Separation culture method of cordyceps sinensis fungi
Through improved purification and culture methods, combined with specific temperature and soil leaching solution, the problems of low spore survival rate and long cycle in the isolated culture of Cordyceps sinensis fungi were solved, and efficient and stable mycelium growth and yield improvement were achieved.
Patent Information
- Application Number
- CN202510581864.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-07
- Publication Date
- 2025-08-08
AI Technical Summary
In the prior art, the isolation and culture method of Cordyceps sinensis fungi has technical bottlenecks in spore purification, germination and medium optimization, resulting in low spore survival rate, long culture cycle and unstable yield.
The purification, germination and culture of multiple fungi were carried out using improved purification methods and medium formulations, combined with specific temperature control and soil leaching solution, ascospore purification, germination and culture of multiple fungi, including ascospore fungi, zombi tissue fungi, celestial fungi and in vitro fungi mycelial culture.
It improves the spore germination rate, shortens the culture cycle, enhances the growth stability and yield of mycelium, reduces the risk of pollution, and ensures the purity and stability of the bacterial species.
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field related to Cordyceps sinensis fungi, and in particular to a method for isolating and culturing Cordyceps sinensis fungi. Background Art
[0002] Methods for collecting, purifying, germinating, and culturing ascospores of Cordyceps sinensis, as well as methods for culturing dead insect tissue, stroma mycelium, and extrastroma mycelium, were studied. Thirty-six fungal strains with identical growth characteristics were cultured using these four methods. One strain (H1) grew stroma similar to natural Cordyceps sinensis on culture medium, thus confirming that the isolated fungus is the fungus that forms Cordyceps sinensis.
[0003] Cordyceps sinensis (Berk) Sacc is a medicinal and edible fungus and the most important species in the genus Cordyceps.
[0004] However, due to the difficulty in isolating the Cordyceps sinensis fungus, no reports have been found so far. Although Nobuo Yachi (1980) reported on the artificial cultivation of 80 species in the genus Cordyceps, no reports have been made on this species.
[0005] In summary, traditional cultivation methods still face technical bottlenecks in spore purification, germination, and culture medium optimization. In particular, the survival rate of ascospores and parasite tissues from different sources is low during cultivation, resulting in unstable yields and long production cycles. Therefore, improving Cordyceps sinensis cultivation technology, especially improving spore germination rates and promoting rapid mycelial growth, has become an urgent issue, and a new solution to these problems is needed. Summary of the Invention
[0006] The object of the present invention is to provide a method for isolating and culturing Cordyceps sinensis fungi to solve the technical problems raised in the background technology.
[0007] To achieve the above object, the present invention provides the following technical solution: a method for isolating and culturing Cordyceps sinensis fungi, comprising at least the following steps:
[0008] S1: Collecting ascospores of Cordyceps sinensis. During the collection process, a paper bag is placed over the stroma of naturally grown, immature Cordyceps sinensis. When the stroma matures, the ascospores are ejected and attached to the paper bag. After removing the paper bag, the ascospores in the bag are dried in the shade and stored in a refrigerator at 0-4°C for later use.
[0009] S2: To collect Cordyceps sinensis, dig up fresh Cordyceps sinensis that has not yet matured, place it together with the soil around it in a sealed box, bring it back to the laboratory, and store it in a refrigerator at 0℃-4℃ until use;
[0010] S3: prepare the culture medium for separation;
[0011] S4: Soil immersion: Soil near the fresh Cordyceps sinensis in S2 is immersed in the liquid;
[0012] S5: Cultivating multiple types of Cordyceps sinensis fungi, including at least germination and cultivation of ascospore fungi, cultivation of dead insect tissue fungi, cultivation of stroma fungal hyphae, and cultivation of fungal hyphae outside the insect body.
[0013] Furthermore, the separation medium in S3 comprises at least 8 g of polypeptone, 110 g of glucose, 0.8 g of yeast extract, 0.4 g of magnesium sulfate (MgSO4.7H2O), 1.5 g of dipotassium hydrogen phosphate (K2HPO4.3H2O), 25 g of agar powder, 800 ml of distilled water and 20 ml of vitamin solution;
[0014] The preparation process of the separation culture medium is as follows: polypeptone, glucose, yeast extract, magnesium sulfate, dipotassium hydrogen phosphate, 25 grams of agar powder and distilled water are mixed, and then heated and boiled to dissolve to obtain a base liquid, and the base liquid is sterilized at 118° C. for 35 minutes. When the sterilized base liquid is cooled to about 50° C., a corresponding amount of vitamin solution is added and mixed, and the mixture is injected into a sterile plate to obtain the separation culture medium.
