Preparation method of pure mycelium, pure mycelium and application

Abstract

The invention discloses a preparation method of pure mycelia, the pure mycelia and application. The method comprises the following steps: S1, preparing a mycelia solid culture medium; S2, preparing a mycelia paste culture medium; S3, forming the mycelia paste culture medium and fermenting mycelia; according to the method, a continuous, compact and thickness-controllable pure mycelium layer is formed on the surface of a pasty fluid culture medium in a manner of combining solid culture and pasty fluid culture, and a mycelium biological material with excellent physical properties can be obtained without adopting a bracket or an adhesive material. The preparation method has the advantages of simple steps, no need of a complex sterilization mode, short preparation period, low cost, capability of forming large-area pure mycelia at one time, suitability for large-scale industrial production, mild preparation conditions, greenness and environmental protection. The prepared pure mycelium can be applied to mycelium leather or mycelium meat, does not need an adhesive when being used for the mycelium leather, is excellent in material physical performance, does not need chemical or plant adhesives when being used for the mycelium meat, and is good in taste and high in nutrition.

Description

A method for preparing pure mycelium, pure mycelium and its applications Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a method for preparing pure mycelium, the pure mycelium, and its applications. Background Technology

[0002] Mycelium refers to an aggregate of hyphae, composed of many branching filamentous hyphae. Hyphae are formed by the germination and reproduction of spores and are filamentous structures composed of tubular cells. Under suitable culture conditions, individual tubular hyphae can be artificially induced to intertwine and aggregate into dense, sheet-like structures. Currently, mycelial materials mainly fall into two categories: pure mycelial materials and mycelial composite materials. Pure mycelial materials exist in a planar morphology, naturally grown from pure mycelium, and can be processed into flexible materials similar to animal leather, i.e., "mycelial leather," which is currently widely used in clothing, footwear, and other fields. Compared to traditional animal leather, "mycelial leather" has advantages such as being renewable, having low carbon emissions, a short production cycle, and high controllability; compared to traditional synthetic leather, "mycelial leather" does not have the pollution problems associated with chemical materials such as polyurethane (PU) / polyvinyl chloride (PVC), making it a green and environmentally friendly material. Besides its application in materials that replace leather, packaging, and construction, pure mycelial materials can also be used to replace protein foods. The mycelial protein in mycelial biomaterials is rich in a complete amino acid profile, high in protein and dietary fiber, with high nutrient density and low fat content. Compared to artificial meat made from soy protein, it has no beany taste, a texture closer to real meat, and higher nutritional value. Compared to traditional animal husbandry, mycelial growth is shorter, requiring only a few days, and does not rely on large areas of land and pastures, significantly saving water resources and reducing greenhouse gas emissions, thus addressing the environmental pressure caused by methane and other emissions from animal husbandry.

[0003] Currently, many existing technologies have disclosed mycelial materials and preparation methods, but the existing technologies still have the following shortcomings: (1) The mycelial content in the mycelial materials is low, and they rely on composite materials or adhesives. For example, the mycelial materials disclosed in patent documents CN114071992A, CN114127278B, and CN120209598B are all composite materials, and the proportion of mycelium in the composite material is low. In addition, adhesives or scaffold materials are required. Patent document WO2020136448A1 discloses a composite material of mycelium and textiles. This material has the problem of uneven mycelial thickness and low mycelial content, making it difficult to form a large area of ​​pure mycelium. (2) Limited cultivation methods. Currently, mycelium is mainly prepared by liquid culture, such as the preparation method disclosed in patent document CN119506103A, which uses liquid culture medium to cultivate the mycelium film. However, the mycelium film is thin and the liquid culture medium is easily contaminated. (3) Difficult to industrialize. Currently, the traditional mycelium preparation process cannot meet the requirements of large area, controllable thickness and no need for complex sterilization conditions, and cannot be industrialized on a large scale.

[0004] In view of this, it is necessary to further improve the existing methods for preparing pure mycelium. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is that existing mycelial preparation methods are difficult to form large-area pure mycelia, have low mycelial content, are difficult to cultivate, are easy to contaminate the culture medium, and are difficult to meet the requirements of industrial production. Therefore, the present invention proposes a method for preparing pure mycelia that can form large-area pure mycelial layers with controllable thickness, with mild preparation conditions, simple preparation conditions, and can be produced on a large scale for industrial production, as well as pure mycelia and their applications.

[0006] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: The first aspect of the present invention provides a method for preparing pure mycelium, which includes the following steps: S1, preparing a mycelial solid culture medium, providing solid culture medium pulverized material, adjusting the moisture content of the solid culture medium pulverized material to a preset range to obtain a moist solid culture medium, stacking and fermenting the moist solid culture medium and sterilizing it; inoculating the sterilized moist solid culture medium with fungal strains, wherein the fungal strains account for 5%-10% of the moist solid culture medium by mass percentage, and culturing to obtain a mycelial solid culture medium; S2, preparing... Prepare mycelial paste culture medium by dispersing the mycelial solid culture medium and adding 1%-10% of gelatinous material (by weight percentage) to the dispersed mycelial solid culture medium to obtain a premix. Mix the premix with 2-5 times its weight of sterile water to form a mycelial paste culture medium. S3: Mycelial paste culture medium formation and mycelial fermentation: Place the mycelial paste culture medium in a container for cultivation, allowing mycelia to grow into a pure mycelial layer on the surface of the paste. S4: Dehydration treatment: Treat the pure mycelial layer until the water content is no higher than 50%, thus obtaining pure mycelia.

[0007] Preferably, in step S1, the pore size of the solid culture medium pulverized material is no greater than 1 mm; the solid culture medium pulverized material includes at least one of sawdust, mushroom residue, rice bran, palm meal, coffee grounds, sugarcane bagasse powder, and distiller's grains powder, as well as at least one of corn flour, wheat bran, soybean meal, and cassava flour; the fungal strain is at least one of Ganoderma lucidum strain and Pleurotus ostreatus strain.

