Method for maintaining vigor of edible mushroom strain

Abstract

The invention discloses a method for maintaining the vitality of an edible mushroom strain, and relates to the technical field of edible mushroom strain preservation. The method comprises the following core steps: screening detoxified and purified strains for low-temperature domestication, and determining a low-temperature sensitive threshold based on the condition that the growth rate at low temperature is greater than or equal to 50% of the normal-temperature rate; preparing an improved culture medium which is formed by mixing wood flour, vermiculite powder and potato dextrose agar, and embedding the slow-release nutrient particles into the improved culture medium; the improved culture medium is used for short-term preservation at 4-6 DEG C, paraffin-nano coating composite protection is used for middle-term preservation, and a low-temperature dormancy awakening mechanism and nitrogen stress are combined for long-term preservation; during passage management, new hyphae with the number of splitting times smaller than or equal to 10 are selected for sterile operation; and finally, carrying out activity detection through molecular level monitoring, such as SSR (Simple Sequence Repeat) markers, and carrying out targeted improvement on strains with insufficient activity. The method can effectively maintain hypha activity, hereditary stability and fruiting performance, is standard in operation and high in applicability, and provides a reliable seed source for large-scale cultivation of shiitake mushrooms.

Description

Technical Field

[0001] The invention relates to the technical field of edible fungus strain preservation, in particular to a method for maintaining the vitality of edible fungus shiitake mushroom strains. Background Art

[0002] In the field of edible mushroom culture, maintaining the vitality of the shiitake mushroom is the key to large-scale cultivation. However, traditional methods of preservation and management of mushrooms have many limitations.

[0003] First, during long-term subculture or storage, shiitake mushroom strains experience slowed growth rates and degraded fruiting performance, such as decreased biological efficiency and abnormal fruiting body morphology. Mycelial resistance weakens, significantly reducing survival rates in adverse environments like high temperatures and drought. Genetic mutation increases, leading to the loss of desirable traits. Second, traditional preservation methods, such as solely cryopreservation, fail to account for the varying tolerances of different strains to low temperatures, which can lead to cold damage and a sudden drop in viability in some strains. Subculture in conventional culture media results in uneven nutrient release, leading to excessive mycelial growth in the early stages and growth restriction in the later stages. Furthermore, the subculture process can easily introduce contaminants. Limited adaptability of preservation techniques: Traditional preservation methods struggle to address short-, medium-, and long-term storage needs and lack the ability to adapt to individual strains. Environmental fluctuations, such as sudden temperature changes and nutrient imbalances, can easily compromise nutrient viability. Inadequate cold adaptability: Shiitake mushroom strains vary in their sensitivity to low temperatures. Traditional acclimation methods lack scientific threshold determination and systematic mechanisms for activating freezing stress, resulting in a susceptibility to suppression during low-temperature storage or environmental changes. Summary of the Invention

