A method for cultivating selenium-enriched north cordyceps sinensis

Abstract

The present application relates to the cultivation technology field of north Ophiocordyceps sinensis, and specifically discloses a cultivation method of selenium-rich north Ophiocordyceps sinensis, which adopts a mode of gradient selenium domestication combined with liquid strain culture and solid substrate cultivation, realizes the gradual adaptation of the strain to the selenium environment through the gradient selenium domestication of the primary mother strain and the secondary liquid original strain, and then carries out selenium-rich cultivation by taking rice as the core solid substrate, and realizes the efficient absorption and conversion of selenium by the mycelium and fruiting body of north Ophiocordyceps sinensis by cooperating with the standardized regulation of temperature, humidity, light and ventilation environment. The present application effectively avoids the inhibitory effect of high selenium on the north Ophiocordyceps sinensis strain, and takes into account the strain activity and selenium enrichment efficiency. The selenium-rich north Ophiocordyceps sinensis cultivated has stable selenium content and high medicinal value, and the cultivation process is standardized and highly repeatable, and is suitable for large-scale industrialization and popularization.

Description

Technical Field

[0001] This invention belongs to the field of artificial cultivation technology of Cordyceps militaris, specifically involving a cultivation method for selenium-enriched Cordyceps militaris, and more particularly involving a highly efficient cultivation method for selenium-enriched Cordyceps militaris by achieving selenium adaptation of the strain through gradient selenium domestication and combining liquid strain culture with solid substrate cultivation. Background Technology

[0002] Cordyceps militaris, also known as North Cordyceps or Cordyceps militaris, is an important medicinal fungus of the Cordyceps genus. Its chemical composition and medicinal effects are similar to those of natural Cordyceps sinensis. It contains active ingredients such as cordycepin, cordyceps polysaccharides, amino acids, and various minerals. It has a variety of pharmacological effects such as immune regulation, anti-oxidation, and anti-fatigue. It is a high-quality substitute for natural Cordyceps sinensis and has broad application prospects in the fields of medicine, health products, and food.

[0003] Selenium is an essential trace element for the human body, participating in the synthesis and metabolism of various enzymes. It has important physiological functions such as antioxidation, enhancing immunity, and preventing cardiovascular diseases. Selenium deficiency can lead to various diseases. Introducing selenium into the cultivation process of Cordyceps militaris allows the plant to absorb and transform selenium into organic selenium during its growth. This not only enhances the medicinal value of Cordyceps militaris but also provides a high-quality natural carrier for supplementing the human body with organic selenium. Therefore, selenium-enriched Cordyceps militaris has become a research hotspot in the field of Cordyceps militaris cultivation.

[0004] Currently, the cultivation technology of selenium-enriched Cordyceps militaris still has many shortcomings. Most cultivation methods directly add high concentrations of selenium sources to the culture medium, making it difficult for the Cordyceps militaris strains to adapt to the selenium environment. This significantly inhibits mycelial growth and can even lead to strain death. Although some methods employ selenium acclimatization techniques, the acclimatization methods are simplistic and fail to achieve gradual adaptation. This results in low selenium tolerance of the strains, poor selenium accumulation efficiency, and large fluctuations and instability in the selenium content of the cultivated Cordyceps militaris. Furthermore, the lack of standardized control over environmental factors such as temperature, humidity, light, and ventilation during cultivation easily leads to abnormal fruiting body differentiation and poor growth, affecting not only the appearance quality of Cordyceps militaris but also further reducing selenium accumulation efficiency. In addition, existing cultivation methods mostly use solid-state inoculum, resulting in low strain activity, slow germination after inoculation, and susceptibility to contamination by other microorganisms during large-scale cultivation, leading to low success rates and failing to meet the needs of industrial production.

