Leather-like bio-composite material, method for its preparation and use thereof

Abstract

A method for preparing a leather-like biological composite material, comprising culturing a culture mixture comprising a fungal species and a nutrient substrate in a culture device; pressing a flat plate against an upper surface of mycelium in a direction, continuing the culturing; removing the flat plate, and tightly attaching a mesh-like membrane to the upper surface of the mycelium, continuing the culturing; pressing the flat plate against the upper surface of the mycelium in a direction, continuing the culturing until a mycelium-membrane composite is formed in which the mycelium completely covers the mesh-like membrane; repeating the foregoing steps at least once; and separating the composite from the nutrient substrate, and post-processing the composite. A leather-like biological composite material prepared by the method, and use thereof in preparing light textile products.

Description

[0001] This application claims priority to Chinese patent application CN202111570984.3, filed December 21, 2021, entitled "Leather-like Biocomposite Material, Preparation Method Thereof and Use Thereof", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to a leather-like biocomposite material, its preparation method, and its uses. More specifically, this invention relates to a method for preparing a leather-like biocomposite material made from fungal hyphae, the leather-like biocomposite material prepared by this method, and the use of this composite material in the preparation of textile products. Background Technology

[0003] Traditional leather comes from animals. Animal leather production is complex, requiring a large amount of resources to raise livestock and can take several years. Resource-related production crises and price fluctuations are common, and there are also potential ethical issues.

[0004] Another source of traditional leather is synthetic materials. Synthetic materials are usually derived from non-renewable resources or can only be obtained from limited natural resources, which is detrimental to sustainable development. Many of these materials also generate significant waste during production, and their waste often harms the environment and nature after use. Therefore, new materials that can be produced without time or space constraints, are green and sustainable, and are also biodegradable and pollution-free are attracting considerable attention.

[0005] Mycelium, an integral part of the life cycle of certain fungi, is one of nature's best decomposers. It can attach to culture media based on biological and agricultural waste, digest them, and promote mycelial growth, self-assembling into intricate hyphal networks. Compared to livestock feed, mycelium can grow within days. Furthermore, unlike leather made from synthetic fibers, this material is made from mycelium, requiring no extraction from petroleum, making it environmentally friendly. It also meets a wider range of aesthetic and functional needs from consumers, making it a biomaterial that is ethically and environmentally responsible. Summary of the Invention

[0006] One of the technical problems to be solved by this invention is to provide a leather-like biocomposite material that can replace animal leather. Another technical problem to be solved by this invention is to improve the feel, texture, and appearance of the resulting leather-like biocomposite material. A further technical problem to be solved by this invention is to improve the mechanical properties of the resulting leather-like biocomposite material, including elongation at break, tensile strength, and tear strength. Finally, a method for preparing the leather-like biocomposite material with the above improvements is provided.

[0007] On one hand, the present invention provides a method for preparing a leather-like biocomposite material, comprising the following steps:

[0008] i) Place the culture mixture containing fungal strains and nutrient substrate in a culture device and culture the fungal strains until the mycelia of the fungal strains fill the entire nutrient substrate, with the mycelia growing to a height of 0.5-1 cm and being evenly distributed;

[0009] ii) Press a non-permeable plate flat on the upper surface of the hyphae in one direction and continue to cultivate until the hyphae on the side of the plate grow to a thickness of 0.1-5 mm and the formed hyphae completely cover the entire nutrient substrate, and a hyphal membrane structure is formed on the surface.

[0010] iii) Remove the non-permeable plate, attach a mesh-like membrane tightly to the upper surface of the hyphae, and continue culturing until the hyphae completely penetrate the mesh-like membrane and grow to a height of 0.5-1 cm and are evenly distributed without forming a hyphal membrane structure;

[0011] iv) Press the non-permeable plate flat on the upper surface of the hyphae in one direction and continue to cultivate until the hyphae grow to a thickness of 1-5 mm and form a hypha-membrane complex that completely covers the mesh membrane, and the surface forms a hypha membrane structure again.

[0012] v). Repeat steps iii) and iv) above at least once; and

[0013] vi) Separate the composite from the nutrient matrix, and post-process the composite to obtain a leather-like biocomposite material.

[0014] On the other hand, the present invention provides a leather-like biocomposite material prepared by the method of the present invention.

[0015] Furthermore, this invention provides the use of the leather-like biocomposite material of this invention in the preparation of textile products.

[0016] The method for preparing the leather-like biocomposite material of this invention does not require the use of animal- or chemically derived materials, making it economical and environmentally friendly, and applicable to a wide range of industrial and residential scenarios. More importantly, the method utilizes the property of fungal hyphae to self-assemble into intricate hyphal networks to prepare biomaterials with excellent mechanical properties and biodegradability. The leather-like biocomposite material obtained by the method of this invention has a uniform texture, a soft and delicate feel, and high mechanical strength. Attached Figure Description

[0017] Figure 1 A schematic flowchart of the method for preparing the leather-like biocomposite material of the present invention:

[0018] Figure 1A - A schematic flowchart of the method for preparing leather-like biocomposite materials according to the present invention; and

[0019] Figure 1 B - Another schematic diagram of the process for preparing the leather-like biocomposite material of the present invention.

[0020] Figure 2 : Schematic diagram of the culture apparatus used in the preparation method of leather-like biocomposite material of the present invention.

[0021] Figure 3 : Entity diagrams of the culture states corresponding to some steps in the preparation method of the leather-like biocomposite material of Example 1 of this invention:

[0022] Figure 3 A- A solid image of a culture mixture of fungal strains and nutrient substrate;

[0023] Figure 3 B - After a period of cultivation, the mycelium of the strain grows to the point of filling the entire nutrient substrate;

[0024] Figure 3 C shows a physical image of the mycelial growth being strictly controlled to reach a height of 0.6 cm and be evenly distributed in step 5 above;

[0025] Figure 3 D shows a physical image of the flat plate used in step 6 above;

[0026] Figure 3 E shows a physical image of the final state of the hyphae flattened in one direction in step 6 above.

[0027] Figure 3 F shows a physical image of the final state of the plate flat pressing culture in step 6 above, until the hyphae at the edge are 1.5 mm thick and the formed hyphae completely cover the entire nutrient substrate, and a hyphal membrane structure is formed on the surface, as well as a magnified image of its part.

[0028] Figure 3 G shows a physical image of the final state of the pure cotton gauze with an average pore size of 100 μm tightly attached to the upper surface of the hyphae in step 7 above.

[0029] Figure 3 H shows a physical image of the hyphae in step 7 above, where the hyphae are controlled to grow until they completely penetrate the mesh-like membrane and reach a height of 0.7 cm and are evenly distributed, and at this time the hyphae do not form a membrane structure.

[0030] Figure 3 I shows a physical image of the hyphae in step 8 above, in which the hyphae finally form a hyphae-membrane complex that completely covers the mesh-like membrane, with a hyphae membrane structure formed on the surface;

[0031] Figure 3 J shows a physical image of the leather-like biocomposite material obtained after the aforementioned step 11 processing steps.

[0032] Figure 4 Comparison of physical samples of mycelial membrane structures and non-mycelial membrane structures obtained by the method of this invention.

[0033] Figure 5 Here is a physical image of the mycelial layer structure obtained through roller pressing and plate pressing:

[0034] Figure 5 A shows a photograph of the mycelial layer structure after a light, single rolling process;

[0035] Figure 5 B shows a photograph of the mycelial layer structure after moderate, secondary rolling.

[0036] Figure 5 C shows a photograph of the mycelial layer structure after heavy, multiple rolling processes; and

[0037] Figure 5 D shows a physical image of the mycelial layer structure obtained using the plate pressing method of the present invention.

[0038] Figure 6 : A physical image of the leather-like biocomposite material obtained by the method of this invention after post-processing, which was sent to Huace Testing for mechanical performance testing.

[0039] Figure 7 The images show physical images of the leather-like biocomposite material obtained by the method of this invention after post-processing, and a hat made using this leather-like biocomposite material. Detailed Implementation

[0040] Exemplary embodiments will be described in more detail below with reference to the accompanying drawings. The drawings are for illustrative purposes only and are not intended to limit the scope of the invention.

[0041] On the one hand, the present invention provides a method for preparing a leather-like biocomposite material. Figure 1 A is a schematic flowchart of the method for preparing leather-like biocomposite materials according to the present invention.

