Fuscoporia lilacinus as well as culture method and application thereof

Abstract

The invention provides a Fuscoporia lilacinus strain as well as a culture method and application thereof, and relates to the technical field of microorganisms. According to the method, biological characteristics and culture conditions of the Fuscoporia lilacinus are defined, the technical problem of artificial culture of the Fuscoporia lilacinus is solved, artificial culture of the Fuscoporia lilacinus is realized for the first time, and a new variety and a new culture way are provided for new resource utilization of rare edible and medicinal fungi in China. The invention provides a Fuscoporia lilacinus strain culture formula and a solid-liquid coupled liquid strain culture method for strain propagation, and mycelia grow fast and are strong in vitality. According to the invention, functional evaluation is carried out on the active ingredients of the sporocarp of the Fuscoporia lilacinus, the extract with the blood sugar reducing activity effect is obtained, and a new raw material source is provided for research and development of medicines and health-care products.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and in particular to a strain of *Porphyra yezoensis*, its culture method, and its application. Background Technology

[0002] Currently, edible and medicinal fungi have become the fifth largest industry in the planting sector, accounting for more than 75% of the world's edible fungi production. With the development of the social economy and the enhancement of people's health awareness, health management is shifting from disease treatment to disease prevention, leading to a year-on-year increase in market demand for medicines and health products made from green and renewable raw materials such as edible and medicinal fungi. Fungal resources are a prerequisite for the high-quality development of the edible and medicinal fungi industry. Currently, nearly 1,000 species of edible and medicinal fungi have been reported, and more than 400 species have been identified as having medicinal effects. However, only about 80 species of edible and medicinal fungi have been artificially cultivated, and only about 30 species have achieved large-scale cultivation.

[0003] Pale yellowish-brown Inonotus obliquus ( Fuscoporia gilva ) belongs to the family Leucobacteriaceae ( Fuscoporia ) genus *Inonotus* Fuscoporia This fungus, widely distributed worldwide, is a decay fungus found on wood. It plays a vital role in the carbon cycle and possesses medicinal properties such as spleen-tonifying, dampness-removing, stomach-strengthening, and anti-tumor effects. In traditional Chinese medicine, its wild fruiting bodies are used as *Sanghuang* for tumor prevention. Currently, wild resources of *Inonotus obliquus* are limited. Research on *Inonotus obliquus* mainly focuses on its distribution, identification, and phylogeny, while research on its wild domestication, resource characteristics, and applications remains incomplete, and artificial cultivation and application have not yet been achieved. Summary of the Invention

[0004] To realize the artificial cultivation and resource application of wild *Inonotus obliquus*, the purpose of this invention is to provide *Inonotus obliquus* and its cultivation method, and to extract and evaluate the active ingredients of its fruiting body, providing a new type of renewable and green raw material for the research and development of drugs and health products with hypoglycemic effects.

[0005] On the one hand, this application provides a strain of pale yellowish-brown Flocculation Fungus ( Fuscoporia gilva XYS3 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42288.

[0006] On the other hand, this application also provides a method for culturing *Inonotus obliquus*, the method comprising: Step 1: Inoculate the pale yellow-brown *Inonotus obliquus* into the mother culture medium and culture it until the mycelium has covered the plate. Then stop the culture to obtain the mother culture. The culture conditions include: culture temperature 16℃-31℃, pH 3-9, and culture in the dark; preferably, pH 4-8. Step 2: Inoculate the mother culture into a liquid culture medium for cultivation to obtain a liquid culture spawn; the cultivation conditions include: cultivation temperature 16℃-31℃, pH 3-9, 100-200 r / min, and cultivation in the dark for 5-20 days; preferably, pH 4-8; Step 3: Inoculate the liquid culture medium with 1%-5% of the liquid culture medium to allow for mycelial growth and fruiting.

[0007] Furthermore, the mother culture medium contains a carbon source and / or a nitrogen source.

[0008] Furthermore, the carbon source comprises one or more of glucose, sucrose, lactose, maltose, and mannitol.

[0009] Preferably, the carbon source is mannitol.

[0010] Furthermore, the nitrogen source comprises one or more of peptone, beef extract, yeast extract, ammonium sulfate, and ammonium nitrate.

[0011] Preferably, the nitrogen source is yeast extract.

[0012] The upper or lower limit of the culture temperature and its range can be any value or range of 16℃, 17℃, 18℃, 19℃, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, and 31℃.

[0013] The culture pH and its upper or lower limit can be any value or range of 3, 4, 5, 6, 7, 8, or 9.

[0014] Preferably, the culture conditions in step one are a culture temperature of 25℃-28℃ and a culture pH of 5-6.

[0015] Furthermore, the culture conditions in step one are a culture temperature of 28°C and a culture pH of 6.

[0016] Preferably, the mother culture medium further includes one or more of potato, potassium dihydrogen phosphate, magnesium sulfate, and vitamin B1.

[0017] More preferably, the mother culture medium comprises, by weight percentage: 10-30% potato, 1-5% mannitol, 0.1-0.5% yeast extract, 0.1-0.5% potassium dihydrogen phosphate, 0.1-0.5% magnesium sulfate, and 0.001-0.005% vitamin B1.

[0018] More preferably, the mother culture medium comprises, by weight percentage: 20% potato, 2% mannitol, 0.3% yeast extract, 0.2% potassium dihydrogen phosphate, 0.1% magnesium sulfate, and 0.001% vitamin B1.