[0008] Preferably, the moisture content of the moist solid culture medium is 60%-65%; the fermentation time is 12-24 hours, and after fermentation, the pH value of the moist culture medium is adjusted to 6.5-7.5; the sterilization treatment is high-pressure steam sterilization, and the sterilization time is 1-2 hours; the mycelial solid culture medium is obtained by mixing the fungal strain with the moist solid culture medium and culturing at 20-25°C for 3-7 days.

[0009] Preferably, the gelling material is at least one selected from guar gum, xanthan gum, gum arabic, and locust bean gum; after the premix is ​​mixed with the sterile water, it is stirred evenly at a speed of 2000-22000 rpm, and the resulting mixture has a viscosity of 1*10. 4 -1*10 5 mPa·s.

[0010] Preferably, in step S3, after the mycelial paste culture medium is placed in the container, the thickness is controlled to be 10-50 mm; the mycelial paste placed in the container is statically cultured for 5-30 days under the conditions of temperature 20-25℃, relative humidity 80%-100%, carbon dioxide concentration 0-3%, and oxygen concentration 18%-21%.

[0011] Preferably, step S3 further includes controlling the concentration of carbon dioxide and oxygen through ventilation and humidifying with a dry fog nozzle with an aperture of no more than 10 μm.

[0012] Preferably, step S4 includes: flattening the pure mycelium layer, blowing dry the free moisture on the surface of the pure mycelium layer, drying at 40-55℃ for 24-48 hours, and the thickness of the pure mycelium layer is 15-100mm.

[0013] A second aspect of the present invention provides a pure mycelium, which is obtained by the preparation method described above.

[0014] A third-party aspect of this invention provides the application of the pure mycelium described above in mycelial leather or mycelial flesh.

[0015] Preferably, when the pure mycelium is applied to mycelial leather, the pure mycelium is soaked in 2-10% glycerol for 12-24 hours, cross-linked with 1-5% citric acid at 120-160℃ for 2-10 minutes, and hot-pressed at 60-80℃ and 0.5-5MPa for 30-60 seconds. Finally, it is washed with water and dried at 40-60℃ until the moisture content is less than 10%. When the pure mycelium is applied to mycelial flesh, the pure mycelium is cold-pressed and compacted at 0.2-1.2MPa.

[0016] Compared with the prior art, the above-mentioned technical solution of the present invention has the following advantages: The method for preparing pure mycelium provided by the present invention includes the following steps: S1, preparing a solid mycelial culture medium; S2, preparing a mycelial paste culture medium; S3, forming the mycelial paste culture medium and fermenting the mycelium; S4, dehydration treatment. Through the combination and synergistic effect of solid culture and paste culture, a continuous, dense, and controllable thickness pure mycelial layer is formed on the surface of the paste culture medium. In the pure mycelial layer, the mycelial content is greater than 99%. Without the use of scaffolds or adhesive materials, mycelial biomaterials with excellent physical properties can be obtained. This preparation method is simple, does not require complex sterilization methods, has a short preparation cycle, low cost, and can form a large area of ​​pure mycelium in one step, making it suitable for large-scale industrial production. At the same time, the preparation conditions are mild, which is in line with the trend of green manufacturing. The pure mycelium obtained by this preparation method can be used in mycelial leather or mycelial meat. When used in mycelial leather, no adhesive is required, and the material has excellent physical properties. When used in mycelial meat, no chemical or plant adhesives are required, resulting in good taste and high nutritional value. Detailed Implementation

[0017] To make the content of this invention easier to understand, the invention will be further described in detail below according to specific embodiments.

[0018] Example 1 This example provides a method for preparing pure mycelium, including the following steps: S1. Preparation of mycelial solid culture medium: S11. Preparation of sterilized solid culture medium: Provide solid culture medium pulverized material, which includes at least one of sawdust, mycelial residue, rice bran, palm meal, coffee grounds, sugarcane bagasse powder, distiller's grains powder, corn flour, wheat bran, soybean meal, and cassava flour. Specifically, in this example, the solid culture medium pulverized material, by weight, includes 60 parts sawdust, 30 parts mycelial residue, 10 parts rice bran, and 20 parts wheat bran. After cleaning the sawdust, mycelial residue, rice bran, and wheat bran, pulverize them to a pore size of no more than 1 mm. Mix the sawdust, mycelial residue, and rice bran evenly according to the above weight parts, add water to adjust the moisture content to 60-65%, pile and ferment for 12 hours. After fermentation, detect the pH value and adjust the pH value to 6.5-7.5. Add wheat bran to the fermented mixture and mix thoroughly to obtain a moist solid culture medium. The moist solid culture medium was subjected to high-pressure steam sterilization. The conditions for high-pressure steam sterilization were: sterilization temperature of 121℃, sterilization pressure of 0.15MPa, and sterilization time of 2h. After cooling, sterilized solid culture medium was obtained.

[0019] S12. Under aseptic conditions, fungal strains are inoculated onto the sterilized moist solid culture medium. In this embodiment, Ganoderma lucidum strains are used as fungal strains. The inoculation amount of Ganoderma lucidum strains is 10% of the mass of the moist solid culture medium. After inoculation, the mixture is mixed evenly to obtain pre-culture material.

[0020] S13. Solid culture: Place the pre-culture material obtained in step S12 into a sterile bag and culture for 7 days at a temperature of 20-25℃, a relative humidity of not less than 60%, and a carbon dioxide concentration of less than 700ppm to obtain mycelial solid culture medium. In the mycelial solid culture medium, the mycelium has completely wrapped the solid culture medium.

[0021] S2. Preparation of mycelial paste culture medium: S21. Completely disperse the mycelial solid culture medium and pass it through a 16-mesh sieve without damaging the pore size of the original solid culture medium powder. Add 9% of the gelling material to the dispersed mycelial solid culture medium by mass percentage to obtain a premix. In this embodiment, the gelling material includes xanthan gum accounting for 3% of the mass of the mycelial solid culture medium and locust bean gum accounting for 6% of the mass of the mycelial solid culture medium by mass percentage.

