Coriaria nepalensis liquid-solid two-phase coupling culture method and application thereof in rapidly judging correct introduction of coriaria nepalensis

Abstract

The invention relates to the technical field of edible mushroom culture and identification, in particular to a coriaria sinica liquid-solid double-phase coupling culture method and application thereof in the aspect of quickly judging the introduction correctness of coriaria sinica. According to the method disclosed by the invention, by constructing a liquid-solid double-phase coupling culture system and combining regulation and control of a sugar agar layer, low-temperature stimulation and the like, rapid judgment on the coriaria sinica strain characters is realized based on the obtained sporocarp, the introduction disorder phenomenon is avoided, the strain activity can be judged through the state of the sporocarp, and the efficiency and benefits of links such as cultivation and production are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of edible mushroom cultivation and identification, and specifically to a method for liquid-solid biphasic coupling cultivation of Coriolus versicolor and its application in rapidly determining the correct introduction of Coriolus versicolor. Background Art

[0002] Coriolus versicolor belongs to the Basidiomycota, Agaricomycetes, Agaricales, and Lentinula genus, and is a rare wild edible mushroom. Its uniqueness lies in its ability to grow on the dead branches of Coriaria nepalensis shrubs containing coriamyrtin throughout the whole plant, hence its name.

[0003] In the market consumption side, Coriolus versicolor with specific traits is more sought after. Coriolus versicolor products with small stipes, dense and tender gills, thin caps that open quickly, and a faint and elegant aroma often receive higher market recognition and economic benefits. However, in the production side, due to the large disparity in the professional theoretical level and practical experience of producers, as well as multiple introductions and decentralized production of various levels of strains, the strains in the hands of different producers vary greatly, and the situations of the same name for different species and different names for the same species often occur. Even some producers are not very clear about the variety names and basic characteristics of the strains. Based solely on the appearance and growth state of the strains (mycelia) obtained during introduction, it is difficult to accurately predict whether the fruiting bodies cultivated from them meet the trait characteristics preferred by the market. If there are various factors such as the degradation of the vitality of the introduced strains and the mismatch of variety traits, it will directly lead to many problems such as poor cultivation efficiency.

[0004] The traditional procedure for further clarifying the characteristics of introduced varieties through cultivation experiments is cumbersome and time-consuming. The entire cycle from strain preparation to mushroom fruiting on the mushroom sticks usually takes about 180 days. If a batch of mushroom sticks are made in the traditional way to judge whether the traits of the introduced variety or the Coriolus versicolor strains planned for production are of high quality and meet the market demand, the time cycle and the cost of various consumables are extremely high. This not only seriously delays the production plan but also increases the production cost and the uncertainty of market supply.

[0005] The prior art CN 113455289A discloses a method for rapidly detecting the fruiting performance of tissue-isolated strains of edible mushrooms. By inoculating Lentinula edodes strains into large test tubes containing potato dextrose agar (PDA) medium and giving appropriate growth temperature and light stimuli, the fruiting performance of the strains is determined by the number and uniformity of the fruiting bodies after differentiation. However, the application of this technology for Coriolus versicolor has significant limitations. First, there are significant physicochemical property differences between the medium used in the method and the main lignocellulose substrate used in actual production, and the ecological simulation degree is insufficient. The fruiting body differentiation characteristics formed in the test tube cannot accurately map to the fruiting body development law in actual production. Second, the cycle compression is not sufficient: although the mycelium culture cycle is shortened, if a complete fruiting evaluation is still required, a long time of waiting is needed. These uncertain factors seriously affect the prediction results and cannot effectively guide the cultivation and introduction of Coriolus versicolor and other work.

