Tunnel fermentation-based tricholoma matsutake culture medium and tricholoma matsutake semi-artificial

Abstract

The invention belongs to the technical field of edible fungus cultivation, and particularly relates to a tricholoma matsutake culture medium based on tunnel fermentation and a tricholoma matsutake semi-artificial cultivation method. The tricholoma matsutake culture medium comprises 45-55% of wheat straw, 10-20% of soybean meal, 19-21% of Korean pine leaves and 14-16% of leaf mold; a secondary tunnel fermentation process is adopted to prepare the feed additive, high throughput is adopted in primary fermentation, and low throughput is adopted in secondary fermentation. The semi-artificial cultivation method of the tricholoma matsutake comprises the following steps: inoculating a tricholoma matsutake strain into a liquid culture medium, fermenting to obtain a liquid strain, then mixing the liquid strain with the tricholoma matsutake culture medium, and pre-culturing to obtain an inoculation material; burying the inoculation material in a tree pit, covering a mixture of decomposed pine needles and black sandy soil and living moss on the inoculation material, and culturing. The tricholoma matsutake culture medium is high in fermentation efficiency and low in fermentation energy consumption, the mycelium planting rate is high when the tricholoma matsutake culture medium is used for tricholoma matsutake culture, and the tricholoma matsutake semi-artificial cultivation method is high in mycorrhiza forming rate, high in annual yield and low in sporocarp malformation rate.

Description

Technical Field

[0001] The invention belongs to the technical field of edible fungus cultivation, and particularly relates to a matsutake mushroom culture medium based on tunnel fermentation and a semi-artificial matsutake mushroom cultivation method. Background Art

[0002] Matsutake is an ectomycorrhizal fungus found on trees such as pine and oak. Fresh matsutake is umbrella-shaped and brightly colored, with a brown cap and a white stipe, both covered with fibrous hairs and scales. The flesh is white, tender, and thick, with a fine texture and a rich, distinctive aroma. Matsutake contains 18 amino acids, 14 essential trace elements, 49 active nutrients, 5 unsaturated fatty acids, 8 vitamins, 2 glycoproteins, a rich source of dietary fiber, and several active enzymes. It also contains three valuable active substances: double-stranded pine mushroom polysaccharides, pine mushroom polypeptides, and pine mushroom alcohol, making it a valuable natural medicinal fungus.

[0003] However, matsutake mushrooms are extremely demanding in their growing environment and their growth process is extremely slow, typically requiring 5 to 6 years. Current cultivation methods suffer from poor adaptability of culture media. Traditional matsutake culture media often rely on pure pine sawdust, lacking a mature fermentation process, resulting in a mycelial colonization rate of less than 40%. Furthermore, liquid cultures are also relatively inactive, with conventional liquid culture cycles being long (>15 days) and pellet diameters exceeding 2mm, impacting inoculation uniformity. Furthermore, existing understory cultivation methods are relatively extensive, with direct burial methods resulting in inefficient mycelial-root contact and a mycorrhizal synthesis rate of less than 25%.

[0004] Patent CN108124691A discloses a tunnel fermentation composting process for Agaricus bisporus culture medium. This process involves mixing straw, chicken manure, and soybean meal, then fermenting them four times in separate fermentation tunnels at precise temperatures, achieving a primary fermentation. The mixture is then transferred to another fermentation tunnel, where the temperature is raised for a period of time, pasteurized, and then cooled and fermented four times in a row, achieving a secondary fermentation. Discharging the compost after conditions are met completes the tunnel fermentation composting process. This process boasts high composting efficiency, complete nutrient conversion, and thorough sterilization, significantly reducing pest and disease contamination in Agaricus bisporus cultivation and resulting in high mushroom yields. However, it is only applicable to saprophytes and does not optimize the carbon-nitrogen ratio for the symbiotic properties of matsutake mushrooms.

[0005] Patent CN105505786A provides a method for cultivating a liquid matsutake spawn, comprising preparing a liquid matsutake spawn culture medium, inoculating a stock matsutake seed into the liquid matsutake spawn culture medium for sealed cultivation, and, after mycelium from the stock matsutake seed germinates, shaking the liquid matsutake spawn culture medium once daily until a liquid matsutake spawn is obtained. This invention uses sucrose and other substances as the primary carbon source, which can lead to excessive mycelial growth and inhibit the expression of symbiotic genes.

[0006] Therefore, how to improve the fermentation efficiency and mycelium colonization rate of Matsutake culture medium, while increasing the yield of artificial cultivation of Matsutake and reducing the fruiting body deformity rate, is a technical problem that needs to be solved urgently. Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides a matsutake culture medium based on tunnel fermentation, which has high fermentation efficiency, low fermentation energy consumption, and a high mycelium colonization rate when used for matsutake cultivation; the present invention also provides a semi-artificial cultivation method for matsutake, which has a high mycorrhizal formation rate, high annual yield, and low fruiting body deformity rate.