[0004] In response to the problems raised by the above background technology, the present invention proposes a method for maintaining the vitality of edible mushroom shiitake mushroom strains, and the specific scheme is as follows: A method for maintaining the vitality of edible mushroom shiitake mushroom strains comprises the following steps: S1. Initial strain screening: Select virus-free strains, which are purified strains obtained by tissue separation or single spore separation, and meet the requirements of white, dense, uniform mycelium, free of foreign bacteria and pigment secretion, strong mycelium climbing ability, and fast feed consumption; S2. Adaptively acclimate the virus-free strain obtained in S1 to low temperature; set four temperature gradients of 0°C, 3°C, 5°C, and 8°C in a constant temperature incubator, record the expansion of colony diameter, calculate the growth rate, screen the temperature boundary of "growth rate at low temperature ≥ 50% of the normal temperature rate", and generate a personalized "low temperature sensitive threshold interval"; based on the sensitive threshold interval, perform 5-8 pulse treatments at the upper temperature of the threshold interval, with each pulse lasting 2 hours and an interval of 24 hours; after the pulse stimulation, add antifreeze protectants in stages, add 0.05% trehalose to the culture medium before the first pulse, and add 0.05% trehalose after the last pulse; after strengthening the activation effect, reduce the temperature from room temperature to the upper temperature of the threshold interval on the 1st to 3rd day, and gradually reduce it to the lower temperature on the 4th to 7th day, inoculate the strain after adaptive low temperature acclimatization into PDA culture medium, monitor the mycelial growth rate and measure the colony diameter every day, and screen single colonies with "growth rate decrease of ≤30% at low temperature"; S3. Culture medium preparation: using an edible fungus modified culture medium, the modified culture medium is a mixed culture medium of wood powder, vermiculite powder and potato dextrose agar, and embedded with slow-release nutrient particles, the slow-release nutrient particles are gelatin microspheres embedded with yeast extract; S4. Preservation of strains: Select the preservation method according to the preservation period. For short-term preservation, use edible strain modified culture medium at 4-6°C for 3-6 months. For medium-term preservation, use paraffin-nano coating composite protection. For long-term preservation, use low temperature 1.5°C dormancy awakening mechanism and nitrogen adversity preservation. S5. Subculture management: In a sterile environment, use a peptide nucleic acid probe labeled with the telomere-targeted fluorescent dye Cy3 to stain hyphae. Quantitative analysis of telomere fluorescence intensity using a microscopic imaging system indirectly assesses the number of divisions. Only new hyphae with a division number ≤ 10 are selected for transfer. For transfer, select new hyphae 0.5–2 cm from the bevel edge. S6. Regular vitality testing: New molecular level monitoring: Detect the genetic stability of the strain through SSR molecular markers, detect 5 to 8 highly polymorphic sites, and the mutation rate of polymorphic sites ≤1% is considered normal vitality. On this basis, introduce methylation-sensitive amplified polymorphism technology to detect the DNA methylation level of the mycelial genome, and issue an early warning when the mutation rate exceeds 0.5%.

[0005] S7. For edible fungi with insufficient activity, the following methods can be adopted to improve their activity based on the above operations: add 2g / L yeast extract and appropriate amount of B vitamins to the modified culture medium; adopt the "gradient temperature increase + nutrient pulse" strategy in the early stage of recovery of subculture management, first increase the temperature to 5℃ at 2℃ / hour, and then add 1% glucose solution to stimulate growth.

[0006] Preferably, in step S2, the growth rate calculation formula is growth rate = (colony diameter on the 7th day - colony diameter on the 3rd day) / 4 days; the number of pulses is dynamically adjusted according to the tolerance of the strain. If the mycelium shrinks or secretes pigment after the pulse, the number of pulses is reduced to 5 times; if the tolerance is good, the number of pulses is increased to 8 times.

[0007] Preferably, in step S2, the cryoprotectant is replaced by 0.2% proline instead of 0.1% trehalose, that is, 0.1% proline is added before the first pulse and after the last pulse respectively.

[0008] Preferably, in step S3, the formula of the improved culture medium is that the total volume of wood powder and vermiculite powder is in a volume ratio of (1-5):1 to the potato dextrose agar medium, the volume ratio of the wood powder to the vermiculite powder is 1:1, and the wood powder with a "porous microsphere structure" with a particle size of 20-40 mesh is obtained by low-temperature freeze-drying technology pretreatment, the vermiculite powder particle size is passed through an 80-mesh sieve, 20g of glucose, 2g of yeast extract, 250g of potato, 18g of agar, and 1000g of sterile water; the low-temperature freeze-drying technology pretreatment is first rapid freezing at -20°C to -40°C, and then increasing the temperature and pressure under vacuum conditions to allow the ice to sublime and dry.

[0009] Preferably, in step S4, the operation of preserving the liquid paraffin includes: forming a layer of nano-scale chitosan film with a concentration of 0.5% on the surface of the mycelium, pouring the pretreated liquid paraffin onto the slope covered with mycelium to cover the mycelium layer by 1 to 2 cm, and storing at 4°C; the pretreatment of the liquid paraffin is: 500 mL of paraffin plus 10 g of filtered anhydrous calcium chloride, sterilizing at 121°C for 2 hours, the pH value after sterilization is in the range of natural pH or 5.5-6.5, and taking the upper clear liquid after cooling.

[0010] Preferably, in step S4, the operation of the low-temperature 1.5°C dormancy awakening mechanism is as follows: before storage under nitrogen stress, a "resurrection signal molecule", i.e., 50 μM cAMP, is added to the bacterial suspension; during resuscitation, "gradient temperature increase + nutrient pulse" is adopted, first heating to 5°C at 2°C / hour, and then adding 1% glucose solution.