[0006] However, the prior art CN 110423794B discloses a method for rapidly predicting the fruiting ability of Lentinula edodes strains. A method uses guaiacol as a chromogenic substrate, inoculates the pre-cultured Lentinula edodes mycelium on it, and after the enzymatic reaction, uses spectrophotometry to read the OD value of the chromogenic substrate to detect the continuous change of the enzyme activity of a large number of strains to determine the fruiting ability of Lentinula edodes. However, this technology uses a 465 nm wavelength for detection, which essentially captures the chromogenic reaction of guaiacol to generate tetramethoxybiphenylquinone (λmax = 465 nm) catalyzed by PPO / POD. This wavelength is only sensitive to copper-containing oxidase (PPO) and heme peroxidase (POD), but six characteristic enzymes such as β-glucosidase (the activity peak lags behind PPO by many days) and chitinase (directly related to mycelial knotting) involved in the critical period of Coriaria nepalensis fruit body formation are not included in the detection system, and there is a long time window difference between the peak period of enzyme activity (rapid mycelial growth) and the critical regulation period of fruiting performance (primordium differentiation), with obvious problems of incomplete enzyme system coverage, unstable prediction ability, and inability to observe the traits of the final product, i.e., the fruit body, and also unable to guide the work of introduction and cultivation.

[0007] Therefore, it is urgent to develop a method that can quickly and accurately determine the variety traits of Coriaria nepalensis, avoid misintroduction, assist in identifying the vitality of strains, greatly shorten the cultivation cycle, and improve production efficiency and product quality, which is of crucial significance for promoting the sustainable development of the Coriaria nepalensis industry. Summary of the Invention

[0008] In order to solve the above technical problems existing in the prior art, the present invention provides a liquid-solid biphasic coupling culture method for Coriaria nepalensis and its application in quickly determining the correct introduction of Coriaria nepalensis, specifically as follows:

[0009] A liquid-solid biphasic coupling culture method for Coriaria nepalensis includes the following steps:

[0010] (1) Liquid fermentation:

[0011] Prepare a liquid fermentation broth, dispense 50 - 200 mL / bottle into tissue culture flasks, and sterilize with a high-pressure steam sterilizer at 115°C - 126°C for 5 - 60 min by moist heat sterilization; after sterilization, inoculate 1 - 50 mycelial blocks with a diameter of φ3 - 10 mm in a super-clean environment, transfer to a constant temperature shaker, and shake and ferment at 10°C - 30°C and 60 - 200 rpm / min for 5 - 20 d;

[0012] (2) Solid matrix pretreatment:

[0013] Select bamboo chips and wood chips as solid substrates; rinse the wood chips clean with water, remove large bamboo pieces from the bamboo chips and cut them into small sections, then rinse clean; boil the bamboo and wood chips separately in about 2 - 10 times the volume of water for 5 - 60 minutes and repeat 1 - 10 times; drain and spread them out, then dry them in a forced air drying oven at 45 - 105 °C for 4 - 36 hours; then put them into containers and place them in a high-pressure steam sterilizer for moist heat sterilization at 121 - 126 °C for 15 - 180 minutes.

[0014] (3) Addition of solid substrate:

[0015] Take out the tissue culture bottles that have completed fermentation from the shaker, add a mixture of 5 - 50% bamboo chips and 50 - 95% wood chips in a laminar flow hood, and stir up and down to mix the fermented mycelial balls, wood chips, and bamboo chips evenly.

[0016] (4) Spawn-running culture:

[0017] After the bamboo and wood chips are mixed evenly, let them stand for 5 - 150 minutes. Wait for them to fully absorb the fermentation broth, then pour out the excess liquid. On the premise of ensuring air permeability, slightly flatten and compact the bamboo and wood chips, then seal the tissue culture bottles, and transfer them to a constant temperature incubator at 10 - 30 °C for dark static spawn-running culture for 4 - 30 days.

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

[0019] Prepare a sugar agar solution, perform moist heat sterilization at 115 - 126 °C for 10 - 60 minutes, take it out and place it in a laminar flow hood, cool it to 30 - 55 °C, pour a 0.5 - 5 mm thick sugar agar layer onto the surface of the internal substrate of the tissue culture bottles that have completed spawn-running. After it solidifies, use an inoculation needle to puncture holes to increase the air permeability of the substrate, and then put it back in the dark at 10 - 30 °C for culturing for 5 - 10 days until the surface of the sugar agar layer is covered with mycelia.