[0015] Furthermore, the vitamin solution comprises at least 100 mg of thiamine hydrochloride, 150 mg of calcium vanadate, 26 mg of para-aminobenzoic acid, 110 mg of choline chloride, 80 mg of inositol, 80 mg of niacinamide, 3.5 mg of folic acid, 1 mg of biotin, and 500 ml of distilled water;
[0016] The vitamin solution is prepared by mixing and dissolving thiamine hydrochloride, calcium vanadate, p-aminobenzoic acid, choline chloride, inositol, nicotinamide, folic acid, biotin and distilled water, and then sterilizing and filtering.
[0017] Furthermore, the S4 at least includes the following steps:
[0018] Take 1000g of soil near fresh Cordyceps sinensis, add 1000ml of distilled water, and boil for 5 minutes;
[0019] The boiled soil liquid was filtered, and the filtrate was sterilized at 115°C for 30 minutes to obtain a soil base liquid;
[0020] Add 9 parts of sterile distilled water to 1 part of soil base liquid to make a 1:10 soil extract.
[0021] Furthermore, the germination and cultivation of the ascospore fungus includes three steps: purification of ascospores, germination of ascospores and separation of single ascospores.
[0022] The purification of the ascospores is as follows: 100 paper bags with ascospores are taken, immersed in a 1:10 soil extract, and after soaking for 2 hours, the ascospores are scraped off the paper bags with a knife, collected in a centrifuge tube, and centrifuged for 10 minutes at 1000 rpm. The supernatant is discarded, and the tube is repeatedly washed five times with soil extract, then washed three times with 10% and 20% decoction sugar solutions, respectively, and then centrifuged with a 50% sucrose solution for 30 minutes to collect the spores floating on the liquid surface. Finally, the tube is washed three times with soil extract and then placed in a -10°C refrigerator for standby use.
[0023] The germination of the ascospores is as follows: taking a small amount of ascospores purified in the purification step, mixing 100 units / ml of gentamicin with soil extract in a ratio of 1:1, placing the purified ascospores in the 1:1 prepared soil extract, and placing the purified ascospores at a room temperature of 15° C. to 20° C. for 3-6 days, so that the ascospores can germinate;
[0024] The single ascospore separation is to take a suspension of ascospores that have germinated three times, drop it on a 2% agar plate, pick a single germinated ascospore with a thinner inoculation needle under a 100x microscope, inoculate it on a separation medium, culture it at room temperature of 15°C-20°C, and observe its growth frequently under a microscope.
[0025] Furthermore, the culturing of the dead insect tissue fungus comprises at least the following steps:
[0026] Take fresh Cordyceps sinensis, wash it, and place it in 0.1% mercuric chloride solution for 5 minutes for disinfection;
[0027] Then, the mercuric chloride was washed off with sterile distilled water, and under aseptic conditions, the outer skin was cut off, avoiding the intestinal tract, and the white tissue was cut and crushed, and then the fragments were spread on the separation medium prepared in S3;
[0028] Incubate at room temperature of 15℃-20℃.
[0029] Furthermore, the cultivation of stroma fungus hyphae comprises at least the following steps:
[0030] Take the immature fruiting bodies of Cordyceps sinensis, that is, before ascospores are formed, wash them, and place them in 0.1% mercuric chloride solution for 5 minutes for disinfection;
[0031] Wash off the mercuric chloride, cut it open, and take out the white mycelium bundles;
[0032] Inoculate on the culture medium, press a portion into the agar, and culture at room temperature of 15℃-20℃.
[0033] Furthermore, the culturing of the extracellular fungal hyphae comprises at least the following steps:
[0034] If you dig up a Cordyceps sinensis specimen that has not been taken out of the ground and remove the soil around the insect, you will see a layer of white mycelium surrounding the dead insect.
[0035] Place the white mycelium in a moist dish and place it in a 5°C refrigerator for 2 months. The white mycelium will grow slowly.
