A strain of Hemipoloma TS-10 and its application in the production of planar mycelial leather

Abstract

This invention discloses a strain of *Hemipolomyces cerevisiae* TS-10 and its application in the production of planar mycelial leather, belonging to the field of new materials technology. This invention directly inoculates intact mycelial balls using liquid pre-culture, eliminating the need for spore collection or mycelial disruption steps, further shortening the solid-state culture cycle and increasing efficiency by more than 50% compared to traditional spore inoculation. Compared to traditional static culture on sawdust substrates (20-25 days), the dynamic control of this invention compresses primordia formation time to 8-12 days, shortening the cycle by 40%, greatly improving the production efficiency of planar mycelium. After the first harvest of planar mycelial skin, the supplemented gallic acid solution acts as an intermediate for lignin degradation, making the culture substrate more similar to a natural decaying environment, stimulating the continued reproduction of *Hemipolomyces cerevisiae*, achieving recyclable culture, and improving the biomass utilization rate of agricultural waste.

Description

Technical Field

[0001] This invention relates to the field of new materials technology, specifically to a strain of Hemipoloma TS-10 and its application in the production of planar mycelial leather. Background Technology

[0002] Against the backdrop of the global transition from a linear economy to a circular economy, the development of eco-friendly and renewable material solutions has become an urgent need. Traditional petroleum-based polymers and animal leather face severe environmental sustainability challenges in their production, use, and recycling, necessitating the search for novel bio-based alternatives. Fungi, due to their unique biological characteristics and diverse substrate utilization capabilities, have become a popular source of unique pure / composite mycelial materials as a "vegetarian alternative" to animal leather in countries such as the United States, Norway, the Netherlands, and Italy. Therefore, the development and utilization of mycelium will largely provide new solutions for sustainable biomaterials research and development, becoming an ideal platform for developing bio-based materials, thereby impacting existing production and lifestyle patterns.

[0003] Fungi are ubiquitous in the biological world, especially filamentous fungi. Their hyphae are composed of cross-linked filamentous cells, achieving rapid growth through polarized tip extension and branching, with an efficiency comparable to cell division in animals and plants. Research has found that fungal-based composite mycelial materials possess environmentally friendly characteristics such as sustainable production and natural biodegradability. They can utilize agricultural and forestry byproducts as growth substrates, achieving waste recycling and shortening the carbon turnover cycle. Furthermore, pure mycelial materials composed of fungal mycelial biomass also possess characteristics such as thermal insulation, corrosion resistance, high mechanical strength, good flame retardancy, high plasticity, and air permeability and insulation, making them a new favorite for interdisciplinary research and a novel biomaterial with broad development and application prospects.

[0004] Blood-red Fungi ( Pycnoporus sanguineus As a white-rot basidiomycete, *Hemiberlesia javanica* is currently mainly used in the fields of medicine and biodegradation due to its diverse bioactive substances and enzyme activities. However, because *Hemiberlesia javanica* is difficult to induce fruiting bodies under natural conditions, has a low primordia formation rate and a long growth cycle, and its natural mycelium has insufficient tensile strength and toughness, making it difficult to meet industrial applications, its application in the field of biomaterials development is still relatively limited. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a strain of Heme Fungi TS-10 and its application in the production of planar mycelial leather.

[0006] Specifically, the present invention relates to the following technical solutions: In a first aspect, the present invention provides a strain of *Hematoxylinum* (…). Pycnoporus sanguineusTS-10, this strain was deposited on June 19, 2025 at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with the biological accession number: CGMCC NO.42037.

[0007] The blood-red dense-pore fungus ( Pycnoporus sanguineus TS-10, isolated from the Taishan Scenic Area in Tai'an City, Shandong Province, has the following characteristics: It is easy to cultivate, grows relatively quickly, and has relatively broad requirements for substrates. The hyphae exhibit a relatively dense state, intertwining to form a relatively complex network structure. This high hyphal density means that it can provide a good foundation of strength and toughness when producing mycelial leather. At the same time, the relatively fine hyphae of TS-10 indicate a large specific surface area, which facilitates full contact and reaction with various chemical reagents during the leather-making process. The fine hyphae also better mimic the delicate texture of fibers in natural leather, making the resulting mycelial leather closer to traditional leather in appearance and feel.

[0008] In a second aspect, the present invention provides the above-mentioned Heme-red Fungi ( Pycnoporus sanguineus Application of TS-10 in the production of planar mycelium and / or mycelium leather.