[0020] (6) Ripening culture and promoting the formation of the mycelial membrane:

[0021] Place the tissue culture bottles with the sugar agar poured and spread on the surface of the culture medium in a constant temperature incubator at 10 - 30 °C for culturing for 7 - 20 days (no need for dark culture), to promote the rapid growth of the mycelia. When tumor-like substances can be observed with the naked eye, the ripening is completed.

[0022] (7) Low-temperature stimulation:

[0023] Place the tissue culture bottles in a refrigerator at 2 - 10 °C for 4 - 8 days to complete the low-temperature stimulation treatment.

[0024] (8) Inducing fruiting:

[0025] The transferred tissue culture bottles are placed in a room temperature environment with weak light. Using sterile instruments, holes are punched in the sugar agar layer, and a small amount of sterile water is sprayed to induce rapid differentiation of the primordium. After the appearance of the fruiting body primordium, the lid is slightly opened to increase ventilation for natural growth for 5 - 15 days. By judging the traits of the grown fruiting body (such as whether the cap has warts, the color of the stipe, the thickness and length of the mushroom stick, etc.), it is determined whether its phenotype meets the requirements.

[0026] Further, in the step (1), the liquid fermentation broth formula (g / L) is as follows: potato powder 1 - 25 g, glucose 3 - 30 g, maltose 3 - 30 g, peptone 0.5 - 10 g, corn steep powder 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] Further, in the step (2), the sawdust is miscellaneous sawdust, but it should not contain oily substances, such as pine wood, etc.

[0028] Further, in the step (2), the bamboo chips are waste from bamboo product processing, regardless of the bamboo species. Adding bamboo chips is to ensure the air permeability and overall water retention of the cultivation substrate.

[0029] Further, in the step (5), the sugar agar formula (g / L) is as follows: glucose 5 - 15 g, sucrose 5 - 15 g, and agar 8 - 25 g.

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

[0031] Further, in the step (8), when inducing fruiting for natural growth, the temperature needs to be maintained at 10 - 28 °C and the relative humidity at 60 - 95%.

[0032] Further, the maximum temperature of the room temperature environment in the step (8) does not exceed 35 °C.

[0033] Further, the punching depth in the step (8) is 1 - 3 cm.

[0034] The described method for liquid - solid biphasic coupling culture of Coriaria nepalensis Wall. realizes rapid determination of the traits of Coriaria nepalensis Wall. strains based on the obtained fruiting bodies by constructing a liquid - solid biphasic coupling culture system, combining sugar agar layer regulation, low - temperature stimulation, etc., avoiding the phenomenon of incorrect introduction. Moreover, the viability of the strains can be judged through the state of the fruiting bodies, significantly improving the efficiency and benefits of cultivation production and other links.

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

[0036] (1) When the invention is used for the liquid fermentation of Coriolus versicolor, parameters such as the inoculation amount and the culture rotation speed selected can, when providing sufficient centrifugal force and dissolved oxygen, promote the formation of small and dense mycelial pellets during fermentation, greatly increasing the mycelial biomass and the effective contact area (the contact area between the strain and the substrate), and thus shortening the spawn-running period.

[0037] (2) The invention combines liquid fermentation with solid-state cultivation to form a brand-new Coriolus versicolor cultivation system. By using the liquid fermentation stage to rapidly propagate the mycelium and then adding solid bamboo sawdust, while ensuring the nutrient source, the actual cultivation parameters are simulated, enabling the mycelium to reach the physiologically mature state in a short time. This two-phase coupling cultivation method solves the bottleneck problems of long traditional cultivation cycle and low efficiency.

[0038] (3) In the invention, a sugar agar layer is laid. During after-ripening, the carbon-nitrogen ratio of the surface substrate is adjusted, which is beneficial to the differentiation of fruiting bodies. In addition, the drying and water loss of the agar layer also promote the formation of a mycelial membrane, and the dehydrated sugar agar layer also forms a physical defense barrier against miscellaneous bacteria contamination.

[0039] (4) The invention conducts a low-temperature stimulation treatment on the spawn-run and after-ripened Coriolus versicolor, further ensuring the differentiation of fruiting bodies.