[0042] like Figure 1As shown in step A, the preparation method of the leather-like biocomposite material of the present invention includes: taking an appropriate amount of a culture mixture containing fungal strains and a nutrient substrate and culturing it in a culture device until the hyphae on the upper surface of the nutrient substrate grow to a height of 0.5-1 cm and are evenly distributed (step D). Using a plate, flatten the upper surface of the hyphae in one direction, and culturing until the hyphae on the side of the plate that is in close contact with the plate grow to a thickness of 0.1-5 mm and the formed hyphae completely cover the entire nutrient substrate, forming a hyphal membrane structure on the surface (step E). Uncovering the plate and attaching a mesh-like membrane tightly to the upper surface of the hyphae, continuing to culture until the hyphae completely penetrate the mesh-like membrane and grow to a height of 0.5-1 cm and are evenly distributed, without forming a membrane structure (step F). Again, using a plate, flatten the upper surface of the hyphae in one direction, and continuing to culture until the hyphae grow to a thickness of 1-5 mm and form a hypha-membrane composite that completely covers the mesh-like membrane, with a hyphal membrane structure forming on the surface again (step G). Repeating steps F→G at least once.

[0043] Separate the mycelium-membrane complex (containing a layer of mesh membrane) obtained in step G from the nutrient substrate attached to its lower part from the upper mesh membrane (step H). Optionally, since the nutrient substrate separated in step H has already been infiltrated by cultivated microorganisms during the previous culture process, this nutrient substrate can be placed in a culture device, and then the operation of steps D→H can be repeated.

[0044] The mycelium-membrane composite obtained after separation in step H is post-processed (step I) to prepare a leather-like biocomposite material.

[0045] In one embodiment, the present invention provides a method for preparing a leather-like biocomposite material, comprising the following steps:

[0046] i) Place the culture mixture containing fungal strains and nutrient substrate in a culture device and culture the fungal strains until the mycelia of the fungal strains fill the entire nutrient substrate, with the mycelia growing to a height of 0.5-1 cm and being evenly distributed;

[0047] ii) Press a non-permeable plate flat on the upper surface of the hyphae in one direction and continue to cultivate until the hyphae on the side of the plate grow to a thickness of 0.1-5 mm and the formed hyphae completely cover the entire nutrient substrate, and a hyphal membrane structure is formed on the surface.

[0048] iii) Remove the non-permeable plate, attach a mesh-like membrane tightly to the upper surface of the hyphae, and continue culturing until the hyphae completely penetrate the mesh-like membrane and grow to a height of 0.5-1 cm and are evenly distributed without forming a membrane structure;

[0049] iv) Press the non-permeable plate flat on the upper surface of the hyphae in one direction and continue to cultivate until the hyphae grow to a thickness of 1-5 mm and form a hypha-membrane complex that completely covers the mesh membrane, and the surface forms a hypha membrane structure again.

[0050] v). Repeat steps iii) and iv) above at least once; and

[0051] vi) Separate the composite from the nutrient matrix, and post-process the composite to obtain a leather-like biocomposite material.

[0052] In this invention, the fungal strain in step i) can be cultured by placing a certain amount of a culture mixture containing fungal strains and nutrient substrate in a culture device, wherein the volume of the mixture in the culture device does not exceed 60% of the volume of the culture device, the mixture is compacted and flattened in the culture device, and then cultured in the dark for 3-7 days, for example, 3 days, 4 days, 5 days, 6 days or 7 days, under the conditions of a culture temperature of 25-35°C, a carbon dioxide concentration of 3-7%, an oxygen concentration of ≤20% and a relative humidity of ≥40%.

[0053] In one embodiment, in step i), the fungal strain is cultured under the following conditions: the nutrient substrate is a nutrient substrate containing lignin or its derivatives; the volume of the culture mixture does not exceed 60% of the volume of the culture device; the culture time is 3-7 days; the culture temperature is 25-35°C; the culture is kept in the dark; the carbon dioxide concentration is 3-7%; the oxygen concentration is ≤20%; and the relative humidity is ≥40%, until the mycelium of the fungal strain fills the entire nutrient substrate. In one embodiment, the nutrient substrate is selected from one or more of the following substances: straw, rice husks, firewood, bark, peanut shells, corn cobs, branches, bark, sawdust, and mixtures thereof.

[0054] In one embodiment, in step i), the height of the mycelial growth is selected from the range of 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1.0 cm, and any two of the aforementioned values. In one embodiment, in step i), the height of the mycelial growth is 0.5 cm, 0.6 cm, or 0.7 cm.

[0055] By controlling the growth state of the hyphae in steps ii)-iv), such as growth height and density, the hyphae self-assemble into an intricate hyphal network, fully enveloping and intertwining with the mesh membrane to form a robust, biodegradable material. The mesh membrane encapsulating the hyphae-membrane composite provides necessary structural support, further enhancing the composite's mechanical properties, such as tensile strength.

[0056] In step ii) of the method of the present invention, the hyphae are kept under a non-porous plate and pressed for a period of time until the hyphae adhering to one side of the plate grow to a thickness of 0.1-5 mm and completely cover the entire nutrient substrate, forming a hyphal membrane structure on the surface. In one embodiment, a non-porous plate is pressed flat along one direction onto the upper surface of the hyphae, and cultivation continues until the hyphae adhering to the plate grow to a thickness of 0.1-5 mm and completely cover the entire nutrient substrate, forming a hyphal membrane structure on the surface. After plate pressing cultivation, the upward growth space of the hyphae is blocked, and they instead grow laterally close to the plate, intricately knotting into a film-forming plane. This hyphal membrane facilitates the separation between the lower nutrient substrate and the upper hyphal composite material in subsequent steps. At the same time, the composite material grown on this hyphal membrane structure achieves a uniform, fine, and smooth state on both sides. Achieving a fine state on both sides simultaneously meets the requirements for subsequent industrial applications of the material, without the need for further processing / refinement.

[0057] In one embodiment, in step ii) of the method of the present invention, the thickness to which the hyphae grow is selected from 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, 2.1 mm, 2.2 mm, 2.3 mm, and 2. The thickness of the hyphae is 4mm, 2.5mm, 2.6mm, 2.7mm, 2.8mm, 2.9mm, 3mm, 3.1mm, 3.2mm, 3.3mm, 3.4mm, 3.5mm, 3.6mm, 3.7mm, 3.8mm, 3.9mm, 4mm, 4.1mm, 4.2mm, 4.3mm, 4.4mm, 4.5mm, 4.6mm, 4.7mm, 4.8mm, 4.9mm, 5mm, and any two of the aforementioned values. In one embodiment, in step ii) of the method of the present invention, the thickness of the hyphae is 1mm to 2mm. In one embodiment, in step ii) of the method of the present invention, the thickness of the hyphae is 1mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm, or 2mm.

[0058] In step iii) of the method of the present invention, it is necessary to control the hyphae to completely penetrate the mesh-like membrane and grow to a height of 0.5-1 cm and be evenly distributed, with the hyphae appearing upright or branched and growing in a state where they are not connected to each other. In one embodiment, the non-perforated plate is removed, a mesh-like membrane is tightly attached to the upper surface of the hyphae, and cultivation continues until the hyphae completely penetrate the mesh-like membrane and grow to a height of 0.5-1 cm and are evenly distributed, with the hyphae appearing upright or branched and growing in a state where they are not connected to each other, and no hyphal membrane structure is formed.

[0059] In one embodiment, in step iii) of the method of the present invention, the height of mycelial growth needs to be controlled within a range of 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1 cm, and any two of the aforementioned values. In one embodiment, in step iii) of the method of the present invention, the height of mycelial growth needs to be controlled to be between 0.5 cm and 0.8 cm. In one embodiment, in step iii) of the method of the present invention, the height of mycelial growth needs to be controlled to be 0.5 cm, 0.6 cm, 0.7 cm, or 0.8 cm.

[0060] In step iv) of the method of the present invention, the hyphae need to be kept growing under the condition of being flattened on a non-porous plate for a period of time until the hyphae grow to a thickness of 1-5 mm and form a mesh-like membrane completely covered by the hyphae, with no visible gaps or unevenness, and a smooth and delicate surface. In one embodiment, the non-porous plate is flattened on the upper surface of the hyphae in one direction and cultured until the hyphae grow to a thickness of 1-5 mm and form a mesh-like membrane completely covered by the hyphae, with no visible gaps or unevenness, and a smooth and delicate surface, and a hyphal membrane structure is formed on the surface again. The re-formation of the hyphal membrane structure increases the mechanical strength of the hyphae-membrane composite and makes the appearance smoother and the feel more delicate.

[0061] In one embodiment, in step iv) of the method of the present invention, the thickness of the mycelium is selected from the range of 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3 mm, 3.1 mm, 3.2 mm, 3.3 mm, 3.4 mm, 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4 mm, 4.1 mm, 4.2 mm, 4.3 mm, 4.4 mm, 4.5 mm, 4.6 mm, 4.7 mm, 4.8 mm, 4.9 mm, 5 mm, and any two of the aforementioned values. In one embodiment, in step iv) of the method of the present invention, the thickness of the mycelium is 1 mm to 2 mm. In one embodiment, in step iv) of the method of the present invention, the mycelium grows to a thickness of 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm or 2 mm.