[0008] The tunnel fermentation-based matsutake culture medium of the present invention comprises the following raw materials in percentage by mass: 45-55% wheat straw, 10-20% soybean meal, 19-21% Korean pine leaves, and 14-16% leaf mold; wherein the wheat straw is wheat straw soaked and drained in lime water, and the Korean pine leaves are Korean pine leaves sterilized by ultraviolet irradiation;

[0009] The tunnel fermentation-based matsutake culture medium is prepared by the following steps:

[0010] (1) Pile: Mix the above raw materials in proportion, adjust the moisture content to 65-68%, transfer them into the fermentation tunnel, and pile them into a pile;

[0011] (2) Primary fermentation: Hot air at 30-45°C is introduced into the fermentation tunnel with a ventilation volume of 0.8-1.2m 3 Air / (h·ton of material), maintain the pore oxygen concentration of the pile ≥12% for 4-6 days;

[0012] When the pile temperature is greater than 65℃, increase the ventilation volume to 1.5-2.0m 3 / (h·ton of material), lasting 10-30min;

[0013] Turn the pile once every 20-30 hours and increase the ventilation volume to 2.2-2.8m 3 / (h·ton of material), lasting 20-40min;

[0014] (3) Secondary fermentation: After the first fermentation is completed, the temperature of the pile is controlled at 45-50℃, thermophilic actinomycetes are inoculated, and the ventilation volume is 0.5-0.8m 3 Air / (h·ton of material), maintain the pore oxygen concentration of the pile at 8-10% for 2-3 days;

[0015] After inoculation of thermophilic actinomycetes, pulse ventilation was performed every 5-7 hours with a ventilation volume of 0.4-0.6m 3 / (h·ton of material), lasting 15-25min;

[0016] When the ammonia concentration is detected to be greater than 200ppm, the instantaneous ventilation volume is increased to 1.3-1.6m 3 / (h·ton of material), lasting 50-60min;

[0017] The maturity index of the matsutake culture medium is: C / N ratio (20-25):1, pH value 6.5-7.0, and ammonia nitrogen concentration <0.2 mg / g.

[0018] The wheat straw is crushed into 2-3 cm segments, soaked in lime water with a pH value of 9.5-10.5 for 2-3 hours, drained and set aside. The porosity of the wheat straw is ≥60%, and the function is to provide a cellulose skeleton.

[0019] The soybean meal is dried at 75-85° C. and crushed into 1-2 mm for later use. The crude protein content of the soybean meal is ≥45%, and the soybean meal is used as a high nitrogen source.

[0020] The red pine leaves are sterilized by ultraviolet irradiation for 5-15 minutes and then used for later use. The function of the leaves is to release terpene symbiotic signal substances.

[0021] The leaf mold is passed through a 2mm sieve and is ready for use. The moisture content of the leaf mold is 25-35%, and the function of the leaf mold is to introduce beneficial microbial communities.

[0022] Preferably, the pile height in step (1) is 1.2-1.6 m.

[0023] Preferably, the inoculum amount of thermophilic actinomycetes in step (3) is 0.8-1.2%.

[0024] The semi-artificial cultivation method of Matsutake mushrooms of the present invention comprises the following steps:

[0025] (1) Inoculation: Inoculate the matsutake strain into a liquid culture medium and ferment for 8-10 days to obtain a liquid strain;

[0026] (2) Cultivation: Mix the liquid spawn into the matsutake culture medium and pre-cultivate in a dark and ventilated environment for 2-4 days to obtain the inoculum; bury the inoculum in the tree pit and cover it with two layers: the lower layer is a mixture of decomposed pine needles and black sand, and the upper layer is living moss, and then spray the symbiotic inducer;

[0027] (3) Cultivation: Maintain the temperature of the covering layer between 5-25°C, maintain the humidity of the inoculum at 60-65% during the mycelium expansion period, and maintain the humidity of the inoculum at 70-75% during the primordium differentiation period. When the diameter of the cap of the matsutake fruiting body reaches 3-5 cm, pick it.

[0028] In step (1), the liquid culture medium formula is: peptone 7.0-9.0 g / L, yeast extract 4.5-5.5 g / L, lactose 14.0-16.0 g / L, potassium dihydrogen phosphate 1.4-1.6 g / L, magnesium sulfate 0.4-0.6 g / L, potassium nitrate 1.5-2.5 g / L, calcium chloride 0.15-0.25 g / L, vitamin B1 0.008-0.012 g / L, and pine needle extract 1.8-2.2 g / L.

[0029] Preferably, the liquid culture medium formula is: peptone 8.0g / L, yeast extract 5.0g / L, lactose 15.0g / L, potassium dihydrogen phosphate 1.5g / L, magnesium sulfate 0.5g / L, potassium nitrate 2.0g / L, calcium chloride 0.2g / L, vitamin B1 0.01g / L, and pine needle extract 2g / L.

[0030] Among them, the pine needle extract is the liquid obtained by boiling red pine leaves in water for 20-40 minutes, and the mass ratio of red pine leaves to water is 5:100.

[0031] In step (1), the inoculation amount of the matsutake mushroom is 4-6%, preferably 5%.

[0032] In step (1), during the fermentation culture, a 500-800 L liquid fermentation tank is used, sterilized under high pressure at 121° C. for 40-50 min, cooled to 24° C., inoculated with the matsutake strain, and then cultured at 24-25° C. and 180 rpm for 8-10 days.

[0033] In step (1), the diameter of the pellet in the liquid culture is 0.5-1.0 mm (microscopic examination), the mycelial biomass is ≥15 g / L (dry weight), and the pH value is 5.8-6.2.

[0034] In step (2), the matsutake culture medium is a matsutake culture medium based on tunnel fermentation prepared by the above-mentioned preparation method of the present invention, and the mass ratio of liquid strain to matsutake culture medium is 1:(8-12).

[0035] In step (2), during the light-proof and ventilated pre-culture, the temperature is maintained at 24-26°C, preferably 25°C.

[0036] In step (2), the pit is located near the root system of the pine tree, with a diameter of 40-50 cm and a depth of 30-40 cm. The bottom of the pit is left with a thickness of 5-10 cm of original soil. Preferably, the pit is located within the canopy projection of Yunnan pine / red pine, with a slope of 15°-30° and a soil organic matter content of ≥3%.

