the continued culture is 20℃~24℃, the total carbon dioxide concentration of the continued culture is ≤2000ppm, and the air humidity of the continued culture is 60~70%.

[0010] Preferably, before step (1), the method further includes inoculating Cordyceps militaris onto potato dextrose agar slant medium and culturing it for 8-10 days to obtain Cordyceps militaris mycelium, wherein the culture temperature is 22-25℃.

[0011] Preferably, after obtaining the high-selenium and high-cordycepin mycelium in step (2), the process further includes slicing, drying and packaging.

[0012] Preferably, the drying method is as follows: the sliced ​​high-selenium and high-cordycepin mycelium is placed in an oven and baked for 16-20 hours to obtain mycelium dry powder; the baking temperature is 60-70℃, and the moisture content of the mycelium dry powder is ≤7%; the packaging method is as follows: the mycelium dry powder is bagged with plastic bags, each bag containing 25kg, sealed, and transferred to a warehouse for storage until use.

[0013] The present invention also provides Cordyceps militaris mycelium cultured by the aforementioned culture method.

[0014] The present invention also provides the application of the Cordyceps militaris mycelium in the fields of food, cosmetics, medicine, and feed.

[0015] Preferably, the food is a functional food or a health product.

[0016] Violet leaf shepherd's purse is a wild vegetable belonging to the genus *Capsella* in the Brassicaceae family. This plant is native to the Shuanghe area of ​​Xintang, Enshi, Hubei Province, where local residents have a long-standing tradition of consuming it. Violet leaf shepherd's purse is rich in protein, soluble sugars, amino acids, vitamins, and other essential nutrients. It has a delicious flavor and is used in traditional medicine for relieving coughs, treating chronic bronchitis, childhood diarrhea, and injuries. It possesses a super-accumulation capacity for selenium; when the soil selenium content is 12 mg / kg, the plant's average selenium content exceeds 1000 mg / kg, demonstrating an exceptionally high accumulation coefficient, far surpassing other plants. Therefore, it is known as the "King of Selenium Accumulation" among plants.

[0017] The beneficial effects of this invention are as follows: (1) The culture medium formula used in this invention has safe and inexpensive raw materials, low cost, simple production process, and strong production stability, which is conducive to the standardized preparation and promotion of Cordyceps militaris mycelium and has significant economic value.

[0018] (2) Viola yedoensis is a common vegetable with an organic selenium content of over 80%. It is non-toxic and has high safety. This invention uses it as a plant selenium-enriched raw material, which is beneficial for the absorption of selenium by Cordyceps militaris mycelium.

[0019] (3) The selenium-enriched corydalis leaf powder or selenium-enriched corydalis leaf extract used in this invention is in the form of dry powder, which is easy to store and use in precise quantities, thus ensuring the convenience and stability of production.

[0020] (4) The cordycepin content in the Cordyceps militaris mycelium cultured in this invention is high, ranging from 1.77% to 2.48%. The total selenium content in the Cordyceps militaris mycelium (including the culture medium) is 0.76 to 191.45 mg / kg, with organic selenium content exceeding 99%. The cordycepin content is 17707.3 to 24769 mg / kg, the adenosine content is 110 to 154.5 ug / g, and the protein content is 25.4 to 26.8 g / 100g. The selenium content meets the requirements for edible fungi, and the main effective components such as protein and adenosine in the selenium-enriched Cordyceps militaris mycelium are significantly increased, significantly improving the effective nutritional components of the Cordyceps militaris mycelium, and possessing important economic and social value.

[0021] (5) The high-selenium and high-cordycepin Cordyceps militaris mycelium of the present invention meets the selenium nutrient fortifier standard and can be used as a series of products such as selenium-enriched functional foods, cosmetics, health products, and selenium-enriched probiotics, providing reliable technical support for the development of the selenium industry in China. Detailed Implementation

[0022] This invention provides a method for culturing Cordyceps militaris mycelium with high selenium and high cordycepin content, comprising the following steps: (1) Cordyceps militaris mycelium was inoculated into liquid culture medium and shaken for 4-7 days to obtain Cordyceps militaris liquid seeds; (2) Add the plant selenium source to the solid culture medium, sterilize it, and obtain the Cordyceps militaris selenium-enriched solid culture medium. Add the Cordyceps militaris liquid seeds to the Cordyceps militaris selenium-enriched solid culture medium and continue to culture for 30-40 days to obtain Cordyceps militaris mycelium with high selenium and high cordycepin content.

