Method for rejuvenating hirsutella hepialid strain

Abstract

The invention discloses a method for rejuvenating a hirsutella hepialid strain, which specifically comprises the following steps of: step 1, inoculating a degenerated hirsutella hepialid strain into an extractable stroma culture medium, and culturing until aerial hyphae are generated on the surface of a bacterial colony; 2, performing variable-temperature stimulation on the bacterial colonies grown in the step 1, and producing sporocarp on the bacterial colonies; step 3, taking out the sporocarp in the step 2, performing tissue isolation, inoculating the sporocarp into a PDA culture medium, and culturing until the diameter of a hirsutella hepialid colony is 3cm; and 4, inoculating the hirsutella hepialid strain in the step 3 into a shake flask liquid culture medium with the inoculation amount of about 1cm < 2 > of bacterial colony, filtering and collecting mycelium after culture is completed, and drying and detecting. Compared with a strain before rejuvenation, the strain rejuvenated by the method disclosed by the invention has the advantages that the mycelium yield and the content of active ingredients in bacterial powder are remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of rejuvenation of degenerated edible and medicinal fungus strains, and particularly relates to a method for rejuvenating Hirsutella hepiali strains. Background Art

[0002] Cordyceps Sinensis is a complex of entomogenous fungi parasitic in the larvae of Hepialus armoricanus Oberthur and the corpses of its host Hepialus armoricanus Oberthur larvae, with the effects of tonifying the kidney and lung, resolving phlegm and relieving cough, etc. It is a precious Chinese medicinal material with a long medicinal history. Due to its specific growth environment requirements, its output is very limited. Coupled with long-term over-excavation, the ecological environment has been damaged, and the quantity of this precious medicinal material has become increasingly scarce. With the continuous increase in the demand for Cordyceps, wild Cordyceps has become increasingly in short supply.

[0003] Microbial strain degeneration refers to the phenomenon that after the degenerated cells in the microbial population reach a certain level in quantity, the deterioration of the original excellent production traits, the loss of genetic markers, and the change of typical characteristics occur. There are many reasons for the decline of microbial strains, such as the quantitative change to qualitative change of spontaneous mutations of strains, induced mutations by environmental factors, continuous subculture (the more times of subculture, the more likely spontaneous mutations, especially negative mutations) occur, the invasion of other microorganisms, improper culture and preservation conditions, etc.; poor culture conditions will not only induce the emergence of degenerated cells, but also promote the rapid reproduction of degenerated cells, greatly exceeding the normal cells in quantity, resulting in the decline of the strain. Inappropriate preservation conditions will enable microbial cells to still maintain a certain metabolic activity, increasing the possibility of variation and degeneration.

[0004] When a microbial strain shows degeneration, it is necessary to use rejuvenation methods to restore its original excellent traits. Generally speaking, the method that can be adopted is to carry out pure culture separation, and then measure its typical traits and production performance and other indicators, and screen out a few individuals that have not degenerated from the degenerated population. The probability of screening out the target strain by this method is low, the screening workload is large, and it consumes a large amount of labor and time. Therefore, it is necessary to find a new rejuvenation method. Summary of the Invention

[0005] In view of the above problems, the present invention provides a simple, easy-to-implement and effective method for rejuvenating Hirsutella hepiali strains. After rejuvenation by this method, the production capacity of Hirsutella hepiali can quickly return to the production level of the original primary strain.