[0011] Preferably, in step S4, the nitrogen stress preservation operation includes: taking mycelium in the logarithmic growth phase, adding PDA liquid culture medium containing 10% glycerol, packaging into cryo tubes, and gradient cooling, i.e., maintaining at -20°C for 2 hours → maintaining at -80°C for 12 hours → liquid nitrogen, and storing in -196°C liquid nitrogen.

[0012] Preferably, in step S5, the sterile environment is a clean bench, and the inoculation tools are sterilized by burning and cooled before use.

[0013] Preferably, in step S6, the mycelium growth rate is detected by transferring the strain to a plate of an improved edible fungus culture medium, culturing at 25°C, measuring the mycelium diameter within 7 days, and the daily growth of healthy strains is ≥0.5 cm and the edges are neat.

[0014] Preferably, in step S6, the fruiting test is specifically as follows: the fungus is inoculated into a sawdust culture medium, cultured at 25° C. for 30 days, and then transferred to a fruiting room. Fungus with normal vitality should bud in 7 to 10 days, and the fruiting body should be complete in morphology.

[0015] Preferably, in step S4, the liquid paraffin is checked every 12 months during storage, and if the paraffin is turbid, it is re-switched.

[0016] The experimental operation also includes establishing a strain file, marking the strain name, storage date and number of passages.

[0017] The experimental operation also includes environmental control steps: all operations are carried out in a Class 100 clean bench (wind speed 0.3-0.5m / s, ultraviolet light irradiation for 30 minutes), plasma sterilization technology is used in the storage area, and high-frequency plasma (10-30kHz) is used to decompose microorganisms (including spores) in the air in real time; the temperature fluctuation of the storage equipment is ≤±1°C; sensors monitor the concentration of microorganisms in the environment in real time (≤1CFU / m 3 ), ensure sterility throughout the operation.

[0018] The threshold interval is determined by calculating the growth rate, screening the temperature boundary of "growth rate at low temperature ≥ 50% of the normal temperature rate", and using this as the boundary to determine the "low temperature sensitive threshold interval" of the strain.

[0019] Guiding low-temperature acclimation: Based on the determined sensitive threshold range, set a "short-term low-temperature pulse" (repeated 5 to 8 times) to activate the antifreeze stress pathway of the mycelium; subsequently, combine antifreeze protectants (such as 0.1% trehalose) and a gradual cooling strategy to screen out single colonies with excellent growth performance under low temperatures (growth rate decrease ≤ 30%).

[0020] Compared with the prior art, the advantages of the present invention are as follows: (1) The present invention introduces a growth rate calculation formula and uses "growth rate at low temperature ≥ 50% of the normal temperature rate" as the temperature boundary to determine a personalized low-temperature sensitive threshold range. By activating the antifreeze stress pathway through "short-term low-temperature pulses", the low-temperature adaptation characteristics of different strains are accurately matched, overcoming the drawback of the traditional domestication method that lacks specificity and improving the efficiency and effect of low-temperature domestication of shiitake mushroom strains.

[0021] (2) The culture medium of the present invention can continuously provide nutrition for mycelial growth and meet the needs of mycelial growth at different stages.

[0022] (3) The short-term, medium-term and long-term preservation methods of the present invention respectively use improved culture medium, paraffin-nanocoating composite protection, low-temperature dormancy awakening mechanism and nitrogen adversity preservation, which can effectively maintain the vitality of the strain and reduce the risk of strain degeneration.

[0023] (4) The present invention detects the number of mycelial cell divisions through strict subculture management and only selects new mycelium with a division number of ≤10 for transfer, thereby ensuring the quality of the transferred mycelium, reducing the degradation of the strain caused by subculture, and achieving precise control of the strain vitality.

[0024] (5) The present invention adds molecular-level monitoring, which can timely eliminate deteriorated strains and reduce cultivation risks. At the same time, there are special improvement methods for strains with insufficient activity, such as re-determining the low-temperature sensitivity threshold range, adding nutrients, and adopting specific recovery strategies, which can effectively improve the activity of strains and provide personalized improvement measures. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a system architecture diagram for maintaining the vitality of edible mushroom shiitake mushroom strains proposed by the present invention. DETAILED DESCRIPTION

[0026] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0027] Example 1: Comprehensive application of the low-temperature sensitive shiitake mushroom variety '091' S1. Initial strain screening: In a clean bench (controlled air speed of 0.3-0.5 m / s, UV sterilization for 30 minutes), screen for samples with dense, white mycelium free of contaminants. Mycelium climbing ability should be assessed as "strong" (feeding time ≤ 48 hours) to ensure initial quality.