[0023] In this invention, the liquid culture medium in step (1), preferably using water as a solvent, contains the following raw materials at the following final concentrations: glucose 15-25 g / L, peptone 2-3 g / L, yeast extract 0.5-1.5 g / L, potassium dihydrogen phosphate 1-3 g / L, magnesium sulfate 0.5-1.5 g / L, and vitamin B1 0.2-1 g / L. More preferably, it contains the following raw materials at the following final concentrations: glucose 20 g / L, peptone 2.5 g / L, yeast extract 1 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate 1 g / L, and vitamin B1 0.5 g / L. The temperature of the shaking culture is preferably 22-25°C, more preferably 24°C, and the shaking speed is preferably 130-150 r / min, more preferably 140 r / min.

[0024] In this invention, the plant selenium source in step (2) is preferably selenium-enriched Corydalis leaf powder or selenium-enriched Corydalis leaf extract; the selenium content of the plant selenium source is preferably 1000~2000 mg / kg, more preferably 1200~1800 mg / kg, and even more preferably 1500 mg / kg, the organic selenium content of the plant selenium source is preferably ≥80%, the plant selenium source is sieved before use, the sieve is preferably 80~100 mesh, more preferably 100 mesh; the selenium content in the Cordyceps militaris selenium-enriched solid culture medium is preferably 0~120 mg / kg, more preferably 20~100 mg / kg, and even more preferably 60 mg / kg.

[0025] In this invention, the solid culture medium in step (2) is composed of mixed dry matter and water, and the weight ratio of the mixed dry matter and water is preferably 1:1.2~1.4, more preferably 1:1.3; the mixed dry matter is preferably composed of raw materials in the following weight ratio: corn flour: wheat bran: soybean meal: corn cob: light calcium carbonate = 40~60:20~30:5~10:5~10:1~3, more preferably composed of raw materials in the following weight ratio: corn flour: wheat bran: soybean meal: corn cob: light calcium carbonate = 50:25:7:7:2; the pupae The preferred ratio of liquid seeds of Cordyceps militaris to selenium-enriched solid culture medium is 8-12 mL:400 g, more preferably 10 mL:400 g; the pH of the solid culture medium is preferably 7.5-8.5, more preferably 8.0; the continued culture is preferably aseptic culture, the temperature of the continued culture is preferably 20℃-24℃, more preferably 22℃, the total carbon dioxide concentration of the continued culture is preferably ≤2000 ppm, more preferably 1000 ppm, and the air humidity of the continued culture is preferably 60-70%, more preferably 70%.

[0026] In this invention, before step (1), the method further includes inoculating Cordyceps militaris onto potato dextrose agar slant medium and culturing it for 8 to 10 days, more preferably 9 days, to obtain Cordyceps militaris mycelium. The culture temperature is preferably 22 to 25°C, more preferably 24°C.

[0027] In this invention, after obtaining the high-selenium and high-cordycepin mycelium of Cordyceps militaris in step (2), the steps of slicing, drying and packaging are also preferred.

[0028] In this invention, the drying method is as follows: the sliced ​​high-selenium and high-cordycepin mycelium is placed in an oven and baked for 16-20 hours, more preferably 18 hours, to obtain mycelium dry powder; the baking temperature is preferably 60-70℃, more preferably 65℃, and the moisture content of the mycelium dry powder is preferably ≤7%; the packaging method is as follows: the mycelium dry powder is bagged in plastic bags, each bag containing 25kg, sealed, and transferred to a warehouse for storage until use.

[0029] The present invention also provides Cordyceps militaris mycelium cultured by the aforementioned culture method.

[0030] The present invention also provides the application of the Cordyceps militaris mycelium in the fields of food, cosmetics, medicine, and feed.

[0031] In this invention, the food is a functional food or a health product.

[0032] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1

[0034] (1) Preparation of primary strains

[0035] Prepare PDA slant culture medium (potato dextrose agar slant culture medium), dispense into test tubes, seal with silicone stoppers, sterilize at 121℃ for 45 min, and cool to below 25℃ after slanting. Perform aseptic inoculation in a clean bench, inoculating the original Cordyceps militaris spawn into the above PDA medium, and incubate at 24℃ in the dark for 8 days. After the mycelium matures, obtain Cordyceps militaris mycelium for later use.