[0006] To achieve the above object, the present invention adopts the following technical solutions: A method for rejuvenating Hirsutella hepiali fungi, comprising the following steps: (1) Inoculate the degenerated Hirsutella hepiali strain on a solid medium capable of producing stroma, and inoculate about 1 cm 21 to 2 mycelium blocks of a certain size are cultured until aerial hyphae appear on the colony surface; (2) The colony obtained in step (1) is subjected to a temperature change stimulation of 4 - 14°C to grow primordia of fruiting bodies, and then cultured at 15 - 20°C for 25 - 35 days to obtain fruiting bodies; Fruiting bodies with a length of 3 - 10 cm can be harvested. The harvested fruiting bodies are similar in shape to those of wild Cordyceps sinensis, being grayish-brown and club-shaped; (3) The fruiting bodies obtained in step (2) are subjected to tissue isolation and then inoculated into an enriched PDA medium, and cultured at 15 - 20°C for 45 - 60 days until the colony diameter of Hirsutella hepiali reaches 3 cm; (4)The Hirsutella hepiali colony obtained in step (3) is inoculated into a shake flask liquid medium, and 1 cm 2 solid strain is inoculated for every 30 - 40 mL of liquid medium, and cultured until the medium is covered with mycelial balls. The mycelium is filtered, collected, dried, and then the mycelium yield is calculated and the content is determined.

[0007] Furthermore, the formula of the solid medium capable of producing stroma in step (1) is: glucose 20.0 g, potato 200.0 g, agar powder 20.0 g, yeast extract powder 0.5 - 1.5%, peptone 1.0 - 2.0%, milk powder 0.5 - 1.0%, KH2PO4 0.1 - 0.3%, MgSO4·7H2O 0.1 - 0.2%, silkworm pupa powder 0.5 - 1.5%, VB1 0.01%, H2O 1000.0 mL, natural pH. The preparation method is: Wash and peel the potatoes, boil them until soft, then filter with a gauze. Add glucose, yeast extract powder, peptone, milk powder, KH2PO4, MgSO4·7H2O, silkworm pupa powder, and VB1 to the filtrate. Additionally, boil the agar powder until thick, and then combine the two and make up the volume to 1.0 L, sterilize at 121°C for 30 minutes for later use.

[0008] Furthermore, the culture conditions in step (1) are to culture at 15 - 20°C for 45 - 60 days until the colony diameter reaches 3 - 5 cm.

[0009] Furthermore, the temperature change stimulation of 0 - 15°C in step (2) means that the colony is placed in an incubator, stimulated and cultured at 0°C for 12 hours every day, then heated to 15°C and cultured for 12 hours, and cyclically stimulated and cultured for 45 - 60 days until primordia of fruiting bodies appear on the colony surface.

[0010] Furthermore, the culture process of the primordia of fruiting bodies in step (2) also includes irradiating with red and blue light with an illumination intensity of 200 LX - 500 LX for no less than 12 hours every day, and ventilating for half an hour every morning and evening.

[0011] Further, the specific steps of tissue separation in step (3) are as follows: After the well-grown fruiting bodies are rinsed with sterile water and surface-disinfected with 75% alcohol solution, under sterile conditions, the surface layer of the fruiting bodies is removed with a surgical blade that has been flame-sterilized, and then cut into tissue blocks with a diameter of 3-5 mm.

[0012] Further, the formula of the enriched PDA medium in step (3) is as follows: 20.0 g of glucose, 200.0 g of potato, 20.0 g of agar powder, 1.0 - 1.5% of peptone, 0.5 - 1.0% of milk powder, 0.2 - 0.5% of KH2PO4, 0.1 - 0.3% of MgSO4·7H2O, 0.01 - 0.02% of VB1, 1000.0 mL of H2O, natural pH. The preparation method is as follows: Wash the potato, peel it, boil it until it becomes mushy, then filter it with a gauze. Add glucose, peptone, milk powder, KH2PO4, MgSO4·7H2O, and VB1 to the filtrate. Separately, boil the agar powder with cold water until it becomes thick, then combine and make up the volume to 1000 mL, and sterilize it at 121°C for 30 minutes for standby.

[0013] Further, the formula of the shake-flask liquid medium in step (4) is as follows: 30 g of glucose, 10.0 g of peptone, 5.0 g of yeast extract powder, 5.0 g of milk powder, 2.0 g of KH2PO4, 1.0 g of MgSO4·7H2O, 0.1 g of VB1. After dissolving each component in water and combining them, make up the volume to 1.0 L, adjust the pH to 6.0 - 6.5, dispense it into 250 mL shake flasks, with a filling volume of 100 mL, and sterilize it at 121°C for 30 minutes for standby. For the determination results of the mycelium yield and main components, three repeated experiments are set, and the results are averaged.