[0005] To address the aforementioned technical challenges, there is an urgent need to develop a method for cultivating selenium-enriched Cordyceps militaris that balances strain activity and selenium enrichment efficiency. This method would enhance the strain's selenium tolerance through scientific gradient selenium acclimatization techniques, combining the advantages of liquid culture and solid substrate cultivation with standardized environmental control. This would enable Cordyceps militaris to efficiently and stably absorb and transform selenium, cultivating selenium-enriched Cordyceps militaris with stable selenium content and high medicinal value. Simultaneously, it would improve the repeatability and scalability of the cultivation process. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for cultivating selenium-enriched Cordyceps militaris, solving problems such as high selenium inhibiting fungal growth, low selenium enrichment efficiency, unstable selenium content, poor fruiting body quality, and low cultivation success rate in existing methods. This invention employs a gradient selenium acclimatization combined with liquid spawn culture and solid substrate cultivation, enabling the fungus to gradually adapt to the selenium environment, improving its selenium tolerance and activity. Combined with standardized cultivation environment control and harvesting and preservation methods, it balances fungal activity and selenium enrichment efficiency, cultivating selenium-enriched Cordyceps militaris with stable selenium content and high medicinal value. Furthermore, the cultivation process is standardized, with low contamination rates and high reproducibility, making it suitable for large-scale industrial promotion.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for cultivating selenium-enriched Cordyceps militaris, comprising three steps: gradient domestication of selenium-enriched Cordyceps militaris strain, cultivation and selenium enrichment of selenium-enriched Cordyceps militaris, and harvesting and selenium content assurance; the gradient domestication adopts a low-selenium start-up and a gradually increasing sodium selenite concentration gradient, divided into two steps: primary mother strain selenium domestication and secondary liquid original strain selenium domestication; cultivation and selenium enrichment use rice as a solid substrate, with sodium selenite selenium source added to the substrate, divided into three stages: culture medium preparation, inoculation culture, and fruiting body cultivation; harvesting and selenium content assurance includes fruiting body harvesting, secondary cultivation, and drying and preservation, with full control of sodium selenite concentration and aseptic operation, to achieve efficient absorption and transformation of selenium by Cordyceps militaris mycelium and fruiting bodies, and to cultivate selenium-enriched Cordyceps militaris with stable selenium content.

[0008] Furthermore, the culture medium for selenium domestication of the primary mother seed, calculated per 1000 mL, is formulated as follows: 200 g potato, 20 g glucose, 5 g peptone, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 10 mg vitamin B1, 10 mg sodium selenite, and 1000 mL water.

[0009] Furthermore, the preparation and cultivation process of the primary mother culture for selenium domestication is as follows: potatoes are peeled, boiled until soft, filtered to extract juice, the remaining components are added and dissolved, and then dispensed into test tubes. The tubes are then sterilized at 121℃ for 20 minutes and arranged as slant cultures. Under aseptic conditions, the original mother culture of Cordyceps militaris is inoculated and cultured at a constant temperature of 23-25℃ in the dark for 7-10 days. When the mycelium has grown all over the slant and is white and dense, the low-selenium domesticated mother culture is obtained.

[0010] Furthermore, the culture medium for secondary liquid original seed selenium acclimatization, calculated per 1000 mL, has the following formula: 30 g glucose, 10 g peptone, 5 g yeast extract, 2 g potassium dihydrogen phosphate, 1 g magnesium sulfate, 20 mg sodium selenite, and 1000 mL water, with the pH value of the culture medium adjusted to 6.0–6.5.

[0011] Furthermore, the preparation and cultivation process of the secondary liquid primary culture for selenium acclimatization is as follows: the culture medium is placed into a 500mL Erlenmeyer flask with a liquid volume of 200mL, and sterilized at 121℃ for 30 minutes; after cooling to 25℃, 3-5 pieces of primary acclimatization mother culture mycelial blocks are inoculated, with the mycelial block size being 1cm²; the Erlenmeyer flask is placed on a shaker and cultured at 150r / min and 24℃ in the dark for 5-7 days until the culture medium becomes turbid and the mycelial balls become dense, thus obtaining a liquid primary culture adapted to the selenium environment.

[0012] Furthermore, the formula for each bag of selenium-enriched solid culture medium is as follows: 40g rice, 3g silkworm pupa powder, 1g peptone, 30mg sodium selenite, and 50mL water, with a pH value of natural.

[0013] Furthermore, the preparation process of selenium-enriched solid culture medium is as follows: mix rice, silkworm pupa powder, and peptone evenly, add a solution of dissolved sodium selenite, and stir until the rice fully absorbs the water; pack into 15cm×30cm polypropylene cultivation bags, press them firmly, and seal them; sterilize at 121℃ for 40 minutes or at 100℃ for 8 hours, and cool to room temperature before use.