[0040] (5) By constructing a liquid-solid two-phase coupling cultivation system, combining the regulation of the sugar agar layer, low-temperature stimulation, etc., the invention realizes the rapid determination of the traits of Coriolus versicolor strains based on the obtained fruiting bodies, avoids the phenomenon of incorrect introduction, and can also judge the viability of the strain through the state of the fruiting body, significantly improving the efficiency and benefits of cultivation production and other links.

[0041] (6) 1. Traditional cultivation requires about 180 days to complete the fruiting verification of the strain, while the invention can complete the determination of the fruiting body traits in only about 60 ± 10 days by optimizing the cultivation process. The cycle is shortened by about 66.67%, and the efficiency is increased by about 200%, effectively solving the problems of long time consumption and lagging production plan in traditional cultivation methods.

[0042] (7) The liquid-solid coupling cultivation is adopted to reduce the input of consumables for making mushroom sticks and large-scale cultivation; at the same time, by accurately obtaining candidate strains through the fruiting body traits and phenotypes, ineffective cultivation caused by introduction deviation, strain degeneration or inconsistent traits is avoided, and the cost is significantly reduced.

[0043] (8) Liquid fermentation promotes the rapid accumulation of mycelial biomass through liquid propagation with high dissolved oxygen and high rotation speed, laying a foundation for subsequent solid-state cultivation. In addition, based on the integrated innovation of the regulation of the carbon-nitrogen ratio of the substrate surface layer by the sugar agar layer, the application of the physical antibacterial barrier and low-temperature stimulation, the efficiency is greatly improved. Description of the Drawings

[0044] Figure 1Solid matrices that have been rinsed and dried, wood chips (left), bamboo chips (right).

[0045] Figure 2 It is the liquid fermentation state of the Corybas mycobacterium Le.Msy-01 strain.

[0046] Figure 3 It is the feeding state of the Corybas mycobacterium Le.Msy-01 strain.

[0047] Figure 4 It is the state of the surface mycelium during after-ripening growth.

[0048] Figure 5 It is the state during primordium differentiation in the tissue culture bottle (red arrow: effective differentiated tumor-like structures that can continue to grow and develop into fruiting bodies; blue arrow: ineffective differentiated tumor-like structures that cannot grow and develop into fruiting bodies; yellow arrow: the surface mycelium gradually turns into a brown mycelial membrane).

[0049] Figure 6 It is the fruiting body of the Corybas mycobacterium Le.Msy-01 strain.

[0050] Figure 7 It is the fruiting body of the Corybas mycobacterium obtained by wild purification.

[0051] Figure 8 It is the fruiting body of the Corybas mycobacterium purified by the institution. Detailed implementation methods

[0052] The following combines specific implementation methods to further limit the technical solutions of the present invention, but the scope of protection required is not limited to the description made.

[0053] Example 1: Using the Corybas mycobacterium Le.Msy-01 as the test strain, this strain is a wild species collected and domesticated in the wild by Guizhou Biotechnology Research and Development Base Co., Ltd., and the preservation number in the China Center for Type Culture Collection is CCTCC NO: M20242211.

[0054] 1. Liquid fermentation

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

[0056] Dispense 100 mL per bottle into glass tissue culture bottles with a specification of 5.6×7.3×11.5 cm (mouth diameter × chest diameter × height), and sterilize in a high-pressure steam sterilizer at 121 °C for 15 minutes.

[0057] Transfer the tissue culture bottles after sterilization to the laminar flow hood and irradiate them with ultraviolet light for 30 min. Use a puncher to dig out 13 pieces of fresh solid Le.Msy-01 Coriaria nepalensis Wall. strains with a diameter of φ = 4 mm growing on PDA medium, inoculate them into the tissue culture bottles, and place them in a constant temperature shaker for shaking fermentation at 26 °C and 135 rpm for 15 d.

[0058] 2. Pretreatment of solid substrate

[0059] Add bamboo chips and sawdust to about 5 times their volume of water respectively, rinse to remove dust, then drain. Boil them again with about 5 times their volume of water for 30 min and remove the filtrate. Repeat the boiling treatment 3 times. After filtering, spread out the drained bamboo chips and sawdust and dry them in a forced air drying oven at 105 °C for 12 h ( Figure 1 ).

