Liquid-solid two-phase coupling culture method of coriolus versicolor and application thereof to rapid identification of introduction of coriolus versicolor

Abstract

The present application relates to edible mushroom culture identification technical field, specifically a liquid-solid dual-phase coupling culture method of Coriolus hirsutus and application thereof in rapidly identifying correct introduction of Coriolus hirsutus.The method of the present application constructs a liquid-solid dual-phase coupling culture system, combines with sugar agar layer regulation, low-temperature stimulation, etc., realizes rapid determination of Coriolus hirsutus strain characteristics based on the obtained fruiting body, avoids introduction confusion phenomenon, and can also judge strain activity through the fruiting body state, significantly improves the efficiency and benefit of cultivation production and other links.

Description

A liquid-solid two-phase coupled culture method for *Aureobasidium sarmentosum* and its application in rapidly determining the correctness of *Aureobasidium sarmentosum* introduction. Technical Field

[0001] This invention relates to the field of edible fungi cultivation and identification technology, specifically a liquid-solid two-phase coupled culture method for *Aspergillus oryzae* and its application in rapidly determining the correctness of *Aspergillus oryzae* introduction. Background Technology

[0002] Mushroom (Lentinula edodes) belongs to the phylum Basidiomycota, class Agaricales, order Agaricales, and genus Lentinus. It is a rare wild edible fungus. Its unique feature is that it can grow on the dead branches of the Mushroom shrub, which contains toxins, hence its name.

[0003] In the consumer market, *Mucuna spp.* (also known as Masang mushroom) with specific characteristics is highly sought after. Products with small stems, dense and tender gills, thin caps that open quickly, and a delicate, elegant aroma often enjoy higher market acceptance and economic benefits. However, on the production side, due to significant differences in the professional theoretical level and practical experience of producers, as well as multiple introductions and the dispersed production of different strains, the strains held by different producers vary greatly. Cases of the same name for different species and the same species having different names frequently occur, and some producers are even unclear about the varietal name and basic characteristics of the strains. Relying solely on the appearance and growth state of the mycelium obtained at the time of introduction makes it difficult to accurately predict whether the cultivated fruiting bodies will meet the characteristics preferred by the market. If the introduced strain has factors such as degenerated vitality or incorrect varietal characteristics, it will directly lead to poor cultivation efficiency and other problems.

[0004] Traditional methods for further clarifying the characteristics of introduced varieties through cultivation trials are cumbersome and time-consuming, typically taking about 180 days from spawn preparation to mushroom cultivation. If a batch of mushroom logs is produced using traditional methods to determine whether the introduced variety or the planned *Mallotus simonii* strain is of high quality and meets market demand, the time commitment and the cost of various consumables are extremely high. This not only severely delays production plans but also increases production costs and market supply uncertainty.

[0005] Existing technology CN 113455289 A discloses a method for rapidly detecting the fruiting performance of edible fungi tissue-isolated strains. This method involves inoculating shiitake mushroom spawn into large test tubes containing potato dextrose agar (PDA) medium and providing appropriate growth temperature and light stimulation. After fruiting buds differentiate, the fruiting performance of the strain is determined by the number and uniformity of the buds. However, this technology has significant limitations in its application to *Agaricus simulans*. First, the culture medium used in this method differs significantly in physicochemical properties from the lignocellulosic matrix used in actual production, resulting in insufficient ecological simulation. The differentiation characteristics of the fruiting buds formed in the test tubes cannot accurately map to the development patterns of fruiting bodies in actual production. Second, the cycle compression is insufficient: although the mycelial culture cycle is shortened, a complete fruiting evaluation still requires a long waiting period. These uncertainties severely affect the prediction results and cannot effectively guide the cultivation and introduction of *Agaricus simulans*.