[0019] In a preferred embodiment, the mother culture medium comprises: 20% potato, 2% mannitol, 0.3% yeast extract, 0.2% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 0.001% vitamin B1, 1.5% agar, and the remainder is water.

[0020] The mother culture medium used in this application enables efficient cultivation of *Inonotus obliquus*, with a daily mycelial growth rate of 1.8-5.31 mm / d. This means that the mother culture medium used in this application can promote the growth of *Inonotus obliquus* and increase its growth rate.

[0021] Further, the liquid culture medium contains plant extracts; preferably, the plant extracts include poplar flower extract and / or sea buckthorn extract; more preferably, poplar flower extract; even more preferably, the concentration of the poplar flower extract is 0.01%-1% by weight of the liquid culture medium; preferably, 0.3%.

[0022] The concentration of the poplar flower extract, including its upper or lower limits, can be any value or range of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%.

[0023] This application demonstrates for the first time that adding poplar flower extract or sea buckthorn extract to liquid culture medium can promote the growth of *Inonotus obliquus*, improve the morphology of *Inonotus obliquus*, and increase the biomass of *Inonotus obliquus*.

[0024] The liquid culture medium used in this application enables efficient cultivation of *Polyporus pachycarpus*, with a biomass of ≥0.8 g / 100 mL and a maximum of 1.12 g / 100 mL.

[0025] Preferably, the liquid culture medium further includes one or more of mannitol, corn flour, yeast extract, wheat bran powder, peptone, potassium dihydrogen phosphate, magnesium sulfate, and vitamin B1.

[0026] More preferably, by weight percentage, the liquid culture medium comprises: 1-5% mannitol, 1-5% corn flour, 0.1-0.5% yeast extract, 0.1-0.5% wheat bran, 0.1-0.5% peptone, 0.1-0.5% potassium dihydrogen phosphate, 0.1-0.5% magnesium sulfate, 0.001-0.005% vitamin B1, and 0.01%-1% poplar flower extract.

[0027] More preferably, the liquid culture medium comprises, by weight percentage: 3% mannitol, 1% corn flour, 0.5% yeast extract, 0.5% wheat bran powder, 0.5% peptone, 0.2% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 0.001% vitamin B1, and 0.3% poplar flower extract.

[0028] In a preferred embodiment, the liquid culture medium comprises: 3% mannitol, 1% corn flour, 0.5% yeast extract, 0.5% wheat bran powder, 0.5% peptone, 0.2% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 0.001% vitamin B1, 0.3% poplar flower extract, and the balance being water.

[0029] The upper or lower limit of the culture temperature and its range can be any value or range of 16℃, 17℃, 18℃, 19℃, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, and 31℃.

[0030] The culture pH and its upper or lower limit can be any value or range of 3, 4, 5, 6, 7, 8, or 9.

[0031] The upper or lower limit of the cultivation rotation speed and its range can be any value or range of 100 r / min, 110 r / min, 120 r / min, 130 r / min, 140 r / min, 150 r / min, 160 r / min, 170 r / min, 180 r / min, 190 r / min, and 200 r / min.

[0032] The upper or lower limit of the cultivation time and its range can be any value or range of 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, and 20 days.

[0033] Furthermore, the culture conditions in step two include: a culture temperature of 28°C, a culture pH of 6, a speed of 150 r / min, and culture in the dark for 7-8 days.

[0034] Furthermore, the bag cultivation medium contains plant extracts; preferably, the plant extracts include poplar flower extract and / or sea buckthorn extract; more preferably, poplar flower extract; even more preferably, the concentration of the poplar flower extract is 0.01%-1% by weight of the bag cultivation medium; preferably, 0.3%.

[0035] Preferably, the bag cultivation medium further includes one or more of the following: sawdust, chestnut shells, wheat bran, soybean meal, quicklime, gypsum, and mannitol.

[0036] More preferably, by weight percentage, the bag cultivation medium comprises: 10-50% sawdust, 10-50% chestnut shells, 10-50% wheat bran, 1-5% soybean meal, 1-5% quicklime, 1-5% gypsum, 0.01%-1% poplar flower extract, and 1-5% mannitol.

[0037] More preferably, the bag cultivation medium comprises, by weight percentage: 40% sawdust, 40% chestnut shells, 15% wheat bran, 1% soybean meal, 1% quicklime, 1% gypsum, 0.3% poplar flower extract, and 1.7% mannitol.

[0038] More preferably, the moisture content of the bag cultivation medium is 55%-60%.

[0039] Those skilled in the art can obtain the above-mentioned culture medium using conventional methods, such as stirring and mixing, or by sterilizing the culture medium using known methods. They can also use existing techniques to control the aseptic state of the inoculation environment to ensure experimental accuracy; no specific limitations are made here. The sterilization temperature can be 121℃-125℃, and the sterilization time can be 1-2 hours.

[0040] Furthermore, the inoculation amount in step three is 1%.

[0041] Furthermore, the cultivation step in step three includes: (1) Mycelium management: Inoculate the liquid culture medium with 1%-5% of the liquid culture medium, culture temperature 28℃, culture humidity 40%-60%, carbon dioxide concentration not higher than 4000 ppm, culture for 25-30 days, then lower the temperature to 25℃ and culture for 10-15 days; (2) Budding stage: 300-500 Lux diffused light, carbon dioxide concentration of 800-1000 ppm, humidity of 80%-90%, temperature controlled at 18℃-28℃. After the mycelium twists and forms mycelial clumps, fruiting management is carried out. (3) Mushroom management: 300-500 Lux diffused light, carbon dioxide concentration of 800-1000 ppm, humidity of 80%-90%, culture at 25℃-28℃, and harvest the fruiting bodies when the pale yellow growth ring of the fruiting bodies disappears.