[0022] S22. Add 2 parts by weight of sterile water to the premix. Place the resulting mixture in a high-speed mixer and stir at 22,000 rpm to ensure thorough mixing of the aqueous phase and the premix, forming a fluid mycelial paste culture medium. The viscosity of the mycelial paste culture medium should be controlled at 1*10. 5 mPa·s.

[0023] S3. Mycelial paste culture medium forming and mycelial fermentation: S31. Place the mycelial paste culture medium in a mold or shallow dish container and gently shake it to make the paste smooth. In this embodiment, the paste thickness is controlled at 30mm.

[0024] S32. Place the container containing the mycelial paste culture medium in an environment with a temperature of 20-22℃, a relative humidity of 80%-100%, a carbon dioxide concentration of 0-3%, and an oxygen concentration of 18%-21% and let it stand for 20 days. During this period, intermittent ventilation is carried out using a micro fan to adjust the carbon dioxide and oxygen concentrations. The fan speed should not exceed 0.2m / s. When the ambient humidity decreases due to ventilation, water is added and humidified through a dry fog nozzle with an aperture of no more than 10μm. After cultivation, the mycelium grows upward on the surface of the paste culture medium to form a uniform, dense, and continuous pure mycelial layer. In this embodiment, the thickness of the pure mycelial layer is 75-80mm.

[0025] S4. Dehydration treatment: Press the pure mycelium layer with a plate to flatten the aerial mycelium, dry the free water on the surface of the pure mycelium layer by low-speed ventilation, and then slowly dry it at 40°C for 48 hours to reduce the water content of the pure mycelium to about 50%.

[0026] This embodiment also provides a pure mycelium, which is prepared by the above method.

[0027] This embodiment also provides an application of the above-mentioned pure mycelium, which is applied to mycelial leather.

[0028] The above-mentioned pure mycelium is applied to mycelial leather. The pure mycelium is soaked in 10% glycerol for 24 hours to plasticize it, then crosslinked with 5% citric acid at 160°C for 10 minutes, then hot-pressed at 80°C and 5MPa for 60 seconds, and finally washed with water and dried at 60°C until the moisture content is less than 10% to obtain pure mycelial leather. Pure mycelial leather can be further used in packaging, clothing, automobiles, home furnishings and other fields.

[0029] The method for preparing pure mycelium provided in this embodiment combines primary basal culture on solid culture medium with secondary culture on paste culture medium through a synergistic effect. This results in a continuous and dense pure mycelium layer on the surface of the paste culture medium, with a mycelium content greater than 99%. Through secondary culture on the paste culture medium, the thickness of the pure mycelium layer can be controlled between 15-100 mm, and different thicknesses can meet different application requirements. Compared with traditional mycelium culture methods, this method does not rely on composite materials, scaffolds, or adhesives, nor does it require strict and complex sterilization methods. The preparation method is simple, has a short preparation cycle (only a few days), and is low in cost. It can produce large-area pure mycelium in a single step, making it suitable for large-scale industrial production. Furthermore, the preparation conditions are mild and environmentally friendly, saving water resources and reducing greenhouse gas emissions, aligning with the trend of green manufacturing. The pure mycelium obtained by this method can be used in mycelial leather. When used in mycelial leather, no adhesive is required, and the material exhibits excellent physical properties.

[0030] Example 2 This example provides a method for preparing pure mycelium, including the following steps: S1, preparing mycelial solid culture medium: S11, preparing sterilized solid culture medium: providing solid culture medium pulverized material, which includes at least one of sawdust, mycelial residue, rice bran, palm meal, coffee grounds, sugarcane bagasse powder, distiller's grains powder, corn flour, wheat bran, soybean meal, and cassava flour. Specifically, in this example, the solid culture medium pulverized material, by weight, includes 65 parts palm meal, 35 parts coffee grounds, and 20 parts soybean meal. After cleaning the palm meal, coffee grounds, and soybean meal, pulverize them to a pore size of no more than 1 mm. Mix the palm meal and coffee grounds evenly according to the above weight proportions, add water to adjust the moisture content to 60-65%, pile them up for fermentation for 24 hours, detect the pH value after fermentation, and adjust the pH value to 6.5-7.5. Add soybean meal to the fermented mixture and mix thoroughly to obtain a moist solid culture medium. The moist solid culture medium was subjected to high-pressure steam sterilization. The conditions for high-pressure steam sterilization were: sterilization temperature of 121℃, sterilization pressure of 0.15MPa, and sterilization time of 1h. After cooling, sterilized solid culture medium was obtained.

[0031] S12. Under aseptic conditions, fungal strains are inoculated onto the sterilized moist solid culture medium. In this embodiment, oyster mushroom strains are used. The inoculation amount of oyster mushroom strains is 5% of the mass of the moist solid culture medium. After inoculation, the mixture is mixed evenly to obtain the pre-culture material.

[0032] S13. Solid culture: Place the pre-culture material obtained in step S12 into a sterile bag and culture it for 4 days at a temperature of 20-25℃, a relative humidity of not less than 60%, and a carbon dioxide concentration of less than 700ppm to obtain mycelial solid culture medium. In the mycelial solid culture medium, the mycelium has completely wrapped the solid culture medium.

[0033] S2. Preparation of mycelial paste culture medium: S21. Completely disperse the mycelial solid culture medium, and pass it through a 16-mesh sieve without damaging the pore size of the original solid culture medium powder. Add 8% of the gelling material to the dispersed mycelial solid culture medium by mass percentage to obtain a premix. In this embodiment, the gelling material is locust bean gum.

[0034] S22. Add 5 parts by weight of sterile water to the premix. Place the resulting mixture in a high-speed mixer and stir at 22,000 rpm to ensure thorough mixing of the aqueous phase and the premix, forming a fluid mycelial paste culture medium. The viscosity of the mycelial paste culture medium should be controlled at 5 x 10⁻⁶. 4 mPa·s.

[0035] S3. Mycelial paste culture medium forming and mycelial fermentation: S31. Place the mycelial paste culture medium in a mold or shallow dish container and wait for 60 seconds until the paste flows smoothly. In this embodiment, the paste thickness is 20 mm.