[0006] Existing technology CN 110423794 B discloses a method for rapidly predicting the fruiting ability of shiitake mushroom strains. This method uses a chromogenic matrix containing guaiacol, inoculates pre-cultured shiitake mushroom mycelia onto it, and after an enzymatic reaction, uses spectrophotometry to read the OD value of the chromogenic matrix to detect the continuous changes in enzyme activity of a large number of strains to determine the fruiting ability of shiitake mushrooms. However, this technique uses a 465nm wavelength for detection, essentially capturing the colorimetric reaction of guaiacol to tetra-o-methoxybiphenylquinone (λmax=465nm) under PPO / POD catalysis. This wavelength is only sensitive to copper-containing oxidase (PPO) and heme peroxidase (POD), but six characteristic enzymes involved in the critical period of fruiting body formation of *Mycorrhiza rubra*, including β-glucosidase (whose activity peak lags behind PPO by several days) and chitinase (which is directly related to mycelial entanglement), were not included in the detection system. Furthermore, there is a long time window difference between the peak enzyme activity period (rapid mycelial growth) and the critical period for regulating fruiting performance (primordium differentiation), indicating a significant problem of incomplete enzyme system coverage. This results in unstable predictive ability and makes it impossible to observe the characteristics of the final product, i.e., the fruiting body, and also fails to guide the introduction and cultivation of this fungus.

[0007] Therefore, it is urgent to develop a method that can quickly and accurately determine the characteristics of *Mallotus simonii* varieties, avoid incorrect introductions, identify strain viability, significantly shorten the cultivation cycle, and improve production efficiency and product quality. This is of vital importance to promoting the sustainable development of the *Mallotus simonii* industry. Summary of the Invention

[0008] To address the aforementioned technical problems in the existing technology, this invention provides a liquid-solid two-phase coupled culture method for *Aureobasidium sarmentosum* and its application in rapidly determining the correctness of *Aureobasidium sarmentosum* introduction, as detailed below:

[0009] A method for liquid-solid two-phase coupled culture of *Aureobasidium monnieri* includes the following steps:

[0010] (1) Liquid fermentation:

[0011] Prepare the liquid fermentation broth, dispense 50-200 mL / bottle into tissue culture flasks, and autoclave at 115-126°C for 5-60 min. After sterilization, inoculate 1-50 pieces with a diameter of [missing information] in a clean environment. 3-10 mm mycelial blocks were transferred to a constant temperature shaker and fermented at 10-30℃ and 60-200 rpm / min for 5-20 days.

[0012] (2) Solid matrix pretreatment:

[0013] Bamboo shavings and wood shavings were selected as solid substrates. The wood shavings were rinsed clean with water, and the bamboo shavings were cut into small pieces and rinsed clean after removing large pieces. The bamboo and wood shavings were boiled in water of about 2 to 10 times their volume for 5 to 60 minutes, and this process was repeated 1 to 10 times. After draining, the shavings were spread out and dried in a forced-air drying oven at 45 to 105°C for 4 to 36 hours. Then, they were placed in containers and placed in a high-pressure steam sterilizer for moist heat sterilization at 121 to 126°C for 15 to 180 minutes.

[0014] (3) Addition of solid matrix:

[0015] Remove the fermented tissue culture bottle from the shaker and add a mixture of 5-50% bamboo chips and 50-95% sawdust to the clean bench. Stir the fermented mycelial balls, sawdust, and bamboo chips thoroughly.

[0016] (4) Mycelium growth culture:

[0017] After mixing the bamboo and wood chips, let them stand for 5-150 minutes to fully absorb the fermentation liquid. Then, pour off the excess liquid. While ensuring ventilation, slightly flatten and compact the bamboo and wood chips, seal the culture bottle, and transfer it to a constant temperature incubator at 10-30℃ to allow the bacteria to grow in the dark for 4-30 days.

[0018] (5) Lay the sugar agar layer:

[0019] Prepare sugar agar solution, sterilize by moist heat at 115~126℃ for 10~60 min, remove and place in a clean bench, cool to 30~55℃, pour a 0.5~5 mm thick sugar agar layer onto the surface of the substrate inside the tissue culture bottle after mycelial growth is complete, and after solidification, prick holes with an inoculation needle to increase substrate aeration, return to 10~30℃ under dark conditions and culture for 5~10 days until the surface of the sugar agar layer is covered with mycelium;

[0020] (6) Post-ripening culture and mycelial film formation:

[0021] Place tissue culture flasks with sugar agar poured onto the surface of the culture substrate in a constant temperature incubator at 10-30℃ for 7-20 days (do not culture in complete darkness) to promote rapid mycelial growth. When nodule-like structures are visible to the naked eye, the after-ripening is complete.