[0036] (2) Preparation of secondary strains

[0037] Prepare CYM comprehensive culture medium (liquid culture medium), dispense into Erlenmeyer flasks, seal with silicone stoppers, sterilize at 121℃ for 45 min, and cool to below 25℃ before use. Perform aseptic inoculation in a laminar flow hood, inoculating the Erlenmeyer flasks with seven pea-sized pieces of Cordyceps militaris mycelium. After standing for 12 h, place in a constant temperature shaker and incubate at 24℃ and 150 rpm in the dark for 5 days to obtain Cordyceps militaris liquid seed. The CYM comprehensive culture medium (liquid culture medium) contains the following raw materials in final concentrations using water as a solvent: glucose 20 g / L, peptone 2.5 g / L, yeast extract 1 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate 1 g / L, and vitamin B1 0.5 g / L.

[0038] (3) Preparation of solid culture medium

[0039] The weight ratio of the mixed dry matter to water is 1:1.4; Preparation of mixed dry matter: The weight ratio of corn flour, wheat bran, soybean meal, corn cob and light calcium carbonate is 60:30:5:5:3; Water and the raw materials of the mixed dry matter were thoroughly mixed according to the above proportions to obtain a solid culture medium (the pH of the solid culture medium was 8). The solid culture medium was then placed into sterilization bags at a rate of 400 grams per bag, sealed, and then placed in a sterilization cabinet for high-temperature sterilization. The sterilization temperature was set to 123°C, the sterilization pressure to 0.135 MPa, and the effective sterilization time to 2 hours. After sterilization, the bags were cooled to room temperature and then placed in a clean bench for ultraviolet and ozone sterilization for 45 minutes to obtain a sterilized solid culture medium. The amount of selenium source added to the solid culture medium was 0.

[0040] (4) Solid culture of Cordyceps militaris

[0041] Add 10 ml of the Cordyceps militaris liquid seed obtained in step (2) to the prepared 400 g solid culture medium for selenium enrichment culture. During the mycelium growth period, the temperature of the culture was controlled at 24℃, the air humidity was 70%, the total carbon dioxide concentration was 1000 ppm, and the culture was carried out in complete darkness for 30 days. After the fungal mycelium fermentation was completed, Cordyceps militaris mycelium product with high selenium and high cordycepin was obtained. The Cordyceps militaris mycelium was further processed by slicing, drying, and packaging to obtain mycelium powder (a mixture of mycelium and culture medium). The drying method is as follows: the sliced ​​high-selenium and high-cordycepin mycelium is placed in an oven and baked for 20 hours to obtain mycelium dry powder; the baking temperature is 60℃ and the moisture content of the mycelium dry powder is 6.8%; the packaging method is as follows: the mycelium dry powder is bagged with plastic bags, each bag containing 25kg, sealed, and transferred to a warehouse for storage until use.

[0042] Example 2

[0043] (1) Preparation of primary strains

[0044] Prepare PDA slant culture medium, dispense into test tubes, seal with silicone stoppers, sterilize at 121℃ for 45 min, and cool to below 25℃ after slanting. Perform aseptic inoculation in a clean bench, inoculating the original Cordyceps militaris spawn into the above PDA culture medium, and incubate at 24℃ in the dark for 8 days. After the mycelium matures, obtain Cordyceps militaris mycelium for later use.

[0045] (2) Preparation of secondary strains

[0046] Prepare CYM comprehensive culture medium (liquid culture medium), dispense into Erlenmeyer flasks, seal with silicone stoppers, sterilize at 121℃ for 45 min, and cool to below 25℃ before use. Perform aseptic inoculation in a laminar flow hood, inoculating the Erlenmeyer flasks with seven pea-sized pieces of Cordyceps militaris mycelium. After standing for 12 h, place in a constant temperature shaker and incubate at 24℃ and 150 rpm in the dark for 7 days to obtain Cordyceps militaris liquid seed. The CYM comprehensive culture medium (liquid culture medium) contains the following raw materials in final concentrations using water as a solvent: glucose 20 g / L, peptone 2.5 g / L, yeast extract 1 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate 1 g / L, and vitamin B1 0.5 g / L.

[0047] (3) Preparation of selenium-enriched solid culture medium for Cordyceps militaris

[0048] The solid culture medium is composed of mixed dry matter and water, with a weight ratio of 1:1.3 for the mixed dry matter and water; the mixed dry matter is composed of raw materials in the following weight ratio: corn flour: wheat bran: soybean meal: corn cob: light calcium carbonate = 55:27:8:7:3. The plant selenium source is selenium-enriched corydalis leaf powder, which is sieved through a 100-mesh sieve and has a selenium content of 1000 mg / kg and an organic selenium content of 85.7%.