[0036] At this time, use white gold to pick up a small amount of mycelium, inoculate it on the separation culture medium, and culture it at room temperature of 15-20℃.
[0037] Compared with the prior art, the present invention has the following beneficial effects:
[0038] 1. The method of the present invention improves the spore germination rate: through improved purification and culture methods, the germination rate of ascospores reaches 20%-50%, which is higher than the traditional method and ensures the subsequent mycelial growth;
[0039] 2. Optimizing culture conditions: By using a 1:10 soil extract solution and combining it with specific temperature control (15°C-20°C), the growth of Cordyceps sinensis mycelium is effectively promoted. The colony morphology conforms to the characteristics of natural growth and is highly stable.
[0040] 3. The cultivation cycle of the method of the present invention is greatly shortened: through the improved culture medium formula and environmental control, the growth rate of mycelium is significantly improved, and the colonies reach 2-6 mm in 2 months, and colonies with a diameter of 1-2 cm can be formed in 3-4 months, shortening the cultivation cycle and increasing the yield;
[0041] 4. The method of the present invention can prevent contamination and competition from foreign bacteria: by adding gentamicin to inhibit the growth of foreign bacteria, the success rate of culture is significantly improved, the risk of contamination is reduced, and the stability of the experiment is guaranteed;
[0042] 5. The present invention enables efficient strain isolation: through the isolation and culture of single ascospores, 35 different fungal strains were successfully isolated from multiple sources, and it was proved that these strains have similar growth characteristics to natural Cordyceps sinensis, ensuring the purity and stability of the strains. DETAILED DESCRIPTION
[0043] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.
[0044] A method for isolating and culturing Cordyceps sinensis fungi comprises at least the following steps:
[0045] S1: Collecting Cordyceps sinensis ascospores. In this embodiment, the location for collecting Cordyceps sinensis ascospores is Naqu, Tibet. In different embodiments, naturally produced Cordyceps sinensis ascospores from other locations may be used. During the collection process, a paper bag is placed over the stroma of naturally grown immature Cordyceps sinensis. After the stroma matures, the ascospores are ejected and attached to the paper bag. After the paper bag is removed, the ascospores in the paper bag are dried in the shade and stored in a refrigerator at 0-4°C for later use.
[0046] S2: Harvesting Cordyceps sinensis. In this embodiment, immature fresh Cordyceps sinensis was harvested from Nagqu, Tibet. In different embodiments, naturally produced Cordyceps sinensis ascospores from other locations may be used. Unripe fresh Cordyceps sinensis was dug up, placed in a sealed box along with the surrounding soil, brought back to the laboratory, and stored in a refrigerator at 0°C-4°C until use.
[0047] S3: prepare the culture medium for separation;
[0048] S4: Soil immersion: Soil near the fresh Cordyceps sinensis in S2 is immersed in the liquid;
[0049] S5: Cultivating multiple types of Cordyceps sinensis fungi, including at least germination and cultivation of ascospore fungi, cultivation of dead insect tissue fungi, cultivation of stroma fungal hyphae, and cultivation of fungal hyphae outside the insect body.
[0050] The separation medium in S3 comprises at least 8 g of polypeptone, 110 g of glucose, 0.8 g of yeast extract, 0.4 g of magnesium sulfate (MgSO4.7H2O), 1.5 g of dipotassium hydrogen phosphate (K2HPO4.3H2O), 25 g of agar powder, 800 ml of distilled water, and 20 ml of vitamin solution;
[0051] The preparation process of the separation culture medium is to mix polypeptone, glucose, yeast extract, magnesium sulfate, dipotassium hydrogen phosphate, 25 grams of agar powder and distilled water, then heat and boil to dissolve to obtain a basic liquid, and then sterilize the basic liquid at 118°C for 35 minutes. When the sterilized basic liquid is cooled to about 50°C, add the corresponding amount of vitamin solution and mix well, and inject it into a sterile plate to obtain the separation culture medium.