[0022] (7) Low temperature stimulation:

[0023] Place the tissue culture flasks at 2-10℃ for 4-8 days to complete the low-temperature stimulation treatment;

[0024] (8) Inducing mushroom growth:

[0025] The transferred tissue culture bottles were placed in a low-light environment at room temperature. Sterile instruments were used to make holes in the sugar agar layer, and a small amount of sterile water was sprayed to induce rapid differentiation of primordia. After the fruiting body primordia appeared, the caps were slightly opened to increase ventilation and the plants were allowed to grow naturally for 5-15 days. The phenotype of the fruiting bodies was determined by judging the characteristics of the grown fruiting bodies (e.g., whether the cap has warts, the color of the stipe, the thickness and length of the club, etc.).

[0026] Further, in step (1), the liquid fermentation broth formula (g / L) is as follows: potato extract 1~25 g, glucose 3~30 g, maltose 3~30 g, peptone 0.5~10 g, corn extract 1~15 g, magnesium sulfate 0.5~10 g, potassium dihydrogen phosphate 0.5~10 g, vitamin B1 5~400 mg, and agar 0.5~12 g.

[0027] Furthermore, in step (2), the wood chips are mixed wood chips, but they must not contain oily substances, such as pine wood.

[0028] Furthermore, in step (2), bamboo shavings are waste products from bamboo product processing. Regardless of the type of bamboo, adding bamboo shavings is to ensure the air permeability and overall water retention of the cultivation substrate.

[0029] Furthermore, in step (5), the sugar agar formulation (g / L) is: glucose 5~15 g, sucrose 5~15 g, and agar 8~25 g.

[0030] Furthermore, in step (6), while the mycelium on the sugar agar layer on the surface of the cultivation substrate is observed to grow densely during the after-ripening growth, the cap of the tissue culture bottle is slightly opened to increase ventilation for 2-10 days, so that the sugar agar layer loses water quickly, forms a protective layer, and induces the rapid appearance of the mycelial film.

[0031] Furthermore, in step (8), when inducing the natural growth of mushrooms, it is necessary to maintain a temperature of 10~28℃ and a relative humidity of 60~95%.

[0032] Furthermore, the maximum ambient temperature in step (8) shall not exceed 35°C.

[0033] Furthermore, the drilling depth in step (8) is 1~3 cm.

[0034] The aforementioned liquid-solid two-phase coupling culture method for *Amanita muscaria* constructs a liquid-solid two-phase coupling culture system, combined with sugar agar layer regulation and low temperature stimulation, to achieve rapid determination of the characteristics of *Amanita muscaria* strains based on the obtained fruiting bodies, avoiding the phenomenon of incorrect introduction. Furthermore, the state of the fruiting bodies can also be used to determine the viability of the strain, significantly improving the efficiency and benefits of cultivation and production processes.

[0035] Compared with the prior art, the technical effects of this invention are reflected in:

[0036] (1) In the liquid fermentation of Mycorrhiza rubra, the parameters such as the inoculum amount and the number of culture rotations selected in this invention can promote the fermentation of small and dense mycelial balls when sufficient centrifugal force and dissolved oxygen are provided, which greatly increases the mycelial biomass and effective contact area (contact area between the strain and the substrate), thereby shortening the inoculum growth cycle.

[0037] (2) This invention combines liquid fermentation with solid-state culture to form a novel Mycorrhiza rubra culture system. By rapidly proliferating mycelium during the liquid fermentation stage and then adding solid bamboo and wood chips, the nutrient source is ensured while simulating actual cultivation parameters, allowing the mycelium to reach physiological maturity in a short time. This dual-phase coupled culture method solves the bottleneck of long cultivation cycle and low efficiency in traditional cultivation.

[0038] (3) In this invention, a sugar agar layer is laid, which adjusts the carbon-nitrogen ratio of the surface matrix during post-ripening, which is beneficial to the differentiation of fruiting bodies. In addition, the drying and dehydration of the agar layer also promotes the formation of the bacterial film, and the dehydrated sugar agar layer also forms a physical defense barrier against contamination by miscellaneous bacteria.