[0062] In this invention, the term "hyphae membrane structure" refers to a complete and integral structure of hyphae, with a smooth and flat upper surface, forming a membrane-like structure. Conversely, if hyphae are dispersed or densely distributed, exhibiting a fluffy appearance and lacking an integral structure, this state of hyphae is referred to as a non-membrane-forming hyphae structure. A physical image of a hyphae membrane structure can be found in [reference needed]. Figure 4 .

[0063] In this invention, unexpectedly, compared to the simpler roller pressing method, the flattening operation achieves superior texture, feel, appearance, and mechanical properties in the final single-layer mycelium-membrane composite. Unbound by any theoretical constraints, the inventors believe that by employing flattening during mycelial growth, regardless of differences in growth direction or state, the growth of these mycelia can be controlled on a single plane, which is crucial for the formation of the mycelial membrane structure. Furthermore, flattening can significantly increase the density of the mycelia within the mycelium-membrane composite and the overall thickness of the composite, resulting in a finer mycelial membrane structure on the surface. This allows the final single-layer mycelium-membrane composite to achieve excellent mechanical properties.

[0064] In one embodiment, in steps ii) and iv), the non-porous plate can be pressed flat onto the upper surface of the hyphae in a random direction. In another embodiment, in steps ii) and iv), the non-porous plate can be pressed flat onto the upper surface of the hyphae in a direction such that the hyphae, after being pressed down, do not intersect each other. Through the flattening method of the present invention, regardless of differences in the growth direction and state of each hyphae, the growth of these hyphae can be controlled on a single plane by the flattening operation of the plate, and their growth can be knotted into a uniform state.

[0065] In one embodiment, in steps ii) and iv), the non-porous plate is pressed flat against the upper surface of the hyphae in the same direction. Compared to hyphae-membrane composites obtained by changing the orientation of the plate during the pressing process, the hyphae-membrane composites obtained by keeping the non-porous plate pressed flat against the upper surface of the hyphae in the same direction exhibit superior mechanical properties, such as superior elongation at break, tensile strength, and tear strength. Without being bound by any theory, the inventors believe that keeping the non-porous plate pressed flat against the upper surface of the hyphae in the same direction ensures that the hyphae have the same orientation, resulting in a hyphae-membrane composite with a uniform texture, a smooth and delicate feel, and superior mechanical properties in the same orientation as the hyphae.

[0066] In this invention, the culture conditions for the fungal strains in steps ii)-iv) can be the same or different. In one embodiment, in step ii), a non-permeable plate is pressed flat onto the upper surface of the mycelium and cultured continuously for 3-7 days at a temperature of 25-35°C, in the dark, with a carbon dioxide concentration of 3-7%, an oxygen concentration ≤20%, and a relative humidity ≥40%. In one embodiment, in step iii), a mesh-like film is tightly adhered to the upper surface of the mycelium and cultured continuously for 3-7 days at a temperature of 25-35°C, in the dark, with a carbon dioxide concentration of 3-7%, an oxygen concentration ≤20%, and a relative humidity ≥40%. In one embodiment, in step iv), a non-permeable plate is pressed flat onto the upper surface of the mycelium and cultured continuously for 3-7 days at a temperature of 25-35°C, in the dark, with a carbon dioxide concentration of 3-7%, an oxygen concentration ≤20%, and a relative humidity ≥40%.

[0067] In this invention, the non-porous plate can be selected from materials that are not easily adhered to and digested by fungal hyphae. In one embodiment, the non-porous plate is a plastic plate. In another embodiment, the non-porous plate is a glass plate.

[0068] In this invention, the mesh membrane can be selected from materials with a certain pore size and a certain thickness. In one embodiment, the mesh membrane has a thickness of 0.1 mm to 5 mm, preferably 0.5 mm to 4 mm, preferably 0.5 mm to 3 mm, preferably 0.5 mm to 2 mm, preferably 0.5 mm to 1 mm, and preferably 0.5 mm. In another embodiment, the mesh membrane has an average pore size of 1 μm to 5 mm, preferably 10 μm to 5 mm, and preferably 100 μm to 5 mm. Appropriate selection of the pore size and thickness of the mesh membrane ensures that a sufficient number of hyphae pass through it, thereby guaranteeing the texture and mechanical strength of the hyphae-membrane composite.

[0069] In this invention, the mesh membrane can be made of a biodegradable material, such as a fibrous material. In one embodiment, the mesh membrane is a fibrous material. In another embodiment, the mesh membrane is selected from one or more of the following: gauze, nylon woven fabric, and natural fiber fabric. The fibrous material can be partially consumed or biodegraded by the hyphae, and the substances secreted by the hyphae bind the fibers together with the hyphae to form a composite material with better physical properties, thereby improving the mechanical strength of the hyphae-membrane composite.

[0070] In this invention, the material that can be used as a nutrient matrix is ​​a nutrient matrix containing lignin or its derivatives. In one embodiment, the nutrient matrix is ​​selected from one or more of the following substances: straw, rice husks, firewood, bark, peanut shells, corn cobs, branches, bark, sawdust, and mixtures thereof.

[0071] In this invention, steps iii) and iv) are repeated at least once to ensure that the mycelium-membrane complex contains at least one layer of mesh membrane after the final separation of the mycelium-membrane complex from the nutrient matrix. In one embodiment, the method of this invention further includes: in step v), repeating the aforementioned steps iii) and iv) several times until a complex containing a multi-layered structure is formed. In one embodiment, a mesh membrane of different materials can be used each time steps iii) and iv) are repeated. In one embodiment, in step v), steps ii)-v) are repeated 1-5 times, for example, once, twice, three times, four times, or five times. In one embodiment, steps ii)-v) are repeated once or twice.

[0072] In this invention, the step of separating the composite from the nutrient matrix in step vi) can be achieved by separating the second mesh-like membrane from the hyphae located below it (retaining the second mesh-like membrane and the hyphae that wrap around the membrane and are partially or mostly located above it), thereby separating the hyphae-membrane composite from the nutrient matrix to obtain a hyphae-membrane composite containing one mesh-like membrane. In this separation step, the removed nutrient matrix contains the first layer (i.e., the bottom layer) of the mesh-like membrane. By discarding the first mesh-like membrane, the surface of the resulting hyphae-membrane composite containing the second mesh-like membrane is smoother, and the resulting hyphae-membrane composite containing the second mesh-like membrane has hyphae that completely cover the mesh-like membrane. The overall appearance presents a delicate, uniform, and smooth hyphae arrangement, thereby ensuring that the leather-like biocomposite material prepared from this hyphae-membrane composite has a soft and delicate appearance and feel.

[0073] In one embodiment, steps ii)-v) are repeated once, thereby obtaining a mycelium-membrane composite containing two mesh-like membranes before the separation step. The mesh-like membranes encapsulating the mycelium-membrane composite can improve its mechanical properties. If necessary, steps ii)-v) can be repeated multiple times to prepare a mycelium-membrane composite containing multiple mesh-like membranes, which can further improve the mechanical strength of the mycelium-membrane composite. Furthermore, by using mesh-like membranes of different materials and by utilizing different numbers of stacked mycelial layers, mycelium-membrane composites with different appearances and mechanical properties can be produced. This further enriches the application range of the leather-like biocomposite materials prepared by the method of the present invention.

[0074] In one embodiment, the method of the present invention further includes placing the nutrient substrate separated in step vi) back into the culture device and culturing it, and then repeating steps ii)-v). Since the separated nutrient substrate has already been infiltrated by fungi and their hyphae during the previous culture process, it can be directly placed back into the culture device for culture after separation without the need to add fungal strains again or only a small amount of fungal strains are needed. Therefore, the method of the present invention greatly saves raw materials and can achieve the circular preparation of leather-like biocomposite materials in a green and environmentally friendly manner.

[0075] Figure 1 B is another schematic flowchart of the method for preparing the leather-like biocomposite material of the present invention. Figure 1 In B, with Figure 1 The same reference numerals in the figures have the same meaning, and will not be repeated here.

[0076] like Figure 1 As shown in B, the method for preparing the leather-like biocomposite material of the present invention further includes the step of preparing a fungal strain for step D. Specifically, as... Figure 1 As shown in step B, the method for preparing the leather-like biocomposite material of the present invention further includes preparing a fungal mother culture (step A), preparing the fungal mother culture into a secondary culture (secondary liquid culture, corresponding to step B1; or secondary solid culture, corresponding to step B2), and then preparing the secondary culture into a cultivation culture (step C). Optionally, the cultivation culture can be further expanded to prepare a cultivation expansion culture (step C1).

[0077] A certain volume of cultured inoculum or culture expansion inoculum, along with some of its accompanying nutrient substrate, can be placed in a culture device for cultivation until the mycelium of the inoculum grows to fill the entire nutrient substrate (step D). Alternatively, secondary liquid inoculum can be directly added to a culture device pre-filled with nutrient substrate at an inoculation rate of 1-10% for cultivation. Figure 1 (The process is shown by the dashed line in section B).