[0049] The water and dry matter were thoroughly mixed according to the above proportions. Then, the plant selenium source was added and the mixture was stirred again to obtain a solid culture medium (pH 8). The solid culture medium was packed into sterilization bags at 400g / bag, sealed, and then placed in a sterilization cabinet for high-temperature sterilization. The sterilization temperature was set to 123℃, the sterilization pressure to 0.135MPa, and the effective sterilization time to 3 hours. After sterilization, the bags were cooled to room temperature and placed in a clean bench for ultraviolet and ozone sterilization for 45 minutes to obtain sterilized Cordyceps militaris selenium-enriched solid culture medium with a selenium content of 20mg / kg.

[0050] (4) Solid culture of selenium-enriched Cordyceps militaris mycelium

[0051] Add 10 ml of the Cordyceps militaris liquid seed obtained in step (2) to the prepared 400 g selenium-enriched solid culture medium for selenium enrichment culture. During the mycelial growth period, the temperature for continued culture should be controlled at 22℃, the air humidity at 60%, the total carbon dioxide concentration at 1000 ppm, and cultured in complete darkness for 40 days. Once the fungal mycelial fermentation is complete, a high-selenium and high-cordycepin Cordyceps militaris mycelial product is obtained. The Cordyceps militaris mycelium was further processed by slicing, drying, and packaging to obtain mycelium powder (a mixture of mycelium and culture medium). The drying method is as follows: the sliced ​​high-selenium and high-cordycepin mycelium is placed in an oven and baked for 20 hours to obtain mycelium dry powder; the baking temperature is 60℃ and the moisture content of the mycelium dry powder is 6.7%; the packaging method is as follows: the mycelium dry powder is bagged with plastic bags, each bag containing 25kg, sealed, and transferred to a warehouse for storage until use.

[0052] Example 3

[0053] (1) Preparation of primary strains

[0054] Prepare PDA slant culture medium, dispense into test tubes, seal with silicone stoppers, sterilize at 121℃ for 45 min, and cool to below 25℃ after slanting. Perform aseptic inoculation in a clean bench, inoculating the original Cordyceps militaris spawn into the above PDA culture medium, and incubate at 24℃ in the dark for 9 days. After the mycelium matures, obtain Cordyceps militaris mycelium for later use.

[0055] (2) Preparation of secondary strains

[0056] Prepare CYM comprehensive culture medium (liquid culture medium), dispense into Erlenmeyer flasks, seal with silicone stoppers, sterilize at 121℃ for 45 min, and cool to below 25℃ before use. Perform aseptic inoculation in a laminar flow hood, inoculating the Erlenmeyer flasks with seven pea-sized pieces of Cordyceps militaris mycelium. After standing for 12 h, place in a constant temperature shaker and incubate at 24℃ and 150 rpm in the dark for 6 days to obtain Cordyceps militaris liquid seed. The CYM comprehensive culture medium (liquid culture medium) contains the following raw materials in final concentrations using water as a solvent: glucose 20 g / L, peptone 2.5 g / L, yeast extract 1 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate 1 g / L, and vitamin B1 0.5 g / L.

[0057] (3) Preparation of selenium-enriched solid culture medium for Cordyceps militaris

[0058] The solid culture medium is composed of mixed dry matter and water, with a weight ratio of 1:1.2 for the mixed dry matter and water; the mixed dry matter is composed of raw materials in the following weight ratio: corn flour: wheat bran: soybean meal: corn cob: light calcium carbonate = 50:25:8:8:2. The plant selenium source is selenium-enriched corydalis leaf powder, which is sieved through a 100-mesh sieve and has a selenium content of 1200 mg / kg and an organic selenium content of 87.3%.

[0059] The water and dry matter were thoroughly mixed according to the above proportions. Then, a plant selenium source was added, and the mixture was stirred again to obtain a solid culture medium (pH 7.8). The solid culture medium was packed into sterilization bags at 400g / bag, sealed, and then placed in a sterilization cabinet for high-temperature sterilization. The sterilization temperature was set to 123℃, the sterilization pressure to 0.135MPa, and the effective sterilization time to 2.5h. After sterilization, the bags were cooled to room temperature and placed in a clean bench for ultraviolet and ozone sterilization for 45min to obtain sterilized Cordyceps militaris selenium-enriched solid culture medium. The selenium content in the sterilized Cordyceps militaris selenium-enriched solid culture medium was 40mg / kg.