[0039] (4) The present invention subjected the mycelium and post-ripening of the mycelium to low-temperature stimulation treatment, which further ensured the differentiation of the fruiting bodies.

[0040] (5) This invention constructs a liquid-solid dual-phase coupling culture system, combined with sugar agar layer regulation, low temperature stimulation, etc., to achieve rapid determination of the characteristics of the *Amanita muscaria* strain based on the obtained fruiting body, avoiding the phenomenon of incorrect introduction, and the viability of the strain can also be judged by the state of the fruiting body, which significantly improves the efficiency and benefits of cultivation and production.

[0041] (6) Traditional cultivation requires about 180 days to complete the fruiting verification of the strain, while the present invention optimizes the cultivation process and only requires about 60±10 days to complete the determination of fruiting body characteristics, shortening the cycle by about 66.67% and increasing efficiency by about 200%, effectively solving the problems of time-consuming and delayed production planning in traditional cultivation methods.

[0042] (7) Liquid-solid coupling culture reduces the material input for the production of mushroom sticks and large-scale cultivation; at the same time, candidate strains are accurately obtained through fruiting body characteristics and phenotypes, avoiding ineffective cultivation caused by introduction deviation, strain degeneration or inconsistency of characteristics, and significantly reducing costs.

[0043] (8) Liquid fermentation promotes rapid accumulation of mycelial biomass through high dissolved oxygen and high rotation speed liquid propagation, laying the foundation for subsequent solid-state culture. In addition, the efficiency is greatly improved by the integrated innovation of technologies such as the adjustment of carbon-nitrogen ratio on the substrate surface by sugar agar layer, the application of physical antibacterial barriers, and low temperature stimulation. Attached Figure Description

[0044] Figure 1 shows the rinsed and dried solid matrix: wood chips (left) and bamboo chips (right).

[0045] Figure 2 shows the liquid fermentation state of strain Le.Msy-01 of *Trichoderma equisetifolia*.

[0046] Figure 3 shows the feeding status of strain Le.Msy-01 of *Trichoderma equinophorum*.

[0047] Figure 4 shows the state of the surface mycelium during post-ripening growth.

[0048] Figure 5 shows the state of primordia differentiation in tissue culture bottles (red arrow: effectively differentiated nodules that can continue to grow and develop into fruiting bodies; blue arrow: ineffectively differentiated nodules that cannot grow and develop into fruiting bodies; yellow arrow: surface hyphae gradually change color to brown film).

[0049] Figure 6 shows the fruiting body of the Le.Msy-01 strain of Mycobacterium equisetifolium.

[0050] Figure 7 shows the fruiting bodies of *Mycorrhiza rubra* obtained from wild purification.

[0051] Figure 8 shows the fruiting bodies of *Amanita muscaria* purified by the college. Detailed Implementation

[0052] The technical solution of the present invention will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description made.

[0053] Example 1: The strain Le.Msy-01 was used as the test strain. This strain was a wild species collected and domesticated in the field by Guizhou Biotechnology Research and Development Base Co., Ltd., and its accession number at China Center for Type Culture Collection is CCTCC NO: M20242211.

[0054] 1. Liquid fermentation

[0055] Prepare the liquid fermentation broth: Weigh out 8 g potato extract powder, 15 g glucose, 5 g maltose, 5 g peptone, 2 g corn extract powder, 3 g magnesium sulfate, 0.5 g potassium dihydrogen phosphate, 40 mg vitamin B1, and 5 g agar, and dissolve them in pure water to a final volume of 1000 mL.

[0056] Dispense 100 mL / bottle into glass culture bottles measuring 5.6 × 7.3 × 11.5 cm (mouth diameter × breast diameter × height), and autoclave at 121°C for 15 min.

[0057] Transfer the sterilized tissue culture bottles to a clean bench and irradiate with ultraviolet light for 30 minutes. Use a hole punch to cut out a diameter... Thirteen pieces of fresh Le.Msy-01 solid inoculum, each 4 mm in size, grown on PDA medium, were inoculated into tissue culture flasks and fermented in a constant temperature shaker at 26°C and 135 rpm / min for 15 days.