[0042] Furthermore, the pale yellow-brown porphyria is pale yellow-brown porphyria ( Fuscoporia gilva XYS3 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42288.

[0043] Using the method described in this application, the yield of *Porphyra yezoensis* per bottle can reach 15-20 g.

[0044] On the other hand, this application also provides a light yellowish-brown Inonotus var. praecox culture medium, wherein the culture medium contains poplar flower extract and / or sea buckthorn extract; preferably, the culture medium contains poplar flower extract; more preferably, the concentration of poplar flower extract is 0.01%-1% by weight of the culture medium; preferably, 0.3%.

[0045] Preferably, the culture medium for *Inonotus obliquus* is a liquid culture medium for *Inonotus obliquus* and / or a bag culture medium for *Inonotus obliquus*.

[0046] In a preferred embodiment, the *Polyporus pachycarpa* liquid culture medium comprises: 1-5% mannitol, 1-5% corn flour, 0.1-0.5% yeast extract, 0.1-0.5% wheat bran powder, 0.1-0.5% peptone, 0.1-0.5% potassium dihydrogen phosphate, 0.1-0.5% magnesium sulfate, 0.001-0.005% vitamin B1, and 0.01%-1% poplar flower extract.

[0047] In a preferred embodiment, the light yellowish-brown *Polygonum multiflorum* bag cultivation medium comprises: 10-50% sawdust, 10-50% chestnut shells, 10-50% wheat bran, 1-5% soybean meal, 1-5% quicklime, 1-5% gypsum, 0.01%-1% poplar flower extract, and 1-5% mannitol.

[0048] Furthermore, the pale yellow-brown porphyria is pale yellow-brown porphyria ( Fuscoporia gilva XYS3 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42288.

[0049] On the other hand, this application also provides a light yellow-brown pore culture medium for *Polyporus fusiforme*, the culture medium comprising: 20% potato, 2% mannitol, 0.3% yeast extract, 0.2% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 0.001% vitamin B1, 1.5% agar, and the remainder being water.

[0050] Furthermore, the pale yellow-brown porphyria is pale yellow-brown porphyria ( Fuscoporia gilva XYS3 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42288.

[0051] On the other hand, this application also provides the application of the *Inonotus obliquus* culture medium in the cultivation of *Inonotus obliquus*, wherein the cultivation of *Inonotus obliquus* includes increasing the yield of *Inonotus obliquus*, increasing the growth rate of *Inonotus obliquus*, improving the growth characteristics of *Inonotus obliquus*, and increasing the nutrient content of *Inonotus obliquus*.

[0052] Furthermore, the pale yellow-brown porphyria is pale yellow-brown porphyria ( Fuscoporia gilva XYS3 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42288.

[0053] On the other hand, this application also provides the use of the described culture method, the described culture medium, or the described *Inonotus obliquus* in the preparation of products containing *Inonotus obliquus*.

[0054] Furthermore, the pale yellow-brown porphyria is pale yellow-brown porphyria ( Fuscoporia gilva XYS3 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42288.

[0055] Furthermore, the product containing *Paecilomyces paeonii* contains active substances, including one or more of polysaccharides, flavonoids, and polyphenols.

[0056] On the other hand, this application also provides the application of the culture method, the culture medium, or the *Paecilomyces pachycarpa* in the preparation of hypoglycemic products.

[0057] Furthermore, the pale yellow-brown porphyria is pale yellow-brown porphyria ( Fuscoporia gilva XYS3 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42288.

[0058] Furthermore, the blood sugar lowering product contains blood sugar lowering active substances, which include one or more of polysaccharides, flavonoids, and polyphenols.

[0059] Furthermore, the blood glucose reduction is achieved by inhibiting the activity of α-glucosidase and / or α-amylase.

[0060] On the other hand, this application also provides the use of the described culture method, the described culture medium, or the described *Polyporus pachycarpus* in the preparation of products that inhibit α-glucosidase and / or α-amylase activity.

[0061] On the other hand, this application also provides a solution containing the aforementioned pale yellowish-brown Flocculation Fungus ( Fuscoporia gilva XYS3 products.

[0062] Preferably, the product includes a hypoglycemic product, an α-glucosidase activity inhibitory product, and / or an α-amylase activity inhibitory product.

[0063] Preferably, the product further includes physiologically acceptable excipients or media.

[0064] Those skilled in the art may add excipients as needed, or prepare the product into the required dosage form, and no specific limitations are made in this application.

[0065] In a preferred embodiment, the product may be a light yellowish-brown lyophilized powder of *Polyporus fusilli*.

[0066] On the other hand, this application also provides a method for extracting pale yellowish-brown Flouria ( Fuscoporia gilva The method for extracting active substances from the fruiting bodies of the pale yellow-brown *Potamogeton crispus* is as follows: drying the fruiting bodies at 40℃-80℃, pulverizing them to 60-100 mesh, adding the extractant at a material-to-liquid ratio of 1:(10-20) g / mL, refluxing at 60℃-90℃ for 1-5 h, and collecting the supernatant by centrifugation.