[0060] (4) Solid-state selenium-enriched culture of Cordyceps militaris

[0061] Add 10 ml of the Cordyceps militaris liquid seed obtained in step (2) to the prepared 400 g selenium-enriched solid culture medium for selenium enrichment culture. During the mycelial growth period, the temperature for continued culture should be controlled at 23℃, the air humidity at 65%, the total carbon dioxide concentration at 1000 ppm, and cultured in complete darkness for 35 days. After the fungal mycelial fermentation is completed, a high-selenium and high-cordycepin Cordyceps militaris mycelial product is obtained. The Cordyceps militaris mycelium was further processed by slicing, drying, and packaging to obtain mycelium powder (a mixture of mycelium and culture medium). The drying method is as follows: the sliced ​​high-selenium and high-cordycepin mycelium is placed in an oven and baked for 18 hours to obtain mycelium dry powder; the baking temperature is 65℃ and the moisture content of the mycelium dry powder is 6.8%; the packaging method is as follows: the mycelium dry powder is bagged with plastic bags, each bag containing 25kg, sealed, and transferred to a warehouse for storage until use.

[0062] Example 4

[0063] (1) Preparation of primary strains

[0064] Prepare PDA slant culture medium, dispense into test tubes, seal with silicone stoppers, sterilize at 121℃ for 45 min, and cool to below 25℃ after slanting. Perform aseptic inoculation in a clean bench, inoculating the original Cordyceps militaris spawn into the above PDA culture medium, and incubate at 24℃ in the dark for 10 days. After the mycelium matures, obtain Cordyceps militaris mycelium for later use.

[0065] (2) Preparation of secondary strains

[0066] Prepare CYM comprehensive culture medium (liquid culture medium), dispense into Erlenmeyer flasks, seal with silicone stoppers, sterilize at 121℃ for 45 min, and cool to below 25℃ before use. Perform aseptic inoculation in a clean bench, inoculating the Erlenmeyer flasks with seven pea-sized pieces of Cordyceps militaris mycelium. After standing for 12 h, place in a constant temperature shaker and incubate at 24℃ and 140 rpm in the dark for 5 days to obtain Cordyceps militaris liquid seed. The CYM comprehensive culture medium (liquid culture medium) contains the following raw materials in final concentrations using water as a solvent: glucose 20 g / L, peptone 2.5 g / L, yeast extract 1 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate 1 g / L, and vitamin B1 0.5 g / L.

[0067] (3) Preparation of selenium-enriched solid culture medium for Cordyceps militaris

[0068] The solid culture medium is composed of mixed dry matter and water, with a weight ratio of 1:1.3 for the mixed dry matter and water; the mixed dry matter is composed of raw materials in the following weight ratio: corn flour: wheat bran: soybean meal: corn cob: light calcium carbonate = 50:25:7:7:2. The plant selenium source is selenium-enriched corydalis leaf powder, which is sieved through a 100-mesh sieve and has a selenium content of 1500 mg / kg and an organic selenium content of 89.5%.

[0069] The water and dry matter were thoroughly mixed according to the above proportions. Then, a plant selenium source was added and the mixture was stirred again to obtain a solid culture medium (pH 7.9). The solid culture medium was packed into sterilization bags at 400g / bag, sealed, and then placed in a sterilization cabinet for high-temperature sterilization. The sterilization temperature was set to 123℃, the sterilization pressure to 0.135MPa, and the effective sterilization time to 3 hours. After sterilization, the bags were cooled to room temperature and placed in a clean bench for ultraviolet and ozone sterilization for 45 minutes to obtain sterilized Cordyceps militaris selenium-enriched solid culture medium with a selenium content of 60mg / kg.

[0070] (4) Solid-state selenium-enriched culture of Cordyceps militaris

[0071] Add 10 ml of the Cordyceps militaris liquid seed obtained in step (2) to the prepared 400 g selenium-enriched solid culture medium for selenium enrichment culture. During the mycelial growth period, the temperature for continued culture should be controlled at 24℃, the air humidity at 70%, and the total carbon dioxide concentration at 1000 ppm. After 38 days of culture under completely dark conditions, the fungal mycelial fermentation is completed, and the high-selenium and high-cordycepin Cordyceps militaris mycelial product is obtained. The Cordyceps militaris mycelium was further processed by slicing, drying, and packaging to obtain mycelium powder (a mixture of mycelium and culture medium). The drying method is as follows: the sliced ​​high-selenium and high-cordycepin mycelium is placed in an oven and baked for 18 hours to obtain mycelium dry powder; the baking temperature is 65℃ and the moisture content of the mycelium dry powder is 6.6%; the packaging method is as follows: the mycelium dry powder is bagged with plastic bags, each bag containing 25kg, sealed after packaging, and transferred to a warehouse for storage until use.