[0058] 2. Solid matrix pretreatment

[0059] Bamboo and wood chips were rinsed in about 5 times their volume of water to remove dust, then drained. They were then boiled again in about 5 times their volume of water for 30 minutes to remove the filtrate. The boiling process was repeated 3 times. After the bamboo and wood chips were drained, they were spread out and dried in a forced-air drying oven at 105°C for 12 hours (Figure 1).

[0060] The dried bamboo and wood chips are placed into high-temperature resistant containers and sealed. They are then sterilized by high-pressure steam at 121°C for 30 minutes. After sterilization, they are temporarily stored in a clean bench for later use.

[0061] 3. Addition of solid matrix

[0062] Take out the tissue culture bottle (Figure 2) after fermentation in the constant temperature shaker. Add sterilized bamboo shavings (40%) and wood shavings (60%) (i.e., a mixture of 40 g bamboo shavings and 60 g wood shavings) to the ultra-clean workbench until it is level with the fermentation liquid. Stir and mix well, and let stand for 30 min for the bamboo and wood shavings to absorb the fermentation liquid.

[0063] 4. Mycelium culture

[0064] Further, pour off any excess fermentation liquid that has not been absorbed by the bamboo and wood chips, and slightly compact the mixture to ensure that the bamboo and wood chips have suitable pore space for mycelial growth and development. After sealing the tissue culture bottle, transfer it to a 24°C constant temperature incubator and let it grow in the dark for 8 days (Figure 3). Observe that the mycelial absorption of the substrate is good before proceeding to the next step.

[0065] 5. Lay a layer of sugar agar.

[0066] Further, the tissue culture flasks that have been incubated for 8 days are placed in a clean bench. After cleaning the mycelium growing on the inner wall, the flasks are compacted slightly again, and a thin layer of sugar agar about 3 mm thick is poured in.

[0067] The sugar agar formula used above (g / L) is: 7.5 g glucose, 7.5 g sucrose, and 15 g agar. The prepared sugar agar should be sterilized by moist heat at 115℃ for 15 min. After sterilization, cool it until it is no longer hot to the touch before use.

[0068] 6. Post-ripening growth and film formation

[0069] Further, the tissue culture bottles covered with a sugar agar layer on the bamboo and wood chip substrate were placed back into a 24°C incubator for continued cultivation. After 10 days, the mycelium grew to an extremely dense size (Figure 4). The caps of the tissue culture bottles were slightly opened for 3 days to allow the sugar agar layer to rapidly lose water. The next step was then performed.

[0070] 7. Low temperature stimulation

[0071] Furthermore, the tissue culture bottles covered with mycelium were placed at 4°C for 5 days for low-temperature stimulation culture.

[0072] 8. Fruiting induction

[0073] Furthermore, the tissue culture bottles subjected to low-temperature stimulation were transferred to room temperature and placed in a location away from direct sunlight. The top cap was slightly unscrewed, and a hole about 1.5 cm deep was made in the sugar agar layer using a sterile pipette tip. A small amount of sterile water was sprayed on the hole, and after 3 days of natural growth, a large number of fruiting body primordia appeared on the sugar agar layer.

[0074] Furthermore, the cells were transferred to a temperature of 20°C and a relative humidity of 90% for further cultivation (Figure 5).

[0075] Mature fruiting bodies can be obtained in about 53.0±2.7 days of the entire growth and development cycle. The characteristics and phenotypes of the fruiting bodies are then observed and described (Table 1, Figure 6).

[0076] Table 1. Observation of fruiting body characteristics and phenotype of *M. malaisei* strain Le.Msy-01

[0077]

[0078] Example 2: The experiment was conducted using a strain of *Aureobasidium monnieri* collected in the wild. Therefore, the strain was purified before liquid fermentation to simulate rapid verification after field collection.