[0078] In one embodiment, the culture mixture cultured in step i) of the present invention can be obtained by a method selected from the following:

[0079] i-1) The fungi are cultured on slant medium to prepare a mother culture, the mother culture is transferred to liquid medium for further culture to prepare a secondary liquid culture, and the secondary liquid culture is transferred to a new solid medium for further culture to prepare a liquid culture, wherein the liquid culture is used as the fungal strain cultured in step i).

[0080] i-2) The fungi are cultured on slant medium to prepare a mother culture, the mother culture is transferred to a solid medium for further culture to prepare a secondary solid culture, and the secondary solid culture is transferred to a new solid medium for further culture to prepare a solid culture, wherein the solid culture is used as the fungal strain cultured in step i).

[0081] i-3) The fungi are cultured on slant medium to prepare a mother culture, the mother culture is transferred to liquid medium for further culture to prepare a secondary liquid culture, and the secondary liquid culture is transferred to a new solid medium for further culture to prepare a liquid culture strain. The liquid culture strain is further expanded to prepare a liquid culture expanded strain, which is used as the fungal strain cultured in step i).

[0082] i-4) The fungi are cultured on slant culture medium to prepare a mother culture, the mother culture is transferred to a solid culture medium for further culture to prepare a secondary solid culture, and the secondary solid culture is transferred to a new solid culture medium for further culture to prepare a solid culture strain. The solid culture strain is further expanded to prepare a solid culture expanded strain, which is used as the fungal strain cultured in step i); and / or

[0083] i-5) The fungi are cultured on slant medium to prepare a mother culture, and the mother culture is transferred to liquid medium for further culture to prepare a secondary liquid culture, which is used as the fungal culture in step i).

[0084] In one embodiment, in the aforementioned methods i-1)-i-5), the solid culture medium is a culture medium containing lignin or its derivatives as a nutrient matrix. In one embodiment, the nutrient matrix is ​​selected from one or more of the following substances: straw, rice husks, firewood, bark, peanut shells, corn cobs, branches, bark, sawdust, and mixtures thereof. In one embodiment, the nutrient matrix composition is: 40% by weight straw, 5% by weight rice husks, 5% by weight sawdust, and 50% by weight water. In one embodiment, the nutrient matrix composition is: 45% by weight corn cobs, 5% by weight straw, and 50% by weight water. In one embodiment, the nutrient matrix composition is: 45% by weight straw, 5% by weight sawdust, and 50% by weight water.

[0085] In one embodiment, in method i-1), the mother culture is obtained under the following conditions: PDA slant medium, culture time 5-10 days, and culture temperature 25-35℃; the secondary liquid culture is obtained under the following conditions: PDA liquid medium, shake flask speed 100-200 rpm, culture time 5-10 days, and culture temperature 25-35℃; the secondary liquid culture is transferred to a new solid medium at an inoculum of 1-10% for further culture to prepare liquid culture culture.

[0086] In one embodiment, in method i-2), the mother culture is cultured under the following conditions: PDA slant medium, culture time 5-10 days, and culture temperature 25-35°C; the secondary solid culture is obtained by culturing under the following conditions: a solid medium containing lignin or its derivatives as a nutrient matrix, culture time 10-20 days, and culture temperature 25-35°C; the secondary solid culture is transferred to a new solid medium at an inoculum of 1-10% for further culture to prepare a solid culture strain.

[0087] In one embodiment, in method i-3), the mother culture is cultured under the following conditions: PDA slant medium, culture time 5-10 days, and culture temperature 25-35℃; the secondary liquid culture is obtained by culture under the following conditions: PDA liquid medium, shaking speed 100-200 rpm, culture time 5-10 days, and culture temperature 25-35℃; the secondary liquid culture is transferred to a new solid medium at an inoculum of 1-10% for further culture to prepare a liquid culture strain; the liquid culture expansion strain is obtained by culture under the following conditions: the liquid culture strain is transferred again to a new solid medium at an inoculum of 1-10% for further culture for 5-10 days, and culture temperature 25-35℃.

[0088] In one embodiment, in method i-4), the mother culture is cultured under the following conditions: PDA slant medium, culture time 5-10 days, and culture temperature 25-35°C; the secondary solid culture is obtained by culture under the following conditions: a solid medium containing lignin or its derivatives as a nutrient matrix, culture time 10-20 days, and culture temperature 25-35°C; the secondary solid culture is transferred to a new solid medium at an inoculum of 1-10% for further culture to prepare a solid culture expansion culture; the solid culture expansion culture is obtained by culture under the following conditions: the solid culture culture is transferred again to a new solid medium at an inoculum of 1-10% for further culture, culture time 5-10 days, and culture temperature 25-35°C.

[0089] In one embodiment, in method i-5), the culture device in step i) has a solid culture medium containing lignin or its derivatives as a nutrient matrix; the mother culture is cultured under the following conditions: PDA slant medium, culture time 5-10 days, and culture temperature 25-35°C; the secondary liquid culture is obtained by culture under the following conditions: PDA liquid medium, shaking speed 100-200 rpm, culture time 5-10 days, and culture temperature 25-35°C; the secondary liquid culture is used as the fungal culture in step i) at an inoculum of 1-10%.

[0090] In one embodiment, in the aforementioned methods i-1)-i-5), the solid culture medium is a culture medium containing lignin or its derivatives as a nutrient matrix. In one embodiment, the nutrient matrix is ​​selected from one or more of the following substances: straw, rice husks, firewood, bark, peanut shells, corn cobs, branches, bark, sawdust, and mixtures thereof.

[0091] In this invention, compared to directly using uncultured fungal strains, the advantage of using pre-cultured strains lies in the rapid and thorough activation of the strains, whether in liquid or solid seed preparation. This ensures high activity during growth in the nutrient substrate, significantly shortening the growth cycle of the leather-like biocomposite material and facilitating its industrialization. Furthermore, the fully activated strains are evenly and appropriately distributed in the nutrient substrate, promoting uniform mycelial growth during composite formation. This contributes to the uniform texture, soft and delicate feel, and excellent mechanical properties of the resulting leather-like biocomposite material.

[0092] In this invention, a further step of post-treatment of the mycelial-membrane complex obtained after separation is included. In one embodiment, in step vi), the post-treatment includes treating the complex with an organic or inorganic reagent, followed by drying the complex. In one embodiment, the post-treatment includes removing surface proteins from the complex using ethanol and moisturizing the complex with glycerol, and the drying is heat drying, freeze drying, or air drying. In one embodiment, the post-treatment includes immersing the complex in a mixed solution of ethanol (50%-70%) and glycerol at a ratio of 15-20:1 (v / v) for 8-24 hours, followed by heat treatment at 50-80°C for 10-60 minutes.

[0093] The fungal species used in the method of the present invention are selected from fungi whose vegetative body is hyphae, preferably vigorous fungi whose vegetative body is hyphae, and more preferably fungi of the class Basidiomycetes. In one embodiment, the fungal species of the present invention is a fungi whose vegetative body is hyphae. In one embodiment, the fungal species of the present invention is a lower filamentous fungus whose vegetative body is hyphae, a common edible fungus whose vegetative body is hyphae, or a higher fungus whose vegetative body is hyphae. In one embodiment, the fungal species of the present invention is a fungi of the class Basidiomycetes. In one embodiment, the fungal strain of the present invention is selected from one or more of the following: Agaricus bisporus, Pleurotus ostreatus, Lentinula edodes, Hericium erinaceus, Pleurotus ferulae Lanzi, Flammulina velutipes, Auriclaria polytricha, Ganoderma lucidum, Trametes versicolor, and Maitakemushroom.

[0094] Figure 2 This is a schematic diagram of the culture apparatus used in the preparation method of the present invention. Figure 2 As shown, the culture device 1 of the present invention includes a receiving portion and a lid for sealing the receiving portion. The culture device 1 further includes a movable plate 2, which fits tightly against all four sides of the receiving portion of the culture device 1, dividing the space of the receiving portion into upper and lower parts. Optionally, the culture device 1 further includes a nutrient substrate 3 and a mesh membrane 4.

[0095] In one embodiment, the culture device can be a plastic box. The volume of this culture device can be determined according to the required size of the biocomposite material to be produced. In one embodiment, the volume of the culture device is (1-5) m × (1-5) m × (0.05-0.1) m, preferably (1-2.5) m × (1-2.5) m × (0.05-0.08) m. In one embodiment, the depth of the culture device is 0.05-0.1 m, preferably 0.05-0.08 m.

[0096] In the culture device 1, the plate 2 needs to be a non-porous plate, and it needs to be made of a material that is not easily adhered to and digested by fungal hyphae. In one embodiment, the non-porous plate is a plastic plate. In another embodiment, the non-porous plate is a glass plate.