[0072] Example 5

[0073] (1) Preparation of primary strains

[0074] Prepare PDA slant culture medium, dispense into test tubes, seal with silicone stoppers, sterilize at 121℃ for 45 min, and cool to below 25℃ after slanting. Perform aseptic inoculation in a clean bench, inoculating the original Cordyceps militaris spawn into the above PDA culture medium, and incubate at 24℃ in the dark for 8 days. After the mycelium matures, obtain Cordyceps militaris mycelium for later use.

[0075] (2) Preparation of secondary strains

[0076] Prepare CYM comprehensive culture medium (liquid culture medium), dispense into Erlenmeyer flasks, seal with silicone stoppers, sterilize at 121℃ for 45 min, and cool to below 25℃ before use. Perform aseptic inoculation in a laminar flow hood, inoculating the Erlenmeyer flasks with seven pea-sized pieces of Cordyceps militaris mycelium. After standing for 12 h, place in a constant temperature shaker and incubate at 24℃ and 150 rpm in the dark for 4 days to obtain Cordyceps militaris liquid seed. The CYM comprehensive culture medium (liquid culture medium) contains the following raw materials in final concentrations using water as a solvent: glucose 20 g / L, peptone 2.5 g / L, yeast extract 1 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate 1 g / L, and vitamin B1 0.5 g / L.

[0077] (3) Preparation of selenium-enriched solid culture medium for Cordyceps militaris

[0078] The solid culture medium is composed of mixed dry matter and water, with a weight ratio of 1:1.4 for the mixed dry matter and water; the mixed dry matter is composed of raw materials in the following weight ratio: corn flour: wheat bran: soybean meal: corn cob: light calcium carbonate = 45:23:10:8:1.5; The plant selenium source is selenium-enriched corydalis leaf powder, which is sieved through a 100-mesh sieve and has a selenium content of 1800 mg / kg and an organic selenium content of 83.4%.

[0079] The water and dry matter were thoroughly mixed according to the above proportions. Then, the plant selenium source was added and the mixture was stirred again to obtain a solid culture medium (pH 7.8). The solid culture medium was packed into sterilization bags at 400g / bag, sealed, and then placed in a sterilization cabinet for high-temperature sterilization. The sterilization temperature was set to 123℃, the sterilization pressure to 0.135MPa, and the effective sterilization time to 2.5h. After sterilization, the bags were cooled to room temperature and placed in a clean bench for ultraviolet and ozone sterilization for 45min to obtain sterilized Cordyceps militaris selenium-enriched solid culture medium with a selenium content of 80mg / kg.

[0080] (4) Solid-state selenium-enriched culture of Cordyceps militaris

[0081] Add 10 ml of the Cordyceps militaris liquid seed obtained in step (2) to the prepared 400 g selenium-enriched solid culture medium for selenium enrichment culture. During the mycelial growth period, the temperature for continued culture should be controlled at 23℃, the air humidity at 70%, and the total carbon dioxide concentration at 1000 ppm. After 38 days of culture under completely dark conditions, the fungal mycelial fermentation is completed, and the high-selenium and high-cordycepin Cordyceps militaris mycelial product is obtained. The Cordyceps militaris mycelium was further processed by slicing, drying, and packaging to obtain mycelium powder (a mixture of mycelium and culture medium). The drying method is as follows: the sliced ​​high-selenium and high-cordycepin mycelium is placed in an oven and baked for 16 hours to obtain mycelium dry powder; the baking temperature is 70℃ and the moisture content of the mycelium dry powder is 6.6%; the packaging method is as follows: the mycelium dry powder is bagged with plastic bags, each bag containing 25kg, sealed, and transferred to a warehouse for storage until use.

[0082] Example 6

[0083] (1) Preparation of primary strains

[0084] Prepare PDA slant culture medium, dispense into test tubes, seal with silicone stoppers, sterilize at 121℃ for 45 min, and cool to below 25℃ after slanting. Perform aseptic inoculation in a clean bench, inoculating the original Cordyceps militaris spawn into the above PDA culture medium, and incubate at 24℃ in the dark for 9 days. After the mycelium matures, obtain Cordyceps militaris mycelium for later use.