[0036] S32. Place the container containing the mycelial paste culture medium in an environment with a temperature of 23-25℃, a relative humidity of 80-100%, a carbon dioxide concentration of 0-3%, and an oxygen concentration of 18-21% and let it stand for 15 days. During this period, intermittent ventilation is carried out using a micro fan to adjust the carbon dioxide and oxygen concentrations. The fan speed should not exceed 0.2m / s. When the ambient humidity decreases due to ventilation, water is added and humidified through a dry fog nozzle with an aperture of no more than 10μm. After cultivation, the mycelium grows upward on the surface of the paste culture medium to form a uniform, dense, and continuous pure mycelial layer. In this embodiment, the thickness of the pure mycelial layer is 66-70mm.

[0037] S4. Dehydration treatment: Press the pure mycelium layer with a plate to flatten the aerial mycelium, dry the free water on the surface of the pure mycelium layer by low-speed ventilation, and then slowly dry it at 55°C for 48 hours to reduce the water content of the pure mycelium to about 50%.

[0038] This embodiment also provides a pure mycelium, which is prepared by the above method.

[0039] This embodiment also provides an application of the above-mentioned pure mycelium, which is applied to mycelial meat.

[0040] The aforementioned pure mycelium, when applied to mycelial meat, mimics the structure of animal muscle fibers. This process includes a pressing step: the pure mycelium is cold-pressed at 0.2 MPa to compact it, forming a stable structure without the addition of any adhesives, thickeners, or cross-linking agents. This process makes the mycelial fibers more uniform in arrangement and increases their density, forming a longitudinal striation structure similar to animal muscle, thus making the chewiness and layered structure of mycelial meat more closely resemble that of animal meat.

[0041] Example 3 This example provides a method for preparing pure mycelium, including the following steps: S1, preparing mycelial solid culture medium: S11, preparing sterilized solid culture medium: providing solid culture medium pulverized material, which includes at least one of sawdust, mycelial residue, rice bran, palm meal, coffee grounds, sugarcane bagasse powder, distiller's grains powder, corn flour, wheat bran, soybean meal, and cassava flour. Specifically, in this example, the solid culture medium pulverized material, by weight, includes 55 parts sawdust, 45 parts palm meal, and 20 parts cassava flour. After cleaning the sawdust, palm meal, and cassava flour, pulverize them to a pore size of no more than 1 mm. Mix the sawdust and palm meal evenly according to the above weight proportions, add water to adjust the moisture content to 60-65%, pile and ferment for 12 hours. After fermentation, detect the pH value and adjust the pH value to 6.5-7.5. Add cassava flour to the fermented mixture and mix thoroughly to obtain a moist solid culture medium. The moist solid culture medium was subjected to high-pressure steam sterilization. The conditions for high-pressure steam sterilization were: sterilization temperature of 121℃, sterilization pressure of 0.15MPa, and sterilization time of 2h. After cooling, sterilized solid culture medium was obtained.

[0042] S12. Under aseptic conditions, fungal strains are inoculated onto the sterilized moist solid culture medium. In this embodiment, Ganoderma lucidum strains are used as fungal strains. The inoculation amount of Ganoderma lucidum strains is 5% of the mass of the moist solid culture medium. After inoculation, the mixture is mixed evenly to obtain pre-culture material.

[0043] S13. Solid culture: Place the pre-culture material obtained in step S12 into a sterile bag and culture for 3 days at a temperature of 20-25℃, a relative humidity of not less than 60%, and a carbon dioxide concentration of less than 700ppm to obtain mycelial solid culture medium. In the mycelial solid culture medium, the mycelium has completely wrapped the solid culture medium.

[0044] S2. Preparation of mycelial paste culture medium: S21. Completely disperse the mycelial solid culture medium, and pass it through a 16-mesh sieve without damaging the pore size of the original solid culture medium powder. Add 1% of a gelling material to the dispersed mycelial solid culture medium by mass percentage to obtain a premix. In this embodiment, the gelling material is gum arabic.

[0045] S22. Add 5 times the weight of sterile water to the premix. Place the resulting mixture in a high-speed mixer and stir at 2000 rpm to ensure thorough mixing of the aqueous phase and the premix, forming a fluid mycelial paste culture medium. The viscosity of the mycelial paste culture medium should be controlled at 1*10. 4 mPa·s.

[0046] S3. Mycelial paste culture medium forming and mycelial fermentation: S31. Place the mycelial paste culture medium in a mold or shallow dish container and wait for 30 seconds until the paste flows smoothly. In this embodiment, the paste thickness is 5 mm.

[0047] S32. Place the container containing the mycelial paste culture medium in an environment with a temperature of 20-22℃, a relative humidity of 80-100%, a carbon dioxide concentration of 0-3%, and an oxygen concentration of 18-21% and let it stand for 5 days. During this period, intermittent ventilation is carried out using a micro fan to adjust the carbon dioxide and oxygen concentrations. The fan speed should not exceed 0.2m / s. When the ambient humidity decreases due to ventilation, water is added and humidified through a dry fog nozzle with an aperture of no more than 10μm. After cultivation, the mycelium grows upward on the surface of the paste culture medium to form a uniform, dense, and continuous pure mycelial layer. In this embodiment, the thickness of the pure mycelial layer is 15-18mm.

[0048] S4. Dehydration treatment: Press the pure mycelium layer with a plate to flatten the aerial mycelium, dry the free water on the surface of the pure mycelium layer by low-speed ventilation, and then slowly dry it at 45°C for 48 hours to reduce the water content of the pure mycelium to about 50%.

[0049] This embodiment also provides a pure mycelium, which is prepared by the above method.

[0050] This embodiment also provides an application of the above-mentioned pure mycelium, which is applied to mycelial leather.