[0079] 1. Purification of wild-type strains

[0080] Fruiting bodies of *Cinnamomum camphora* were collected from dead branches of *Cinnamomum camphora* trees in the wild. Under aseptic conditions, the mycelium was separated using tissue isolation and placed on potato dextrose agar (PDA) medium. The culture was maintained at 18°C ​​for 3 days to restore the physiological activity of the mycelium within the mycelium. The mycelium appearing around the mycelium blocks on the plates was then transferred to fresh PDA medium and purified at 23°C for 6 days. This process was repeated twice before proceeding to the next step.

[0081] 2. Liquid fermentation

[0082] Weigh out 5 g potato extract powder, 10 g glucose, 10 g maltose, 3 g peptone, 3 g corn extract powder, 5 g magnesium sulfate, 1.5 g potassium dihydrogen phosphate, 100 mg vitamin B1, and 8 g agar. Dissolve and bring the volume to 1000 mL to prepare the fermentation broth. Dispense 80 mL portions into tissue culture flasks and sterilize at 121℃ for 30 min before use.

[0083] Furthermore, the aseptic excavation diameter =Inoculate 5 pieces of 10 mm inoculum and ferment at 25℃ and 100 rpm / min for 10 days.

[0084] 3. Solid matrix pretreatment

[0085] Rinse bamboo and wood chips separately in about twice their volume of water to remove dust and drain. Boil them in about twice their volume of water for 10 minutes and remove the filtrate. Repeat this process twice. Dry at 80℃ for 10 hours and sterilize in a sealed container at 126℃ for 15 minutes before use.

[0086] 4. Addition of solid matrix

[0087] Furthermore, add bamboo shavings (50%) and wood shavings (50%) to the ultra-clean environment and mix well until they are level with the fermentation liquid. After stirring and mixing, let it stand for 60 minutes for the bamboo and wood shavings to absorb the fermentation liquid.

[0088] 5. Mycelium culture

[0089] Further, pour off the excess fermentation liquid and compact it slightly. After sealing, transfer it to a 23°C incubator for 10 days to allow the mycelium to grow properly. Once the mycelium is observed to be consuming the substrate well, proceed to the next step.

[0090] 6. Lay a layer of sugar agar.

[0091] Furthermore, in the ultra-clean environment, the culture medium, after 10 days of incubation, is slightly compacted again, and a layer of sugar agar about 5 mm thick is poured in.

[0092] The above-mentioned sugar agar formula (g / L) is: 10 g glucose, 10 g sucrose, 20 g agar. Sterilize at 121℃ for 30 min and cool until it is not hot to the touch before use.

[0093] 7. Post-ripening growth and film formation

[0094] Furthermore, return the bottle to 23°C and continue culturing for 10 days. Once the mycelium has grown to an extremely dense size, slightly open the bottle cap for 5 days to allow the sugar agar layer to rapidly lose water before proceeding to the next step of processing.

[0095] 8. Low temperature stimulation

[0096] Furthermore, it was refrigerated at 4°C for 7 days to induce low-temperature stimulation.

[0097] 9. Fruiting Induction

[0098] Furthermore, the agar was moved to room temperature and protected from direct sunlight. The lid was slightly opened to allow ventilation. Holes about 1.0 cm deep were drilled in sterile equipment, and a small amount of sterile water was sprayed on. After 3 days of growth, fruiting body primordia appeared on the sugar agar layer.

[0099] Furthermore, the cells were transferred to a temperature of 20°C and a relative humidity of 95% for further cultivation.

[0100] Mature fruiting bodies can be obtained in about 60.0±2.5 days of the entire growth and development cycle. The characteristics and phenotypes of the fruiting bodies are then observed and described (Table 2, Figure 7).

[0101] Table 2. Observation of the cultivation characteristics and phenotypes of wild-type purified *Mallotus spp.* strains.

[0102]

[0103] Example 3: An example was conducted using a purified strain of *Mallotus erythrorhizon* purchased from a third-party institution.

[0104] 1. Liquid fermentation of strains

[0105] Weigh out 5 g potato extract powder, 15 g glucose, 10 g maltose, 3 g peptone, 1 g corn extract powder, 2 g magnesium sulfate, 2 g potassium dihydrogen phosphate, 150 mg vitamin B1, and 1 g agar. Make up to 1000 mL to prepare fermentation broth. Dispense 120 mL / bottle into tissue culture bottles and sterilize at 121℃ for 15 min.