[0085] (2) Preparation of secondary strains

[0086] Prepare CYM comprehensive culture medium (liquid culture medium), dispense into Erlenmeyer flasks, seal with silicone stoppers, sterilize at 121℃ for 45 min, and cool to below 25℃ before use. Perform aseptic inoculation in a laminar flow hood, inoculating the Erlenmeyer flasks with seven pea-sized pieces of Cordyceps militaris mycelium. After standing for 12 h, place in a constant temperature shaker and incubate at 24℃ and 150 rpm in the dark for 5 days to obtain Cordyceps militaris liquid seed. The CYM comprehensive culture medium (liquid culture medium) contains the following raw materials in final concentrations using water as a solvent: glucose 20 g / L, peptone 2.5 g / L, yeast extract 1 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate 1 g / L, and vitamin B1 0.5 g / L.

[0087] (3) Preparation of selenium-enriched solid culture medium for Cordyceps militaris

[0088] The solid culture medium is composed of mixed dry matter and water, with a weight ratio of 1:1.2; the mixed dry matter is composed of raw materials in the following weight ratio: corn flour: wheat bran: soybean meal: corn cob: light calcium carbonate = 40:20:10:10:1.5; The plant selenium source is selenium-enriched corydalis leaf powder, which is sieved through a 100-mesh sieve and has a selenium content of 2000 mg / kg and an organic selenium content of 87.9%.

[0089] The water and dry matter were thoroughly mixed according to the above proportions. Then, the plant selenium source was added and the mixture was stirred again to obtain a solid culture medium (pH 7.9). The solid culture medium was packed into sterilization bags at 400g / bag, sealed, and then placed in a sterilization cabinet for high-temperature sterilization. The sterilization temperature was set to 123℃, the sterilization pressure to 0.135MPa, and the effective sterilization time to 2.5h. After sterilization, the bags were cooled to room temperature and placed in a clean bench for ultraviolet and ozone sterilization for 45min to obtain sterilized Cordyceps militaris selenium-enriched solid culture medium. The selenium content in the sterilized Cordyceps militaris selenium-enriched solid culture medium was 100mg / kg.

[0090] (4) Solid-state selenium-enriched culture of Cordyceps militaris

[0091] Add 10 ml of the Cordyceps militaris liquid seed obtained in step (2) to the prepared 400 g selenium-enriched solid culture medium for selenium enrichment culture. During the mycelial growth period, the temperature for continued culture should be controlled at 24℃, the air humidity at 70%, and the total carbon dioxide concentration at 1000 ppm. After 36 days of culture under completely dark conditions, the fungal mycelial fermentation is completed, and the high-selenium and high-cordycepin Cordyceps militaris mycelial product is obtained. The Cordyceps militaris mycelium was further processed by slicing, drying, and packaging to obtain mycelium powder (a mixture of mycelium and culture medium). The drying method is as follows: the sliced ​​high-selenium and high-cordycepin mycelium is placed in an oven and baked for 20 hours to obtain mycelium dry powder; the baking temperature is 60℃ and the moisture content of the mycelium dry powder is 6.7%; the packaging method is as follows: the mycelium dry powder is bagged with plastic bags, each bag containing 25kg, sealed, and transferred to a warehouse for storage until use.

[0092] Example 7

[0093] The mycelial powders obtained in Examples 1-6 were passed through a 100-mesh sieve to obtain selenium-enriched Cordyceps militaris mycelial powder. The total selenium content was then determined according to GB 5009.93-2017, extracted using GB 1903.22-2016, and the organic selenium content was determined according to GB 5009.93 hydride atomic fluorescence spectrometry. Cordycepin and adenosine contents were determined according to NY / T 2116-2012, and protein content was determined according to GB 5009.5-2016, the first method for the determination of protein in food, using the Kjeldahl method. The results are shown in Table 1 below. Table 1

[0094] As shown in Table 1, the Cordyceps militaris mycelium cultured in this invention has a high cordycepin content of 1.77-2.48%, a total selenium content of 0.76-191.45 mg / kg (including the culture medium), an organic selenium content of over 99%, a cordycepin content of 17707.3-24769 mg / kg, an adenosine content of 110-154.5 ug / g, and a protein content of 25.4-26.8 g / 100g. The selenium content meets the requirements for edible fungi, and the main effective components such as protein and adenosine in the selenium-enriched Cordyceps militaris mycelium are significantly increased, significantly improving the effective nutritional components of the Cordyceps militaris mycelium and possessing significant economic and social value.