[0014] Furthermore, the inoculation and cultivation process is as follows: Under aseptic conditions, 10-15 mL of secondary selenium-enriched liquid inoculum is inoculated into each bag of cultivation medium. The cultivation bag is gently shaken to ensure that the inoculum is evenly distributed on the surface of the rice substrate. The inoculated cultivation bags are placed in a constant temperature incubation room at 20-22℃ and cultured for 15-20 days under conditions of darkness, relative humidity of 60%-65%, and good ventilation. Once the mycelium has covered the entire culture medium and a white mycelial film has formed on the surface, the process is transitioned to the fruiting body induction stage.

[0015] Further, the environmental control for fruiting body cultivation is as follows: after the mycelium has filled the bag, open the cultivation bag seal and provide 12 hours of diffused light daily, with a light intensity of 1000-1500 lx; ​​control the temperature at 20-23℃, maintaining a day-night temperature difference of 3-5℃; increase the relative humidity to 80%-90%, and spray with sterile water 2-3 times a day to avoid water accumulation in the substrate; ventilate 2-3 times a day for 30 minutes each time, and control the carbon dioxide concentration; after the primordia have formed, continue cultivation for 25-30 days, and when the fruiting bodies grow to 8-12cm, with a swollen and full top and a bright orange-yellow color, the fruiting body cultivation is complete.

[0016] Furthermore, the harvesting and selenium content assurance process is as follows: the fruiting bodies are cut off from the base with sterile scissors to complete the harvest. After harvesting, sterile water containing 5 mg / L sodium selenite is added to the original substrate. After culturing for 10 to 15 days, a second batch of fruiting bodies can be harvested. The harvested fruiting bodies are dried at a low temperature below 60℃ until the moisture content is ≤10%. After drying, they are sealed in packaging and stored in a cool and dry place.

[0017] Compared with the prior art, the present invention has the following significant advantages: (1) Gradient selenium acclimatization to gradually improve the selenium tolerance of the strain and avoid high selenium inhibition: The present invention adopts a low selenium starting (10 mg / L) and gradually increasing (20 mg / L→30 mg / L) sodium selenite concentration gradient, and carries out selenium acclimatization in two steps: primary mother strain and secondary liquid original strain. This allows the Cordyceps militaris strain to gradually adapt to the selenium environment, effectively avoiding the inhibitory effect of directly adding high concentrations of selenium source on the strain in the existing technology, ensuring the activity and growth rate of the strain, and significantly improving the selenium tolerance and selenium absorption and conversion capacity of the strain, laying a solid foundation for efficient selenium enrichment in the subsequent cultivation stage.

[0018] (2) Liquid culture improves strain activity and cultivation success rate: The secondary domestication of this invention adopts liquid culture mode. Compared with traditional solid strains, the mycelial balls of liquid strains are dense, highly active and germinate quickly. After inoculation, they can spread and grow rapidly on solid substrate, shortening the mycelial culture cycle. At the same time, liquid strains have good uniformity and no dead corners after inoculation, which can ensure uniform mycelial growth, effectively reduce the probability of contamination by miscellaneous bacteria, improve the cultivation success rate of selenium-enriched Cordyceps militaris, and are suitable for large-scale industrial inoculation.

[0019] (3) Solid substrate cultivation combined with gradient increase of selenium source to achieve efficient and stable enrichment of selenium: This invention uses rice as the core solid substrate, combined with silkworm pupa powder, peptone and other auxiliary materials to provide sufficient and balanced nutrition for the growth of fruiting bodies. At the same time, the concentration of selenium source in the cultivation stage is increased to 30mg / L to achieve gradient increase of selenium domestication. On the basis of early domestication, the strain can efficiently absorb and transform selenium elements, so that selenium elements are fully accumulated in mycelium and fruiting bodies. The selenium content of the selenium-enriched Cordyceps sinensis cultivated is stable and without obvious fluctuations, and the proportion of organic selenium is high, which significantly improves the medicinal value.

[0020] (4) Standardized environmental control improves the quality and yield of fruiting bodies: This invention standardizes and precisely controls environmental factors such as light, temperature, humidity and ventilation during the fruiting body cultivation stage, and clarifies the optimal light time, light intensity, temperature, humidity and ventilation frequency, which effectively stimulates the formation and differentiation of fruiting body primordia, avoids abnormal growth of fruiting bodies, and produces fruiting bodies with thick stipes, swollen and full tips, and bright orange-yellow color, with excellent appearance quality; at the same time, moderate diurnal temperature difference and sufficient nutrient supply promote nutrient accumulation in fruiting bodies, increase the yield of fruiting bodies, and, in conjunction with secondary cultivation technology, further increase the total yield of Cordyceps militaris and increase economic benefits.