[0060] Put the dried bamboo chips and sawdust into heat-resistant containers respectively, seal them, sterilize them with high-pressure steam at 121 °C for 30 min, and store them temporarily in the laminar flow hood for use after sterilization.

[0061] 3. Addition of solid substrate

[0062] Take out the tissue culture bottles after fermentation culture in the constant temperature shaker ( Figure 2 ), add sterilized bamboo chips (40%) and sawdust (60%) (i.e., a mixture of 40 g of bamboo chips and 60 g of sawdust) in the laminar flow hood until it is flush with the fermentation broth. Stir and mix evenly, then let it stand for 30 min for the bamboo chips and sawdust to absorb the fermentation broth.

[0063] 4. Spawn running culture

[0064] Furthermore, pour out the excess fermentation broth that has not been absorbed by the bamboo chips and sawdust, slightly compact it to ensure that there is appropriate pore space for the mycelium to grow and develop. After sealing the tissue culture bottles, transfer them to a constant temperature incubator at 24 °C for dark static spawn running culture for 8 d ( Figure 3 ), and when observing that the mycelium has a good condition of consuming the substrate, proceed to the next step.

[0065] 5. Laying the sugar agar layer

[0066] Furthermore, place the tissue culture bottles after 8 d of spawn running culture in the laminar flow hood. After cleaning the mycelium growing on the inner wall, slightly compact it again and pour a thin sugar agar layer about 3 mm thick.

[0067] The sugar agar formula (g / L) used above: glucose 7.5 g, sucrose 7.5 g, agar 15 g. Sterilize the prepared sugar agar by moist heat at 115 °C for 15 min, and use it after cooling to a temperature that is not too hot to touch.

[0068] 6. Ripening growth and promoting the formation of the mycelial film

[0069] Further, place the tissue culture flask with a layer of sugar agar on the bamboo sawdust substrate back into the constant temperature incubator at 24 °C for continued cultivation. After 10 days, it can be seen that the mycelium grows extremely densely ( Figure 4 ), slightly open the tissue culture flask cap for 3 days to allow the sugar agar layer to lose water quickly. Proceed to the next step of treatment.

[0070] 7. Low-temperature stimulation

[0071] Further, place the tissue culture flask full of mycelium in the refrigerator at 4 °C for 5 days for low-temperature stimulation cultivation.

[0072] 8. Fruiting induction

[0073] Further, transfer the tissue culture flask after low-temperature stimulation to room temperature conditions, place it in a place avoiding direct sunlight, slightly unscrew the top lid, use a sterile pipette tip to make holes about 1.5 cm deep in the sugar agar layer, spray a small amount of sterile water, and when natural growth for 3 days, a large number of fruiting body primordia appear on the sugar agar layer.

[0074] Further, transfer it to the conditions of 20 °C and 90% relative humidity for cultivation ( Figure 5 ).

[0075] The entire growth and development cycle takes about 53.0 ± 2.7 days to obtain mature fruiting bodies, and then carry out the observation and description of the fruiting body traits and phenotypes (Table 1, Figure 6 ).

[0076] Table 1 Observation of the fruiting body traits and phenotypes of the Coriolus versicolor strain Le.Msy-01

[0077]

[0078]

[0079] Example 2: Conducted with the Coriolus versicolor strain collected in the wild. Therefore, the strain was purified before liquid fermentation to simulate the rapid verification after field collection.

[0080] 1. Purification of wild strain

[0081] Collect the fruiting bodies of Coriolus versicolor growing on the dead branches of Coriaria nepalensis in the wild, cut the flesh under sterile conditions by tissue isolation method and place it on the potato dextrose agar (PDA) medium, cultivate at a constant temperature of 18 °C for 3 days to restore the physiological activity of the mycelium in the flesh. Transfer the mycelium that appears around the fungal block in the petri dish to a new PDA medium again, purify and cultivate at 23 °C for 6 days, repeat for 2 generations, and proceed to the next step of treatment.