[0095] As shown in the above embodiments, this invention provides a method for cultivating Cordyceps militaris mycelium with high selenium and cordycepin content, and its application. The cultivation method involves: inoculating Cordyceps militaris mycelium into a liquid culture medium and shaking it for 4-7 days to obtain liquid seeds; adding a plant selenium source to a solid culture medium, sterilizing it to obtain a selenium-enriched solid culture medium for Cordyceps militaris; adding the liquid seeds to the selenium-enriched solid culture medium for further cultivation for 30-40 days to obtain high-selenium and high-cordycepin mycelium. The cultivation method of this invention is simple, uses inexpensive and safe raw materials, and produces mycelium with total selenium content of 0.76-191.45 mg / kg, organic selenium exceeding 99%, and cordycepin content of 17707.3-24769 mg / kg. The high content and high yield of various effective components, including total selenium, organic selenium, and cordycepin, facilitates the standardized promotion and application of the selenium industry in multiple fields and has broad market prospects.

[0096] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements can be made without departing from the principle of the present invention, and these improvements should also be considered within the scope of protection of the present invention.

Claims

1. A method for culturing Cordyceps militaris mycelium with high selenium and high cordycepin content, characterized in that, Includes the following steps: (1) Cordyceps militaris mycelium was inoculated into liquid culture medium and shaken for 4-7 days to obtain Cordyceps militaris liquid seeds; (2) Add the plant selenium source to the solid culture medium, sterilize it, and obtain the Cordyceps militaris selenium-enriched solid culture medium. Add the Cordyceps militaris liquid seeds to the Cordyceps militaris selenium-enriched solid culture medium and continue to culture for 30-40 days to obtain Cordyceps militaris mycelium with high selenium and high cordycepin content.

2. The cultivation method according to claim 1, characterized in that, The liquid culture medium in step (1) contains the following raw materials in a final concentration using water as a solvent: glucose 15~25g / L, peptone 2~3g / L, yeast extract 0.5~1.5g / L, potassium dihydrogen phosphate 1~3g / L, magnesium sulfate 0.5~1.5g / L, and vitamin B1 0.2~1g / L; the temperature of the shaking culture is 22~25℃, and the shaking speed is 130~150r / min.

3. The cultivation method according to claim 2, characterized in that, The plant selenium source mentioned in step (2) is selenium-enriched Corydalis leaf powder or selenium-enriched Corydalis leaf extract; the selenium content of the plant selenium source is 1000~2000mg / kg, the organic selenium content of the plant selenium source is ≥80%, the plant selenium source is sieved before use, and the sieve mesh size is 80~100 mesh; the selenium content in the Cordyceps militaris selenium-enriched solid culture medium is 0~120 mg / kg.

4. The cultivation method according to claim 3, characterized in that, The solid culture medium in step (2) consists of mixed dry matter and water, with a weight ratio of 1:1.2~1.

4. The mixed dry matter is composed of the following raw materials in the following weight ratio: corn flour: wheat bran: soybean meal: corn cob: light calcium carbonate = 40~60:20~30:5~10:5~10:1~3. The ratio of Cordyceps militaris liquid seeds to Cordyceps militaris selenium-enriched solid culture medium is 8~12mL:400g. The pH of the solid culture medium is 7.5~8.

5. The continued culture is aseptic, the temperature of the continued culture is 20℃~24℃, the total carbon dioxide concentration of the continued culture is ≤2000ppm, and the air humidity of the continued culture is 60~70%.

5. The cultivation method according to claim 1, characterized in that, Before step (1), the method further includes inoculating Cordyceps militaris onto potato dextrose agar slant medium and culturing it for 8-10 days to obtain Cordyceps militaris mycelium, wherein the culture temperature is 22-25℃.

6. The cultivation method according to claim 5, characterized in that, After obtaining the high-selenium and high-cordycepin mycelium in step (2), the process also includes slicing, drying and packaging.

7. The cultivation method according to claim 6, characterized in that, The drying method is as follows: the sliced ​​high-selenium and high-cordycepin mycelium is placed in an oven and baked for 16-20 hours to obtain mycelium dry powder; the baking temperature is 60-70℃, and the moisture content of the mycelium dry powder is ≤7%; the packaging method is as follows: the mycelium dry powder is bagged in plastic bags, each bag containing 25kg, sealed, and transferred to a warehouse for storage until use.

8. Cordyceps militaris mycelium cultured by the culture method according to any one of claims 1 to 7.

9. The application of Cordyceps militaris mycelium as described in claim 8 in the fields of food, cosmetics, medicine, and feed.

10. The application according to claim 9, characterized in that, The food products mentioned are functional foods or health products.

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