[0037] In step (2), when the inoculum is buried in the tree pit, the bottom of the inoculum is close to the tree roots, and the top of the inoculum is 10-20 cm above the ground.

[0038] In step (2), the mixture of decomposed pine needles and black sand is covered to a thickness of 5-10 cm, has a pH value of 6.0-6.5, and the mass percentages of decomposed pine needles and black sand are 30-40% and 70-60%, respectively.

[0039] In step (2), the living moss is of the genus Glauca, and the covering thickness is 5-10 cm.

[0040] In step (2), the symbiotic inducer is preferably an aqueous solution containing 0.05% strigolactone and 0.1% trehalose.

[0041] In step (3), when temperature control is performed, a sunshade net can be built when the temperature is high in summer, and straw mats can be covered on the surface to keep warm when the temperature is low in winter; when humidity control is performed, the humidity can be increased by spraying.

[0042] In step (3), during the culture process, pest control operations can be carried out if necessary, such as spraying a Bacillus preparation once a month, and controlling green mold spots by local injection of 0.5% cinnamaldehyde ethanol solution.

[0043] In step (3), when picking, the diameter of the Matsutake fruiting body cap reaches 3-5 cm. At this time, the fruiting body cap is not fully expanded, and the gills are light yellow. Use bamboo strips to cut along the base of the stipe to avoid disturbing the mycelium. After harvesting, fill the covering layer immediately and spray 0.1% glucose solution.

[0044] In step (3), the harvested matsutake mushrooms are pre-cooled at 4° C. for 2 hours and then vacuum-packed to maintain an oxygen concentration of ≤1%. The waste generated after harvesting is backfilled into the tree pit to promote mycelial germination in the next season.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] (1) The present invention adjusts the raw material composition of the matsutake culture medium and optimizes its fermentation process conditions, so that the preparation method achieves the best efficiency-energy consumption balance, with high fermentation efficiency and low fermentation energy consumption, high mycelium colonization rate, and low ammonia nitrogen residue;

[0047] (2) The present invention adjusts the raw material composition of the matsutake culture medium, improves the liquid spawn culture process and cultivation method, and uses a mixture of decomposed pine needles and black sand soil, as well as living moss as the covering layer, thereby greatly improving the mycorrhizal formation rate and annual yield per pit of matsutake mushrooms and reducing the fruiting body deformity rate. DETAILED DESCRIPTION

[0048] The present invention will be further described below with reference to the following examples. The raw materials used in the examples, unless otherwise specified, are all commercially available conventional raw materials; the process methods used in the examples, unless otherwise specified, are all conventional methods in the art.

[0049] The preservation information of the Tricholoma matsutake strains used in the examples is as follows:

[0050] Classification and nomenclature of the deposited biological materials (strains): Lentinula edodes;

[0051] Depository: China General Microbiology Center (CGMCC);

[0052] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0053] Deposit date: April 3, 2025;

[0054] Deposit number: CGMCC No.41881.

[0055] Example 1

[0056] A tunnel fermentation-based matsutake culture medium comprises the following raw materials in percentage by mass: 50% wheat straw, 15% soybean meal, 20% red pine leaves, and 15% leaf mold;

[0057] The wheat straw is crushed into 2-3 cm segments, soaked in lime water with a pH value of 10±5 for 2 hours, drained and set aside, and its porosity is ≥60%;

[0058] Soybean meal is dried at 80℃ and crushed into 1-2mm pieces for later use. Its crude protein content is ≥45%;

[0059] The Korean pine leaves were sterilized by ultraviolet irradiation for 10 minutes before use;

[0060] The leaf mold is passed through a 2mm sieve and has a moisture content of 30%.

[0061] The tunnel fermentation-based matsutake culture medium is prepared by the following steps:

[0062] (1) Stacking: Mix the above raw materials according to the proportion, adjust the moisture content to 65%, transfer them into the fermentation tunnel, and stack the materials into a pile with a height of 1.5m;

[0063] (2) Primary fermentation: Hot air at 40°C is introduced into the fermentation tunnel with a ventilation volume of 1.0 m 3 Air / (h·ton of material), maintain the pore oxygen concentration of the pile ≥12% for 5 days;

[0064] When the pile temperature is greater than 65°C, increase the ventilation volume to 1.5m 3 / (h·ton of material), lasting 20min;

[0065] Turn the pile once every 24 hours and increase the ventilation volume to 2.5m3 / (h·ton of material), lasting 30min;

[0066] (3) Secondary fermentation: After the first fermentation is completed, the temperature of the pile is controlled between 45-50℃, and thermophilic actinomycetes are inoculated with an inoculation volume of 1% and a ventilation volume of 0.6m 3 Air / (h·ton of material), maintain the pore oxygen concentration of the pile between 8-10% for 3 days;

[0067] After inoculation of thermophilic actinomycetes, pulse ventilation was performed every 6 hours with a ventilation volume of 0.5m 3 / (h·ton of material), lasting 20min;

[0068] When the ammonia concentration is detected to be greater than 200ppm, the instantaneous ventilation volume is increased to 1.5m 3 / (h·ton of material), lasting 60min;

[0069] The maturity index of the matsutake culture medium is: C / N ratio (20-25):1, pH value 6.5-7.0, and ammonia nitrogen concentration <0.2 mg / g.