[0097] The plate 2 in the cultivation device 1 needs to have a certain weight. A plate with sufficient weight can better achieve the flattening effect; plates that are too light or too heavy cannot produce dense, flattened mycelia. If the plate is too light, the growing mycelia will not be dense and compact enough; if the plate is too heavy, the burden on mycelial growth will increase, the thickness will decrease, and it may even hinder mycelial growth to some extent, making it impossible to obtain a sufficient amount of mycelia per unit area, thus failing to form dense mycelia that meet the requirements of subsequent processing. Selecting a suitable plate weight for mycelial growth can be done using existing techniques. Testing has shown that when the pressure exerted by the plate on the mixed culture is 10-20 Pa, preferably 10-15 Pa, it can balance the flattening / compacting effect of the mycelia with the maintenance of growth. In one embodiment, the pressure exerted by the plate on the mixed culture is 10-20 Pa, preferably 10-15 Pa. In another embodiment, the pressure exerted by the plate on the mixed culture is 10 Pa, 11 Pa, 12 Pa, 13 Pa, 14 Pa, or 15 Pa.

[0098] In one embodiment, a culture mixture containing fungal strains and nutrient substrate 3 is placed in a culture device 1 and the fungal strain is cultured until the mycelia of the fungal strain fill the entire nutrient substrate; then, a plate 2 is pressed flat in one direction onto the upper surface of the mycelia, and culture continues until the mycelia on the side of the plate adhering to the plate grow to a thickness of 0.1-5 mm and the formed mycelia completely cover the entire nutrient substrate, forming a mycelial membrane structure on the surface; then, the plate 2 is removed, a mesh membrane 4 is adhered to the upper surface of the mycelia, and culture continues until the mycelia completely penetrate the mesh membrane 4 and grow to a height of 0.5-1 cm and are evenly distributed, without forming a membrane structure; the plate 2 is pressed flat in one direction onto the upper surface of the mycelia, and culture continues until the mycelia grow to a thickness of 1-5 mm and form a mycelial-membrane complex that completely covers the mesh membrane 4, forming a mycelial membrane structure on the surface; the above steps are repeated at least once, for example, 3 times, to obtain a mycelial-membrane complex 5 with a lower nutrient substrate 3 and completely covered by three mesh membranes 4; then, from Figure 2 At the mesh-like membrane shown in Figure b (reference numeral a represents the first mesh-like membrane; reference numeral b represents the second mesh-like membrane), the mesh-like membrane 4b is separated from the hyphae located below it (the second mesh-like membrane and the hyphae that wrap around the membrane and are partially or mostly located above it are retained), thereby separating the hyphae-membrane complex from the nutrient substrate located below it.

[0099] In the method of this invention, the separation step ensures that the mycelium-membrane complex is not separated from the underlying nutrient substrate from the first mesh membrane. For example, if the aforementioned step is repeated four times, and a mycelium-membrane complex with the underlying nutrient substrate and completely covered by the four mesh membranes is obtained, the bottom two mesh membranes can be discarded, and the mycelium-membrane complex can be separated from the underlying nutrient substrate from the third mesh membrane. Therefore, in one embodiment, when the aforementioned step is repeated three times, the mycelium-membrane complex is separated from the underlying nutrient substrate from the second mesh membrane. In another embodiment, when the aforementioned step is repeated four times, the mycelium-membrane complex is separated from the underlying nutrient substrate from the second or third mesh membrane. By discarding the first mesh membrane, the surface of the resulting mycelium-membrane complex containing the upper mesh membrane is smoother, and the overall appearance exhibits a delicate, uniform, and smooth mycelial arrangement. The separated lower nutrient substrate, including the first mesh membrane, can also be placed back into the culture device and cultured. By repeating the method of the present invention, the self-circulation of the method of the present invention can be achieved, which greatly reduces the cost of the method of the present invention and is environmentally friendly.

[0100] On the other hand, the present invention provides a leather-like biocomposite material prepared by the method of the present invention. The leather-like biocomposite material prepared by the method of the present invention has excellent appearance, including uniform hyphal distribution and a soft and delicate hand feel, as well as excellent mechanical properties, including excellent elongation at break and tear tension. The elongation at break and the breaking strength of the leather-like biocomposite material prepared by the method of the present invention can be tested according to the standard test method for textiles (ASTM D5034-09(2017)). The tear strength of the leather-like biocomposite material prepared by the method of the present invention can be tested according to the test method for fabric tear strength (BS EN ISO 13937-2:2000). In one embodiment, the elongation at break of the leather-like biocomposite material prepared by the method of the present invention is 20-40%, preferably 25-35%. In one embodiment, the breaking strength of the leather-like biocomposite material prepared by the method of the present invention is 45-60 lb, preferably 50-57 lb. In one embodiment, the leather-like biocomposite material prepared by the method of the present invention has a tear strength of 6-25 (N), preferably 9-20 (N).

[0101] Furthermore, this invention provides the use of the leather-like biocomposite material prepared according to the method of this invention in the preparation of textile products. The leather-like biocomposite material of this invention is similar to leather in appearance, feel, texture, and mechanical properties, and therefore can be used as a biocomposite material in the field of textile products. In one embodiment, the textile product is selected from one or more of the following: hats, shoes, clothing, and handbags. In one embodiment, the textile product is a leather substitute.

[0102] Example

[0103] The present invention will be further described below through specific embodiments.

[0104] Example 1 - Preparation of the leather-like biocomposite material of the present invention

[0105] I. Strain Acquisition and Identification

[0106] The *Trametes versicolor* strain used in this embodiment was a commercially available *Trametes versicolor* slant strain purchased from the Shenzhen Agricultural Products Wholesale Market. The identification method for this strain is based on [1] Tie Weifang, Jia Junzhong, Zhang Ye. Cultivation and strain identification of *Pleurotus ostreatus* [J]. Food Safety Guide, 2017(27): 83-84. DOI: 10.16043 / h.cnki.cfs.2017.27.065. After obtaining the strain, its genomic DNA was extracted, and then the 18S rDNA fragment was amplified by PCR. After sequencing, it was compared with GeneBank and identified as *Trametes versicolor*.

[0107] II. Preparation of Leather-like Biocomposite Materials

[0108] The preparation of leather-like biocomposite materials involves the following steps:

[0109] 1. Purchase commercially available Trametes versicolor slant culture from an agricultural wholesale market (confirmed as Trametes versicolor by 18S identification). Use an inoculation spatula to take a small piece and inoculate it into the center of PDA slant culture medium (1L potato extract, 20g glucose, 3g KH2PO4, 1.5g MgSO4·7H2O, 15g agar, 10.05g vitamin B1). Place it in a 25℃ incubator and incubate at a constant temperature for 7 days until the mycelium covers the slant culture medium, thus preparing the mother culture.

[0110] 2. Add 5 mL of PDA liquid culture medium (1 L potato extract, 20 g glucose, 3 g KH2PO4, 1.5 g MgSO4·7H2O, 0.05 g vitamin B1) to the mycelial slant. Use a sterile inoculation spatula to gently scrape the upper layer of mycelium into the liquid culture medium. Add all 5 mL of the culture medium containing mycelium to a 250 mL shake flask containing 45 mL of fresh PDA culture medium. Incubate at 25°C and 120 rpm for 5 days until mycelial balls are produced. Add sterile glass beads (5 mm) to the surface of the culture medium and shake the flask vigorously to break the mycelial balls until no visible lumps of mycelium are visible. This yields the secondary liquid culture.

[0111] 3. At a 5% inoculation rate, take 50 mL of secondary liquid inoculum and evenly transfer it into a cultivation bag containing 1 kg of sterile nutrient substrate (40% straw, 5% rice husk, 5% sawdust, and 50% water). Incubate aseptically at 25°C for 15 days until the mycelium covers the surface of the nutrient substrate to obtain the liquid culture.

[0112] 4. Transfer 500g of liquid culture spawn to a 10kg new sterile nutrient substrate (40% straw, 5% rice husk, 5% sawdust, and 50% water) cultivation bag at a 5% inoculation rate. Incubate aseptically at 25℃ for 10 days until the mycelium covers the surface of the nutrient substrate to obtain the liquid culture expansion strain.