[0082] 2. Liquid fermentation

[0083] Weigh 5 g of potato extract powder, 10 g of glucose, 10 g of maltose, 3 g of peptone, 3 g of corn steep powder, 5 g of magnesium sulfate, 1.5 g of potassium dihydrogen phosphate, 100 mg of vitamin B1, and 8 g of agar. Dissolve and make up the volume to 1000 mL to prepare the fermentation broth. Aliquot 80 mL into tissue culture flasks and sterilize at 121 °C for 30 min for later use.

[0084] Further, aseptically dig out 5 pieces of the strain with a diameter of φ = 10 mm and inoculate. Ferment at 25 °C and 100 rpm for 10 d.

[0085] 3. Solid matrix pretreatment

[0086] Add bamboo chips and wood chips to about 2 times the volume of water respectively, rinse to remove dust, drain, boil with about 2 times the volume of water for 10 min and remove the filtrate. Repeat the treatment 2 times. Dry at 80 °C for 10 h, seal and sterilize at 126 °C for 15 min for later use.

[0087] 4. Addition of solid matrix

[0088] Further, add bamboo chips (50%) and wood chips (50%) in a super-clean environment, mix well and use until it is level with the fermentation broth. After stirring and mixing evenly, let it stand for 60 min for the bamboo and wood chips to absorb the fermentation broth.

[0089] 5. Spawn-running culture

[0090] Further, pour out the excess fermentation broth and slightly compact it. After sealing, transfer it to a 23 °C static environment for spawn-running culture for 10 d. After observing that the mycelium has a good feeding condition, proceed to the next step.

[0091] 6. Laying the sugar agar layer

[0092] Further, slightly compact the culture with 10 d of spawn-running again in a super-clean environment, and pour a layer of sugar agar layer about 5 mm thick.

[0093] The sugar agar formula (g / L) used above: 10 g of glucose, 10 g of sucrose, 20 g of agar. Sterilize at 121 °C for 30 min and cool until it is not hot to the touch before use.

[0094] 7. After-ripening growth and promoting the formation of the mycelium membrane

[0095] Further, put it back to 23 °C and continue to culture for 10 d. After seeing that the mycelium grows extremely dense, slightly open the bottle cap for 5 d to make the sugar agar layer quickly lose water, and proceed to the next treatment.

[0096] 8. Low-temperature stimulation

[0097] Further, place it in a refrigerator at 4 °C for 7 d for low-temperature stimulation.

[0098] 9. Fruiting induction

[0099] Furthermore, transfer to room temperature conditions and avoid direct sunlight. Slightly open the lid for ventilation. Use a sterile instrument to make holes about 1.0 cm deep, spray a small amount of sterile water. When growing for 3 days, primordia of fruiting bodies appear on the sugar agar layer.

[0100] Furthermore, transfer to the conditions of 20 °C and 95% relative humidity and continue culturing.

[0101] The entire growth and development cycle is about 60.0 ± 2.5 days to obtain mature fruiting bodies, and then observe and describe the traits and phenotypes of the fruiting bodies (Table 2, Figure 7 ).

[0102] Table 2 Observation on Cultivation Traits and Phenotypes of Wild Purified Coriaria sinica Strains

[0103]

[0104]

[0105] Example 3: Use the purified Coriaria sinica strain purchased from a third-party institution to carry out the example.

[0106] 1. Liquid fermentation of the strain

[0107] Weigh 5 g of potato powder, 15 g of glucose, 10 g of maltose, 3 g of peptone, 1 g of corn steep powder, 2 g of magnesium sulfate, 2 g of potassium dihydrogen phosphate, 150 mg of vitamin B1, and 1 g of agar, make up the volume to 1000 mL to prepare the fermentation broth, divide it into 120 mL per bottle in tissue culture flasks and sterilize at 121 °C for 15 min.

[0108] After sterilization, take 10 pieces of the strain with a diameter of φ = 6 mm growing on PDA in a super-clean environment, and carry out shaking fermentation for 10 days under the conditions of 27 °C and 135 rpm / min after inoculation.