[0070] Example 2

[0071] A tunnel fermentation-based matsutake culture medium comprises the following raw materials in percentage by mass: 45% wheat straw, 20% soybean meal, 21% red pine leaves, and 14% leaf mold;

[0072] The wheat straw is crushed into 2-3 cm segments, soaked in lime water with a pH value of 10±5 for 3 h, drained and set aside, and its porosity is ≥60%;

[0073] Soybean meal is dried at 85℃ and crushed into 1-2mm pieces for later use. Its crude protein content is ≥45%;

[0074] The Korean pine leaves were sterilized by ultraviolet irradiation for 5 minutes before use;

[0075] The leaf mold is passed through a 2mm sieve and has a moisture content of 25%.

[0076] The tunnel fermentation-based matsutake culture medium is prepared by the following steps:

[0077] (1) Stacking: Mix the above raw materials according to the proportion, adjust the moisture content to 68%, transfer them into the fermentation tunnel, and stack the materials into a pile with a height of 1.2m;

[0078] (2) Primary fermentation: Hot air at 30°C is introduced into the fermentation tunnel for a fermentation period of 4 days with a ventilation volume of 1.2 m 3 Air / (h·ton of material), maintain the oxygen concentration in the pile pores ≥12%;

[0079] When the pile temperature is greater than 65°C, increase the ventilation volume to 2.0m 3 / (h·ton of material), lasting 10min;

[0080] Turn the pile once every 20 hours and increase the ventilation volume to 2.2m 3 / (h·ton of material), lasting 40min;

[0081] (3) Secondary fermentation: After the first fermentation is completed, the temperature of the pile is controlled between 45-50℃, and thermophilic actinomycetes are inoculated with an inoculation volume of 0.9% and a ventilation volume of 0.8m 3 Air / (h·ton of material), maintain the pore oxygen concentration of the pile between 8-10% for 3 days;

[0082] After inoculation of thermophilic actinomycetes, pulse ventilation was performed every 7 hours with a ventilation volume of 0.6 m 3 / (h·ton of material), lasting 15min;

[0083] When the ammonia concentration is detected to be greater than 200ppm, the instantaneous ventilation volume is increased to 1.6m 3 / (h·ton of material), lasting 50min;

[0084] The maturity index of the matsutake culture medium is: C / N ratio (20-25):1, pH value 6.5-7.0, and ammonia nitrogen concentration <0.2 mg / g.

[0085] Example 3

[0086] A tunnel fermentation-based matsutake culture medium comprises the following raw materials in percentage by mass: 55% wheat straw, 10% soybean meal, 19% red pine leaves, and 16% leaf mold;

[0087] The wheat straw is crushed into 2-3 cm segments, soaked in lime water with a pH value of 10±5 for 2 hours, drained and set aside, and its porosity is ≥60%;

[0088] Soybean meal is dried at 75°C and crushed to 1-2 mm for later use. Its crude protein content is ≥45%.

[0089] The Korean pine leaves were sterilized by ultraviolet irradiation for 15 minutes before use;

[0090] The leaf mold is passed through a 2mm sieve and has a moisture content of 35%.

[0091] The tunnel fermentation-based matsutake culture medium is prepared by the following steps:

[0092] (1) Stacking: Mix the above raw materials according to the proportion, adjust the moisture content to 66%, transfer them into the fermentation tunnel, and stack the materials into a pile with a height of 1.6m;

[0093] (2) Primary fermentation: Hot air at 45°C was introduced into the fermentation tunnel for a fermentation period of 6 days with a ventilation volume of 0.8 m 3 Air / (h·ton of material), maintain the oxygen concentration in the pile pores ≥12%;

[0094] When the pile temperature is greater than 65°C, increase the ventilation volume to 1.5m 3 / (h·ton of material), lasting 30min;

[0095] Turn the pile once every 30 hours and increase the ventilation volume to 2.8m 3 / (h·ton of material), lasting 20min;

[0096] (3) Secondary fermentation: After the first fermentation is completed, the temperature of the pile is controlled between 45-50℃, and thermophilic actinomycetes are inoculated with an inoculation volume of 1.2% and a ventilation volume of 0.4m 3 Air / (h·ton of material), maintain the pore oxygen concentration of the pile between 8-10% for 2 days;

[0097] After inoculation of thermophilic actinomycetes, pulse ventilation was performed every 5 hours with a ventilation volume of 0.4 m 3 / (h·ton of material), lasting 25min;

[0098] When the ammonia concentration is detected to be greater than 200ppm, the instantaneous ventilation volume is increased to 1.3m 3 / (h·ton of material), lasting 60min;

[0099] The maturity index of the matsutake culture medium is: C / N ratio (20-25):1, pH value 6.5-7.0, and ammonia nitrogen concentration <0.2 mg / g.

[0100] Example 4

[0101] This example uses the matsutake culture medium prepared in Example 1 to carry out semi-artificial cultivation of matsutake, including the following steps:

[0102] (1) Inoculation: The liquid culture medium formula is: peptone 8.0 g / L, yeast extract 5.0 g / L, lactose 15.0 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate 0.5 g / L, potassium nitrate 2.0 g / L, calcium chloride 0.2 g / L, vitamin B1 0.01 g / L, pine needle extract 2 g / L; wherein, the pine needle extract is the liquid obtained by boiling red pine leaves in water for 30 minutes, and the mass ratio of red pine leaves to water is 5:100;

[0103] Fermentation was performed in a 500 L liquid fermenter, with the liquid culture medium added to the liquid fermenter, autoclaved at 121° C. for 45 minutes, cooled to 24° C., inoculated with a 5% inoculum of Tricholoma matsutake, and then cultured at 25° C. and 180 rpm for 8 days to obtain a liquid culture with a pellet diameter of 0.5-1.0 mm (microscopic examination), a mycelial biomass of ≥15 g / L (dry weight), and a pH of 5.8-6.2.