[0021] (5) Scientific harvesting and preservation methods ensure that the selenium content and active ingredients are not lost: The present invention adopts a sterile harvesting method to avoid matrix damage. Secondary cultivation is achieved by supplementing sterile water with low concentration of selenium source, which increases the yield while ensuring the selenium content of the second batch of fruiting bodies is stable. The low temperature drying method below 60℃ is adopted to effectively avoid the damage of high temperature to the organic selenium structure and active ingredients such as cordycepin and cordyceps polysaccharide. After drying, the fruiting bodies are sealed and stored in a cool and dry place to prevent the fruiting bodies from absorbing moisture and becoming moldy and the selenium element from volatilizing and being lost, thus ensuring that the selenium content and medicinal value of selenium-enriched Cordyceps sinensis remain stable during storage.

[0022] (6) The cultivation method is standardized and highly reproducible, making it suitable for large-scale promotion: The cultivation method of this invention has clear steps, and the formula, process parameters and environmental control indicators of each stage are clear and specific. There are no ambiguous operations. The concentration of selenium source and aseptic operation are strictly controlled throughout the process, resulting in low contamination rate and high success rate of cultivation. At the same time, the culture medium raw materials and cultivation equipment used are all conventional and readily available materials and equipment, which are inexpensive and do not require special high-end equipment. The reproducibility is strong, making it suitable for large-scale and industrialized promotion by small and medium-sized enterprises and planting bases, and it has broad market application prospects. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1 A method for cultivating selenium-enriched Cordyceps militaris, the specific steps of which are as follows: Gradual domestication of selenium-enriched Cordyceps militaris strains (1) Selenium domestication of primary mother culture: Prepare culture medium according to 1000mL formula, take 200g of potato, peel and boil until soft, filter and extract juice, add 20g of glucose, 5g of peptone, 1g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, 10mg of vitamin B1 and 10mg of sodium selenite, add water to make up to 1000mL, dispense into test tubes and autoclave at 121℃ for 20 minutes, arrange into slant; under sterile conditions, inoculate with the original mother culture of Cordyceps militaris, and culture at 24℃ in the dark for 8 days. The mycelium grows all over the slant and is white and dense, thus obtaining the low selenium domestication mother culture.

[0025] (2) Secondary liquid original seed selenium acclimatization: Prepare culture medium according to the formula of 1000mL, add 30g glucose, 10g peptone, 5g yeast extract, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, and 20mg sodium selenite, add water to make up to 1000mL, and adjust the pH to 6.2; put into 500mL Erlenmeyer flasks, fill with 200mL of liquid, autoclave at 121℃ for 30 minutes, cool to 25℃ and inoculate 4 1cm² primary acclimatization mother seed mycelial blocks; culture at 150r / min and 24℃ in the dark for 6 days. The culture medium is turbid and the mycelial balls are dense, thus obtaining the secondary liquid original seed.

[0026] Cultivation and Selenium Enrichment of Selenium-Enriched Cordyceps militaris (1) Preparation of selenium-enriched solid culture medium: Take 40g of rice, 3g of silkworm pupa powder and 1g of peptone per bag and mix them evenly. Add 50mL of aqueous solution containing 30mg of sodium selenite and stir until the rice fully absorbs the water. Pack into 15cm×30cm polypropylene cultivation bags, press and seal them, sterilize at 121℃ for 40 minutes, and cool to room temperature for later use.

[0027] (2) Inoculation and mycelial culture: Under aseptic conditions, 12 mL of secondary liquid original culture was inoculated into each bag and shaken to distribute the culture evenly; it was placed in a constant temperature incubation room at 21℃, protected from light, with a relative humidity of 62%, and ventilated once a day for 20 minutes each time. After 18 days of culture, the mycelium covered the culture medium and formed a white mycelial film.

[0028] (3) Fruiting body cultivation and selenium enrichment regulation: Unseal the opening and provide 12 hours of diffused light per day with a light intensity of 1200 lx; ​​control the temperature at 22℃ with a day-night temperature difference of 4℃; maintain a relative humidity of 85% and spray with sterile water twice a day; ventilate twice a day for 30 minutes each time; after the primordia are formed, cultivate for 28 days until the fruiting body grows to about 10cm, with the top swollen and full, and the color bright orange-yellow.