[0014] Compared with the prior art, the advantages of the present invention are as follows: (1) The test materials are easy to obtain, the culture medium is simple, the cost is low, and the workload is reduced; (2) After rejuvenation, the Hirsutella hepiali strain grows vigorously, and both the shake-flask fermentation yield and the final quality of the bacterial powder are significantly improved. Specific Embodiments

[0015] In order to make the content of the present invention easier to understand, the following further illustrates the technical solutions of the present invention in combination with specific embodiments, but the present invention is not limited thereto.

[0016] The degenerated Hirsutella hepiali strain in the following embodiments is as follows: (1) After continuous subculture for 3 - 5 times, the growth of the strain colonies is slow. Under the condition of the same culture time, the colony diameter decreases significantly compared with the previous stage. The colony color changes from white to yellowish-brown or brownish-yellow, and the growth point is not obvious.

[0017] (2)After continuous subculture for 5 times, the index contents of adenosine and mannitol decreased by about 30%.

[0018] The following are the components of the culture medium and the preparation method involved in the following examples: The formula for the solid medium capable of producing stroma is: glucose 20.0 g, potato 200.0 g, yeast extract powder 5.0 g, peptone 10.0 g, milk powder 5.0 g, KH2PO4 2.0 g, MgSO4·7H2O 1.0 g, silkworm pupa powder 5 g, VB1 0.1 g, agar powder 20.0 g, H2O 1000.0 mL, natural pH. The preparation method is: Wash and peel the potato, cook it until soft in water, then filter it with a gauze. Add glucose, yeast extract powder, peptone, milk powder, KH2PO4, MgSO4·7H2O, silkworm pupa powder, and VB1 to the filtrate. Separately, boil the agar powder in water until thick, and then combine the two and make up the volume to 1.0 L, sterilize at 121 °C for 30 minutes and reserve for use.

[0019] The formula for the enriched PDA medium is: glucose 20.0 g, potato 200.0 g, peptone 10.0 g, milk powder 5.0 g, KH2PO4 2.0 g, MgSO4·7H2O 1.0 g, VB1 0.1 g, agar powder 20.0 g, H2O 1000.0 mL, natural pH. The preparation method is: Wash and peel the potato, cook it until soft in water, then filter it with a gauze. Add glucose, peptone, milk powder, KH2PO4, MgSO4·7H2O, and VB1 to the filtrate. Separately, boil the agar powder in cold water until thick, combine them and make up the volume to 1.0 L, sterilize at 121 °C for 30 minutes and reserve for use.

[0020] The formula for the shake flask liquid medium is: glucose 30 g, peptone 10.0 g, yeast extract powder 5.0 g, milk powder 5.0 g, KH2PO4 2.0 g, MgSO4·7H2O 1.0 g, VB1 0.1 g. Dissolve each component in water and then combine and make up the volume to 1.0 L, adjust the pH to 6.0 - 6.5, dispense into 250 ml shake flasks, with a filling volume of 100 ml, sterilize at 121 °C for 30 minutes and reserve for use.

[0021] Example 1 Step 1: Inoculate the degenerated Hirsutella hepiali strain into 30 - 40 mL of the medium capable of producing stroma, inoculate 1 fungal block with a size of about 1.0 cm 2 in diameter, culture at 15 °C for 55 days, aerial hyphae will be produced on the surface of the colony, and the colony diameter is 4.3 cm, reserve for use; Step 2: Subject the grown colonies to temperature variation stimulation at 0 - 15°C for 45 days. After the formation of fruiting body primordia, transfer them to a 15°C environment for culturing for 25 days to harvest fruiting bodies with a length of 3.0 - 5.0 cm. The harvested fruiting bodies are similar in morphology to those of wild Cordyceps sinensis, being grayish-brown and rod-shaped. The specific steps for temperature variation stimulation are as follows: Stimulate and culture at 0°C for 12 hours every day, then raise the temperature to 15°C for 12 hours of culture. The cultivation process of the fruiting body primordia also includes irradiating with red and blue light (3:1) at an illuminance of 300 LX for 12 hours every day, and ventilating for half an hour in the morning and evening every day.