[0051] The above-mentioned pure mycelium was applied to mycelial leather. The pure mycelium was soaked in 2% glycerol for 12 hours to plasticize it. Then, it was cross-linked with 1% citric acid at 120°C for 2 minutes. Next, it was hot-pressed at 60°C and 0.5 MPa for 30 seconds. Finally, it was washed with water and dried at 40°C until the moisture content was less than 10% to obtain pure mycelial leather.

[0052] Example 4 This example provides a method for preparing pure mycelium, including the following steps: S1, preparing mycelial solid culture medium: S11, preparing sterilized solid culture medium: providing solid culture medium pulverized material, which includes at least one of sawdust, mycelial residue, rice bran, palm meal, coffee grounds, sugarcane bagasse powder, distiller's grains powder, corn flour, wheat bran, soybean meal, and cassava flour. Specifically, in this example, the solid culture medium pulverized material, by weight, includes 70 parts sugarcane bagasse powder, 30 parts distiller's grains powder, and 20 parts corn flour. After cleaning the sugarcane bagasse powder, distiller's grains powder, and corn flour, pulverize them to a pore size of no more than 1 mm. Mix the sugarcane bagasse powder and distiller's grains powder evenly according to the above weight parts, add water to adjust the moisture content to 60-65%, pile and ferment for 24 hours. After fermentation, detect the pH value and adjust the pH value to 6.5-7.5. Add corn flour to the fermented mixture and mix thoroughly to obtain a moist solid culture medium. The moist solid culture medium was subjected to high-pressure steam sterilization. The conditions for high-pressure steam sterilization were: sterilization temperature of 121℃, sterilization pressure of 0.15MPa, and sterilization time of 1h. After cooling, sterilized solid culture medium was obtained.

[0053] S12. Under aseptic conditions, fungal strains are inoculated onto the sterilized moist solid culture medium. In this embodiment, oyster mushroom strains are used. The inoculation amount of oyster mushroom strains is 10% of the mass of the moist solid culture medium. After inoculation, the mixture is stirred evenly to obtain the pre-culture medium.

[0054] S13. Solid culture: Place the pre-culture material obtained in step S12 into a sterile bag and culture for 7 days at a temperature of 20-25℃, a relative humidity of not less than 60%, and a carbon dioxide concentration of less than 700ppm to obtain mycelial solid culture medium. In the mycelial solid culture medium, the mycelium has completely wrapped the solid culture medium.

[0055] S2. Preparation of mycelial paste culture medium: S21. Completely disperse the mycelial solid culture medium, and pass it through a 16-mesh sieve without damaging the pore size of the original solid culture medium powder. Add 10% of the gelling material to the dispersed mycelial solid culture medium by mass percentage to obtain a premix. In this embodiment, the gelling material is guar gum.

[0056] S22. Add 3 times the weight of sterile water to the premix. Place the resulting mixture in a high-speed mixer and stir at 15,000 rpm to ensure thorough mixing of the aqueous phase and the premix, forming a fluid mycelial paste culture medium. The viscosity of the mycelial paste culture medium should be controlled at 8*10. 4 mPa·s.

[0057] S3. Mycelial paste culture medium forming and mycelial fermentation: S31. Place the mycelial paste culture medium in a mold or shallow dish container and gently shake it to help the paste become flat. In this embodiment, the paste thickness is 50mm.

[0058] S32. The container containing the mycelial paste culture medium is placed in an environment with a temperature of 23-25℃, a relative humidity of 85%, a carbon dioxide concentration of 0-3%, and an oxygen concentration of 18-21% and is statically cultured for 30 days. During this period, intermittent ventilation is performed using a micro fan to adjust the carbon dioxide and oxygen concentrations. The fan speed does not exceed 0.2 m / s. When the ambient humidity decreases due to ventilation, water is added and humidified through a dry fog nozzle with an aperture of no more than 10 μm. After cultivation, the mycelia grow upward on the surface of the paste culture medium, forming a uniform, dense, and continuous pure mycelial layer. In this embodiment, the thickness of the pure mycelial layer is 92-100 mm. During the continuous 30-day cultivation, the pure mycelial structure formed on the surface of the paste remains continuous and dense. No fruiting bodies or fruiting body primordia are observed, and the mycelial morphology is always dominated by vegetative growth.

[0059] S4. Dehydration treatment: Press the pure mycelium layer with a plate to flatten the aerial mycelium, dry the free water on the surface of the pure mycelium layer by low-speed ventilation, and then slowly dry it at 50°C for 48 hours to reduce the water content of the pure mycelium to about 50%.

[0060] This embodiment also provides a pure mycelium, which is prepared by the above method.

[0061] The aforementioned pure mycelium, when applied to mycelial meat, mimics the structure of animal muscle fibers. This process includes a pressing step: the pure mycelium is cold-pressed at 1.2 MPa to compact it, forming a stable structure without the addition of any adhesives, thickeners, or cross-linking agents. This process makes the mycelial fibers more uniform in arrangement and increases their density, forming a longitudinal striation structure similar to animal muscle, thus making the chewiness and layered structure of mycelial meat more closely resemble that of animal meat.

[0062] Comparative Example 1 This comparative example provides a method for preparing pure mycelium, which is basically the same as that in Example 1, except that the solid culture medium pulverized material in step S11 includes 65 parts bean stalks, 20 parts corn stalks and 15 parts wheat stalks, and the pore size of the solid culture medium pulverized material is no greater than 1 mm and is processed through a 16-mesh sieve.

[0063] Comparative Example 2 provides a method for preparing pure mycelium, which is basically the same as that in Example 1, except that the solid culture medium powder in step S11 includes 65 parts bean stalks, 20 parts corn stalks and 15 parts wheat stalks, and the pore size of the solid culture medium powder is greater than 1 mm.

[0064] Comparative Example 3 provides a method for preparing pure mycelium, which is basically the same as that in Example 1, except that the pore size of the solid culture medium powder is greater than 1 mm.

[0065] Comparative Example 4 provides a method for preparing pure mycelium, which is basically the same as that in Example 1, except that in step S21, no gelatinous material is added to the dispersed mycelium solid culture medium.