[0067] Preferably, the light yellowish-brown fruiting bodies of *Polyporus flocculationensis* are dried at 60°C, pulverized to 80 mesh, and extracted with an extractant at a material-to-liquid ratio of 1:15 g / mL. The mixture is then refluxed at 80°C for 3 h, and the supernatant is collected by centrifugation.

[0068] Preferably, the extractant can be water or an organic solvent; more preferably, the organic solvent can be ethanol; even more preferably, the concentration of the ethanol is 65%.

[0069] In a preferred embodiment, the method includes: drying the pale yellowish-brown fruiting bodies of *Polyporus flocculationensis* at 60°C, pulverizing them to 80 mesh, adding water at a material-to-liquid ratio of 1:15 g / mL, refluxing at 80°C for 3 h, and centrifuging to collect the supernatant.

[0070] In a preferred embodiment, the method includes: drying the pale yellowish-brown fruiting bodies of *Potamogeton crispus* at 60°C, pulverizing them to 80 mesh, adding 65% ethanol at a material-to-liquid ratio of 1:15 g / mL, refluxing at 80°C for 3 h, and centrifuging to collect the supernatant.

[0071] Preferably, the active substance includes polysaccharides, flavonoids and / or polyphenols.

[0072] Preferably, the polysaccharide is greater than or equal to 20 mg / g; more preferably, it is greater than or equal to 40 mg / g.

[0073] Preferably, the flavonoid is greater than or equal to 4 mg / g; more preferably, it is greater than or equal to 20 mg / g.

[0074] Preferably, the polyphenol is greater than or equal to 4 mg / g; more preferably, it is greater than or equal to 10 mg / g.

[0075] The present invention has the following beneficial effects: This invention clarifies the biological characteristics and culture conditions of *Inonotus obliquus*, solves the technical problems of its artificial culture, and realizes the artificial cultivation of *Inonotus obliquus* for the first time, providing a new variety and a new cultivation method for the new resource utilization of rare edible and medicinal fungi in my country.

[0076] This invention provides a culture formula for *Inonotus obliquus* and a solid-liquid coupled liquid culture method for strain propagation, resulting in rapid mycelial growth and colonization with strong vitality. This invention evaluated the active components of the pale yellow-brown Inonotus obliquus fruiting body and obtained an extract with hypoglycemic activity, providing a new source of raw materials for the research and development of pharmaceuticals and health products. Attached Figure Description

[0077] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments of this application and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1 The images show morphological observations of strain XYS3. In Figure d, the microscope magnification is 20*40x, and in Figure e, the microscope magnification is 20*100x. Figure 2 Phylogenetic tree diagram of strain XYS3; Figure 3 The results of the effect of carbon source on mycelial growth are shown in the figure. In the figure, a is a schematic diagram of the effect of carbon source on mycelial growth, and b is a statistical graph of the effect of carbon source on mycelial growth rate. Figure 4 The results of the effect of nitrogen source on mycelial growth are shown in the figure. In the figure, a is a schematic diagram of the effect of nitrogen source on mycelial growth, and b is a statistical graph of the effect of nitrogen source on mycelial growth rate. Figure 5 The results of the effect of pH on mycelial growth are shown in the figure. In the figure, a is a schematic diagram of the effect of pH on mycelial growth, and b is a statistical graph of the effect of pH on mycelial growth rate. Figure 6 The results of the effect of temperature on mycelial growth are shown in the figure. In the figure, a is a schematic diagram of the effect of temperature on mycelial growth, and b is a statistical graph of the effect of temperature on mycelial growth rate. Figure 7 The effect of different exogenous added factors on the yield of pale yellow-brown prawn mycelium is shown in the figure. From left to right, the liquid culture media are Formula 4, Formula 4 + Sophora japonica flower extract, Formula 4 + Hippophae rhamnoides extract, Formula 4 + Populus tomentosa flower extract, Formula 4 + Cymbidium faberi flower extract, and Formula 4 + Mulberry leaf extract. Figure 8 The figure above shows the effect of different exogenous added factors on the growth of liquid microbial strains. From left to right, they are: Formula 4, Formula 4 + Tree Flower Extract, Formula 4 + Mulberry Leaf Extract, Formula 4 + Poplar Flower Extract, Formula 4 + Sophora Flower Extract, and Formula 4 + Sea Buckthorn Extract. Figure 8 The diagram below shows the optimal liquid culture medium formulation, from left to right: Formulation 4 and Formulation 4 + Poplar Flower Extract. Figure 9 Figures showing mycelial yield in liquid fermentation broth at different culture times; Figure 10 The diagram shows the mycelial morphology after 8 days of culture and the mycelial morphology after 9 days of culture. The magnification is 20*100. Figure 11 The image shows the morphological characteristics of liquid bacterial strains under optimized conditions. The left image is a top view, and the right image is a front view. Figure 12 The image shows the growth of *Inonotus obliquus* XYS3 on an artificial culture medium. From left to right, the images show the mycelial growth, mycelial color change, and fruiting body formation.

[0078] Preservation of biological materials: A pale yellowish-brown porphyria ( Fuscoporia gilva XYS3 was deposited on October 23, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42288. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences. Detailed Implementation

[0079] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0080] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0081] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0082] Unless otherwise specified, in the following embodiments, reagents or instruments whose manufacturers are not indicated are all conventional products that can be purchased commercially.