[0052] The vitamin solution should contain at least 100 mg of thiamine hydrochloride, 150 mg of calcium vanadate, 26 mg of para-aminobenzoic acid, 110 mg of choline chloride, 80 mg of inositol, 80 mg of niacinamide, 3.5 mg of folic acid, 1 mg of biotin, and 500 ml of distilled water;
[0053] The vitamin solution is prepared by mixing and dissolving thiamine hydrochloride, calcium vanadate, p-aminobenzoic acid, choline chloride, inositol, nicotinamide, folic acid, biotin and distilled water, and then sterilizing and filtering to obtain the solution.
[0054] S4 includes at least the following steps:
[0055] Take 1000g of soil near fresh Cordyceps sinensis, add 1000ml of distilled water, and boil for 5 minutes;
[0056] The boiled soil liquid was filtered, and the filtrate was sterilized at 115°C for 30 minutes to obtain a soil base liquid;
[0057] Add 9 parts of sterile distilled water to 1 part of soil base liquid to make a 1:10 soil extract.
[0058] The germination and culture of ascospore fungi include three steps: ascospore purification, ascospore germination and single ascospore separation.
[0059] Purification of ascospores is as follows: 100 paper bags with ascospores are taken and immersed in 1:10 soil extract. After immersion for 2 hours, the ascospores are scraped off the paper bags with a knife and collected in a centrifuge tube. The tubes are centrifuged for 10 minutes at 1000 rpm. The supernatant is discarded and the tubes are repeatedly washed five times with soil extract. The tubes are then washed three times with 10% and 20% decoction sugar solutions respectively. The tubes are then centrifuged for 30 minutes with 50% sucrose solution. The spores floating on the liquid surface are collected and finally washed three times with soil extract. The tubes are then placed in a -10°C refrigerator for later use.
[0060] The germination of ascospores is as follows: a small amount of ascospores purified in the purification step is taken, 100 units / ml of gentamicin is mixed with soil extract in a ratio of 1:1, the purified ascospores are placed in the 1:1 mixed soil extract, and the mixture is placed at room temperature of 15°C-20°C for 3-6 days, and the ascospores can germinate;
[0061] The isolation of single ascospores is to take a suspension of ascospores that have germinated three times, drop it on a 2% agar plate, pick up a single germinated ascospore with a thinner inoculation needle under a 100x microscope, inoculate it on the separation culture medium, and culture it at room temperature of 15℃-20℃, and observe the growth frequently under a microscope.
[0062] The culture of dead insect tissue fungus at least comprises the following steps:
[0063] Take fresh Cordyceps sinensis, wash it, and place it in 0.1% mercuric chloride solution for 5 minutes for disinfection;
[0064] Then, the mercuric chloride was washed off with sterile distilled water, and under aseptic conditions, the outer skin was cut off, avoiding the intestinal tract, and the white tissue was cut and crushed, and then the fragments were spread on the separation medium prepared in S3;
[0065] Incubate at room temperature of 15℃-20℃.
[0066] The cultivation of stroma fungus hyphae comprises at least the following steps:
[0067] Take the immature fruiting bodies of Cordyceps sinensis, that is, before ascospores are formed, wash them, and place them in 0.1% mercuric chloride solution for 5 minutes for disinfection;
[0068] Wash off the mercuric chloride, cut it open, and take out the white mycelium bundles;
[0069] Inoculate on the culture medium, press a portion into the agar, and culture at room temperature of 15℃-20℃.
[0070] The cultivation of fungal hyphae outside the insect body comprises at least the following steps:
[0071] If you dig up a Cordyceps sinensis specimen that has not been taken out of the ground and remove the soil around the insect, you will see a layer of white mycelium surrounding the dead insect.
[0072] Place the white mycelium in a moist dish and place it in a 5°C refrigerator for 2 months. The white mycelium will grow slowly.
[0073] At this time, use white gold to pick up a small amount of mycelium, inoculate it on the separation culture medium, and culture it at room temperature of 15-20℃.
[0074] The specific method proposed based on the above embodiment has the following results:
[0075] Purified ascospores germinate after 3-6 days of incubation in soil extract. Germ tubes emerge from the spores, and the germination rate is generally 20-50%. After 10 days, ascospores transplanted onto culture medium will show elongated, branched, and forming hyphae. After one month, small, colorless colonies will form. After two months, the colonies will reach a diameter of 2-6 mm. After three months, very short, off-white, aerial hyphae will be visible. After four months, the colonies will reach a diameter of 1-2 cm. The raised surface of the colonies will be wrinkled and uneven, ranging from off-white to brownish-yellow. The hyphae at the base will be brown-black, and a brown-black pigment will diffuse into the culture medium. A total of 27 fungal strains were isolated from single ascospores.