[0066] Comparative Example 5 provides a method for preparing pure mycelium, which is basically the same as that in Example 1, except that in step S21, 11% locust bean gum is added to the dispersed mycelium solid culture medium by mass percentage.

[0067] Comparative Example 6 provides a method for preparing pure mycelium, which is basically the same as that in Example 1, except that in step S21, 0.5% of locust bean gum is added to the dispersed mycelium solid culture medium by mass percentage.

[0068] Comparative Example 7 provides a method for preparing pure mycelium, which is basically the same as that in Example 1, except that in step S22, 10 parts by weight of water are added to the premix.

[0069] Comparative Example 8 provides a method for preparing pure mycelium, which is basically the same as that in Example 1, except that in step S22, water with a weight ratio of 1 part is added to the premix.

[0070] Comparative Example 9 provides a method for preparing pure mycelium, which is basically the same as that in Example 1, except that in step S22, the obtained mixture is placed in a high-speed mixer and stirred at a speed of 40,000 rpm to mix the aqueous phase with the premix.

[0071] Comparative Example 10 provides a method for preparing pure mycelium, which is basically the same as that in Example 1, except that in step S22, the obtained mixture is placed in a high-speed mixer and stirred at a speed of 1000 rpm to mix the aqueous phase with the premix.

[0072] Comparative Example 11 provides a method for preparing pure mycelium, which is basically the same as that in Example 1, except that the thickness of the paste is 100 mm.

[0073] Comparative Example 12 provides a method for preparing pure mycelium, which is basically the same as that in Example 1, except that the thickness of the paste is 2 mm.

[0074] Comparative Example 13 provides a method for preparing pure mycelium, which is basically the same as that in Example 1, except that in step S32, the temperature in the culture environment of the mycelium paste culture medium is uncontrolled and fluctuates within the range of 16-29℃.

[0075] Comparative Example 14 provides a method for preparing pure mycelium, which is basically the same as that in Example 1, except that in step S32, the humidity in the culture environment of the mycelium paste culture medium is uncontrolled and fluctuates within the range of 15%-90%.

[0076] Comparative Example 15 provides a method for preparing pure mycelium, which is basically the same as that in Example 1, except that in step S32, the carbon dioxide concentration in the culture environment of the mycelium paste culture medium is uncontrolled and fluctuates within the range of 3%-10%.

[0077] Comparative Example 16 provides a method for preparing pure mycelium, which is basically the same as that in Example 1, except that in step S32, the fraction of the micro fan is greater than 0.2 m / s.

[0078] Comparative Example 17 provides a method for preparing pure mycelium, which is basically the same as that in Example 1, except that in step S32, water is added and humidified through a dry fog nozzle with an aperture greater than 10 μm.

[0079] Comparative Example 18 provides a method for preparing pure mycelium, which is basically the same as that in Example 1, except that in step S4, the pure mycelium is not treated with plate pressing.

[0080] Comparative Example 19 provides a method for preparing pure mycelium, which is basically the same as that in Example 1, except that in step S4, the mycelium is dried by hot air circulation at a temperature of 105°C for 2 hours.

[0081] Comparative Example 20 provides a method for preparing mycelial mats. This preparation method adopts the method disclosed in patent document CN119506103A, including the following steps: 1. Preparation of mycelial slurry: Homogenized liquid culture (Porphyromonas spores) is inoculated into 200 mL of MEB medium at an inoculation rate of 5%, and cultured with shaking at 130 rpm and 25°C for 3 days. Then, it is taken out and inoculated into 1000 mL of MEB medium under aseptic conditions, and cultured in a shake flask for 3-5 days. Then, it is homogenized at 10000 rpm for 20 seconds under aseptic conditions to prepare mycelial mats.

[0082] 2. Preparation of mycelial pads: Under aseptic conditions, the obtained mycelial slurry was mixed with surface liquid culture medium (PDB enriched medium, MEB medium and optimized medium (II-5)), shaken evenly, and poured into a stainless steel dish with an inner diameter of 600mm×400mm, with a liquid layer height of 10mm. The dish was covered with a stainless steel lid with a breathable membrane (with a 1.5cm diameter hole), and the edges of the lid were sealed with plastic wrap. The dish was then incubated at 20-22℃ for 20 days. During the incubation process, the mycelial culture layer was turned upside down to obtain mycelial pads.

[0083] Comparative Example 21 provides a method for preparing Ganoderma lucidum mycelium. This method adopts the method disclosed in patent document CN120209598B, and includes the following steps: 5 mL of Ganoderma lucidum spore suspension is inoculated into a 250 mL Erlenmeyer flask containing 100 mL of MEB medium, and placed in a shaking incubator for 3 days at 25°C and 130 rpm. Then, it is inoculated into a 2 L Erlenmeyer flask containing 800 mL of MEB medium and incubated at a constant temperature of 25°C and 130 rpm for 8 days, and the mycelium is obtained by filtration.

[0084] Comparative Example 22 provides a method for preparing mycelial material. This method adopts the method disclosed in patent document WO2020136448A1, including the following steps: 80 parts by weight of sawdust and 20 parts by weight of wheat bran are mixed evenly, water is added to adjust the moisture content to 60%–65%, and lime is added to adjust the pH to 6.5–7.5. The mixture is sterilized at 121 ℃ and 0.15 MPa for 2 h, cooled to 27–30 ℃, and then inoculated with Ganoderma lucidum spores. After 14 days of cultivation, the spores are allowed to grow and cover the surface of the culture medium. After crushing, the spores are filled into a 600mm*400mm mold, and after a second cultivation for 3 days, the mold is removed. After demolding, a layer of gauze is added to the surface of the culture medium, and the mixture is cultivated a third time for 5 days. The surface mycelium is pressed by hand to ensure close contact with the gauze, and finally separated from the substrate surface to obtain a complex of gauze and mycelium.

[0085] Experimental Example 1: The substrate weight, mycelium weight, size, thickness, water content, mycelium ratio, surface continuity, and appearance of fruiting body primordia in the preparation methods provided in Examples 1-4 and Comparative Examples 1-21 of this application were tested. The test results are shown in Table 1 below.