[0022] Step 3: Select the grown fruiting bodies, rinse them with sterile water, and surface disinfect them with a 75% alcohol solution. Under sterile conditions, use a surgical blade that has been flame-sterilized to remove the surface layer of the fruiting bodies and cut them into tissue blocks with a diameter of 3.0 - 5.0 mm. Transfer them to the center of a Petri dish containing pre-prepared enriched PDA medium, invert it, and culture at 15°C for 55 days until the colony diameter reaches 3.6 cm.

[0023] Step 4: Select the grown Hirsutella hepiali colonies from Step 3 and inoculate them into a shake flask liquid medium. The inoculation amount is approximately 1.0 cm 2 , place it in a shaker at 15°C with a rotation speed of 150 rpm, and culture for 35 days. Filter and collect the mycelia, dry them, calculate the mycelia yield, and conduct component determination. The determination results are shown in Table 1.

[0024] Table 1 Mycelia yield and component determination data of Hirsutella hepiali strains before and after rejuvenation Example 2 Step 1: Inoculate the degenerated Hirsutella hepiali strain into 30 - 40 mL of medium capable of producing stromata. Inoculate 2 mycelial blocks with a size of approximately 1.0 cm 2 , and culture at 18°C for 50 days. Aerial hyphae will be produced on the colony surface, and the colony diameter will be 4.2 cm. Set aside for later use; Step 2: Subject the grown colonies to temperature variation stimulation at 4 - 14°C for 50 days. After the formation of fruiting body primordia, transfer them to an 18°C environment for culturing for 30 days to harvest fruiting bodies with a length of 4.0 - 7.0 cm. The harvested fruiting bodies are similar in morphology to those of wild Cordyceps sinensis, being grayish-brown and rod-shaped. The specific steps for temperature variation stimulation are as follows: Stimulate and culture at 4°C for 12 hours every day, then raise the temperature to 14°C for 12 hours of culture. The cultivation process of the fruiting body primordia also includes irradiating with red and blue light (3:1) at an illuminance of 500 LX for 12 hours every day, and ventilating for half an hour in the morning and evening every day.

[0025] Step 3: Select the well-grown daughter entities, rinse them with sterile water, surface disinfect them with 75% alcohol solution. Under sterile conditions, use a surgical blade that has been flame-sterilized to remove the surface layer of the daughter entities and then cut them into tissue blocks with a diameter of 3.0 - 5.0 mm. Transfer the tissue blocks to the center of a Petri dish containing pre-prepared enriched PDA medium, invert it, and culture it at 18°C for 50 days until the colony diameter reaches 4.3 cm.

[0026] Step 4: Select the well-grown Hirsutella hepiali colony in Step 3, inoculate it into a shake flask liquid medium, and the inoculation amount is about 1.0 cm 2 , place it in a shaker at 18°C with a rotation speed of 180 rpm, culture it for 30 days, filter and collect the mycelium, dry it, calculate the mycelium yield and conduct component determination. The determination results are shown in Table 2.