[0083] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in the fields of microbiology, biochemistry, analytical chemistry, cell culture, and related areas.

[0084] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0085] In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage.

[0086] Example 1: Isolation and Identification of *Polyporus pachychromis* strain Step 1: Preparation of culture medium for bacterial isolation Preparation of PDA enriched medium: 200 g potato, 20 g glucose, 3 g peptone, 2 g potassium dihydrogen phosphate, 1 g magnesium sulfate, 10 mg vitamin B1, 15 g agar powder, 1000 mL distilled water, pH 6.0. Mix well, heat to melt, dispense into containers, sterilize at 121℃ for 25 min, cool, and prepare into plate medium for later use.

[0087] Step 2: Isolation of wild fungi Using a tissue isolation method, the surface of wild-type pale yellowish-brown *Porphyra tenuifolia* fruiting bodies was wiped 3-5 times with 75% alcohol swabs. The bodies were then longitudinally cut with a sterile scalpel, and small tissue pieces (1-2 mm in size) were picked and placed on PDA-enriched medium. The cultures were incubated at 25°C in the dark. After mycelial germination, edge hyphae were aseptically transferred to fresh PDA-enriched medium for purification culture, thus obtaining the mother culture, designated XYS3.

[0088] Step 3: Morphological observation and identification Fresh culture of strain XYS3 was picked from test tube slant and inoculated onto PDA-enriched medium plates. The plates were incubated at 25°C in the dark. Mycelial growth was observed, and colony shape, color, and other characteristics were recorded. Cultured mycelia were also picked and placed on slides for observation of the morphological characteristics of mycelia and basidiospores under an optical microscope. The results are as follows: Figure 1 As shown in the figure. ITS sequence analysis confirmed that the ITS fragment of strain XYS3 is 694 bp in length, as shown in SEQ ID No. 1. Based on this result, a phylogenetic tree was constructed using cluster analysis of strain XYS3, and the results are shown in the figure. Figure 2 As shown.

[0089] SEQ ID No. 1:

[0090] like Figure 1As shown in Figure c, morphological observation indicates that the colonies of strain XYS3 on PDA-enriched medium plates are round and mat-like, with mycelia growing in a fluffy, outward radial pattern and neat edges. The initial mycelia are white, slowly turning pale yellow as they mature, and yellowish-brown after aging. Figure 1 As shown in Figure d, under a microscope, the hyphae are pale yellow, branched and septate, and lack clamp connections; Figure 1 As shown in Figure e, the basidiospores are oval, pale yellow, and have a distinctly thick wall.

[0091] like Figure 2 As shown, cluster analysis revealed that strain XYS3 and Fuscoporia gilva The strain clustered into the same branch, with a sequence similarity of 100%. Based on hyphal morphology, the strain was identified as belonging to the genus *Inonotus glomeratus*. Fuscoporia Murrill pale yellow-brown Inonotus obliquus Fuscoporia gilva .

[0092] Studies have shown that *Inonotus* is a globally distributed genus, with 28 species as of 2020, 13 of which are found in my country. Fungi in this genus not only play an important role in the carbon cycle, but also possess significant medicinal value, such as *Inonotus obliquus* and *Inonotus chrysotrichum*. Currently, research on *Inonotus* fungi mainly focuses on taxonomic studies, while applied research is still in its early stages. Research on its artificial cultivation and product development only involves *Inonotus obliquus*, while the development and utilization of germplasm resources of other fungi in this genus, such as *Inonotus chrysotrichum*, remains a blank area.

[0093] The pale yellowish-brown Inonotus ( Fuscoporia gilva XYS3 was deposited on October 23, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42288. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences.

[0094] Example 2: Optimization of Mother Culture Conditions Propagation of the mother culture: Following the PDA-enriched culture medium preparation method in Example 1, the components in the formula were mixed evenly, heated to dissolve, and sterilized. When the temperature of the culture medium dropped to 45℃-50℃, the culture medium was poured into a sterile petri dish (9 cm in diameter) under aseptic conditions to prepare a plate culture medium. The plate culture medium was then cooled and set aside. Fresh culture of the pale yellow-brown Flocculinus XYS3 mother culture obtained in Example 1 was aseptically picked and placed in the middle of the petri dish. The culture was carried out at 25℃ in the dark. When the mycelium covered the plate culture medium, the colonies on the plate culture medium were punched into mycelial cakes with a diameter of 5 mm using a pre-sterilized punch.

[0095] Optimization of Mother Culture Conditions: Using the PDA-enriched medium from Example 1 as the basal medium, a single-factor experiment was conducted. Fresh cultures of the *Phellinus yunnanensis* XYS3 mother culture obtained in Example 1 were aseptically picked and inoculated into test plates to investigate the effects of different carbon sources (glucose, sucrose, lactose, maltose, mannitol), nitrogen sources (peptone, beef extract, yeast extract, ammonium sulfate, ammonium nitrate), pH (3, 4, 5, 6, 7, 8), and temperatures (16, 19, 22, 25, 28, 31℃) on the mycelial growth of the test strain XYS3. Each treatment was replicated in triplicate. The colony diameter was measured using the cross-crossing method. After the mycelium had fully colonized the plate, the culture was stopped, and the daily mycelial growth rate was calculated to observe the mycelial growth. The calculation of the mycelial growth rate (V) is given by formula (1).