[0028] S2. Data Collection and Threshold Determination: The cold-acclimated strain was inoculated into PDA culture medium. Four temperature gradients (0°C, 3°C, 5°C, and 8°C) were set in a constant-temperature incubator. The colony diameter expansion was recorded and the growth rate was calculated. It was found that at 5°C, the mycelial growth rate decreased by 20%, less than 25%. Therefore, the personalized low-temperature sensitivity threshold range of '091' was determined to be 4-6°C. Seven pulse treatments (each lasting 2 hours, with 24-hour intervals) were performed at the upper temperature of the threshold range (7°C) to activate the mycelial antifreeze stress pathway. Post-pulse observations: Mycelium showed no signs of shrinkage or pigment secretion (good tolerance), so the number of pulses was not reduced to five. 0.05% trehalose was added to the culture medium before the first pulse, and an additional 0.05% trehalose was added after the final pulse (total 0.1%). The temperature was lowered from room temperature (25°C) to the upper limit of the threshold range (7°C) on days 1-3, and then to the target temperature (5°C) at a rate of 0.5°C / day on days 4-7. Colony diameters were measured daily, and growth rate was calculated using the formula: (colony diameter on day 7 - colony diameter on day 3) / 4 days. Individual colonies whose growth rate decreased by ≤25% at low temperature were selected (the growth rate of '091' after acclimation was 0.6 cm / day, a 25% decrease compared to room temperature, meeting the standard).

[0029] S3. Culture medium preparation: Use a modified culture medium with a 1:1 volume ratio of wood flour to vermiculite powder (60% total volume), 20g glucose, 2g yeast extract, 250g potato, 18g agar, and 1000g sterile water. Freeze-dry the wood flour to form 20-40 mesh porous microspheres. Sieve the vermiculite powder through an 80-mesh sieve. Embed the yeast extract in gelatin sustained-release nutrient granules.

[0030] Strain Storage: Select a short-term storage method (target period: 4 months): Inoculate the acclimated strain into the modified culture medium and store at 4°C. Check every 2 months to ensure temperature fluctuations are ≤ ±1°C (using real-time sensor monitoring).

[0031] S5. Subculture Management: In a sterile environment, use a peptide nucleic acid probe labeled with the telomere-targeting fluorescent dye Cy3 to stain hyphae. Quantitative analysis of telomere fluorescence intensity using a microscopic imaging system allows for indirect selection of new hyphae with ≤8 divisions for transfer. Sterilize inoculation tools by burning and cool them before use to avoid contamination.

[0032] S6. Vitality detection and improvement: Regular testing, SSR molecular marker analysis of 5 highly polymorphic sites (variation rate ≤ 0.8%), methylation-sensitive amplified polymorphism technology showed a DNA methylation level variation rate of 0.3%, and no warning was triggered; mushroom fruiting test: inoculated with sawdust culture medium, cultured at 25°C for 30 days, buds appeared in 7 days, the fruiting body morphology was complete, and the biological efficiency reached 85%.

[0033] S7. For insufficient activity: This did not occur in this example, but the contingency plan includes adding 2 g / L yeast extract and B vitamins, or using "gradient temperature increase + nutrient pulse" (raising the temperature at 2°C / hour to 5°C and adding 1% glucose).

[0034] After four months of storage using the above steps, mycelial viability remained strong (growth rate decreased by only 20%), genetic stability was high (SSR mutation rate was 0.8%), and fruiting performance met standards. This method effectively overcomes the low-temperature sensitivity of '091'.

[0035] Example 2: Comprehensive application of the high temperature adaptable shiitake mushroom variety 'High Temperature No. 5' S1. Initial strain screening: Single-spore isolated virus-free strains were used. The screening criteria were the same as S1, but additional attention was paid to the performance of the mycelium in a high-humidity environment (climbing ability was assessed as "extremely strong").

[0036] S2. Low-temperature acclimation: Data collection revealed a wide threshold range (2-10°C). Repeat the pulse temperature at 8°C six times. After adding 0.1% trehalose, cool the mixture to 8°C (target acclimation temperature). Screen for single colonies with a growth rate decrease of ≤20%.