[0009] A third aspect of the present invention provides a method for producing planar mycelia of *Hemiberlesia lataniae*, comprising the following steps: (1) Place the blood-red pore fungus ( Pycnoporus sanguineus TS-10 was inoculated into nano-enhanced PDB liquid medium for liquid pre-culture, and mycelial balls were isolated and collected from the liquid pre-culture. (2) Spread the mycelial balls prepared in step (1) evenly on the surface of the sterilized solid substrate, and then carry out dynamic culture with staged parameter control according to the growth period of Hemoglobinia mesenteroides to produce planar mycelia of Hemoglobinia mesenteroides.

[0010] Preferably, in step (1), the nano-enhanced PDB liquid culture medium is made from the following raw materials in parts by weight: 200 parts peeled potatoes, 20 parts glucose, 5 parts yeast extract, 10 parts corn starch, 1.5 parts potassium dihydrogen phosphate, 0.5 parts magnesium sulfate, 0.01 parts vitamin B1, and 3 parts nanocellulose crystals.

[0011] In the nano-enhanced PDB liquid culture medium of this invention, yeast extract is added to promote rapid mycelial proliferation; corn starch is selected as a slow-release carbon source to stabilize the mycelial ball structure; magnesium sulfate is added to enhance mycelial vigor; vitamin B1 is added to improve spore germination rate; and nanocellulose crystals (CNC) are added to enhance mycelial mechanical strength. Then, the pH of the culture medium is adjusted to 5.5-6.0 to suit *Hemiberlesia oleracea* (…). Pycnoporus sanguineus TS-10 requires a slightly acidic pH.

[0012] Preferably, in step (1), the conditions for liquid pre-culture are: stirring rate 150-180 rpm, 26℃±1℃, culture in the dark for 5-7 days, and dissolved oxygen (DO) ≥30% maintained by aeration during the culture period.

[0013] The main purpose of liquid pre-culture is to rapidly expand the required biomass. Solid inoculation cakes or spore collection have long cycles and suffer from mycelial aging, requiring a large number of inoculation plates, among other practical problems. At the same time, liquid pre-culture can form structurally stable inoculation units (mycelial balls), pre-adapt to a weakly acidic environment (pH 5.5-6.0), and may even activate the lignin-degrading enzyme system.

[0014] In the above-mentioned liquid pre-culture parameters, the stirring rate was adjusted to optimize shear force, promoting uniform formation of mycelial balls and avoiding mycelial breakage due to excessive shearing; dissolved oxygen control in the culture system prevented hypoxia in the mycelial center. Ultimately, the optimized liquid pre-culture conditions yielded a large quantity of dense mycelial-mycelial ball mixtures. The mycelial balls were then separated and collected. When inoculated in a solid substrate, the mycelial balls exhibit uniform distribution, high mycelial activity within the balls, and strong shear resistance, making them less prone to breakage during inoculation.

[0015] Preferably, in step (2), the weight ratio of mycelial balls to solid substrate is 1:(4-6).

[0016] Preferably, in step (2), the solid matrix includes a bottom layer, a middle layer and a top layer, and the mass ratio of the bottom layer, the middle layer and the top layer is (5-6):(3.5-4.5):1, and it is formed by layered laying and filling; The bottom layer consists of sugarcane top particles and eggshell powder; the middle layer consists of pepper seed shells and brewer's grains; and the top layer is an aqueous solution containing nanocellulose and trehalose.

[0017] Furthermore, the eggshell powder is prepared by the following method: The eggshells were calcined at 900℃ for 2 hours and then ground to 200 mesh; then treated with 300W plasma for 10 minutes.

[0018] The above treatment increases the surface micropores of the eggshell powder.

[0019] Furthermore, the pepper seed shells are prepared by the following method: The pepper seed shells are boiled in water to remove volatile oils, dried and crushed to 2-3 mm, treated with alkaline hydrogen peroxide to achieve a lignin degradation loss rate of 15-20%, sterilized, rinsed with water until neutral, and dried until the moisture content is <10%.

[0020] The solid matrix components of this invention are laid out and filled in layers, using density gradients to achieve slow-release nutrient supply. The bottom layer serves as a support layer, consisting of sugarcane top particles and eggshell powder. The fibrous structure of the harvested sugarcane top is loose and contains natural sugars, which provides support and is also more easily degraded by Hemangiomyces than traditional sugarcane bagasse. Replacing the traditionally used calcium carbonate with eggshell powder provides calcium while containing trace amounts of protein membrane residue (0.3-0.5%), promoting mycelial cementation.

[0021] The middle layer serves as a nutrient layer, using the waste shells from pressing peppercorns to replace traditional sawdust as a carbon source. It is rich in lignin (25-30%) and antibacterial volatile oil residues (<0.5%), which can inhibit miscellaneous bacteria and slowly release terpenoids that stimulate the formation of fruiting bodies.

[0022] As a reinforcing layer, the surface layer is coated with an aqueous solution containing nanocellulose and trehalose, which is then sprayed to ensure uniform adhesion to the nutrient layer.