[0106] Sterilization ends in a clean environment with a diameter of [missing information]. Ten pieces of bacterial culture with a diameter of 6 mm were grown on PDA and fermented under shaking conditions at 27°C and 135 rpm / min for 10 days after inoculation.

[0107] 2. Solid matrix pretreatment

[0108] Bamboo and wood chips were rinsed separately in about 8 times their volume of water to remove dust and drain. They were then boiled in about 8 times their volume of water for 30 minutes and the filtrate was removed. This process was repeated once. The chips were dried at 105℃ for 6 hours and then sealed and sterilized at 126℃ for 15 minutes before use.

[0109] 4. Addition of solid matrix

[0110] Further, bamboo shavings (60%) and wood shavings (40%) are added to the ultra-clean environment until they are level with the fermentation liquid. After stirring and mixing, the mixture is left to stand for 15 minutes to allow the bamboo and wood shavings to absorb the fermentation liquid.

[0111] 5. Mycelium culture

[0112] Further, pour off the excess fermentation liquid and compact it slightly. After sealing, transfer it to a 27°C incubator for 14 days to allow the mycelium to grow properly. Once the mycelium is observed to be consuming the substrate well, proceed to the next step.

[0113] 6. Lay a layer of sugar agar.

[0114] Furthermore, in the ultra-clean environment, the culture medium, after 14 days of incubation, is slightly compacted again, and a layer of sugar agar about 2 mm thick is poured in.

[0115] The above-mentioned sugar agar formula (g / L) is: 10 g glucose, 10 g sucrose, 20 g agar. Sterilize at 121℃ for 15 min and cool until it is not hot to the touch before use.

[0116] 7. Post-ripening growth and film formation

[0117] Furthermore, return the bottle to 27°C and continue culturing for 15 days. Once the mycelium has grown to an extremely dense size, slightly open the bottle cap for 5 days to allow the sugar agar layer to rapidly lose water before proceeding to the next step of processing.

[0118] 8. Low temperature stimulation

[0119] Furthermore, it was refrigerated at 4°C for 7 days to induce low-temperature stimulation.

[0120] 9. Fruiting Induction

[0121] Furthermore, transfer to room temperature conditions and avoid direct sunlight, slightly open the lid for ventilation, make holes about 2 cm deep in sterile equipment, spray a small amount of sterile water, and fruiting body primordia appear on the sugar agar layer after 5 days of growth.

[0122] Furthermore, the culture was transferred to a temperature of 20°C and a relative humidity of 85%.

[0123] Mature fruiting bodies can be obtained in about 65.0±2.5 days of the entire growth and development cycle. The characteristics and phenotypes of the fruiting bodies are then observed and described (Table 3, Figure 8).

[0124] Table 3. Observation of the cultivation characteristics and phenotypes of purified strains of *Mallotus spp.*

[0125]