[0109] 2. Pretreatment of the solid matrix

[0110] Add bamboo chips and wood chips to about 8 times the volume of water respectively, rinse to remove dust, drain, boil with about 8 times the volume of water for 30 min and remove the filtrate, and repeat the treatment once. Dry at 105 °C for 6 h, seal and sterilize at 126 °C for 15 min for later use.

[0111] 4. Addition of the solid matrix

[0112] Furthermore, add bamboo chips (60%) and wood chips (40%) in a super-clean environment until it is flush with the fermentation broth, stir and mix evenly, and then let it stand for 15 min for the bamboo and wood chips to absorb the fermentation broth.

[0113] 5. Spawn-running culture

[0114] Furthermore, pour out the excess fermentation broth and slightly compact it. After sealing, transfer it to a static incubation at 27°C for 14 days. After observing that the mycelium has a good growth on the substrate, proceed to the next step.

[0115] 6. Laying a layer of sugar agar

[0116] Furthermore, in a super-clean environment, slightly compact the material that has been incubated for 14 days again, and pour a layer of sugar agar about 2 mm thick.

[0117] The formula of the sugar agar used above (g / L): glucose 10 g, sucrose 10 g, agar 20 g. Sterilize at 121°C for 15 minutes and cool until it is not hot to the touch before use.

[0118] 7. Ripening growth and promoting the formation of the mycelial membrane

[0119] Furthermore, put it back and continue to incubate at 27°C for 15 days. When the mycelium grows extremely dense, slightly open the bottle cap for 5 days to quickly dehydrate the sugar agar layer, and then proceed to the next treatment.

[0120] 8. Low-temperature stimulation

[0121] Furthermore, place it in a refrigerator at 4°C for 7 days for low-temperature stimulation.

[0122] 9. Inducing fruiting

[0123] Furthermore, transfer it to room temperature conditions and avoid direct sunlight. Slightly open the lid for ventilation, make holes about 2 cm deep with a sterile instrument, spray a small amount of sterile water. When growing for 5 days, primordia of fruiting bodies appear on the sugar agar layer.

[0124] Furthermore, transfer it to a cultivation condition of 20°C and 85% relative humidity.

[0125] The entire growth and development cycle takes about 65.0 ± 2.5 days to obtain mature fruiting bodies. Subsequently, observe and describe the traits and phenotypes of the fruiting bodies (Table 3, Figure 8 ).

[0126] Table 3 Observation of cultivation traits and phenotypes of the purified strain of Coriaria nepalensis Wall.

[0127]

[0128] Finally, it should be pointed out that the above embodiments are only relatively representative examples of the present invention. Obviously, the technical solutions of the present invention are not limited to the above embodiments and there can be many variations. All variations that can be directly derived or associated by those of ordinary skill in the art from the disclosed content of the present invention should be considered as within the protection scope of the present invention.