[0023] Preferably, in step (2), the dynamic culture conditions for phased parameter regulation are as follows: Days 0-5: Temperature controlled at 26℃±1℃, humidity at 80%±2%, CO2 concentration at 3.0%~4.0%, and light conditions of blue light:red light = 1:3, light intensity at 50μmol / m² / s, for 16 hours / day; Days 6-12: Temperature controlled at 26℃±1℃, humidity at 80%±2%, CO2 concentration at 3.0%~4.0%, and light conditions of blue light:red light:far-red light = 3:1:1, light intensity at 30μmol / m² / s, 12 h / day; on day 8, spray micron-sized sterilized aerosol containing 0.01% gibberellin and 0.1% trehalose. Days 12-15: Temperature controlled at 22℃±1℃, humidity at 95%±1%, CO2 concentration at 2.5%~3.0%, and light conditions at pure red light intensity at 20μmol / m² / s.

[0024] This invention, based on the three different growth stages of Hemipolome, promotes rapid penetration of mycelia into the matrix by dynamically controlling environmental parameters (temperature, humidity, CO2 concentration, and spectrum) and combining them with exogenous inducers for precise intervention, thereby improving the formation efficiency of Hemipolome fruiting body primordia and efficiently forming planar mycelia.

[0025] Furthermore, the planar mycelium prepared above can be modified to prepare mycelial leather.

[0026] In a preferred embodiment of the present invention, the conditions for the modification treatment are as follows: Planar mycelium is hot-pressed at 80℃ and 5Mpa for 5 minutes, then sprayed with a 5% citric acid solution, heat-treated at 120℃ for 30 minutes, then impregnated with a 5% natural latex solution, dried, and polished to obtain mycelium leather.

[0027] The beneficial effects of this invention are: (1) The present invention directly uses liquid pre-cultured mycelial balls as inoculation units, eliminating the steps of spore collection or mycelial disruption, shortening the solid culture cycle, and improving the efficiency of spore inoculation by more than 50% compared with traditional spore inoculation.

[0028] (2) Compared with the traditional static culture of sawdust substrate (20-25 days), the dynamic regulation of the present invention reduces the primordia formation time to 8-12 days, shortens the cycle by 40%, and greatly improves the production efficiency of planar mycelium.

[0029] (3) After the first flat mycelium is formed and harvested, the gallic acid solution is added as an intermediate for lignin degradation, making the culture medium more similar to the natural decay environment, stimulating the continued reproduction of Hemoglobinia mesenteroides, realizing cyclic culture, and improving the biomass utilization rate of agricultural waste. Attached Figure Description

[0030] Figure 1 Collection and purification of Hemoglobinia mesenteroides TS-10 strain.

[0031] Figure 2 Longitudinal cross-section of the fruiting body of Heme-red Fungi TS-10.

[0032] Figure 3 Schematic diagram of a solid culture box.

[0033] Figure 4 : Schematic diagram of the grid observation method on the surface of the culture box; In the figure: the actual size of the grid is 1cm², ✓: the mycelium is completely covered ░: not completely covered.

[0034] Figure 5 Planar mycelial phenotypes of traditional sawdust substrate static culture (A) and patented method culture (B).

[0035] Figure 6 Microscopic analysis of planar mycelium structure in traditional sawdust substrate static culture (A) and patented method culture (B) (1000x electron microscope image).

[0036] Figure 7TGA and TDG curves of planar mycelia cultured using traditional sawdust substrate static culture and patented method. Detailed Implementation

[0037] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0038] Terminology Explanation: "Mycelium" refers to the free, dispersed state of mycelium, which may be individual filamentous structures that are not tightly clustered together.

[0039] "Mycelial balls" are spherical structures formed by mycelia under specific conditions (such as when they grow in a liquid culture medium, due to the entanglement and aggregation of mycelia).

[0040] To enable those skilled in the art to more clearly understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments. If specific experimental conditions are not specified in the embodiments, they are generally based on conventional conditions or conditions recommended by the reagent company; the reagents, consumables, etc. used in the following embodiments, unless otherwise specified, can be obtained commercially. Wherein: Preparation of modified malt extract-corn flour agar medium (MLYA): First, mix 10 g of corn flour (providing a complex carbon source to enhance mycelial density) with 200 mL of water, incubate at 70℃ for 30 min, filter and collect the supernatant. Then, add 15 g of malt extract (replacing glucose in traditional PDA to promote rapid mycelial growth and spore production), 3 g of yeast extract (supplementing nitrogen source and growth factors), 0.5 g of magnesium sulfate (MgSO4·7H2O) (enhancing mycelial vigor and reducing mycelial autolysis), 1.0 g of potassium dihydrogen phosphate (KH2PO4) (buffering pH and stabilizing the metabolic environment), 0.02 g of vitamin B1 (promoting spore germination), and 18 g of agar. Make up the volume to 1 L with deionized water and adjust the pH to 5.8~6.2 (to suit the slightly acidic preference of Hemothromyces rubrum). Stir magnetically until completely dissolved, boil over high heat 3 times until the culture medium is uniformly viscous (but avoid excessive boiling during operation to prevent component degradation), dispense into test tubes and Erlenmeyer flasks, autoclave at 121℃ for 20 min, after cooling, tilt the test tubes to form a slant, and invert the Erlenmeyer flasks to make a flat culture medium.