[0076] After one week of culturing the insect tissue, hyphae can be seen growing around the tissue fragments, extending into the culture medium below. A grayish-white moss with underdeveloped hyphae can be seen above. Two months later, a colony forms around the tissue fragments. Three to four months later, the colony reaches a diameter of 1 to 2 centimeters. The colony has an uneven, raised surface that ranges from grayish-white to yellow-brown. The colony is firm, with brown-black hyphae at the base, and brown-black pigment radiating outward. Specimens collected from Hualong and Yushu yielded fungi with similar morphology. A total of six fungal strains were isolated from the dead insect tissue.
[0077] 3. Mycelium in the stroma begins to grow after two weeks of culture. Initially, new hyphae grow from the periphery of the stroma, extending downward into the culture medium and upward to form grayish-white aerial hyphae. After two months, colonies form around the original inoculum. Their growth characteristics are similar to those of ascospores and parasite tissue. In addition to hyphae, the ascosphere can also grow and reproduce.
[0078] 4. After one month of culture of mycelium outside the dead insect, mycelium can be seen branching and spreading. After two months, small gray-white colonies are formed, which have the same characteristics as the fungi separated from ascospores and insect tissues.
[0079] The four methods described above yielded a total of 35 fungal strains (27 from ascospores, 6 from dead insect tissue, 1 from stroma, and 1 from extra-insect mycelium). In particular, the fungi cultured from single ascospores were identical to those cultured from dead insect tissue, stroma mycelium, and extra-insect mycelium.
[0080] In addition, after being cultured on the culture medium for 6 months, the "H1" bacteria grew stroma, and its morphology was very similar to that grown in nature.
[0081] Basic information on the isolation of bacteria "H1":
[0082] The strain was collected from Hualong, Qinghai Province. The material for strain isolation was dead insect tissue. The year of isolation was May 24, 1981. The main characteristics of the strain were raised colonies, uneven surfaces, underdeveloped aerial hyphae, grayish white to brownish black in color, diffused brownish black pigment, and slow growth.
[0083] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.
Claims
1. A method for isolating and culturing Cordyceps sinensis fungi, characterized in that: At least the following steps are included: S1: Collecting ascospores of Cordyceps sinensis. During the collection process, a paper bag is placed over the stroma of naturally grown, immature Cordyceps sinensis. When the stroma matures, the ascospores are ejected and attached to the paper bag. After removing the paper bag, the ascospores in the paper bag are dried in the shade and stored in a refrigerator at 0-4°C for later use. S2: To collect Cordyceps sinensis, dig up fresh Cordyceps sinensis that has not yet matured, place it together with the soil around it in a sealed box, bring it back to the laboratory, and store it in a refrigerator at 0℃-4℃ until use; S3: prepare the culture medium for separation; S4: Soil immersion: Soil near the fresh Cordyceps sinensis in S2 is immersed in the liquid; S5: Cultivating multiple types of Cordyceps sinensis fungi, including at least germination and cultivation of ascospore fungi, cultivation of dead insect tissue fungi, cultivation of stroma fungal hyphae, and cultivation of fungal hyphae outside the insect body.
2. The method for isolating and culturing Cordyceps sinensis fungi according to claim 1, wherein: The separation medium in S3 comprises at least 8 g of polypeptone, 110 g of glucose, 0.8 g of yeast extract, 0.4 g of magnesium sulfate, 1.5 g of dipotassium hydrogen phosphate, 25 g of agar powder, 800 ml of distilled water, and 20 ml of vitamin solution; The preparation process of the separation culture medium is as follows: polypeptone, glucose, yeast extract, magnesium sulfate, dipotassium hydrogen phosphate, 25 grams of agar powder and distilled water are mixed, and then heated and boiled to dissolve to obtain a base liquid, and the base liquid is sterilized at 118° C. for 35 minutes. When the sterilized base liquid is cooled to about 50° C., a corresponding amount of vitamin solution is added and mixed, and the mixture is injected into a sterile plate to obtain the separation culture medium.