[0027] Table 2 Mycelium yield and component determination data of Hirsutella hepiali strains before and after rejuvenation Example 3 Step 1: Inoculate the degenerated Hirsutella hepiali strain into 30 - 40 mL of medium that can produce stroma, inoculate 2 mycelial blocks with a size of about 1.0 cm 2 , culture it at 20°C for 45 days. Aerial mycelia will grow on the colony surface, and the colony diameter is 4.6 cm. Set it aside for later use; Step 2: Subject the well-grown colony to temperature variation stimulation at 4 - 14°C for 55 days. After the formation of the stroma primordium, transfer it to 20°C for 35 days to harvest stroma with a length of 5.0 - 10.0 cm. The harvested stroma is similar in morphology to the stroma of wild Cordyceps sinensis, showing a grayish-brown and club-shaped appearance. The specific steps of temperature variation stimulation are as follows: Stimulate and culture it at 4°C for 12 hours every day, then raise the temperature to 14°C for 12 hours. The culture process of the stroma primordium also includes irradiating it with red and blue light (3:1) with an illuminance of 200 LX for 12 hours every day, and ventilating for half an hour in the morning and evening every day.

[0028] Step 3: Select the well-grown stroma, rinse it with sterile water, surface disinfect it with 75% alcohol solution. Under sterile conditions, use a surgical blade that has been flame-sterilized to remove the surface layer of the stroma and then cut it into tissue blocks with a diameter of 3.0 - 5.0 mm. Transfer the tissue blocks to the center of a Petri dish containing pre-prepared enriched PDA medium, invert it, and culture it at 20°C for 45 days until the colony diameter reaches 4.5 cm.

[0029] Step 4: Select the well-grown Hirsutella hepiali colony in Step 3, inoculate it into 30 - 40 mL of shake flask liquid medium, and the inoculation amount is about 1.0 cm 2 , place it in a shaker at 20°C with a rotation speed of 200 rpm, culture it for 25 days, filter and collect the mycelium, dry it, calculate the mycelium yield and conduct component determination. The determination results are shown in Table 3.

[0030] Table 3 Mycelium Yield and Component Determination Data of Hirsutella hepiali Strains before and after Rejuvenation Comparative Example 1 (Rejuvenation at 0°C) Step 1: Inoculate the degenerated Hirsutella hepiali strain into 30 - 40 mL of the medium capable of producing stroma, inoculate 1 mycelium block with a size of about 1.0 cm, and culture it at 15°C for 55 days. Aerial mycelium appears on the colony surface, and the colony diameter is 4.3 cm. Set it aside for later use; 2 Step 2: Subject the well - grown colony to variable - temperature stimulation only at 0°C. The variable - temperature stimulation process also includes irradiating with red and blue light (3:1) with an illuminance of 300 LX for 12 hours every day, and ventilating for half an hour in the morning and evening every day. After culturing for 45 days, no stroma primordium is observed. It takes 390 - 590 days to grow the fruiting body primordium, and then transfer it to 15 - 25°C for culturing for 55 days to harvest fruiting bodies with a length of 3.0 - 5.0 cm. The harvested fruiting bodies are similar in morphology to those of Cordyceps sinensis collected in the wild, being gray - brown and rod - shaped. However, there are only a few individual fruiting bodies, with a very small quantity, an average of 0.3 fruiting bodies per bottle, and the time taken will reach 500 - 700 days. Step 2: Subject the well - grown colony to variable - temperature stimulation only at 0°C. The variable - temperature stimulation process also includes irradiating with red and blue light (3:1) with an illuminance of 300 LX for 12 hours every day, and ventilating for half an hour in the morning and evening every day. After culturing for 45 days, no stroma primordium is observed. It takes 390 - 590 days to grow the fruiting body primordium, and then transfer it to 15 - 25°C for culturing for 55 days to harvest fruiting bodies with a length of 3.0 - 5.0 cm. The harvested fruiting bodies are similar in morphology to those of Cordyceps sinensis collected in the wild, being gray - brown and rod - shaped. However, there are only a few individual fruiting bodies, with a very small quantity, an average of 0.3 fruiting bodies per bottle, and the time taken will reach 500 - 700 days.