[0113] 5. Customize a cultivation device made of plastic with a volume of 1 meter × 1 meter × 0.05 meters, and transfer 40% of the obtained culture medium to the bottom of the device, compacting and flattening it. Figure 3 A) Under conditions of 25℃, 5% carbon dioxide, 10% oxygen, and 75% relative humidity, and incubated in the dark for 4 days, the mycelium will grow until it covers the nutrient substrate. Figure 3 B), continue culturing, and measure the hyphal height at the edge of the nutrient substrate surface in real time until the hyphae reach a height of 0.6 cm and are evenly distributed. Figure 3 C);

[0114] 6. Use a flat plate ( Figure 3 D) Flatten the upper surface of the hyphae in one direction. Figure 3 E), with a pressure of 10 Pa, after culturing for 4 days under the above conditions, the mycelium adhering to one edge of the plate was measured to be 1.5 mm thick, and the formed mycelium completely covered the entire nutrient substrate, with a mycelial membrane structure forming on the surface. Figure 3 F);

[0115] 7. Remove the plate and place a 1mm thick pure cotton gauze with an average pore size of 100um tightly against the upper surface of the mycelium. Figure 3G), continue culturing under the above conditions for 4 days, measuring whether the edge hyphae completely penetrate the mesh-like membrane and grow to a height of 0.7 cm and are evenly distributed, without forming a membrane structure. Figure 3 H);

[0116] 8. Press the surface of the mycelium flat again with a plate in the same direction, and continue culturing under the above conditions for 4 days. Measure when the edge mycelium has grown to a thickness of 1.5 mm and formed a mycelium-membrane complex that completely covers the mesh-like membrane, with a mycelial membrane structure forming on the surface. Figure 3 I);

[0117] 9. Repeat steps 7→8 once;

[0118] 10. Separate the second layer of the mesh-like membrane from the nutrient substrate attached to it below to obtain a single-layer mycelium-membrane complex;

[0119] 11. The composite was soaked in a mixed solution of ethanol (70%) and glycerol at a ratio of 15:1 (v / v) for 24 hours, followed by heat treatment at 60°C in an oven for 15 minutes to obtain a leather-like biocomposite material. Figure 3 J).

[0120] Example 2 - Preparation of the leather-like biocomposite material of the present invention

[0121] I. Strain Acquisition and Identification

[0122] The oyster mushroom strain used in this embodiment was a commercially available oyster mushroom slant culture strain purchased from the Shenzhen Agricultural Products Wholesale Market. The identification method for this strain was the same as in Example 1. After obtaining the strain, its genomic DNA was extracted, and then the 18S rDNA fragment was amplified by PCR. After sequencing, it was compared with GeneBank to confirm that it was oyster mushroom (Pleurotus ostreatus).

[0123] II. Preparation of Leather-like Biocomposite Materials

[0124] The preparation of leather-like biocomposite materials involves the following steps:

[0125] 1. Purchase commercially available oyster mushroom slant culture from an agricultural wholesale market (confirmed as Pleurotus ostreatus by 18S identification). Use an inoculation spatula to take a small piece and inoculate it into the center of PDA slant culture medium (1L potato extract, 20g glucose, 3g KH2PO4, 1.5g MgSO4·7H2O, 15g agar, 0.05g vitamin B1). Place it in a 28℃ incubator and incubate at a constant temperature for 7 days to allow the mycelium to fully grow on the slant culture medium, thus preparing the mother culture.

[0126] 2. Use a sterilized inoculation spatula to take small pieces of mycelium and transfer them to a cultivation bag containing 500g of sterilized nutrient substrate (45% corn cob, 5% straw, and 50% water). Incubate at a constant temperature of 28℃ for 18 days until the mycelium covers the surface of the nutrient substrate to obtain secondary solid inoculum.

[0127] 3. Mix 500g of secondary solid inoculum with 10kg of fresh sterilized nutrient substrate (45% corn cob, 5% straw, 50% water) in a cultivation bag at a 5% inoculation rate. Incubate aseptically at 28℃ for 10 days until the mycelium covers the surface of the nutrient substrate to obtain the cultivar.

[0128] 4. A custom-made plastic cultivation device with a volume of 1.5m × 1.2m × 0.06m was used. The obtained culturing inoculum was transferred to the bottom of the device at 55% of its volume, compacted and flattened. The device was then incubated in the dark for 5 days at a temperature of 28℃, a carbon dioxide concentration of 5%, an oxygen concentration of 12%, and a relative humidity of 80%. The height of the mycelium at the edge of the nutrient substrate was measured. At this point, the mycelium reached a height of 0.5cm and was evenly distributed.

[0129] 5. Press the upper surface of the mycelium flat in one direction with a plate at a pressure of 14 Pa. Incubate for 5 days under the above conditions. Measure the mycelium that is close to the edge of the plate. The mycelium should grow to a thickness of 1 mm and completely cover the entire nutrient substrate. A mycelial membrane structure should be formed on the surface.

[0130] 6. Uncover the plate and place a piece of pure cotton gauze with a thickness of 0.5 mm and an average pore size of 80 μm tightly on the upper surface of the mycelium. Continue to culture for 5 days under the above conditions. Measure the edge mycelium to see if it has completely penetrated the mesh-like membrane and grown to a height of 0.5 cm and is evenly distributed without forming a membrane structure.

[0131] 7. Press the surface of the hyphae flat again with a plate in the same direction, continue to culture, and continue to culture for 5 days under the above conditions. Measure the edge hyphae to grow to a thickness of 1 mm and form a hypha-membrane complex that completely covers the grid-like membrane, with a hypha membrane structure formed on the surface.

[0132] 8. Repeat steps 6→7 twice;

[0133] 9. Separate the second layer of the mesh-like membrane from the nutrient substrate attached to it below to obtain a double-layer mycelium-membrane complex;

[0134] 10. The composite was soaked in a mixed solution of ethanol (70%) and glycerol at a ratio of 19:1 (v / v) for 24 hours, and then heat-treated in an oven at 80°C for 10 minutes to obtain a leather-like biocomposite material.

[0135] The images obtained in each step of this embodiment are similar to those in Embodiment 1 and will not be shown again.

[0136] Example 3 - Preparation of the leather-like biocomposite material of the present invention

[0137] I. Strain Acquisition and Identification

[0138] The shiitake mushroom strain used in this embodiment was a commercially available slant culture of *Lentinula edodes* purchased from the Shenzhen Agricultural Products Wholesale Market. The identification method for this strain was the same as in Example 1. After obtaining the strain, its genomic DNA was extracted, and then the 18S rDNA fragment was amplified by PCR. After sequencing, the fragment was compared with GeneBank data to confirm that it was *Lentinula edodes*.

[0139] II. Preparation of Leather-like Biocomposite Materials

[0140] The preparation of leather-like biocomposite materials involves the following steps:

[0141] 1. Purchase commercially available shiitake mushroom slant culture from an agricultural wholesale market (confirmed as *Lentinula edodes* by 18S identification). Take a small piece with an inoculation spatula and transfer it to the center of a PDA slant culture medium (1L potato extract, 20g glucose, 3g KH2PO4, 1.5g MgSO4·7H2O, 15g agar, 0.05g vitamin B1). Place it in a 26℃ incubator and incubate for 6 days to allow the mycelium to fully grow on the slant culture medium, thus preparing the mother culture.

[0142] 2. Add 5 mL of PDA liquid culture medium (1 L potato extract, 20 g glucose, 3 g KH2PO4, 1.5 g MgSO4·7H2O, 0.05 g vitamin B1) to the mycelial slant. Use a sterile inoculation spatula to gently scrape the upper layer of mycelium into the liquid culture medium. Add all 5 mL of the culture medium containing mycelium to a 250 mL shake flask containing 45 mL of fresh PDA medium. Incubate at 26°C and 120 rpm for 5 days until mycelial balls are produced. Add sterile glass beads (5 mm) to the surface of the culture medium and shake the flask vigorously to break the mycelial balls until no visible lumps of mycelium are visible. This yields the secondary liquid culture.

[0143] 3. A custom-made cultivation device with a volume of 1.1m × 1.1m × 0.05m plastic was used. 10kg of sterilized nutrient substrate (45% straw, 5% sawdust, and 50% water) was transferred to the bottom of the device, compacted and flattened, at 50% of the device's volume. Secondary liquid inoculum was evenly added to the nutrient substrate at a 10% inoculation rate. The device was then incubated in the dark for 20 days at a temperature of 26℃, a carbon dioxide concentration of 6%, an oxygen concentration of 8%, and a relative humidity of 70%. The mycelial height at the edge of the upper surface of the nutrient substrate was measured. At this point, the mycelium reached a height of 0.7cm and was evenly distributed.

[0144] 4. Press the upper surface of the hyphae flat in one direction with a pressure of 12 Pa. Cultivate for 4 days under the above conditions. Measure the hyphae that are close to one edge of the plate. When the hyphae have grown to a thickness of 2 mm and completely covered the entire nutrient substrate, a hyphal membrane structure is formed on the surface.

[0145] 5. Uncover the plate and use a 1mm thick pure cotton non-woven fabric with an average pore size of 120um to adhere tightly to the upper surface of the hyphae. Continue to cultivate for 4 days under the above conditions. Measure the edge hyphae to ensure they completely penetrate the mesh-like membrane and grow to a height of 0.8cm and are evenly distributed without forming a membrane structure.

[0146] 6. Press the surface of the hyphae flat again with a plate in the same direction, continue to culture, and continue to culture for 4 days under the above conditions. Measure the edge hyphae to grow to a thickness of 2 mm and form a hypha-membrane complex that completely covers the grid-like membrane, with a hypha membrane structure formed on the surface.