[0104] (2) Cultivation: The liquid strain was mixed into the matsutake culture medium at a mass ratio of 1:10, and pre-cultured at 25°C in the dark and with ventilation for 3 days to obtain the inoculum;

[0105] Select a site within the projection range of the Yunnan pine / Red pine canopy with a slope of 15°-30° and soil organic matter ≥3%. Dig a tree pit at the root system of the pine tree with a diameter of 45m and a depth of 35cm. Keep a 10cm thick layer of original soil at the bottom of the pit. Bury the inoculum into the tree pit and gently press it so that the bottom of the inoculum is close to the tree roots. The height above the inoculum is 20cm from the ground. Cover the inoculum with a 10cm thick mixture of decomposed pine needles and black sand (the mass ratio of decomposed pine needles to black sand is 3:7), then cover it with a 10cm thick layer of living moss (Glaucophyte). Finally, spray a symbiotic inducer (an aqueous solution containing 0.05% strigolactone and 0.1% trehalose).

[0106] (3) Cultivation: Maintain the temperature of the covering layer between 5-25°C, maintain the humidity of the inoculum between 60-65% during the mycelial expansion period, and maintain the humidity of the inoculum between 70-75% during the primordium differentiation period. During the cultivation process, pest and disease control operations can be carried out if necessary, including spraying Bacillus preparations once a month and controlling green mold spots by local injection of 0.5% cinnamaldehyde ethanol solution;

[0107] When the diameter of the fruiting body cap reaches 3-5cm, it is picked. At this time, the fruiting body cap is not fully unfolded and the gills are light yellow. Use bamboo strips to cut along the base of the stipe to avoid disturbing the mycelium. Immediately after harvesting, fill the covering layer and spray 0.1% glucose solution. After picking, pre-cool at 4℃ for 2h and then vacuum pack, keep the oxygen concentration ≤1%. The waste generated after picking is backfilled into the tree pit to promote mycelium germination in the next season.

[0108] Example 5

[0109] The only difference between this comparative example and Example 4 is that in this comparative example, the matsutake mushrooms were inoculated into the liquid culture medium and cultured for 10 days. The remaining steps were the same as in Example 4.

[0110] Example 6

[0111] This example uses the matsutake culture medium prepared in Example 1 to carry out semi-artificial cultivation of matsutake, including the following steps:

[0112] (1) Inoculation: The liquid culture medium formula is as follows: peptone 7.0 g / L, yeast extract 4.5 g / L, lactose 14.0 g / L, potassium dihydrogen phosphate 1.4 g / L, magnesium sulfate 0.4 g / L, potassium nitrate 1.5 g / L, calcium chloride 0.15 g / L, vitamin B1 0.008 g / L, pine needle extract 1.8 g / L; wherein, the pine needle extract is the liquid obtained by boiling red pine leaves in water for 20 minutes, and the mass ratio of red pine leaves to water is 5:100;

[0113] Fermentation was performed in a 500 L liquid fermenter, with the liquid culture medium added to the liquid fermenter, autoclaved at 121° C. for 40 min, cooled to 24° C., inoculated with a 4% inoculum of Tricholoma matsutake, and then cultured at 24° C. and 180 rpm for 8 days to obtain a liquid culture with a pellet diameter of 0.5-1.0 mm (microscopic examination), a mycelial biomass of ≥15 g / L (dry weight), and a pH of 5.8-6.2.

[0114] (2) Cultivation: The liquid strain was mixed into the matsutake culture medium at a mass ratio of 1:8, and pre-cultured at 24°C in the dark and with ventilation for 4 days to obtain the inoculum;

[0115] Select a site within the projection range of the Yunnan pine / Red pine canopy with a slope of 15°-30° and soil organic matter ≥3%. Dig a tree pit at the root system of the pine tree with a diameter of 45m and a depth of 35cm. Keep a 5cm thick layer of original soil at the bottom of the pit. Bury the inoculum in the tree pit and gently press it so that the bottom of the inoculum is close to the tree roots. The height above the inoculum is 10cm from the ground. Cover the inoculum with a 5cm thick mixture of decomposed pine needles and black sand (the mass ratio of decomposed pine needles to black sand is 4:6), then cover it with a 5cm thick layer of living moss (Glaucophyte). Finally, spray a symbiotic inducer (an aqueous solution containing 0.05% strigolactone and 0.1% trehalose).

[0116] (3) Cultivation: Maintain the temperature of the covering layer between 5-25°C, maintain the humidity of the inoculum between 60-65% during the mycelial expansion period, and maintain the humidity of the inoculum between 70-75% during the primordium differentiation period. During the cultivation process, pest and disease control operations can be carried out if necessary, including spraying Bacillus preparations once a month and controlling green mold spots by local injection of 0.5% cinnamaldehyde ethanol solution;

[0117] When the diameter of the fruiting body cap reaches 3-5cm, it is picked. At this time, the fruiting body cap is not fully unfolded and the gills are light yellow. Use bamboo strips to cut along the base of the stipe to avoid disturbing the mycelium. Immediately after harvesting, fill the covering layer and spray 0.1% glucose solution. After picking, pre-cool at 4℃ for 2h and then vacuum pack, keep the oxygen concentration ≤1%. The waste generated after picking is backfilled into the tree pit to promote mycelium germination in the next season.