[0031] Comparative Example 2 (Rejuvenation at 15°C) Step 1: Inoculate the degenerated Hirsutella hepiali strain into 30 - 40 mL of the medium capable of producing stroma, inoculate 1 mycelium block with a size of about 1.0 cm, and culture it at 15°C for 55 days. Aerial mycelium appears on the colony surface, and the colony diameter is 4.3 cm. Set it aside for later use; 2 Step 2: Subject the well - grown colony to variable - temperature stimulation only at 15°C. The variable - temperature stimulation process also includes irradiating with red and blue light (3:1) with an illuminance of 300 LX for 12 hours every day, and ventilating for half an hour in the morning and evening every day. After culturing for 45 days, no stroma primordium is observed. Continue culturing for 55 days, and still no stroma primordium or stroma bud is observed. Therefore, the rejuvenation effect cannot be achieved. Step 2: Subject the well - grown colony to variable - temperature stimulation only at 15°C. The variable - temperature stimulation process also includes irradiating with red and blue light (3:1) with an illuminance of 300 LX for 12 hours every day, and ventilating for half an hour in the morning and evening every day. After culturing for 45 days, no stroma primordium is observed. Continue culturing for 55 days, and still no stroma primordium or stroma bud is observed. Therefore, the rejuvenation effect cannot be achieved.

[0032] Comparative Example 3 (0°C for 16 h / 15°C for 8 h) Step 1: Inoculate the degenerated Hirsutella hepiali strain into 30 - 40 mL of the medium capable of producing stroma, inoculate 1 mycelium block with a size of about 1.0 cm, and culture it at 15°C for 55 days. Aerial mycelium appears on the colony surface, and the colony diameter is 4.3 cm. Set it aside for later use; 2 Step 2: Subject the well - grown colony to variable - temperature stimulation at 0°C for 16 hours and 15°C for 8 hours. The variable - temperature stimulation process also includes irradiating with red and blue light (3:1) with an illuminance of 300 LX for 12 hours every day, and ventilating for half an hour in the morning and evening every day. After culturing for 45 days, no stroma primordium is observed. Continue culturing for 55 days, and still no stroma primordium or stroma bud is observed. Therefore, the rejuvenation effect cannot be achieved. Step 2: subject the grown colonies to variable temperature stimulation at 0℃16h / 15℃8h. The specific steps of variable temperature stimulation are: stimulate culture at 0℃ for 16 hours every day, then raise the temperature to 15℃ for 8 hours. The variable temperature stimulation process also includes irradiation with red and blue light (3:1) with an illumination of 300LX for 12 hours every day, and ventilation for half an hour every morning and evening. After 130-170 days, the fruiting body primordium grows out, and the fruiting body with a length of 3.0~5.0cm can be harvested after culturing at 15-25℃ for 55-60 days. The harvested fruiting body is similar to the fruiting body of Cordyceps collected in the wild, which is gray-brown and thick rod-shaped. However, the number is small, with an average of 2.6 roots / bottle, and the time spent will reach 240-280 days.

[0033] This group can produce fruiting bodies, but it takes 240-280 days, and the quantity is not large. It can achieve a rejuvenation effect, but the time and workload are also increased.

[0034] Comparative Example 4 (0℃8h / 15℃16h) Step 1: Take the degenerate bat moth Hirsutella strain and inoculate it with 30-40 mL of culture medium that can extract stroma, and inoculate about 1.0 cm 2 One block of the same size was cultured at 15°C for 55 days. Aerial hyphae were produced on the surface of the colony and the colony diameter was 4.3 cm. It was set aside. Step 2: subject the grown colonies to variable temperature stimulation at 0℃8h / 15℃16h. The specific steps of variable temperature stimulation are: stimulate culture at 0℃ for 8 hours every day, then raise the temperature to 15℃ for 16 hours. The variable temperature stimulation process also includes irradiation with red and blue light (3:1) with an illumination of 300LX for 12 hours every day, and ventilation for half an hour every morning and evening. After 50-70 days, the fruiting body primordium grows out, and the fruiting body with a length of 3.0~5.0cm can be harvested after culturing at 15-25℃ for 55-60 days. The harvested fruiting body is similar to the fruiting body of Cordyceps collected in the wild, which is gray-brown and thin in rod shape. However, the number is small, with an average of 3.5 roots / bottle, and the time spent will reach 160-180 days.