[0041] Preparation of nano-enhanced PDB liquid culture medium: 200g of peeled potatoes were weighed and cut into 1cm*1cm*1cm potato chunks. The mixture was boiled in deionized water, filtered, and then the following ingredients were added: 20g glucose, 5g yeast extract (to promote rapid mycelial proliferation), 10g corn starch (to provide a slow-release carbon source and stabilize the mycelial ball structure), 1.5g potassium dihydrogen phosphate (KH2PO4) (to optimize phosphorus metabolism), 0.5g magnesium sulfate (MgSO4·7H2O) (to enhance mycelial vitality), 0.01g vitamin B1 (to improve spore germination rate), and 3g nanocellulose crystals (CNC) (a key innovation, enhancing mycelial mechanical strength). The volume was brought to 1L with deionized water, and the pH was adjusted to 5.5-6.0 (to meet the weakly acidic requirements of *Hemothorax hemangiosum*). The culture medium was autoclaved at 121℃ for 20 min and cooled to 25℃ before use.

[0042] Solid matrix composition and preparation method: The solid matrix used in this application is laid out in layers, consisting of three parts: a bottom layer (support layer), a middle layer (nutrient layer), and a top layer (reinforcing layer), utilizing a density gradient to achieve slow-release nutrient supply. The specific formula and operating method are as follows: Bottom layer: Composed of sugarcane top particles and eggshell powder, with sugarcane top particles accounting for 90% of the bottom layer's weight and eggshell powder accounting for 10%. (The bottom layer is further subdivided into two parts:) The sugarcane top particles have a diameter of 3-5 mm and are dried at 60℃ to break down the waxy layer.

[0043] The preparation method of eggshell powder is as follows: calcination at 900℃ for 2 hours, grinding to 200 mesh; then treatment with 300W plasma for 10 minutes.

[0044] Middle layer: Composed of peppercorn seed husks and brewer's grains, with peppercorn seed husks accounting for 60% of the middle layer's weight and brewer's grains accounting for 40%. (The remaining text appears to be incomplete and requires further context.) The method for preparing Sichuan pepper seed husks is as follows: Boil the pepper seed shells in boiling water for 10 minutes to remove volatile oils, dry and crush them to 2-3 mm, treat them with alkaline hydrogen peroxide (2% H2O2, pH=11.5, 50℃ for 12 h) to achieve a lignin degradation loss rate of 15-20%, then sterilize, rinse with water until neutral, and dry at 60℃ until the moisture content is <10%.

[0045] The brewer's grains are dried at 80℃ until the moisture content is <10%.

[0046] Surface layer: An aqueous solution containing 0.8 wt% nanocellulose and 2 wt% trehalose.

[0047] Preparation method: The bottom layer, middle layer, and top layer are mixed in a weight ratio of 6:4:1. First, the bottom layer is laid, then the middle layer is laid on the bottom layer, and finally, an aqueous solution containing 0.8wt% nanocellulose and 2wt% trehalose is evenly sprayed on the middle layer to form the top layer.

[0048] Sterilization process: After layering the materials into the culture box, ensure that the volume of all contents does not exceed 2 / 3 of the culture box and does not exceed the height of the partition. Sterilize at 121℃ for 60 min (30% shorter than the conventional method, utilizing gradient density differences to achieve selective sterilization and avoiding excessive heat conduction of the stainless steel box leading to matrix carbonization). After sterilization, irradiate with ultraviolet light in a clean bench for 30 min, and then cool to 25℃±1℃ with ventilation.