[0126] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. A method for liquid-solid two-phase coupled culture of *Aureobasidium sarmentosum*, characterized in that, The steps include: (1) Liquid fermentation: Prepare liquid fermentation broth, dispense 80-120 mL / bottle into tissue culture bottles, and sterilize by steam sterilization at 115-126℃ for 15-30 min; after sterilization, inoculate 5-13 pieces with a diameter of 1 mm into a clean environment. (1) 4-10 mm bacterial blocks were transferred to a constant temperature shaker and fermented at 25-27℃ and 100-135 rpm / min for 10-15 days; (2) Solid substrate pretreatment: bamboo chips and wood chips were selected as solid substrates; the wood chips were rinsed clean with water, and the bamboo chips were cleaned by removing large pieces of bamboo and cutting them into small pieces; the bamboo and wood chips were boiled in water of about 2-8 times their volume for 10-30 min, and repeated 1-3 times; after draining, they were spread out and dried in a forced-air drying oven at 80-105℃ for 6-12 h; then they were put into containers and placed in a high-pressure steam sterilizer at 121-126℃ for moist heat sterilization for 15-30 h. min; (3) Addition of solid substrate: Take out the fermented tissue culture bottle from the shaker, add a mixture of 40-60% bamboo chips and 40-60% sawdust in the ultra-clean workbench, stir up and down to mix the fermented mycelial balls, sawdust and bamboo chips; (4) Inoculation culture: After mixing the bamboo and sawdust, let it stand for 15-60 min until it has fully absorbed the fermentation liquid, then pour off the excess liquid, and under the premise of ensuring ventilation, slightly flatten and compact the bamboo and sawdust, then seal the tissue culture bottle, transfer it to a constant temperature incubator at 23-27℃ and let it stand in the dark for inoculation, and culture for 8-14 days; (5) Covering the sugar agar layer: Prepare the sugar agar solution, sterilize it with moist heat at 115-121℃ for 15-30 min, take it out and place it in the ultra-clean workbench, cool it to 30-55℃, pour 2-5 (6) Post-ripening culture and mycelial growth: Place the tissue culture bottle with sugar agar layer of mm thickness on the surface of the substrate inside the culture bottle after mycelial growth is completed. After solidification, use an inoculation needle to prick holes to increase substrate permeability; (7) Post-ripening culture and mycelial growth: Place the tissue culture bottle with sugar agar layer poured on the surface of the substrate in a constant temperature incubator of 23~27℃ for 10~15 days to promote rapid mycelial growth; when the appearance of nodules can be observed with the naked eye, the post-ripening is complete; (8) Low temperature stimulation: Place the tissue culture bottle in a 4℃ environment for 5~7 days to complete the low temperature stimulation treatment; (9) Induction of fruiting: Place the transferred tissue culture bottle in a weak light environment at room temperature, use sterile tools to prick holes on the sugar agar layer and spray a small amount of sterile water to induce rapid differentiation of primordia. After the fruiting body primordia appear, slightly open the lid to increase permeability and wait for the fruiting body to grow naturally; the entire growth and development cycle is 50~67.5 days. By judging the characteristics of the grown fruiting body, it is determined whether its phenotype meets the requirements.

2. The liquid-solid two-phase coupled culture method for *Amanita muscaria* according to claim 1, characterized in that, In step (1), the liquid fermentation broth formula (g / L) is as follows: potato extract powder 5~8g, glucose 10~15g, maltose 5~10g, peptone 3~5g, corn extract powder 1~3g, magnesium sulfate 2~5g, potassium dihydrogen phosphate 0.5~2g, vitamin B1 40~150mg, and agar 1~8g.

3. The liquid-solid two-phase coupled culture method for *Amanita muscaria* according to claim 1, characterized in that, In step (2), the wood chips are mixed wood chips, but they must not contain oily substances.

4. The liquid-solid two-phase coupled culture method for *Amanita muscaria* according to claim 1, characterized in that, In step (2), bamboo shavings are waste products from bamboo product processing.

5. The liquid-solid two-phase coupled culture method for *Amanita muscaria* according to claim 1, characterized in that, In step (5), the sugar agar formula (g / L) is: glucose 7.5~10 g, sucrose 7.5~10 g, and agar 15~20 g.

6. The liquid-solid two-phase coupled culture method for *Amanita muscaria* according to claim 1, characterized in that, In step (6), while the mycelium on the sugar agar layer on the surface of the cultivation substrate is observed to grow densely during the post-ripening growth, the cap of the tissue culture bottle is slightly opened to increase ventilation for 3-5 days, so that the sugar agar layer loses water quickly, forms a protective layer, and induces the rapid appearance of the mycelial film.

7. The liquid-solid two-phase coupled culture method for *Amanita muscaria* according to claim 1, characterized in that, In step (8), when inducing the natural growth of mushrooms, it is necessary to maintain a temperature of 20℃ and a relative humidity of 85~95%.

8. The liquid-solid two-phase coupled culture method for *Amanita muscaria* according to claim 1, characterized in that, The maximum ambient temperature in step (8) shall not exceed 35°C.

9. The method for liquid-solid two-phase coupled culture of *Amanita muscaria* according to claim 1, characterized in that, The drilling depth in step (8) is 1~2 cm.

10. The application of the liquid-solid two-phase coupling culture method of *Aureobasidium sarmentosum* according to any one of claims 1 to 9 in rapidly determining the correctness of *Aureobasidium sarmentosum* introduction.

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