Claims

1. A liquid-solid two-phase coupled culture method of Coriaria officinalis, characterized in that: The steps include: (1) Liquid fermentation: Prepare liquid fermentation liquid, dispense 50-200 mL / bottle into tissue culture bottles, sterilize in a high pressure steam sterilizer at 115°C-126°C for 5-60 min; after sterilization, inoculate 1-50 bacterial blocks with a diameter of φ3-10 mm in an ultra-clean environment, transfer to a constant temperature shaker and ferment at 10-30°C and 60-200 rpm / min for 5-20 days; (2) Solid matrix pretreatment: Select bamboo chips and sawdust as solid matrix; rinse sawdust with clean water, remove large pieces of bamboo chips and cut into small pieces and rinse them clean; place bamboo chips and sawdust in about 2 to 10 times the volume of water and boil for 5 to 60 minutes and repeat 1 to 10 times; after draining, spread them out and place them in a forced air drying oven at 45 to 105 degrees Celsius for 4 to 36 hours; then put them into a container and place them in a high-pressure steam sterilizer for wet heat sterilization at 121 to 126 degrees Celsius for 15 to 180 minutes; (3) Addition of solid matrix: Take out the fermented tissue culture bottle from the shaking table, add a mixture of 5-50% bamboo chips and 50-95% sawdust in the clean bench, and stir up and down to mix the fermented mycelium balls, sawdust and bamboo chips; (4) Bacterial culture: After mixing the bamboo sawdust, let it stand for 5 to 150 minutes to fully absorb the fermentation liquid, then pour out the excess liquid, slightly flatten and compact the bamboo sawdust while ensuring ventilation, seal the tissue culture bottle, transfer it to a constant temperature incubator at 10 to 30°C, avoid light, and let it stand for bacteria growth for 4 to 30 days; (5) Covering sugar agar layer: Prepare sugar agar solution, sterilize with wet heat at 115-126℃ for 10-60min, take it out and place it on a clean bench, cool it to 30-55℃, pour a 0.5-5mm thick sugar agar layer on the surface of the matrix inside the tissue culture bottle after the inoculation is completed, and puncture holes with an inoculation needle to increase the permeability of the matrix after solidification, and return it to 10-30℃ without light for 5-10 days until the surface of the sugar agar layer is covered with hyphae; (6) Post-ripening culture and biofilm promotion: Pour sugar agar on the surface of the culture medium and place the tissue culture bottle in a constant temperature incubator at 10-30℃ for 7-20 days to promote the rapid growth of mycelium. When nodules can be observed with the naked eye, the after-ripening is complete; (7) Low temperature stimulation: Place the tissue culture bottle in a refrigerator at 2-10°C for 4-8 days to complete the low temperature stimulation treatment; (8) Inducing mushroom production: The transferred tissue culture bottle is placed in a low light environment at room temperature. Use a sterile tool to make holes in the sugar agar layer and spray a small amount of sterile water to induce rapid differentiation of primordia. After the fruiting body primordia appear, open the lid slightly to increase air permeability and grow naturally for 5 to 15 days. By judging the characteristics of the grown fruiting bodies, it can be determined whether the phenotype meets the requirements.

2. The liquid-solid two-phase coupled culture method of Coriaria according to claim 1, characterized in that: In the step (1), the liquid fermentation liquid formula (g / L) is: 1-25g potato powder, 3-30g glucose, 3-30g maltose, 0.5-10g peptone, 1-15g corn powder, 0.5-10g magnesium sulfate, 0.5-10g potassium dihydrogen phosphate, 5-400mg vitamin B1, and 0.5-12g agar.

3. The liquid-solid two-phase coupled culture method of Coriaria according to claim 1, characterized in that: In the step (2), the wood chips are mixed wood chips but need not contain oily substances.

4. The liquid-solid two-phase coupled culture method of Coriaria according to claim 1, characterized in that: In the step (2), the bamboo chips are waste from bamboo product processing.

5. The liquid-solid two-phase coupled culture method of Coriaria officinalis according to claim 1, characterized in that: In the step (5), the sugar agar formula (g / L) is: 5-15 g glucose, 5-15 g sucrose, and 8-25 g agar.

6. The liquid-solid two-phase coupled culture method of Coriaria officinalis according to claim 1, characterized in that: In the step (6), when dense mycelium growth is observed on the sugar agar layer on the surface of the cultivation substrate during the post-ripening growth, the cover of the tissue culture bottle is slightly opened to increase air permeability for 2 to 10 days, so that the sugar agar layer loses water quickly, forms a protective layer, and induces the rapid appearance of a biofilm.

7. The liquid-solid two-phase coupled culture method of Coriaria officinalis according to claim 1, characterized in that: In the step (8), when inducing the natural growth of mushrooms, the temperature must be maintained at 10-28° C. and the relative humidity must be maintained at 60-95%.

8. The liquid-solid two-phase coupled culture method of Coriaria according to claim 1, characterized in that: The maximum temperature of the room temperature environment in step (8) does not exceed 35°C.

9. The liquid-solid two-phase coupled culture method of Coriaria officinalis according to claim 1, characterized in that: The drilling depth of step (8) is 1 to 3 cm.

10. Use of the liquid-solid two-phase coupled culture method of Coriaria as claimed in any one of claims 1 to 9 in rapidly determining the correctness of the introduction of Coriaria.

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