[0147] 7. Repeat steps 5 through 6 three times;

[0148] 8. Separate the second layer of the mesh-like membrane from the nutrient substrate attached to it to obtain a three-layer mycelium-membrane complex.

[0149] 9. The composite was soaked in a mixed solution of ethanol (50%) and glycerol at a ratio of 18:1 (v / v) for 24 hours, and then heat-treated in an oven at 70°C for 15 minutes to obtain a leather-like biocomposite material.

[0150] The images obtained in each step of this embodiment are similar to those in Embodiment 1 and will not be shown again.

[0151] Example 4 - Comparative Evaluation of the Mycelial Membrane Structure and Non-Mycelial Membrane Structure of the Present Invention

[0152] To evaluate the effect of non-permeable plate pressing on the formation of mycelial membrane structure and the appearance of mycelial-membrane complex, a single-layer mycelial-membrane complex was prepared using the method described in Example 1. The difference was that only half of the mycelium was subjected to the non-permeable plate pressing operation of the present invention during the entire mycelial growth process, while the other half of the mycelium did not undergo the non-permeable plate pressing operation during the growth process.

[0153] Figure 4 The images show a physical comparison between the mycelial membrane structure obtained by this embodiment and the non-mycelial membrane structure. Figure 4 As shown, Figure 4 The lower part of the mycelium has been treated using the method of this invention during cultivation (through flat pressing with a non-perforated plate). This part of the mycelium has good integrity, a smooth structure, and a smooth, leather-like surface, forming a mycelial membrane structure (see...). Figure 4 (A magnified view in the lower right corner). In contrast, Figure 4The upper hyphae were not subjected to non-permeable plate pressing during cultivation. The hyphae cultivated in this part grew freely towards the vertical plane, forming individual hyphae with an overall villous, non-membrane structure. Magnified observation of this structure reveals that these hyphae are densely distributed and lack a unified structure (see [link to relevant documentation]). Figure 4 (A close-up view in the lower left corner).

[0154] Example 5 - Comparison of the mycelial layer structure obtained by plate pressing according to the present invention and by roller pressing as a comparison Comparison

[0155] This experiment was conducted using the strain and method of Example 1 of this invention, with the only difference being that in steps 6 and 8, instead of using a plate to apply pressure to the surface of the mycelium, a smooth roller was used to apply pressure to the surface of the mycelium by rolling. The experiment was conducted in three parallel groups, with the applied pressure adjusted according to the plate pressure method. Group 1 underwent light, single rolling pressure; Group 2 underwent moderate, double rolling pressure; and Group 3 underwent heavy, multiple rolling pressures. Simultaneously, the experiment of Example 1 was repeated as a control, designated as Group 4.

[0156] Figure 5 Group 1 ( Figure 5 A), group 2( Figure 5 B), Group 3 Figure 5 C) and group 4 Figure 5 D) A physical image of the obtained mycelial layer structure.

[0157] like Figure 5 As shown in Figure A, after a light, single rolling process, the final mycelial film is difficult to form a uniform plane and exhibits obvious unevenness. For example... Figure 5 As shown in Figure B, after moderate secondary rolling, the mycelial layer still exhibits noticeable unevenness. Figure 5 As shown in C, after heavy, repeated rolling, the mycelial layer can be flattened, but the mycelium is uneven, and the appearance is fuzzy and lumpy. Figure 5 As shown in Figure D, after applying pressure using the plate according to the present invention, and ensuring that the plate is pressed in the same direction twice, the mycelial layer eventually forms a smooth, dense, and flawless plane, with a mycelial membrane structure on the surface. Furthermore, experiments revealed that rolling followed by cultivation did not result in mycelial membrane formation, and if the plate was removed after flat pressing and cultivation continued, a mycelial membrane also failed to form. Continuous flat pressing cultivation is beneficial for mycelial membrane formation. These results suggest that, compared to rolling, flat pressing is crucial for achieving a smooth, dense mycelial membrane structure and obtaining a leather-like biocomposite material with a smooth appearance and delicate feel.

[0158] Example 6 - Performance Testing of the Mycelium-Membrane Composite Coated with a Single-Layer Mesh Membrane of the Present Invention

[0159] To evaluate the mechanical properties of the mycelium-membrane composite obtained by the method of the present invention, mechanical property tests including elongation at break, tensile strength and tear strength were performed on the single-layer mycelium-membrane composite prepared by the method described in Example 1.

[0160] The testing was commissioned to CTI, located in Bao'an District, Shenzhen, Guangdong Province. The test report number is (A2210375490101C). Figure 6 The image shows a physical photograph of the leather-like biocomposite material obtained after post-processing using the method of this embodiment of the invention, which was sent to Huace Testing for mechanical property testing. The elongation at break and breaking strength of the leather-like biocomposite material prepared in Example 1 were tested according to the "Standard Test Method for Tension and Elongation at Break of Textiles" (ASTM D5034-09(2017)). The tear strength of the leather-like biocomposite material prepared in Example 1 was tested according to the "Test Method for Tear Strength of Fabrics" (BS EN ISO 13937-2:2000).

[0161] The tear strength test results are shown in Table 1 below.

[0162]

[0163] Note: * = Tear direction is perpendicular to the sample cut.

[0164] The fracture strength test results are shown in Table 2 below.

[0165]

[0166] The results of the elongation at break test are shown in Table 3 below.

[0167]

[0168] Based on the above test results, it can be seen that the composite material obtained by using the method of the present invention, especially the operation of random directional flat pressing of non-perforated flat plate, has good mechanical properties in different directions and excellent overall mechanical properties.

[0169] Example 7 - Comparison of mechanical properties of composite materials obtained by the plate pressing method of the present invention and the comparative roll pressing method.

[0170] The mechanical properties of the monolayer mycelium-membrane composites prepared by the methods in groups 3 and 4 of Example 5 were tested using the test method described in Example 6. The test parameters included tensile strength and tear strength.

[0171] The test results of the breaking strength and tear strength of the two are shown in Table 4 below.

[0172]

[0173] The results show that, compared with the rolling method (even heavy, multiple rolling), the tensile strength and tear strength of the single-layer mycelium-film composite obtained by the plate pressing method of this invention are improved by at least 100% in both the longitudinal and transverse directions, and the overall mechanical properties are significantly improved. This result further demonstrates that the plate pressing method of this invention, while maintaining the same direction of pressure in both plates, is crucial for ultimately improving the mechanical properties of the mycelium-film composite.

[0174] Example 8 - Post-processing of the mycelium-membrane composite of the present invention

[0175] The mycelium-membrane composite obtained was post-treated using the method of Example 1. Specifically, the mycelium-membrane composite was soaked in a mixed solution of ethanol (70%) and glycerol at a ratio of 15:1 (v / v) for 24 hours, and then heat-treated in an oven at 60°C for 15 minutes to obtain a leather-like biocomposite material.

[0176] Figure 7 A photograph of a leather-like biocomposite material obtained by post-processing using the method of Example 1 of this invention is shown, along with a hat made from this leather-like biocomposite material. (Summary) Figure 5 and Figure 7 As can be seen from the physical images of the composite material, the composite material prepared by the method of this invention has a smooth appearance, dense structure, good integrity, and a delicate and smooth feel. It can completely replace animal-derived leather in the production of light textile products.

[0177] The above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. For those skilled in the art, other improvements made based on the above description are still within the scope of protection of this invention.

Claims

1. A method for preparing a leather-like biocomposite material, comprising the following steps: i) Place the culture mixture containing fungal strains and nutrient substrate in a culture device and culture the fungal strains until the mycelia of the fungal strains fill the entire nutrient substrate, with the mycelia growing to a height of 0.5-1 cm and being evenly distributed; ii). Press a non-permeable plate flat on the upper surface of the hyphae in one direction and continue to cultivate until the hyphae on one side of the plate grow to a thickness of 0.1-5 mm and the formed hyphae completely cover the entire nutrient substrate, and a hyphal membrane structure is formed on the surface. iii) Remove the non-permeable plate, attach a mesh-like membrane tightly to the upper surface of the hyphae, and continue culturing until the hyphae completely penetrate the mesh-like membrane and grow to a height of 0.5-1 cm and are evenly distributed without forming a hyphal membrane structure; iv). Press the non-permeable plate flat on the upper surface of the hyphae in one direction and continue to cultivate until the hyphae grow to a thickness of 1-5 mm and form a hypha-membrane complex that completely covers the mesh-like membrane, and the surface forms a hyphae membrane structure again. v). Repeat steps iii) and iv) above 2-5 times to form a complex containing multiple layers; and vi). Separate the composite from the nutrient matrix, and post-process the composite to obtain a leather-like biocomposite material; In steps ii) and iv), the non-perforated plate is pressed flat against the upper surface of the hyphae in the same direction.