[0096] V=D / 2t…………………………………… (1) In formula (1): V represents the daily growth rate of mycelium, mm / d; D represents the diameter of mycelium, mm; t represents the culture time, d.

[0097] 1. Effects of different carbon sources on mycelial growth The experimental method included: using the PDA-enriched medium from Example 1 as a base, replacing glucose in equal amounts with sucrose, lactose, maltose, and mannitol, while keeping the remaining components the same as the PDA-enriched medium. The preparation method was also the same: dissolving each component in the medium with water, sterilizing, and when the medium temperature dropped to 45℃-50℃, pouring the medium into sterile petri dishes (9 cm in diameter) to prepare agar plates with different carbon sources, and cooling. Aseptically picking pre-prepared *Polyporus flocculationii* XYS3 mycelial discs and inoculating them in the center of the agar plates, incubating at 25℃ in the dark, observing mycelial growth, measuring colony diameter using the "cross-cross" method, and calculating the daily mycelial growth rate according to the above formula. The results are shown in Table 1 and... Figure 3 As shown.

[0098] Table 1. Effects of carbon source on mycelial growth of *Inonotus obliquus* strain XYS3

[0099] The results are shown in Table 1 and Figure 3As shown, different carbon sources have a significant impact on the mycelial growth of *Inonotus obliquus* XYS3. Within the experimental range, strain XYS3 could grow on all five carbon source media, with neat colony edges. Regarding growth rate, when maltose, glucose, sucrose, and mannitol were used as carbon sources, the daily mycelial growth rate was between 4.84 and 5.21 mm / d, significantly better than that of lactose medium (4.31 ± 0.14 mm / d). The fastest mycelial growth was observed on the mannitol medium (5.21 ± 0.12 mm / d). In terms of mycelial vigor, when glucose and mannitol were used as carbon sources, their mycelial vigor was similar and significantly better than other carbon sources, resulting in brown colonies and the densest and most vigorous mycelial growth. When sucrose was used as a carbon source, the colony center was brown and relatively dense, but the periphery was grayish-white, and the mycelium was thin and weak. When maltose, lactose, and sucrose were used as carbon sources, the mycelial vigor was the worst, with light-colored colonies and thin, sparse mycelium. Considering both mycelial growth rate and vigor, mannitol was the most suitable carbon source for the mycelial growth of wild-type *Polyporus floribunda* XYS3.

[0100] 2. Effects of different nitrogen sources on mycelial growth The experimental method included: using the PDA-enriched medium from Example 1 as a base, replacing the peptone in the basal medium with equal amounts of yeast extract, beef extract, ammonium sulfate, and ammonium nitrate, respectively. The remaining components and preparation methods were the same as for the PDA-enriched medium. Plate media with different nitrogen sources were prepared according to the experimental method for "the effect of different carbon sources on mycelial growth." Then, pre-prepared pale yellow-brown *Polyporus flocculationii* XYS3 mycelial discs were aseptically picked and inoculated into the center of the plate media. The plates were incubated at 25°C in the dark, and mycelial growth was observed. The colony diameter was measured using the "cross-cross" method, and the daily mycelial growth rate was calculated using the formula described above. The results are shown in Table 2 and... Figure 4 As shown.

[0101] Table 2. Effects of nitrogen source on mycelial growth of *Inonotus obliquus* strain XYS3

[0102] The effects of different nitrogen sources on the mycelial growth of *Inonotus obliquus* XYS3 are shown in Table 2 and 3. Figure 4The results showed that *Inonotus obliquus* XYS3 mycelia grew well on all five nitrogen source media. The daily growth rate of mycelia, from fastest to slowest, was: ammonium sulfate > yeast extract > beef extract > ammonium nitrate > peptone, with a daily growth rate ranging from 5.06 to 5.31 mm / d. Ammonium sulfate medium showed the fastest growth rate (5.31 ± 0.13 mm / d), followed by yeast extract (5.25 ± 0.08 mm / d). Yeast extract provided the best mycelial growth, exhibiting a light yellowish-brown color, dense and robust hyphae with neat edges. Beef extract was the next best source, also showing relatively dense and robust hyphae. In contrast, the hyphae on peptone, ammonium sulfate, and ammonium nitrate media were weaker, sparser, and brown in color. Therefore, considering both mycelial growth rate and vigor, yeast extract is the most suitable nitrogen source for the growth of wild *Inonotus obliquus* XYS3 mycelia.

[0103] 3. Effects of different pH values ​​on mycelial growth The experimental method included: using the PDA-enriched medium from Example 1 as a base, adjusting the pH of the medium to 3, 4, 5, 6, 7, and 8 respectively with 5% dilute hydrochloric acid or 5% sodium hydroxide solution, while maintaining the same preparation method, to obtain plate media with different pH values. Then, aseptically picking pre-prepared pale yellow-brown *Polyporus flocculationii* XYS3 mycelial discs and inoculating them in the center of the plate media, incubating at 25°C in the dark, observing mycelial growth, measuring colony diameter using the "cross-cross" method, and calculating the daily mycelial growth rate according to the above formula. The results are shown in Table 3 and... Figure 5 As shown.