[0049] Example 1: Isolation, purification and performance evaluation of Heme Conococtidae The collection and purification process of Heme Conophytum is as follows: Figure 1 As shown: Fresh, disease-free *Porphyra yezoensis* fruiting bodies were selected from the Taishan Scenic Area in Tai'an City, Shandong Province. The surface dust and impurities were rinsed with sterile water, and then placed in a laminar flow hood. The surface of the fruiting bodies was wiped with 75% ethanol (v / v) and allowed to stand for 30 seconds for preliminary disinfection. Subsequently, a sterile scalpel was used to longitudinally cut along the edge of the cap, selecting the most tender part of the flesh (usually the junction of the cap and stipe). Using a sterile dissecting needle and forceps, the internal hyphae (approximately 2-3 mm³) were quickly picked up, avoiding contact with the potentially contaminated outer pericarp, and immediately transferred to a pre-prepared modified malt extract-corn flour agar (MLYA) slant. After the strain reached the sporulation stage on the MLYA slant (cultured at 25°C in the dark for 9 days), the spore suspension was collected by gently touching the surface of the fruiting body with a sterile moistened cotton swab. The spore suspension was then diluted with sterile water to a concentration of 10. -4 ~10 -5 To prevent spore accumulation, dilute spore solution (50 μL) and spread it onto a MLYA plate. Let it stand for 10 min to allow spore adsorption. Under a 10×10 low-power microscope, gently touch a single spore with a sterile capillary needle (≤0.1 mm in diameter), ensuring only one spore is carried, and transfer it to a slide containing a 0.5% agar block (1 mm³). Transfer the agar block to a new MLYA plate and incubate at 25℃±1℃ in the dark, observing germination daily. Once a single spore has germinated and formed a colony (6 days of incubation), pick edge hyphae and transfer them to an MLYA slant. Repeat streak purification 2-3 times. Verify the purity of the strain by ITS rDNA sequencing to rule out contamination. The ITS rDNA sequencing results are shown in SEQ ID NO.1.

[0050] The purified *Hemerocallis fulva* was named TS-10. The culture performance of *Hemerocallis fulva* TS-10 was investigated, and the results showed that it was easy to culture, grew rapidly, and had relatively broad requirements for substrates, reaching full coverage on PDA plates in about 5 days.

[0051] Scanning electron microscopy was performed on the cross-section of the fruiting body of *Hemiberlesia lataniae* TS-10, and the results are as follows: Figure 2 As shown, the hyphae of TS-10 exhibit a relatively dense state. The hyphae intertwine to form a relatively complex network structure. This high hyphal density means that it can provide a good foundation of strength and toughness when made into mycelial leather. This is because the dense hyphal structure can better withstand external forces, reducing the risk of tearing and breakage, similar to the physical properties imparted by the tightly packed fibers in natural leather. At the same time, the relatively fine hyphae of TS-10 indicate a large specific surface area, which facilitates full contact and reaction with various chemical reagents during the leather-making process. The fine hyphae also better mimic the delicate texture of fibers in natural leather, making the resulting mycelial leather closer to traditional leather in appearance and feel.

[0052] Biopreservation of *Hemiberlesia lataniae* TS-10 is as follows: Reference biomaterial: TS-10 Classification and nomenclature: Hemlockium Pycnoporus sanguineus Preservation period: June 19, 2025 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Collection institution abbreviation: CGMCC Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Registered with the China National Collection Center (CGMCC) No. 42037.

[0053] Example 2: Production of planar mycelium using Hemipoloma TS-10 1. Liquid pre-culture: The mycelial blocks (5×5 mm) of Heme Flococcus TS-10 isolated and purified in Example 1 were inoculated into nano-enhanced PDB liquid medium for liquid pre-culture. The liquid pre-culture conditions were: stirring rate 160 rpm, 26℃±1℃, cultured in the dark for 7 days, and dissolved oxygen (DO) ≥30% was maintained by aeration during the culture period.

[0054] The product obtained from liquid pre-culture was filtered through four layers of sterile gauze to separate and collect mycelial balls, which were then used as inoculation units for subsequent solid-state culture inoculation.

[0055] 2. Solid-state culture box layered directional spectral induction and regulation culture: The sterilized solid substrate is placed in a solid culture box (e.g.) Figure 3 As shown, the solid culture box includes: a culture container and a top cover of the culture container; the culture container is provided with a breathable mesh partition, which utilizes its breathable and water-impermeable properties to ensure gas exchange (such as oxygen entering and carbon dioxide escaping) in the culture container while preventing contamination by external bacteria and leakage of solid substrate, blocking solid substrate, and facilitating the peeling of planar mycelia in the later stage; the top cover of the culture container is provided with a handle and a vent.

[0056] Following a 1:5 ratio of wet weight mycelial balls to solid substrate, the mycelial balls were evenly spread directly on the surface of the solid substrate. The culture container was gently shaken 3-5 times (amplitude ≤30°) to ensure even distribution. The top cover of the culture container was removed, and after sealing, it was quickly transferred to a carbon dioxide incubator with an LED photobioreactor module added to the top layer. Based on the three different growth stages of *Hemipoloma heliotropium*, environmental parameters (temperature, humidity, CO2 concentration, and spectrum) were dynamically adjusted, combined with precise intervention using exogenous inducers, to promote rapid mycelial penetration of the substrate and improve the formation efficiency of *Hemipoloma heliotropium* fruiting body primordia. The specific operating procedure is shown in Table 1.