2. The preparation method according to claim 1, wherein the method further comprises: In step vi), the composite is separated from the nutrient matrix from at least the second mesh membrane; and / or The nutrient matrix separated in step vi) is placed back into the culture device and cultured, and then steps ii)-v) are repeated to achieve cyclic preparation of leather-like biocomposite materials.

3. The preparation method according to claim 1, wherein in step i), the fungal strain is cultured under the following conditions: the nutrient substrate is a nutrient substrate containing lignin or its derivatives, the volume of the culture mixture does not exceed 60% of the volume of the culture device, the culture time is 3-7 days, the culture temperature is 25-35℃, the strain is kept in the dark, the carbon dioxide concentration is 3-7%, the oxygen concentration is ≤20%, and the relative humidity is ≥40%.

4. The preparation method according to claim 3, wherein the nutrient matrix is ​​selected from one or more of the following substances: straw, rice husk, firewood, bark, peanut shell, corn cob, branches, bark, sawdust, and mixtures thereof.

5. The preparation method according to claim 1, wherein... In step ii), a non-permeable plate is pressed flat onto the upper surface of the mycelium and cultured continuously for 3-7 days at a temperature of 25-35℃, in the dark, with a carbon dioxide concentration of 3-7%, an oxygen concentration of ≤20%, and a relative humidity of ≥40%. In step iii), the mesh-like membrane is tightly adhered to the upper surface of the mycelium and continuously cultured for 3-7 days at a temperature of 25-35℃, in the dark, with a carbon dioxide concentration of 3-7%, an oxygen concentration of ≤20%, and a relative humidity of ≥40%; and / or In step iv), a non-permeable plate is pressed flat on the upper surface of the mycelium and cultured for 3-7 days at a temperature of 25-35°C, in the dark, with a carbon dioxide concentration of 3-7%, an oxygen concentration of ≤20%, and a relative humidity of ≥40%.

6. The preparation method according to any one of claims 1-5, wherein the culture mixture cultured in step i) is obtained by a method selected from: i-1) The fungus is cultured on an slant culture medium to prepare a mother culture, the mother culture is transferred to a liquid culture medium for further culture to prepare a secondary liquid culture, and the secondary liquid culture is transferred to a new solid culture medium for further culture to prepare a liquid culture strain, wherein the liquid culture strain is used as the fungal strain cultured in step i). i-2) The fungus is cultured on an slant culture medium to prepare a mother culture, the mother culture is transferred to a solid culture medium for further culture to prepare a secondary solid culture, and the secondary solid culture is transferred to a new solid culture medium for further culture to prepare a solid culture, wherein the solid culture is used as the fungal strain cultured in step i). i-3) The fungus is cultured on a slant medium to prepare a mother culture, the mother culture is transferred to a liquid medium for further culture to prepare a secondary liquid culture, and the secondary liquid culture is transferred to a new solid medium for further culture to prepare a liquid culture strain. The liquid culture strain is further expanded to prepare a liquid culture expanded strain, which is used as the fungal strain cultured in step i). i-4) The fungus is cultured on an slant culture medium to prepare a mother culture; the mother culture is transferred to a solid culture medium for further culture to prepare a secondary solid culture; the secondary solid culture is transferred to a new solid culture medium for further culture to prepare a solid culture spawn; the solid culture spawn is further expanded to prepare a solid culture expanded spawn, which is used as the fungal strain cultured in step i); and / or i-5) The fungus is cultured on an slant culture medium to prepare a mother culture, and the mother culture is transferred to a liquid culture medium for further culture to prepare a secondary liquid culture, which is used as the fungal culture in step i).

7. The preparation method according to claim 6, wherein the culture mixture cultured in step i) is obtained by a method selected from: i-1) In method i-1), the mother culture is obtained under the following conditions: PDA slant medium, culture time 5-10 days, and culture temperature 25-35℃; the secondary liquid culture is obtained under the following conditions: PDA liquid medium, shake flask speed 100-200 rpm, culture time 5-10 days, and culture temperature 25-35℃; the secondary liquid culture is transferred to a new solid medium at an inoculum of 1-10% for further culture to prepare liquid culture culture. i-2) In method i-2), the mother culture is cultured under the following conditions: PDA slant medium, culture time 5-10 days, and culture temperature 25-35℃; the secondary solid culture is obtained by culturing under the following conditions: a solid medium containing lignin or its derivatives as a nutrient matrix, culture time 10-20 days, and culture temperature 25-35℃; the secondary solid culture is transferred to a new solid medium at an inoculum of 1-10% for further culture to prepare a solid culture strain; i-3) In method i-3), the mother culture is cultured under the following conditions: PDA slant medium, culture time 5-10 days, and culture temperature 25-35℃; the secondary liquid culture is obtained by culture under the following conditions: PDA liquid medium, shake flask speed 100-200 rpm, culture time 5-10 days, and culture temperature 25-35℃; the secondary liquid culture is transferred to a new solid medium at an inoculum of 1-10% for further culture to prepare liquid culture culture; the liquid culture expansion culture is obtained by culture under the following conditions: the liquid culture culture is transferred again to a new solid medium at an inoculum of 1-10% for further culture, culture time 5-10 days, and culture temperature 25-35℃; i-4) In method i-4), the mother culture is cultured under the following conditions: PDA slant medium, culture time 5-10 days, and culture temperature 25-35℃; the secondary solid culture is obtained by culture under the following conditions: a solid medium containing lignin or its derivatives as a nutrient matrix, culture time 10-20 days, and culture temperature 25-35℃; the secondary solid culture is transferred to a new solid medium at an inoculum of 1-10% for further culture to prepare a solid culture expansion culture; the solid culture expansion culture is obtained by culture under the following conditions: the solid culture culture is transferred again to a new solid medium at an inoculum of 1-10% for further culture, culture time 5-10 days, and culture temperature 25-35℃; and / or i-5) In method i-5), the culture apparatus in step i) has a solid culture medium containing lignin or its derivatives as a nutrient matrix; the mother culture is cultured under the following conditions: PDA slant medium, culture time 5-10 days, and culture temperature 25-35℃; the secondary liquid culture is obtained by culture under the following conditions: PDA liquid medium, shaking speed 100-200 rpm, culture time 5-10 days, and culture temperature 25-35℃; the secondary liquid culture is used as the fungal culture in step i) at an inoculum of 1-10%.

8. The preparation method according to claim 1, wherein in step vi), the post-treatment includes treating the composite with an organic or inorganic reagent, followed by drying the composite.

9. The preparation method according to claim 8, wherein the post-treatment includes removing surface proteins of the composite using ethanol and moisturizing the composite using glycerol, and the drying is heat drying, freeze drying or natural air drying.

10. The preparation method according to claim 9, wherein the post-treatment comprises immersing the composite in a mixed solution of ethanol (50%-70%) and glycerol at a ratio of 15-20:1 (v / v) for 8-24 hours, followed by heat treatment at a temperature of 50-80°C for 10-60 minutes.

11. The preparation method according to any one of claims 1-5, wherein the mesh membrane has a thickness of 0.1 mm to 5 mm and an average pore size of 1 μm to 5 mm.

12. The preparation method according to claim 11, wherein the thickness is 0.5 mm and the average pore size is 0.1 mm - 5 mm.

13. The preparation method according to claim 12, wherein the mesh membrane is selected from one or more of the following: gauze, nylon woven fabric and natural fiber fabric.

14. The preparation method according to any one of claims 1-5, wherein the fungus is a fungus whose vegetative body is hyphae.

15. The preparation method according to claim 14, wherein the fungus is a Basidiomycetes ( Basidiomycetes ) fungi.

16. The preparation method according to claim 14, wherein the fungus is selected from one or more of the following: Agaricus bisporus (Butyrus pyrifolia) Agaricus bisporus ), oyster mushroom ( Pleurotus ostreatus ),mushroom( Lentinula edodes ), Hericium erinaceus ( Hericium erinaceus ), White Pleurotus eryngii ( Pleurotus ferulae Lanzi ), Enoki mushrooms Flammulina velutipes ), hairy fungus ( Auriclaria polytricha ), Ganoderma lucidum ( Ganoderma lucidum Yunzhi () Trametes versicolor ) and maitake mushroom ( Maitake mushroom ).

17. A leather-like biocomposite material prepared by the method according to any one of claims 1-16.

18. The leather-like biocomposite material according to claim 17, wherein the leather-like biocomposite material has an elongation at break of 25-35% (%), a tensile strength of 50-57 (lb), and a tear strength of 9-20 (N).

19. Use of the leather-like biocomposite material prepared by the method according to any one of claims 1-16 or the leather-like biocomposite material according to claim 17 or 18 in the preparation of textile products.

20. The use according to claim 19, wherein the textile article is selected from one or more of the following: hats, clothing and handbags.

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