[0035] This group can produce fruiting bodies in 160-180 days, and the number is small. The fruiting bodies are relatively thin, which can achieve a rejuvenation effect. Higher technical requirements are required for tissue separation.

[0036] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for rejuvenating the fungus Hirsutella hepiali, characterized in that: It includes the following steps: (1)Inoculate the degenerated strain of Hirsutella hepiali on the solid medium capable of producing stroma, and inoculate 1 to 2 mycelial blocks with a size of about 1 cm, and culture until aerial hyphae are produced on the colony surface; 2 ​ (2) Subject the colonies obtained in step (1) to temperature variation stimulation at 0 - 15°C to grow fruiting body primordia, and then transfer them to culture at 15 - 20°C for 25 - 35 days to obtain fruiting bodies; (3) Conduct tissue separation on the fruiting bodies obtained in step (2), inoculate them into an enriched PDA medium, and culture at 15 - 20°C for 45 - 60 days until the colony diameter of (4) Inoculate the Hirsutella hepiali colony obtained in step (3) into a shake flask liquid medium at an inoculation amount of inoculating 1 cm 2 solid strain per 30 - 40 mL of the liquid medium, and culture until the medium is fully covered with mycelial pellets. 2 ​ 2. The rejuvenation method according to claim 1, wherein: Hirsutella hepiali 3. The rejuvenation method according to claim 1, wherein: reaches 3 cm; 4. The rejuvenation method according to claim 1, characterized in that: The formula of the solid medium capable of producing stromata in step (1) is: glucose 20.0 g, potato 200.0 g, agar powder 20.0 g, yeast extract powder 0.5 - 1.5%, peptone 1.0 - 2.0%, milk powder 0.5 - 1.0%, KH2PO4 0.1 - 0.3%, MgSO4·7H2O 0.1 - 0.2%, silkworm pupa powder 0.5 - 1.5%, VB1 0.01%, H2O 1000.0 mL, natural pH.

5. The rejuvenation method according to claim 1, characterized in that: The culture conditions in step (1) are to culture at 15 - 20°C for 45 - 60 days until the colony diameter reaches 3 - 5 cm.

6. The rejuvenation method according to claim 1, wherein: The temperature variation stimulation at 4 - 14°C in step (2) means putting the colonies into an incubator, stimulating and culturing at 4°C for 12 hours every day, then raising the temperature to 14°C for 12 hours, and cycling the stimulation and culture for 45 - 60 days until fruiting body primordia grow on the colony surface.

7. The rejuvenation method according to claim 1, wherein: The culture process of the fruiting body primordia in step (2) further includes irradiating with light at an illuminance of 200 LX - 500 LX for not less than 12 hours every day, and ventilating for 0.5 hour in the morning and evening every day. The light is red - blue light with a ratio of 3:

1.

8. The rejuvenation method according to claim 1, wherein: The specific steps of tissue separation in step (3) are: after rinsing the well - grown fruiting bodies with sterile water, surface - disinfecting them with 75% alcohol solution, and under sterile conditions, using a surgical blade that has been flame - burned to remove the surface layer of the fruiting bodies and then cutting them into tissue blocks with a diameter of 3 - 5 mm. The formula of the enriched PDA medium in step (3) is: glucose 20.0 g, potato 200.0 g, agar powder 20.0 g, peptone 1.0 - 1.5%, milk powder 0.5 - 1.0%, KH2PO4 0.2 - 0.5%, MgSO4·7H2O 0.1 - 0.3%, VB1 0.01 - 0.02%, H2O 1000.0 mL, natural pH. The formula of the shake - flask liquid medium in step (4) is: glucose 30 g, peptone 1.0 - 2.0%, yeast extract powder 0.5 - 1.0%, milk powder 0.5 - 1.0%, KH2PO4 0.1 - 0.2%, MgSO4·7H2O 0.1 - 0.2%, VB1 0.01%.

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