[0029] Harvesting and Selenium Content Protection (1) Harvesting: Use sterile scissors to cut off the fruiting body from the base and harvest qualified fruiting bodies in batches.

[0030] (2) Secondary cultivation: Add sterile water containing 5 mg / L sodium selenite to the original substrate, cultivate for 12 days, and harvest the second batch of fruiting bodies.

[0031] (3) Drying and preservation: Place the harvested fruiting bodies in a 55℃ drying oven to dry to 8% moisture content, cool and seal them in packaging, and store them in a cool and dry place at 20℃ and 55% relative humidity.

[0032] The selenium-enriched Cordyceps militaris fruiting bodies cultivated in this embodiment have robust stipes and excellent appearance. Testing revealed that the fruiting bodies contained 386 μg / g of selenium, with an organic selenium content of 92.5%, cordycepin content of 1.25%, and cordyceps polysaccharide content of 8.6%, all significantly higher than ordinary Cordyceps militaris. Furthermore, the cultivation process was free from microbial contamination, achieving a success rate of 98%. The second batch of fruiting bodies contained 378 μg / g of selenium, essentially consistent with the first batch, demonstrating good selenium content stability.

[0033] Example 2 A method for cultivating selenium-enriched Cordyceps militaris, the specific steps of which are as follows: Gradual domestication of selenium-enriched Cordyceps militaris strains (1) Selenium domestication of primary mother culture: Prepare culture medium according to 1000mL formula, peel and boil 200g of potatoes until soft, filter and extract juice, add 20g of glucose, 5g of peptone, 1g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, 10mg of vitamin B1 and 10mg of sodium selenite, add water to make up to 1000mL, dispense into test tubes and autoclave at 121℃ for 20 minutes, arrange in slant; aseptically inoculate the original mother culture, and culture at 23℃ in the dark for 10 days to obtain low selenium domesticated mother culture.

[0034] (2) Selenium acclimatization of secondary liquid primary culture: Prepare culture medium according to the formula of 1000mL: glucose 30g, peptone 10g, yeast extract 5g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g, sodium selenite 20mg, add water to make up to 1000mL, adjust pH to 6.0; put into 500mL Erlenmeyer flasks, fill with 200mL of liquid, autoclave at 121℃ for 30 minutes, cool and inoculate with 3 1cm² mycelial blocks; culture at 150r / min and 24℃ in the dark for 7 days to obtain secondary liquid primary culture.

[0035] Cultivation and Selenium Enrichment of Selenium-Enriched Cordyceps militaris (1) Preparation of selenium-enriched solid culture medium: Take 40g of rice, 3g of silkworm pupa powder and 1g of peptone for each bag, mix them, add 50mL of aqueous solution of 30mg sodium selenite, stir evenly; put into polypropylene cultivation bags, press and seal, sterilize at 100℃ and normal pressure for 8 hours, and cool for later use.

[0036] (2) Inoculation and mycelial culture: Under aseptic conditions, inoculate each bag with 10 mL of secondary liquid original culture, shake evenly; place in a constant temperature incubation room at 20℃, avoid light, relative humidity 60%, ventilate twice a day for 20 minutes each time, and incubate for 20 days until the mycelium fills the culture medium and forms a white mycelial film.

[0037] (3) Fruiting body cultivation and selenium enrichment regulation: Unseal the opening, provide 12 hours of diffused light daily with a light intensity of 1000 lx; ​​temperature 20℃, day-night temperature difference 3℃; relative humidity 80%, spray sterile water 3 times a day; ventilate 3 times a day for 30 minutes each time; after the primordia are formed, cultivate for 30 days until the fruiting body grows to 8cm and the top is swollen and plump.

[0038] Harvesting and Selenium Content Protection (1) Harvesting: Use sterile scissors to cut the base of the fruiting body and harvest in batches.

[0039] (2) Secondary cultivation: Add sterile water containing 5 mg / L sodium selenite, cultivate for 15 days, and harvest the second batch of fruiting bodies.

[0040] (3) Drying and preservation: Dry the fruiting bodies in a 60℃ drying oven until the moisture content is 10%, seal the packaging and store them in a cool and dry place at 25℃ and 60% relative humidity.