[0037] S3. Culture medium preparation: Use a modified culture medium consisting of a 1:1 wood flour to vermiculite powder volume ratio (70% total volume), 20g glucose, 2g yeast extract, 250g potato, 18g agar, and 1000g sterile water. Freeze-dry the wood flour to form 20-40 mesh porous microspheres. Sieve the vermiculite powder through an 80-mesh sieve. Embed the microspheres with gelatin sustained-release nutrient granules containing yeast extract and B vitamins.

[0038] Strain Preservation: For medium-term preservation (1-2 years), use a paraffin-nanocoating composite for protection. Spray a 0.5% nano-chitosan film on the mycelial surface and pretreat with liquid paraffin (500 mL of paraffin plus 10 g of anhydrous calcium chloride, sterilize at 121°C for 2 hours to a pH of 5.8). After cooling, collect the clarified liquid to cover a 1.5 cm layer of mycelial layer and store at 4°C. Check the paraffin turbidity every 12 months and transfer if necessary. Preserve under nitrogen stress. Take mycelial cells in the logarithmic growth phase and add PDA liquid medium containing 10% glycerol. Cool the mixture in a gradient manner (-20°C for 2 hours → -80°C for 12 hours → liquid nitrogen at -196°C). Add 50 μM cAMP as a recovery signal molecule.

[0039] S5. Subculture management: In a sterile environment, use telomere-targeted fluorescent dye Cy3-labeled peptide nucleic acid probe to stain hyphae cells, quantitatively analyze telomere fluorescence intensity using a microscopic imaging system, and select new hyphae with ≤6 divisions to reduce the risk of genetic variation.

[0040] S6. Viability detection and improvement: SSR detection of 8 sites, a mutation rate of 1.5% triggered an early warning (exceeding the 1% threshold), and methylation analysis showed a mutation rate of 0.6%; Improvement measures: an additional 2g / L yeast extract was added to the culture medium; a "gradient temperature increase + nutrient pulse" strategy was adopted during recovery (raising the temperature to 5°C at 2°C / hour and adding 1% glucose).

[0041] Through the above steps, the SSR mutation rate was reduced to 0.9% (≤1%), the MSAP mutation rate was reduced to 0.4% (≤0.5%), the budding time of the mushroom test was shortened to 8 days, the fruiting body abnormality rate was reduced to 3%, and the biological efficiency was restored to 80%.

[0042] Throughout this specification, references to terms such as "one embodiment," "example," and "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0043] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the content of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for maintaining the vitality of edible mushroom shiitake mushroom strains, characterized in that: The following steps are involved: S1. Initial strain screening: Select virus-free strains, which are purified strains obtained by tissue separation or single spore isolation, and have white, dense, and uniform mycelium, without foreign bacteria and pigment secretion; S2. Adaptively low-temperature acclimation was performed on the virus-free strain obtained in S1. Four temperature gradients of 0°C, 3°C, 5°C, and 8°C were set in a constant temperature incubator. The expansion of the colony diameter was recorded, the growth rate was calculated, and the temperature boundary of "growth rate at low temperature ≥ 50% of the normal temperature rate" was screened to generate a personalized "low-temperature sensitive threshold interval"; based on the sensitive threshold interval, 5-8 pulse treatments were performed at the upper temperature of the threshold interval, with each pulse lasting 2 hours and an interval of 24 hours; after the pulse stimulation, antifreeze protective agents were added in stages, 0.05% trehalose was added to the culture medium before the first pulse, and 0.05% trehalose was added after the last pulse; after the enhanced activation effect, the temperature was lowered from room temperature to the upper temperature of the threshold interval on days 1-3, and gradually lowered to the lower temperature on days 4-7. The strains after adaptive low-temperature acclimation were inoculated into PDA culture medium, and the mycelial growth rate and colony diameter were measured daily to screen single colonies with a "growth rate decrease of ≤30% at low temperature"; S3. Culture medium preparation: using an edible fungus modified culture medium, the modified culture medium is a mixed culture medium of wood powder, vermiculite powder and potato dextrose agar, and embedded with slow-release nutrient particles, the slow-release nutrient particles are gelatin microspheres embedded with yeast extract; S4. Preservation of strains: Select the preservation method according to the preservation period. For short-term preservation, use edible strain modified culture medium at 4-6°C for 3-6 months. For medium-term preservation, use paraffin-nano coating composite protection. For long-term preservation, use low temperature 1.5°C dormancy awakening mechanism and nitrogen adversity preservation. S5. Subculture management: In a sterile environment, use a peptide nucleic acid probe labeled with the telomere-targeted fluorescent dye Cy3 to stain hyphae. Quantitative analysis of telomere fluorescence intensity using a microscopic imaging system indirectly assesses the number of divisions. Only new hyphae with a division number ≤ 10 are selected for transfer. For transfer, select new hyphae 0.5–2 cm from the bevel edge. S6. Regular vitality testing: The genetic stability of the strain is tested by SSR molecular markers, and 5-8 highly polymorphic sites are tested. The mutation rate of polymorphic sites is ≤1%, which is considered normal vitality. On this basis, methylation-sensitive amplified polymorphism technology is introduced to detect the DNA methylation level of the mycelial genome. When the mutation rate exceeds 0.5%, an early warning is issued.