[0057] Table 1: Dynamic culture parameters of *Hemiberlesia oleracea* mycelium at different growth stages Note: Micron-sized sterilized aerosol containing 0.01% gibberellin + 0.1% trehalose should be sprayed only once on the 8th day of cultivation.

[0058] Preparation method: Gibberellin and trehalose were dissolved in sterile water to achieve a gibberellin concentration of 0.01% and a trehalose concentration of 0.1%. The solution was filtered through a 0.22 μm filter membrane and an ultrasonic nebulizer (1.7 MHz) was used to generate a 5-10 μm aerosol. Spraying volume: 50 mL per square meter of culture area. Its function is to synergistically induce primordium formation; gibberellin promotes hyphal branching; and trehalose provides stress protection.

[0059] After the cultivation stage is completed, the mycelium is peeled off from the top of the breathable mesh layer and dried with hot air at 60°C for 2 hours to control the moisture content of the mycelium at 10%~12%, thus preparing planar mycelium, and then conducting relevant quality tests.

[0060] After harvesting, retain the underlying mycelial network and supplement with 0.1% gallic acid solution (50 ppm) aseptic spray to induce secondary mycelial growth.

[0061] Comparative Example 1: 1. Traditional PDB liquid culture: The mycelial blocks (5×5 mm) of Heme Fungi TS-10 isolated and purified in Example 1 were inoculated into PDB liquid medium (200g potato, 20g glucose, 1L water, 121℃, 20min) and cultured under the following conditions: 120rpm, 28℃, and in the dark for 7 days.

[0062] 2. Static culture in traditional sawdust substrate: Mycelial balls obtained from traditional PDB liquid culture were inoculated into a traditional sawdust substrate (80wt% poplar sawdust, 19wt% wheat bran, 1wt% calcium carbonate). Temperature: constant at 25℃, humidity: constant at 70%, no CO2 control, no light regulation, culture time: 25 days.

[0063] Comparative Example 2: 1. Liquid pre-culture: The conditions for liquid pre-culture are the same as in Example 2.

[0064] 2. Solid-state culture box layered directional spectral induction and regulation culture: The solid substrate was replaced with a substrate consisting only of the "bottom layer" component. The amount of solid substrate in the solid culture box was the same as in Example 2, and the other culture conditions were the same as in Example 2.

[0065] Comparative Example 3: 1. Liquid pre-culture: The conditions for liquid pre-culture are the same as in Example 2.

[0066] 2. Solid-state culture box layered directional spectral induction and regulation culture: The solid substrate was replaced with a mixture of "bottom layer + middle layer" (the weight ratio of the bottom layer to the middle layer was 6:4). The amount of solid substrate in the solid culture box was the same as in Example 2, and the other culture conditions were the same as in Example 2.

[0067] Comparative Example 4: 1. Liquid pre-culture: The conditions for liquid pre-culture are the same as in Example 2.

[0068] 2. Solid-state culture box layered directional spectral induction and regulation culture: The solid substrate was replaced with a mixture of "bottom layer + top layer" (bottom layer to top layer weight ratio of 6:1). The amount of solid substrate in the solid culture box was the same as in Example 2, and the other culture conditions were the same as in Example 2.

[0069] Test Example: Performance Evaluation 1. Quality assessment of mycelial pellets: The compressive strength and solid colonization time of the mycelial pellets obtained from liquid pre-culture in Example 2 and those obtained from conventional PDB liquid culture in Comparative Example 1 were measured using the following methods: Method for determining the compressive strength (kPa) of mycelial pellets: Based on the principle of simulating the mechanical stress borne by the mycelial pellets during solid inoculation, 30 mycelial pellets (diameter 2.0±0.5 mm) after liquid pre-culture were first taken, and the surface moisture was absorbed with sterile filter paper. A micro-force tester equipped with a cylindrical flat-bottomed probe (diameter 2 mm) was used, with a compression rate of 0.5 mm / min and a trigger force of 0.1 mN. The test was stopped when the mycelial pellet ruptured (deformation > 50%). The compressive strength was determined according to the formula: "Compressive strength = maximum breaking force (N) / projected area of ​​mycelial pellet (mm²)". 2 )×10 3 "Perform calculations."