[0041] The selenium-enriched Cordyceps sinensis cultivated in this embodiment is free of deformities and of excellent quality. The test results show that the selenium content of the fruiting bodies is 372 μg / g, the proportion of organic selenium is 91.8%, the content of cordycepin is 1.18%, the content of cordycepin polysaccharide is 8.2%, the contamination rate of miscellaneous bacteria during the cultivation process is only 1%, the cultivation success rate is 97%, and the selenium content of the second batch of fruiting bodies is 365 μg / g, with good selenium content stability.

[0042] Example 3 A method for cultivating selenium-enriched Cordyceps militaris, the specific steps of which are as follows: Gradual domestication of selenium-enriched Cordyceps militaris strains (1) Selenium domestication of primary mother culture: Prepare culture medium according to 1000mL formula, peel and boil 200g of potatoes until soft, filter and extract juice, add 20g of glucose, 5g of peptone, 1g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, 10mg of vitamin B1 and 10mg of sodium selenite, add water to make up to 1000mL, dispense into test tubes, sterilize and place on slant; aseptically inoculate the original mother culture, and culture at 25℃ in the dark for 7 days to obtain low selenium domesticated mother culture.

[0043] (2) Selenium acclimatization of secondary liquid primary culture: Prepare culture medium according to the formula of 1000mL: glucose 30g, peptone 10g, yeast extract 5g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g, sodium selenite 20mg, add water to make up to 1000mL, adjust pH to 6.5; put into Erlenmeyer flasks for sterilization, cool and inoculate 5 1cm² mycelial blocks; culture at 150r / min, 24℃ in the dark for 5 days to obtain secondary liquid primary culture.

[0044] Cultivation and Selenium Enrichment of Selenium-Enriched Cordyceps militaris (1) Preparation of selenium-enriched solid culture medium: Mix 40g of rice, 3g of silkworm pupa powder and 1g of peptone in each bag, add 50mL of aqueous solution containing 30mg of sodium selenite, stir evenly; pack into bags, press and seal, autoclave at 121℃ for 40 minutes, and cool for later use.

[0045] (2) Inoculation and mycelial culture: Under aseptic conditions, inoculate each bag with 15 mL of secondary liquid original culture, shake evenly; place in a constant temperature incubation room at 22℃, avoid light, relative humidity 65%, ventilate once a day for 20 minutes each time, and incubate for 15 days until the mycelium fills the culture medium and forms a white mycelial film.

[0046] (3) Fruiting body cultivation and selenium enrichment regulation: Unseal the opening, provide 12 hours of diffused light daily with a light intensity of 1500 lx; ​​temperature 23℃, day-night temperature difference 5℃; relative humidity 90%, spray sterile water twice a day; ventilate twice a day for 30 minutes each time; after the primordia are formed, cultivate for 25 days until the fruiting body grows to 12cm and the top is swollen and plump.

[0047] Harvesting and Selenium Content Protection (1) Harvesting: Use sterile scissors to cut the base of the fruiting body and harvest in batches.

[0048] (2) Secondary cultivation: Add sterile water containing 5 mg / L sodium selenite, cultivate for 10 days, and harvest the second batch of fruiting bodies.

[0049] (3) Drying and preservation: Dry the fruiting bodies in a 50℃ drying oven until the moisture content is 7%, seal the packaging and store in a cool and dry place at 18℃ and 50% relative humidity.

[0050] The selenium-enriched Cordyceps militaris fruiting bodies cultivated in this embodiment are robust and bright in color. The test results show that the selenium content of the fruiting bodies is 395 μg / g, the proportion of organic selenium is 93.2%, the content of cordycepin is 1.32%, and the content of cordyceps polysaccharide is 9.0%. There is no contamination by miscellaneous bacteria during the cultivation process, and the cultivation success rate is 99%. The selenium content of the second batch of fruiting bodies is 389 μg / g, and the selenium content stability is excellent.

[0051] Comparative test To verify the technical effect of the present invention, a comparative experiment was set up. The control group adopted the existing conventional method of cultivating selenium-enriched Cordyceps militaris, directly adding 30 mg / L sodium selenite to rice solid culture medium without gradient selenium acclimatization. The strain was inoculated with solid original strain, and the environmental control during the cultivation stage was carried out in a conventional manner. The remaining operations were the same as those in Example 1 of the present invention.