[0086] Table 1 In the above tests, surface continuity specifically refers to: continuous mycelium when there is no exposed culture medium material on the surface at harvest time, and discontinuous mycelium otherwise.

[0087] As can be seen from the above test results, compared with Example 1, Comparative Example 1 replaced the solid culture medium powder with straw powder. Although it was possible to obtain continuous pure mycelium, the amount of mycelium was significantly lower than that of the pure mycelium obtained in Example 1 of this application. At the same time, it is difficult to process long fiber materials such as bean stalks and corn stalks into particles with a diameter of no more than 1 mm, which will result in raw material waste and increase production costs.

[0088] Compared with Example 1, in Comparative Examples 2-3, after changing the particle size of the solid culture medium pulverizer to be greater than 1 mm, the mycelia obtained from the culture were unevenly distributed on the surface of the paste culture medium, the matrix particles were obviously exposed, and a continuous pure mycelial layer could not be formed on the surface of the paste, only a loose mycelial network structure was formed.

[0089] Compared to Example 1, Comparative Examples 4-10 varied the proportions of gelatinous material, sterile water, and stirring speed. When the proportion of gelatinous material was reduced, the proportion of sterile water was increased, and the stirring speed was increased, the resulting paste-like culture medium exhibited insufficient structural stability, collapsed during cultivation, and showed significant stratification and water separation after mixing or standing for a period of time. The mycelial growth was discontinuous, and it was impossible to obtain structurally complete pure mycelia. Conversely, increasing the proportion of gelatinous material, decreasing the proportion of sterile water, and decreasing the stirring speed significantly reduced the porosity of the resulting paste-like culture medium, restricted oxygen diffusion, decreased mycelial growth rate, and resulted in localized mycelial aging and blank areas.

[0090] Compared to Example 1, Comparative Examples 11-12 varied the thickness of the paste culture medium. Increasing the thickness resulted in insufficient oxygen exchange near the bottom of the paste culture medium, leading to localized mycelial stagnation and mycelial aging, resulting in a significant decrease in yield. Decreasing the thickness of the paste culture medium significantly reduced the thickness of the mycelial layer on the surface, resulting in a marked decrease in conversion efficiency and increased production costs.

[0091] Compared with Example 1, Comparative Examples 13-17 changed the culture environment of the mycelial paste culture medium. Sudden changes in temperature and humidity, increased wind speed or increased spray particle size can inhibit the growth of mycelia and cause them to emerge from primordia due to unsuitable growth environment. In the environment of high carbon dioxide concentration, although mycelia do not emerge from primordia, the mycelial layer stops growing upward after reaching a certain extent.

[0092] Compared with Example 1, Comparative Example 18 did not use a flat plate to flatten the surface aerial mycelium, and the surface of the pure mycelium obtained had some mycelial spore powder falling off.

[0093] Compared with Example 1, Comparative Example 19 used high-temperature hot air circulation to dry the pure mycelium layer. This drying method caused rapid loss of moisture, and the mycelium showed obvious shrinkage and local breakage after drying.

[0094] Compared with Example 1, the preparation method used in Comparative Example 20 can only produce a pure mycelial layer with a thickness of 1-2 mm, and the liquid culture medium is easily contaminated by other bacteria, requiring complex and strict sterilization methods.

[0095] Compared with Example 1, the preparation method used in Comparative Example 21 can only obtain mycelial balls in liquid shake flasks, and cannot obtain whole pure mycelial bodies.

[0096] Compared with Example 1, the preparation method used in Comparative Example 22 can only obtain a complex of gauze and mycelium with a thickness of about 1-2 mm, and the mycelium content is only about 25%.

[0097] 2. The mycelium obtained by the preparation methods provided in Examples 1, 3 and Comparative Examples 1, 12, 15, 18, 19, 20, 21 and 22 was further prepared into mycelial leather, and the tensile strength, tensile strength at break and tear strength of the prepared mycelial leather were tested.

[0098] The mycelium prepared by the preparation methods provided in Examples 1 and 3 and Comparative Examples 1, 12, 15, 18, and 19 was processed into mycelial leather through the following steps: the mycelial layer was soaked in 10% glycerol for 24 hours to plasticize it, then crosslinked with 5% citric acid at 160°C for 10 minutes, then hot-pressed at 80°C and 5 MPa for 30 seconds, and finally washed with water and dried at 60°C until the moisture content was less than 10% to obtain pure mycelial leather.

[0099] The mycelium prepared by the preparation method provided in Comparative Example 20 was processed into mycelial leather by the following steps: the mycelium was soaked in 12.5% ​​glycerol for 24 hours, then cross-linked with 1.5% genipin, and then hot-pressed at 80°C and 5 MPa for 30 seconds to obtain pure mycelial leather.

[0100] The mycelium prepared by the method provided in Comparative Example 21 was processed into mycelial leather through the following steps: Mycelial balls were mixed with 50 times the amount of water, and then a 40% modified PVA solution was added and mixed. During the stirring process, 2.8 wt% glycerol was added, and after stirring evenly, the mixture was poured into a shallow dish and refrigerated in a refrigerator at 2-4°C for 72 hours. Then, the mixture was placed in an oven at 60°C and dried for 8 hours. After removing the mixture, the mycelial material was peeled off from the shallow dish to obtain a mycelial leather-like material.

[0101] The mycelium prepared by the method provided in Comparative Example 22 was processed into mycelial leather through the following steps: Salt was evenly sprinkled on the surface of the mycelium, and then the mycelium was placed in water and heated to boiling. A humectant was added to retain the moisture on the surface of the mycelium. After storage, the mycelium was ready for moisturizing treatment. A 10% glycerol solution was applied to the upper and lower surfaces of the mycelium and allowed to stand for 30-60 minutes. Finally, it was dried with hot air at 80℃ to obtain the mycelial leather material.