[0118] Example 7

[0119] This example uses the matsutake culture medium prepared in Example 1 to carry out semi-artificial cultivation of matsutake, including the following steps:

[0120] (1) Inoculation: The liquid culture medium formula is as follows: peptone 9.0 g / L, yeast extract 5.5 g / L, lactose 16.0 g / L, potassium dihydrogen phosphate 1.6 g / L, magnesium sulfate 0.6 g / L, potassium nitrate 2.5 g / L, calcium chloride 0.25 g / L, vitamin B1 0.012 g / L, pine needle extract 2.2 g / L; wherein, the pine needle extract is the liquid obtained by boiling red pine leaves in water for 40 minutes, and the mass ratio of red pine leaves to water is 5:100;

[0121] Fermentation was performed in an 800 L liquid fermenter, wherein the liquid culture medium was added to the liquid fermenter, sterilized by autoclaving at 121° C. for 50 min, cooled to 24° C., and inoculated with a 6% inoculum of Tricholoma matsutake. The culture was then cultured at 25° C. and 180 rpm for 8 days to obtain a liquid culture having a pellet diameter of 0.5-1.0 mm (microscopic examination), a mycelial biomass of ≥15 g / L (dry weight), and a pH of 5.8-6.2.

[0122] (2) Cultivation: The liquid strain was mixed into the matsutake culture medium at a mass ratio of 1:12, and pre-cultured at 26°C in the dark and with ventilation for 2 days to obtain the inoculum;

[0123] Select a site within the projection range of the Yunnan pine / Red pine canopy with a slope of 15°-30° and soil organic matter ≥3%. Dig a tree pit at the root system of the pine tree with a diameter of 45m and a depth of 35cm. Keep a 10cm thick layer of original soil at the bottom of the pit. Bury the inoculum in the tree pit and gently press it so that the bottom of the inoculum is close to the tree roots. The height above the inoculum is 18cm from the ground. Cover the inoculum with a 10cm thick mixture of decomposed pine needles and black sand (the mass ratio of decomposed pine needles to black sand is 3:7), then cover it with an 8cm thick layer of living moss (Glaucophyte). Finally, spray a symbiotic inducer (an aqueous solution containing 0.05% strigolactone and 0.1% trehalose).

[0124] (3) Cultivation: Maintain the temperature of the covering layer between 5-25°C, maintain the humidity of the inoculum between 60-65% during the mycelial expansion period, and maintain the humidity of the inoculum between 70-75% during the primordium differentiation period. During the cultivation process, pest and disease control operations can be carried out if necessary, including spraying Bacillus preparations once a month and controlling green mold spots by local injection of 0.5% cinnamaldehyde ethanol solution;

[0125] When the diameter of the fruiting body cap reaches 3-5cm, it is picked. At this time, the fruiting body cap is not fully unfolded and the gills are light yellow. Use bamboo strips to cut along the base of the stipe to avoid disturbing the mycelium. Immediately after harvesting, fill the covering layer and spray 0.1% glucose solution. After picking, pre-cool at 4℃ for 2h and then vacuum pack, keep the oxygen concentration ≤1%. The waste generated after picking is backfilled into the tree pit to promote mycelium germination in the next season.

[0126] Comparative Example 1

[0127] The only difference between this comparative example and Example 1 is that, in the preparation of the matsutake culture medium, low ventilation was used in both the primary and secondary fermentations, i.e., the ventilation volume during the primary fermentation was 0.5 m 3 Air / (h·ton of material), the ventilation volume during the secondary fermentation process is 0.3m 3 Air / (h·ton of material). The remaining steps are the same as in Example 1.

[0128] Comparative Example 2

[0129] The only difference between this comparative example and Example 1 is that in the preparation of the matsutake culture medium, high ventilation was used in both the primary and secondary fermentations, i.e., the ventilation volume during the primary fermentation was 2.0 m 3 Air / (h·ton of material), the ventilation volume during the secondary fermentation process is 1.0m 3 Air / (h·ton of material). The remaining steps are the same as in Example 1.

[0130] The fermentation effects of Example 1 and Comparative Examples 1-2 were compared. Each test group used 3 tons of material, the sample size of each group was ≥10, and the test was repeated ≥3 times. The results are shown in Table 1. Data are Mean ± SD (n = 10), *P < 0.05, **P < 0.01, ***P < 0.001 (vs Group H), Shapiro-Wilk test (α = 0.05); homogeneity of variance test: Levene's Test; software tool: SPSS 26.0.

[0131] Table 1 Fermentation effect of Example 1 and Comparative Examples 1-2

[0132] project Example 1 Comparative Example 1 Comparative Example 2 Fermentation cycle (days) 8.0±0.3** 12.2±0.5 7.1±0.2** Ammonia nitrogen residue (mg / g) 0.15±0.02* 0.42±0.05 0.10±0.01** Mycelium colonization rate (%) 91.7±2.1*** 61.3±3.8 89.4±2.5*** Energy consumption (kWh / ton) 18.5±1.2 12.3±0.9 32.7±2.8

[0133] As can be seen from Table 1, the fermentation method of Example 1 is the best in terms of efficiency-energy consumption balance. Compared with Comparative Example 1, the fermentation cycle is shortened by 33%, the mycelium colonization rate is increased by 51%, energy is saved by 43% compared with Comparative Example 2, and the ammonia nitrogen residue is not significantly increased (P>0.05).

[0134] In addition, the fermentation effects of Examples 2-3 are as follows: the ammonia nitrogen residue is less than 0.2 mg / g, the energy consumption is less than 20 kWh / ton, and the mycelium colonization rate is higher than 90%.