[0070] Solid colonization time (d) determination method: The determination is mainly based on the principle of quantifying the mycelial expansion efficiency in a solid substrate. First, a 1cm × 1cm grid is divided on the surface of the culture box substrate (e.g., ...). Figure 4 As shown, the observation points were marked, and the mycelial coverage of the grid points was observed with an endoscope at 8:00, 14:00 and 20:00 every day. The criterion was that the mycelium completely penetrated the surface of the grid point (a white mycelial network was visible to the naked eye). When "≥50% of the grid points" reached complete coverage for two consecutive days, it was recorded as the first day of planting completion.

[0071] The results are shown in Table 2.

[0072] Table 2: Comparison of the mass of bacterial pellets obtained from liquid culture The results showed that introducing CNC nanomaterials into the liquid culture of *Hemiberlesia oleracea* physically reinforced the mycelial ball structure, solving the problems of fragility and low inoculation efficiency of traditional mycelial balls. Combined with controllable shear stress (150-180 rpm), the mycelial morphology was optimized, avoiding mycelial breakage caused by excessive shearing (high-speed stirring above 200 rpm). Simultaneously, the increased mycelial branching density expanded the specific surface area, which is beneficial for improving the colonization efficiency of subsequent solid-state culture.

[0073] 2. Phenotypic analysis of planar mycelium: The phenotypes of the planar mycelia prepared in Example 2 and Comparative Example 1 were analyzed, and the results are as follows: Figure 5 As shown, the planar mycelial patches cultured using the traditional substrate and method (Comparative Example 1) exhibit a significant color difference, with alternating light and dark areas (the left side is orange-pink with two dark orange-red circular patches, and the right side is dark orange-red), showing an overall uneven gradient. The surface has a noticeable granular texture, uneven thickness, and is generally thin, with significant edge tearing marks and weak mycelial network cohesion. Figure 5A). The planar mycelium harvested using the method of this patent (Example 2) exhibits a highly uniform color, displaying a saturated orange-red hue overall, without localized spots or gradations, demonstrating uniform spectral density (primarily red light) and inducing agent activity during the cultivation phase. Furthermore, the surface integrity is high, with naturally undulating texture but no particulate impurities, indicating high mycelial density. Only a few brownish spots (possibly natural pigment deposition) are present at the edges, with no tears or exposed white background, indicating a dense mycelial network structure. Figure 5 B).

[0074] The results showed that the planar mycelium of *Hemiberlesia lataniae* harvested using this patent exhibited significantly better color uniformity and structural integrity than that of traditional culture methods (Comparative Example 1).

[0075] 3. Microstructural analysis of planar mycelia: The planar mycelia prepared in Example 2 and Comparative Example 1 were analyzed under an electron microscope for microstructure, and the results are as follows: Figure 4 As shown, under a 1000x electron microscope, the planar mycelium of *Hemangioma repens* cultured in Comparative Example 1 exhibits sparse, disordered, interwoven hyphae on the surface with obvious breaks in some areas and granular impurities (possibly undegraded sawdust residue) adhering to the surface. The mycelial network on the reverse side shows poor continuity, with large blank areas and uneven hyphae thickness, with a diameter variation coefficient ≥35%. The longitudinal section shows a loose interlayer structure with significant differences in hyphae stack thickness (thinnest ≤2μm, thickest ≥8μm), exhibiting an "island-like distribution" with a porosity as high as 40%–45%. Figure 6 A). The optimized *Hemangiospermum erythrorhizon* planar mycelium obtained in Example 2 exhibits a highly dense mycelial arrangement on its surface, forming a "woven" topological structure. The diameter of individual mycelial bundles is uniform (3.5±0.3μm), and the surface is clean and free of impurities. The entire back surface is free of discontinuities, and the mycelia achieve three-dimensional interlocking through branch nodes, resulting in a network integrity of over 98%. The cross-section displays a "gradient compaction" characteristic, with the upper layer of mycelia having an orientation degree of 75% (anisotropy), and the lower layer having a porosity controlled at 20%~25%. Figure 6 B). Therefore, the planar mycelium of *Hemipoloma spp.* harvested using the "matrix gradient design + mycelial directional induction" method of this patent significantly optimizes the microstructural integrity of the mycelium, and its density, purity and mechanical properties far exceed those of traditional processes.

[0076] 4. Mechanical property analysis of planar mycelium: The mechanical properties of the planar mycelia prepared in Example 2 and Comparative Examples 1-4 were analyzed using the following methods: According to the requirements of QB / T 2710-2018 "Determination of Tensile Strength and Elongation of Leather (Physical and Mechanical Tests)", the main steps are as follows: (1) Sample preparation: Use a standard double-blade die (50mm×6.5mm) to cut the sample along the hyphal orientation, retaining the original thickness of 2.0±0.1mm, and do not grind; (2) Pretreatment: Condition the environment at 23±2℃ and 65±5% relative humidity for 48 hours; (3) Test conditions: clamping distance 50mm, stretching speed 100mm / min; (4) Record indicators: tensile strength, elongation at break and elongation at specified load (5N / mm² stress value).