3. The method for isolating and culturing Cordyceps sinensis fungi according to claim 2, wherein: The vitamin solution comprises at least 100 mg of thiamine hydrochloride, 150 mg of calcium vanadate, 26 mg of para-aminobenzoic acid, 110 mg of choline chloride, 80 mg of inositol, 80 mg of niacinamide, 3.5 mg of folic acid, 1 mg of biotin, and 500 ml of distilled water; The vitamin solution is prepared by mixing and dissolving thiamine hydrochloride, calcium vanadate, p-aminobenzoic acid, choline chloride, inositol, nicotinamide, folic acid, biotin and distilled water, and then sterilizing and filtering.
4. The method for isolating and culturing Cordyceps sinensis fungi according to claim 2, wherein: The S4 at least includes the following steps: Take 1000g of soil near fresh Cordyceps sinensis, add 1000ml of distilled water, and boil for 5 minutes; The boiled soil liquid was filtered, and the filtrate was sterilized at 115°C for 30 minutes to obtain a soil base liquid; Add 9 parts of sterile distilled water to 1 part of soil base liquid to make a 1:10 soil extract.
5. The method for isolating and culturing Cordyceps sinensis fungi according to claim 4, wherein: The germination and cultivation of the ascospore fungus includes three steps: purification of ascospores, germination of ascospores and separation of single ascospores. The purification of the ascospores is as follows: 100 paper bags with ascospores are taken, immersed in a 1:10 soil extract, and after soaking for 2 hours, the ascospores are scraped off the paper bags with a knife, collected in a centrifuge tube, and centrifuged for 10 minutes at 1000 rpm. The supernatant is discarded, and the ascospores are repeatedly washed five times with soil extract, then washed three times with 10% and 20% decoction sugar solutions, respectively, and then centrifuged with a 50% sucrose solution for 30 minutes, spores floating on the liquid surface are collected, and finally washed three times with soil extract, and then placed in a -10°C refrigerator for standby use; The germination of the ascospores is as follows: taking a small amount of ascospores purified in the purification step, mixing 100 units / ml of gentamicin with soil extract in a ratio of 1:1, placing the purified ascospores in the 1:1 prepared soil extract, and placing the purified ascospores at a room temperature of 15° C. to 20° C. for 3-6 days, so that the ascospores can germinate; The single ascospore separation is to take a suspension of ascospores that have germinated three times, drop it on a 2% agar plate, pick a single germinated ascospore with a thinner inoculation needle under a 100x microscope, inoculate it on a separation medium, culture it at room temperature of 15°C-20°C, and observe its growth frequently under a microscope.
6. The method for isolating and culturing Cordyceps sinensis fungi according to claim 1, wherein: The culturing of the dead insect tissue fungus at least comprises the following steps: Take fresh Cordyceps sinensis, wash it, and place it in 0.1% mercuric chloride solution for 5 minutes for disinfection; Then, the mercuric chloride was washed off with sterile distilled water, and under aseptic conditions, the outer skin was cut off, avoiding the intestinal tract, and the white tissue was cut and crushed, and then the fragments were spread on the separation medium prepared in S3; Incubate at room temperature of 15℃-20℃.
7. The method for isolating and culturing Cordyceps sinensis fungi according to claim 1, wherein: The cultivation of stroma fungus hyphae comprises at least the following steps: Take the immature fruiting bodies of Cordyceps sinensis, that is, before ascospores are formed, wash them, and place them in 0.1% mercuric chloride solution for 5 minutes for disinfection; Wash off the mercuric chloride, cut it open, and take out the white mycelium bundles; Inoculate on the culture medium, press a portion into the agar, and culture at room temperature of 15℃-20℃.
8. The method for isolating and culturing Cordyceps sinensis fungi according to claim 1, wherein: The culturing of the extra-parasitic fungal hyphae comprises at least the following steps: If you dig up a Cordyceps sinensis specimen that has not been taken out of the ground and remove the soil around the insect, you will see a layer of white mycelium surrounding the dead insect. Place the white mycelium in a moist dish and place it in a 5°C refrigerator for 2 months. The white mycelium will grow slowly. At this time, use white gold to pick up a small amount of mycelium, inoculate it on the separation culture medium, and culture it at room temperature of 15-20℃.