[0104] Table 3. Effect of culture medium pH on mycelial growth of *Polyporus pachycarpus* strain XYS3

[0105] The effects of different initial pH values ​​of the culture medium on the mycelial growth of wild-type *Polyporus pachycarpus* XYS3 are shown in Table 3 and... Figure 5 The results showed that within the experimental range, strain XYS3 could grow at an initial pH of 3.0-8.0. Except for slow and weak mycelial growth at pH 3.0, strain XYS3 adapted well to an environment with a pH of 4.0-8.0, exhibiting good mycelial growth, dense and vigorous hyphae, and neat colony edges. At initial pH values ​​of 5.0 and 6.0, the mycelial growth rate reached a relatively high level, with daily growth rates of 4.98±0.08 mm / d and 5.06±0.10 mm / d, respectively. Then, the mycelial growth rate gradually decreased with increasing initial pH. Considering both mycelial growth rate and vigor, the optimal initial pH for mycelial growth of strain XYS3 was 6.0.

[0106] 4. Effects of different culture temperatures on mycelial growth The experimental methods included: using the PDA-enriched medium from Example 1 as the base formula, and preparing plate culture media according to the experimental method for "the effect of different carbon sources on mycelial growth," then aseptically picking pre-prepared pale yellow-brown *Polyporus flocculationii* XYS3 mycelial discs and inoculating them into the middle of the plate culture media. The media were then cultured in the dark at 16℃, 19℃, 22℃, 25℃, 28℃, and 31℃, respectively. Mycelial growth was observed, and the colony diameter was measured using the "cross-cross" method. The daily mycelial growth rate was calculated according to the above formula. The results are shown in Table 4 and... Figure 6 As shown.

[0107] Table 4. Effects of culture temperature on mycelial growth of *Inonotus obliquus* strain XYS3

[0108] The effects of different culture temperatures on the mycelial growth of wild-type *Inonotus obliquus* XYS3 are shown in [reference needed]. Figure 6 The results showed that there were significant differences in mycelial growth rate and vigor among the tested strains within the culture temperature range of 16℃-31℃. In contrast, when the culture temperature was below 25℃, the mycelial growth of the tested strains was slow, and the mycelia were weak, thin, and sparse. When the culture temperature was between 25℃ and 28℃, the mycelial growth rate reached a relatively high level, with the fastest growth rate (5.23±0.12 mm / d) at 28℃. At this temperature, the mycelial vigor was optimal, with vigorous and dense mycelia, brown colonies, and neat edges. When the culture temperature reached 31℃, the mycelial growth rate decreased significantly, the mycelia were thinner and more prone to aging, and the colonies were dark brown with irregular edges. Therefore, considering both mycelial growth rate and vigor, the optimal temperature for the mycelial growth of wild-type *Polyporus pachycarpus* XYS3 is 28℃.

[0109] In summary, the optimal mother culture medium for wild-type *Polyporus pachycarpus* XYS3 is: 200 g potato, 20 g mannitol, 3 g yeast extract, 2 g potassium dihydrogen phosphate, 1 g magnesium sulfate, 10 mg vitamin B1, 15 g agar powder, and 1000 mL distilled water, with a pH of 6.0. The optimal culture temperature is 28℃.

[0110] Example 3: Optimization of liquid culture conditions Step 1: Propagation of the microbial strain Using the optimal mother culture medium obtained in Example 2 as the mother culture propagation medium, the components of the culture medium were dissolved in water, dispensed into 500 mL Erlenmeyer flasks, sterilized, and then plate culture medium was prepared. Then, fresh culture of the pale yellow-brown Flocculinus XYS3 strain was aseptically inoculated into the middle of the culture medium and cultured in the dark at 28°C. When the mycelium covered the plate, holes were punched with a sterile punch (0.5 cm in diameter) to prepare mycelial sheets of uniform size for later use.

[0111] Step 2: Optimization of liquid culture conditions 1. Preparation of liquid culture medium Formula 1: 200 g potato, 20 g mannitol, 3 g yeast extract, 2 g potassium dihydrogen phosphate, 1 g magnesium sulfate, 10 mg vitamin B1, 1000 mL distilled water, pH 6.0.

[0112] Formula 2: 200 g potato, 20 g mannitol, 3 g yeast extract, 5 g corn flour, 2 g potassium dihydrogen phosphate, 1 g magnesium sulfate, 10 mg vitamin B1, 1000 mL distilled water, pH 6.0.

[0113] Formula 3: 20 g mannitol, 10 g corn flour, 10 g malt extract powder, 2 g potassium dihydrogen phosphate, 1 g magnesium sulfate, 10 mg vitamin B1, 1000 mL distilled water, pH 6.0.

[0114] Formula 4: 20 g mannitol, 10 g corn flour, 5 g yeast extract, 5 g wheat bran powder, 5 g peptone, 2 g potassium dihydrogen phosphate, 1 g magnesium sulfate, 10 mg vitamin B1, 1000 mL distilled water, pH 6.0.

[0115] Formula 5: 30 g mannitol, 10 g soybean meal powder, 2 g potassium dihydrogen phosphate, 1 g magnesium sulfate, 10 mg vitamin B1, 1000 mL distilled water, pH 6.0.

[0116] Sterilize at 121℃ for 30 min, and then prepare the culture medium for later use.

[0117] 2. Effects of different liquid culture media on mycelial growth Aseptically, the light yellowish-brown *Polyporus flocculationii* XYS3 mycelial sheets from step one were picked and cultured in different pre-prepared liquid culture media at 28℃ and 150 r / min under shaking conditions in the dark for 10 days. After the culture was completed, the fermentation broth was filtered through three layers of gauze and washed three times with distilled water. The mycelium was collected, dried at 80℃, and weighed. The dry weight of mycelium obtained per 100 mL of fermentation broth was calculated using the dry weight method. The mycelial biomass in different culture media was investigated, and the results are shown in Table 5.