[0077] Tensile strength (MPa) = Maximum breaking force (N) / [Specimen width (mm) × Thickness (mm)] Elongation at break = (Distance at break - Original gauge length) / Original gauge length × 100% (Original gauge length is the clamping distance of 50mm) The specified load elongation is the elongation value under a specific stress (e.g., 5 N / mm²).

[0078] The results are shown in Table 3.

[0079] Table 3: Results of mechanical property analysis of planar mycelia obtained by different culture methods 5. Thermal stability analysis of planar mycelium: The thermal stability of the planar mycelia prepared in Example 2 and Comparative Example 1 was analyzed, and the results are shown in Table 4. Figure 7 As shown.

[0080] Table 4: Thermal stability data of planar mycelia obtained by different culture methods The results showed that the planar mycelia of *Hemiberlesia lataniae* cultured using the patented method (Example 2) exhibited stronger tolerance to high temperatures, better thermal stability, and a certain degree of improvement in residual carbon content, retaining more skeletal structure at high temperatures. Furthermore, the full width at half maximum (FWHM) data of the DTG curves indicated that, compared to the planar mycelia cultured under conventional methods (Comparative Example 1), the improved planar mycelial material had a more concentrated decomposition rate, suggesting better molecular structure uniformity and a more controllable decomposition process.

[0081] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for producing planar mycelium of *Hemiberlesia lataniae*, characterized in that, Includes the following steps: (1) The Heliotropium indicum with biological accession number: CGMCC NO.42037 ( Pycnoporus sanguineus TS-10 was inoculated into nano-enhanced PDB liquid medium for liquid pre-culture, and mycelial balls were isolated and collected from the liquid pre-culture. (2) Spread the mycelial balls prepared in step (1) evenly on the surface of the sterilized solid substrate, and then carry out dynamic culture with staged parameter control according to the growth period of Hemoglobinia mesenteroides to produce planar mycelia of Hemoglobinia mesenteroides. In step (1), the nano-enhanced PDB liquid culture medium is made from the following raw materials in parts by weight: 200 parts peeled potatoes, 20 parts glucose, 5 parts yeast extract, 10 parts corn starch, 1.5 parts potassium dihydrogen phosphate, 0.5 parts magnesium sulfate, 0.01 parts vitamin B1, and 3 parts nanocellulose crystals; In step (2), the solid matrix includes a bottom layer, a middle layer and a top layer, and the mass ratio of the bottom layer, the middle layer and the top layer is (5-6):(3.5-4.5):1, and it is formed by layering and filling. The bottom layer consists of sugarcane top particles and eggshell powder; the middle layer consists of pepper seed shells and brewer's grains; and the top layer is an aqueous solution containing nanocellulose and trehalose. In step (2), the dynamic culture conditions for phased parameter regulation are as follows: Days 0-5: Temperature controlled at 26℃±1℃, humidity at 80%±2%, CO2 concentration at 3.0%~4.0%, and light conditions of blue light:red light = 1:3, light intensity at 50μmol / m² / s, for 16 hours / day; Days 6-12: Temperature controlled at 26℃±1℃, humidity at 80%±2%, CO2 concentration at 3.0%~4.0%, and light conditions of blue light:red light:far-red light = 3:1:1, light intensity at 30μmol / m² / s, 12 h / day; on day 8, spray micron-sized sterilized aerosol containing 0.01% gibberellin and 0.1% trehalose. Days 12-15: Temperature controlled at 22℃±1℃, humidity at 95%±1%, CO2 concentration at 2.5%~3.0%, and light conditions at pure red light intensity at 20μmol / m² / s.

2. The production method according to claim 1, characterized in that, In step (1), the conditions for liquid pre-culture are: stirring speed 150-180 rpm, 26℃±1℃, culture in the dark for 5-7 days, and maintain dissolved oxygen ≥30% by aeration during the culture period.

3. The production method according to claim 1, characterized in that, In step (2), the weight ratio of mycelial balls to solid substrate is 1:(4-6).

4. The production method according to claim 1, characterized in that, The eggshell powder is prepared by the following method: The eggshells were calcined at 900℃ for 2 hours and then ground to 200 mesh; then treated with 300W plasma for 10 minutes.

5. The production method according to claim 1, characterized in that, The pepper seed shells are prepared by the following method: The pepper seed shells are boiled in water to remove volatile oils, dried and crushed to 2-3 mm, treated with alkaline hydrogen peroxide to achieve a lignin degradation loss rate of 15-20%, sterilized, rinsed with water until neutral, and dried until the moisture content is <10%.

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