[0052] The experimental results showed that the Cordyceps militaris strain in the control group germinated slowly after inoculation, with sparse mycelial growth. After 25 days of culture, it still failed to fully cover the culture medium and was contaminated by a large number of miscellaneous bacteria, with a cultivation success rate of only 65%. The fruiting bodies were abnormally differentiated, with thin and long stipes and no apical swelling, resulting in poor appearance quality. The test results showed that the Cordyceps militaris fruiting bodies cultivated in the control group had a selenium content of only 125 μg / g, an organic selenium content of 68%, a cordycepin content of 0.65%, and a cordyceps polysaccharide content of 4.2%, all of which were significantly lower than those in Example 1 of this invention.

[0053] The cultivation method of Example 1 of this invention, after gradient selenium acclimatization, resulted in rapid germination of the strain, dense mycelial growth, and complete colonization of the culture medium within 18 days. The contamination rate of miscellaneous bacteria was low, and the cultivation success rate reached 98%. The fruiting bodies exhibited excellent appearance quality, with significantly higher selenium content, organic selenium ratio, and active ingredient content compared to the control group. This fully demonstrates that the gradient selenium acclimatization, liquid strain cultivation, and standardized environmental control of this invention can effectively improve the cultivation success rate, selenium enrichment efficiency, and medicinal value of selenium-enriched Cordyceps militaris.

[0054] The present invention discloses a method for cultivating selenium-enriched Cordyceps militaris. The process parameters for each step are clear and specific, and the operation is standardized. The culture medium raw materials used are conventional agricultural and chemical raw materials such as potatoes, rice, glucose, and peptone, which are readily available and inexpensive. The cultivation equipment consists of conventional microbial culture equipment such as high-pressure steam sterilizers, constant temperature incubators, constant temperature shakers, and drying ovens. No special high-end customized equipment is required, making it suitable for industrial production.

[0055] The cultivation method of this invention exhibits low contamination rate and high success rate, resulting in selenium-enriched Cordyceps militaris with stable selenium content, a high proportion of organic selenium, and significantly increased levels of active ingredients such as cordycepin and cordyceps polysaccharides. This leads to high medicinal and economic value, and the total yield can be increased through secondary cultivation, further enhancing economic benefits. Furthermore, the cultivation method of this invention is highly reproducible, enabling large-scale, industrialized cultivation. It is suitable for promotion and application in pharmaceutical and health product raw material planting bases and related enterprises, demonstrating significant industrial applicability and broad market prospects.

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. Technical contents not described in detail in this invention can be implemented by referring to existing technologies or conventional technical means in the field.

[0057] The scope of protection of this invention is determined by the scope of the claims. Any modifications or improvements to the technical solutions based on this invention shall fall within the scope of protection of this invention.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for cultivating selenium-enriched Cordyceps militaris, characterized in that, It includes three steps: gradient domestication of selenium-enriched Cordyceps militaris strains, cultivation and selenium enrichment of selenium-enriched Cordyceps militaris, and harvesting and selenium content protection. The gradient acclimatization adopts a low selenium starting point and a gradually increasing sodium selenite concentration gradient, and is carried out in two steps: primary mother seed selenium acclimatization and secondary liquid original seed selenium acclimatization. The cultivation and selenium enrichment process uses rice as a solid substrate, with sodium selenite added as a selenium source, and is divided into three stages: culture medium preparation, inoculation culture, and fruiting body cultivation. The harvesting and selenium content assurance process includes fruiting body harvesting, secondary cultivation, and drying and preservation. The concentration of sodium selenite and aseptic operation are controlled throughout the process to achieve efficient absorption and transformation of selenium by Cordyceps militaris mycelium and fruiting bodies, thus cultivating selenium-enriched Cordyceps militaris with stable selenium content.

2. The method for cultivating selenium-enriched Cordyceps militaris according to claim 1, characterized in that: The culture medium for selenium domestication of the primary mother seed, calculated per 1000 mL, consists of: 200 g potato, 20 g glucose, 5 g peptone, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 10 mg vitamin B1, 10 mg sodium selenite, and 1000 mL water.