[0135] Comparative Example 3

[0136] The matsutake culture medium used in this comparative example comprises the following raw materials in mass percentages: 55% wheat straw, 15% soybean meal, 15% Korean pine leaves, and 15% leaf mold. The steps for preparing the matsutake culture medium are the same as those in Example 1, and the semi-artificial cultivation method for matsutake is the same as that in Example 4.

[0137] Comparative Example 4

[0138] The matsutake culture medium used in this comparative example comprises the following raw materials in percentage by mass: 45% wheat straw, 15% soybean meal, 25% Korean pine leaves, and 15% leaf mold. The steps for preparing the matsutake culture medium are the same as those in Example 1, and the semi-artificial cultivation method for matsutake is the same as that in Example 4.

[0139] The cultivation effects of the matsutake mushrooms of Example 4 and Comparative Examples 3-4 were compared, with 10 tree pits selected for each group. The results are shown in Table 2. The mycorrhizal formation rate was calculated 90 days after cultivation. α-pinene is a key signal substance for the mycorrhizal symbiosis of matsutake mushrooms, and its content was detected by GC-MS.

[0140] Table 2 Matsutake cultivation effects of Examples 4-6 and Comparative Examples 3-4

[0141] project Example 4 Comparative Example 3 Comparative Example 4 Korean pine leaf percentage (%) 20 15 25 Mycorrhiza formation rate (%) 87.1±4.1 68.3±5.2 72.5±6.0 Annual output per pit (g) 650±85 480±62 520±58 α-Pinene content (μg / g matrix) 28.7±3.2 12.5±1.8 35.4±4.1

[0142] As can be seen from Table 2, Example 4 has the highest mycorrhizal formation rate and annual yield per pit, and the α-pinene content reaches 28.7 μg / g, which can effectively induce mycorrhizae while avoiding high concentration inhibition of growth (when the α-pinene content is greater than 30 μg / g, the mycelial growth rate decreases by about 15%). It can be seen that the addition ratio of Korean pine leaves in the matsutake culture medium has an important influence on the cultivation effect of matsutake.

[0143] In addition, the cultivation effects of Tricholoma matsutake in Examples 5-7 are as follows: the mycorrhizal formation rate is higher than 70%, the annual yield per pit is higher than 620 g, and the α-pinene content is between 25-30 μg / g substrate.

[0144] Comparative Example 5

[0145] The only difference between this comparative example and Example 4 is that in this comparative example, the matsutake mushrooms were inoculated into the liquid culture medium and cultured for 12 days. The remaining steps were the same as in Example 4.

[0146] The mycelium culture effects of Examples 4-5 and Comparative Example 5 were compared using the same batch of Matsutake liquid spawn, and the results are shown in Table 3. The mycelium colonization rate was calculated 30 days after cultivation, and the mycorrhizal formation rate was calculated 90 days after cultivation.

[0147] Table 3 Mycelial culture effects of Examples 4-5 and Comparative Example 5

[0148] project Example 4 Example 5 Comparative Example 5 Mycelium colonization rate (%) 80.2±3.1 78.5±2.8 52.4±6.3 Mycorrhiza formation rate (%) 75.7±4.5 73.2±5.1 51.5±7.2 Mycelial biomass (g / kg substrate) 18.3±1.8 17.6±1.5 12.1±2.0

[0149] As can be seen from Table 3, the optimal window period for liquid culture (peak mycelial activity) is 8-10 days. Overtime culture will lead to aging of the pellets and a significant decrease in the colonization rate (P<0.05).

[0150] Comparative Example 6

[0151] This comparative example differs from Example 4 only in that black sand was used as the covering layer during the cultivation step. Specifically, the inoculum was buried in the tree pit, covered with 18 cm of black sand, and then sprayed with a symbiosis inducer. The remaining steps were the same as in Example 4.

[0152] Comparative Example 7

[0153] This comparative example differs from Example 4 only in that leaf mold is used as a covering layer during the cultivation step. Specifically, the inoculum is buried in the tree pit, covered with 18 cm of leaf mold, and then sprayed with a symbiosis inducer. The remaining steps are the same as in Example 4.

[0154] The cultivation effects of the matsutake mushrooms of Example 4 and Comparative Examples 6-7 were compared. Five tree pits were selected for each group and observed for two consecutive years. The results are shown in Table 4. The mycorrhizal formation rate was calculated 90 days after cultivation.

[0155] Table 4 Matsutake cultivation effects of Example 4 and Comparative Examples 6-7

[0156] project Example 4 Comparative Example 6 Comparative Example 7 Annual output per pit (g) 650±85 320±60 410±55 Average single fruiting body weight (g) 42.5±3.8 28.3±5.2 31.6±4.7 Fruiting body deformity rate (%) 6 34.7 22.5

[0157] As can be seen from Table 4, Example 4 has the highest mycorrhizal formation rate and annual yield per pit, and the α-pinene content reaches 28.7 μg / g, which can effectively induce mycorrhizae while avoiding high concentration inhibition of growth (when the α-pinene content is greater than 30 μg / g, the mycelial growth rate decreases by about 15%). It can be seen that the addition ratio of Korean pine leaves to the matsutake culture medium has an important influence on the cultivation effect of matsutake.