[0102] The performance test data of mycelium leather are shown in Table 2 below: Table 2 The test results above show that the pure mycelium prepared by the method provided in this application has the best properties. After further processing into mycelial leather, it is superior to the comparative example in terms of tensile strength and tear elongation, and the breaking tensile rate fully meets the requirements for leather use. The pure mycelium layer obtained by the preparation method provided in Comparative Example 20 through liquid culture medium has a small thickness, and the mycelial leather further prepared has poor performance, is easily contaminated, and is difficult to promote on a large scale. The mycelial balls prepared by the preparation method provided in Comparative Example 21 have no fibrous structure arrangement of mycelium, and the mycelial leather further prepared has even worse performance. The gauze and mycelial composite material prepared by the preparation method provided in Comparative Example 22 has low mycelial content, poor performance, and insufficient environmental friendliness.

[0103] 3. The pure mycelium provided in Example 2 was cold-pressed and compacted under a pressure of 1.2 MPa to obtain mycelial meat. A stable structure could be formed without the addition of any adhesives, thickeners, or cross-linking agents. The obtained mycelial meat has a more uniform fiber arrangement and increased density, forming a longitudinal striation structure similar to animal muscle. The mycelial meat was cut into pieces of 20cm*4cm*6-10cm and boiled. The mycelial meat had a texture similar to chicken breast.

[0104] The test data for mycelium meat, plant protein meat, and chicken breast provided in the embodiments of this application are shown in Table 3 below: Table 3 The test results above show that the mycelium meat provided in this application embodiment maintains a high protein content while having a significantly lower fat content than commercially available plant-based meat products. It also naturally contains dietary fiber but no cholesterol, demonstrating a superior nutritional structure. Its key textural parameters, such as elasticity and cohesion, are similar to chicken breast and significantly superior to commercially available plant-based meat products, indicating that it has structural characteristics closer to animal meat. Furthermore, without adding any adhesives, the mycelium, relying on its self-formed three-dimensional network structure, achieves stable molding after appropriate dehydration and compaction.

[0105] The mycelium meat provided in this application embodiment is produced by inactivating mycelium and forming it using a purely physical method, without introducing chemical cross-linking agents, so that the product has good food safety while maintaining structural stability.

[0106] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing pure mycelium, characterized in that, The process includes the following steps: S1. Preparing a mycelial solid culture medium, providing a solid culture medium powder, adjusting the moisture content of the solid culture medium powder to a preset range to obtain a moist solid culture medium, stacking and fermenting the moist solid culture medium and sterilizing it; S1. Inoculate the sterilized moist solid culture medium with fungal strains, the fungal strains accounting for 5%-10% of the moist solid culture medium by mass, and culture to obtain a mycelial solid culture medium; S2. Prepare a mycelial paste culture medium by dispersing the mycelial solid culture medium and adding 1%-10% of a gelatinous material by mass to the dispersed mycelial solid culture medium to obtain a premix. Mix the premix with 2-5 times its weight of sterile water evenly to form a mycelial paste culture medium; S3. Form the mycelial paste culture medium and ferment the mycelium. Place the mycelial paste culture medium in a container for cultivation, allowing the mycelium to grow into a pure mycelial layer on the surface of the paste; S4. Dehydration treatment: treat the pure mycelial layer until the water content is no more than 50%, thus obtaining pure mycelium.

2. The preparation method according to claim 1, characterized in that, In step S1, the pore size of the solid culture medium pulverized material is no greater than 1 mm; the solid culture medium pulverized material includes at least one of sawdust, mushroom residue, rice bran, palm meal, coffee grounds, sugarcane bagasse powder, and distiller's grains powder, as well as at least one of corn flour, wheat bran, soybean meal, and cassava flour; the fungal strain is at least one of Ganoderma lucidum strain and Pleurotus ostreatus strain.

3. The preparation method according to claim 2, characterized in that, The moisture content of the moist solid culture medium is 60%-65%; the fermentation time is 12-24 hours, and after fermentation, the pH value of the moist culture medium is adjusted to 6.5-7.5; the sterilization treatment is high-pressure steam sterilization, and the sterilization time is 1-2 hours; the mycelial solid culture medium is obtained by mixing the fungal strain with the moist solid culture medium and culturing at 20-25℃ for 3-7 days.

4. The preparation method according to claim 1, characterized in that, The gelling material is at least one selected from guar gum, xanthan gum, gum arabic, and locust bean gum; the premix is ​​mixed with the sterile water and stirred evenly at a speed of 2000-22000 rpm, resulting in a mixture with a viscosity of 1*10. 4 -1*10 5 mPa·s.

5. The preparation method according to claim 1, characterized in that, In step S3, after the mycelial paste culture medium is placed in the container, the thickness is controlled to be 10-50 mm; the mycelial paste placed in the container is statically cultured for 5-30 days under the conditions of temperature 20-25℃, relative humidity 80%-100%, carbon dioxide concentration 0-3%, and oxygen concentration 18%-21%.

6. The preparation method according to claim 5, characterized in that, Step S3 further includes controlling the concentration of carbon dioxide and oxygen through ventilation and humidifying by using a dry fog nozzle with an aperture of no more than 10 μm.

7. The preparation method according to claim 1, characterized in that, Step S4 includes: flattening the pure mycelium layer, blowing dry the free moisture on the surface of the pure mycelium layer, drying at 40-55℃ for 24-48 hours, and the thickness of the pure mycelium layer is 15-100mm.

8. A pure mycelium, characterized in that, The pure mycelium is prepared by the preparation method according to any one of claims 1-7.

9. The use of the pure mycelium as described in claim 8 in mycelial leather or mycelial flesh.

10. The application according to claim 9, characterized in that, When the pure mycelium is applied to mycelial leather, the pure mycelium is soaked in 2-10% glycerol for 12-24 hours, then cross-linked with 1-5% citric acid at 120-160℃ for 2-10 minutes, and hot-pressed at 60-80℃ and 0.5-5MPa for 30-60 seconds. Finally, it is washed with water and dried at 40-60℃ until the moisture content is less than 10%. When the pure mycelium is applied to mycelial flesh, the pure mycelium is cold-pressed and compacted at 0.2-1.2MPa.

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