3. The method for cultivating selenium-enriched Cordyceps militaris according to claim 2, characterized in that: The preparation and cultivation process of the primary mother culture for selenium domestication is as follows: potatoes are peeled, boiled until soft, filtered to extract juice, the remaining components are added and dissolved, and then dispensed into test tubes. The tubes are sterilized at 121℃ for 20 minutes and arranged as slant cultures. Under aseptic conditions, the original mother culture of Cordyceps militaris is inoculated and cultured at 23-25℃ in the dark for 7-10 days. When the mycelium grows all over the slant culture and is white and dense, the low-selenium domesticated mother culture is obtained.

4. The method for cultivating selenium-enriched Cordyceps militaris according to claim 1, characterized in that: The culture medium for the secondary liquid original seed selenium acclimatization, calculated per 1000 mL, has the following formula: 30 g glucose, 10 g peptone, 5 g yeast extract, 2 g potassium dihydrogen phosphate, 1 g magnesium sulfate, 20 mg sodium selenite, and 1000 mL water, with the pH value of the culture medium adjusted to 6.0–6.

5.

5. The method for cultivating selenium-enriched Cordyceps militaris according to claim 4, characterized in that: The preparation and cultivation process of the secondary liquid primary culture for selenium acclimatization is as follows: the culture medium is placed into a 500mL Erlenmeyer flask with a liquid volume of 200mL, and sterilized at 121℃ for 30 minutes; after cooling to 25℃, 3-5 pieces of primary acclimatization mother culture mycelial blocks are inoculated, with the mycelial block size being 1cm²; the Erlenmeyer flask is placed on a shaker and cultured at 150r / min and 24℃ in the dark for 5-7 days until the culture medium becomes turbid and the mycelial balls become dense, thus obtaining a liquid primary culture adapted to the medium selenium environment.

6. The method for cultivating selenium-enriched Cordyceps militaris according to claim 1, characterized in that: The selenium-enriched solid culture medium, per bag, has the following formula: 40g rice, 3g silkworm pupa powder, 1g peptone, 30mg sodium selenite, and 50mL water, with a pH value of natural.

7. The method for cultivating selenium-enriched Cordyceps militaris according to claim 1, characterized in that: The preparation process of the selenium-enriched solid culture medium is as follows: mix rice, silkworm pupa powder, and peptone evenly, add a solution of sodium selenite, and stir until the rice fully absorbs the water; pack into 15cm×30cm polypropylene cultivation bags, press them firmly, and seal them; sterilize at 121℃ for 40 minutes or at 100℃ for 8 hours, and cool to room temperature before use.

8. The method for cultivating selenium-enriched Cordyceps militaris according to claim 1, characterized in that: The inoculation and cultivation process is as follows: Under aseptic conditions, 10-15 mL of secondary selenium-enriched liquid inoculum is inoculated into each bag of cultivation medium. The cultivation bag is gently shaken to ensure that the inoculum is evenly distributed on the surface of the rice substrate. The inoculated cultivation bags are placed in a constant temperature incubation room at 20-22℃ and cultured for 15-20 days under conditions of light protection, relative humidity of 60%-65%, and good ventilation. Once the mycelium has covered the entire culture medium and a white mycelial film has formed on the surface, the process is transferred to the fruiting body induction stage.

9. The method for cultivating selenium-enriched Cordyceps militaris according to claim 1, characterized in that: The environmental control for fruiting body cultivation is as follows: after the mycelium has filled the bag, open the cultivation bag seal and provide 12 hours of diffused light daily, with a light intensity of 1000-1500 lx; ​​control the temperature at 20-23℃, maintaining a day-night temperature difference of 3-5℃; increase the relative humidity to 80%-90%, and spray with sterile water 2-3 times a day to avoid water accumulation in the substrate; ventilate 2-3 times a day for 30 minutes each time, and control the carbon dioxide concentration; after the primordia have formed, continue cultivation for 25-30 days, and when the fruiting bodies grow to 8-12cm, with a swollen and full top and a bright orange-yellow color, the fruiting body cultivation is completed.

10. The method for cultivating selenium-enriched Cordyceps militaris according to claim 1, characterized in that: The harvesting and selenium content assurance process is as follows: the fruiting bodies are cut off from the base with sterile scissors to complete the harvest. After harvesting, sterile water containing 5 mg / L sodium selenite is added to the original substrate. After culturing for 10 to 15 days, a second batch of fruiting bodies can be harvested. The harvested fruiting bodies are dried at a low temperature below 60°C until the moisture content is ≤10%. After drying, they are sealed in packaging and stored in a cool and dry place.