Claims

1. A Tricholoma matsutake culture medium based on tunnel fermentation, characterized by: The raw materials include the following percentages by mass: 45-55% wheat straw, 10-20% soybean meal, 19-21% Korean pine leaves, and 14-16% leaf mold; wherein the wheat straw is wheat straw soaked and drained in lime water, and the Korean pine leaves are Korean pine leaves sterilized by ultraviolet irradiation; The preparation is obtained by the following steps: (1) Pile: Mix the above raw materials in proportion, adjust the moisture content to 65-68%, transfer them into the fermentation tunnel, and pile them into a pile; (2) Primary fermentation: Hot air at 30-45°C is introduced into the fermentation tunnel with a ventilation volume of 0.8-1.2m 3 Air / (h·ton of material), maintain the pore oxygen concentration of the pile ≥12% for 4-6 days; When the pile temperature is greater than 65℃, increase the ventilation volume to 1.5-2.0m 3 / (h·ton of material), lasting 10-30min; Turn the pile once every 20-30 hours and increase the ventilation volume to 2.2-2.8m 3 / (h·ton of material), lasting 20-40min; (3) Secondary fermentation: After the first fermentation is completed, the temperature of the pile is controlled at 45-50℃, thermophilic actinomycetes are inoculated, and the ventilation volume is 0.5-0.8m 3 Air / (h·ton of material), maintain the pore oxygen concentration of the pile at 8-10% for 2-3 days; After inoculation of thermophilic actinomycetes, pulse ventilation was performed every 5-7 hours with a ventilation volume of 0.4-0.6m 3 / (h·ton of material), lasting 15-25min; When the ammonia concentration is detected to be greater than 200ppm, the instantaneous ventilation volume is increased to 1.3-1.6m 3 / (h·ton of material), lasting 50-60min; The maturity index of the matsutake culture medium is: C / N ratio (20-25):1, pH value 6.5-7.0, and ammonia nitrogen concentration <0.2 mg / g.

2. The tunnel fermentation-based matsutake culture medium according to claim 1, characterized in that: The wheat straw is crushed into 2-3 cm segments, soaked in lime water with a pH value of 9.5-10.5 for 2-3 hours, drained and set aside, and the porosity is ≥60%; The soybean meal is dried at 75-85°C and crushed into 1-2 mm pieces for later use, with a crude protein content of ≥45%; The Korean pine leaves are sterilized by ultraviolet irradiation for 5-15 minutes and then set aside; The leaf mold is passed through a 2 mm sieve and then used for later use, and its moisture content is 25-35%.

3. The tunnel fermentation-based matsutake culture medium according to claim 1, characterized in that: The pile height in step (1) is 1.2-1.6 m; and the thermophilic actinomycete inoculation amount in step (3) is 0.8-1.2%.

4. A semi-artificial cultivation method for Matsutake mushrooms, characterized by: The matsutake culture medium used is prepared by the preparation method according to any one of claims 1 to 3; the semi-artificial matsutake cultivation method comprises the following steps: (1) Inoculation: Inoculate the matsutake strain into a liquid culture medium and ferment for 8-10 days to obtain a liquid strain; (2) Cultivation: Mix the liquid spawn into the matsutake culture medium and pre-cultivate in a dark and ventilated environment for 2-4 days to obtain the inoculum; bury the inoculum in the tree pit and cover it with two layers: the lower layer is a mixture of decomposed pine needles and black sand, and the upper layer is living moss, and then spray the symbiotic inducer; (3) Cultivation: Maintain the temperature of the covering layer between 5-25°C, maintain the humidity of the inoculum at 60-65% during the mycelium expansion period, and maintain the humidity of the inoculum at 70-75% during the primordium differentiation period. When the diameter of the cap of the matsutake fruiting body reaches 3-5 cm, pick it.

5. The semi-artificial cultivation method of Tricholoma matsutake according to claim 4, characterized in that: In step (1), the liquid culture medium formula is: peptone 7.0-9.0 g / L, yeast extract 4.5-5.5 g / L, lactose 14.0-16.0 g / L, potassium dihydrogen phosphate 1.4-1.6 g / L, magnesium sulfate 0.4-0.6 g / L, potassium nitrate 1.5-2.5 g / L, calcium chloride 0.15-0.25 g / L, vitamin B1 0.008-0.012 g / L, and pine needle extract 1.8-2.2 g / L.

6. The semi-artificial cultivation method of Tricholoma matsutake according to claim 4, characterized in that: In step (1), the inoculation amount of the matsutake strain is 4-6%; the liquid strain obtained has a pellet diameter of 0.5-1.0 mm, a mycelial biomass of ≥15 g / L, and a pH value of 5.8-6.

2.

7. The semi-artificial cultivation method of Tricholoma matsutake according to claim 4, characterized in that: In step (2), the mass ratio of the liquid spawn to the matsutake culture medium is 1:(8-12).

8. The semi-artificial cultivation method of Tricholoma matsutake according to claim 4, characterized in that: In step (2), during the light-proof and ventilated pre-culture, the temperature is maintained at 24-26°C.

9. The semi-artificial cultivation method of Tricholoma matsutake according to claim 4, characterized in that: In step (2), the tree pit is located at the root system of the pine tree, and the original soil with a thickness of 5-10 cm is retained at the bottom of the pit; when the inoculum is buried in the tree pit, the bottom of the inoculum is close to the tree roots, and the top of the inoculum is 10-20 cm above the ground.

10. The semi-artificial cultivation method of Matsutake according to claim 4, characterized in that: In step (2), the mixture of decomposed pine needles and black sand has a covering thickness of 5-10 cm, a pH value of 6.0-6.5, and the mass percentages of decomposed pine needles and black sand are 30-40% and 70-60% respectively; the living moss is of the genus Glechoma, and the covering thickness is 5-10 cm.