2. The method according to claim 1, characterized in that In step S2, the growth rate is calculated as follows: growth rate = (colony diameter on day 7 - colony diameter on day 3) / 4 days. The number of pulses is dynamically adjusted according to the strain tolerance. If the mycelium shrinks or secretes pigment after the pulse, the number of pulses is reduced to 5; if the tolerance is good, the number of pulses is increased to 8.

3. The method according to claim 1, characterized in that In step S2, the cryoprotectant replacement scheme is to replace 0.1% trehalose with 0.2% proline, that is, 0.1% proline is added before the first pulse and after the last pulse respectively.

4. The method according to claim 1, wherein In step S3, the volume ratio of the total volume of the vermiculite powder to the potato dextrose agar medium is (1-5):1, the volume ratio of the wood powder to the vermiculite powder is 1:1, and the wood powder with a "porous microsphere structure" having a particle size of 20-40 mesh is obtained by low-temperature freeze-drying technology pretreatment, and the vermiculite powder particle size is passed through an 80-mesh sieve; the potato dextrose agar mixed medium has the following formula: 20g of glucose, 2g of yeast extract, 250g of potatoes, and 18g of agar are added to every 1000g of sterile water; the low-temperature freeze-drying technology pretreatment is first rapid freezing at -20°C to -40°C, and then increasing the temperature and pressure under vacuum conditions to allow the ice to sublime and dry.

5. The method according to claim 1, wherein In step S4, the paraffin-nanocoating composite protection and preservation operation includes: forming a layer of nano-scale chitosan film with a concentration of 0.5% on the surface of the mycelium, pouring the pretreated liquid paraffin onto the slope covered with mycelium to cover the mycelium layer by 1 to 2 cm, and storing at 4°C; the liquid paraffin pretreatment is: 500 mL of paraffin plus 10 g of filtered anhydrous calcium chloride, sterilizing at 121°C for 2 hours, the pH value after sterilization is in the range of natural pH or 5.5-6.5, and taking the upper clear liquid after cooling.

6. The method according to claim 1, characterized in that In step S4, the operation of the low-temperature 1.5°C dormancy awakening mechanism is as follows: before storage under nitrogen stress, a "resuscitation signal molecule", i.e., 50 μM cAMP, is added to the bacterial suspension; during resuscitation, a "gradient temperature increase + nutrient pulse" is used, first raising the temperature to 5°C at 2°C / hour, and then adding 1% glucose solution.

7. The method according to claim 1, characterized in that In step S4, the nitrogen stress preservation operation includes: taking mycelium in the logarithmic growth phase, adding PDA liquid culture medium containing 10% glycerol, packaging into cryo tubes, and gradually cooling, that is, maintaining at -20°C for 2 hours → maintaining at -80°C for 12 hours → liquid nitrogen, and storing in liquid nitrogen at -196°C.

8. The method according to claim 1, characterized in that In step S5, the sterile environment is a clean bench, and the inoculation tools are sterilized by burning and cooled before use.

9. The method according to claim 1, characterized in that For edible fungi with insufficient activity, the following improvements were taken based on the S1-S6 procedures to enhance their activity: 2 g / L yeast extract and an appropriate amount of B vitamins were added to the modified culture medium; a "gradient temperature increase + nutrient pulse" strategy was adopted in the initial recovery stage of subculture management, first raising the temperature to 5°C at 2°C / hour, and then adding 1% glucose solution to stimulate growth.

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