[0118] Table 5. Effects of different liquid culture media on the mycelial growth of *Polyporus pachycarpus*.

[0119] As shown in Table 5, among the five liquid culture media, the mycelial balls in the fermentation broth of Formula 4 were the finest and most uniform, with the highest mycelial yield of 0.85 g / 100 mL.

[0120] 3. Effects of different exogenous additives on mycelial growth Using Formula 4 as the basal culture medium and as a control, 0.2% of extracts of poplar flowers, sophora flowers, mulberry leaves, tree flowers, and sea buckthorn (10:1 ratio) (purchased from Shaanxi Senrui Biotechnology Co., Ltd.) were added to the medium to investigate the effects of different exogenous factors on mycelial growth. The culture method and biomass calculation method were the same as in step 2 above, and the results are as follows: Figure 7 , Figure 8 As shown in Table 6.

[0121] Table 6. Effects of different exogenous additives on the mycelial growth of *Inonotus obliquus*.

[0122] From Table 6, Figure 7 , Figure 8 It can be seen that, within the experimental range, the order of mycelial yield of *Inonotus obliquus* XYS3 from high to low among the six culture media is: poplar flower extract > sea buckthorn extract > formulation 4 > sophora japonica flower extract > *Inonotus obliquus* flower extract > mulberry leaf extract. Compared with formulation 4, poplar flower and sea buckthorn extracts have a certain promoting effect on the mycelial growth of *Inonotus obliquus* XYS3, with poplar flower extract showing the most significant growth promoting effect, the densest mycelium, and the highest mycelial yield (0.927±0.02). In contrast, sophora japonica flower, *Inonotus obliquus* flower, and mulberry leaf extracts have a certain inhibitory effect on the mycelial growth of *Inonotus obliquus* XYS3. It is evident that the optimal exogenous additive in the liquid culture medium for *Polyporus pachycarpa* XYS3 is poplar flower extract. The preliminary formulation of the liquid culture medium for *Polyporus pachycarpa* XYS3 is as follows: 20 g mannitol, 10 g corn flour, 5 g yeast extract, 5 g wheat bran powder, 5 g peptone, 2 g poplar flower extract (10:1), 2 g potassium dihydrogen phosphate, 1 g magnesium sulfate, 10 mg vitamin B1, 1000 mL distilled water, pH 6.0.

[0123] Step 3: Optimization of culture medium formulation based on orthogonal experimental method To determine the optimal liquid culture medium formulation, the experiment was based on the culture medium formulation obtained in step 2.3 above. Four factors were selected: mannitol, yeast extract, peptone, and poplar flower extract (10:1). Three levels were set for each factor in an L9 (3... 4 The orthogonal experiment was conducted using the same cultivation and biomass calculation methods as in step 2. Factors and levels are shown in Table 7, and the results are shown in Table 8. The results showed that the order of influence of the four factors from largest to smallest was A>D>B>C, and the optimal combination was A3B2C3D3. Therefore, the optimal culture medium formula for the liquid culture of *Polyporus pachycarpus* XYS3 was: 30 g mannitol, 10 g corn flour, 5 g yeast extract, 5 g wheat bran powder, 5 g peptone, 3 g poplar flower extract (10:1), 2 g potassium dihydrogen phosphate, 1 g magnesium sulfate, 10 mg vitamin B1, 1000 mL distilled water, pH 6.0.

[0124] Table 7. Four Factors, Three Levels Table

[0125] Table 8 L9 (3) 4 Orthogonal experimental table and results

[0126] Step 4: Optimization of the liquid culture cycle Fresh cultures of wild-type *Polyporus floribunda* XYS3 from step one were aseptically picked and cultured in a pre-prepared optimized liquid medium (30 g mannitol, 10 g corn flour, 5 g yeast extract, 5 g wheat bran powder, 5 g peptone, 3 g poplar flower extract (10:1), 2 g potassium dihydrogen phosphate, 1 g magnesium sulfate, 10 mg vitamin B1, 1000 mL distilled water, pH 6.0) at 28℃ and 150 r / min in the dark. Mycelial biomass in the liquid medium was measured at different culture times, and the biomass calculation method was the same as in step two. The results are shown in Table 9. Figure 9 , Figure 10 , Figure 11 As shown.

[0127] Table 9. Effects of different culture times on the mycelial growth of *Inonotus obliquus*.

[0128] From Table 9, Figure 9 , Figure 10 It can be seen that in the early stage of cultivation, the mycelial growth of *Inonotus obliquus* XYS3 was relatively slow. After the 5th day of cultivation, the mycelial yield increased significantly, reaching a high level (0.95±0.03g / 100mL) on the 7th day. Afterward, the mycelial yield increased slowly, reaching its maximum on the 8th day (1.11±0.04g / 100mL). At this point, the mycelial balls of *Inonotus obliquus* were small, uniform, and abundant, with vigorous mycelial growth. Subsequently, the mycelial yield began to decline, and on the 9th day, the mycelial contents aggregated, and the mycelium tended to autolyze. Considering all factors, the cultivation cycle of *Inonotus obliquus* XYS3 liquid culture is 7-8 days.

[0129] In a preferred embodiment, the liquid culture method includes: aseptically picking light yellow-brown Coleus XYS3 bacterial sheets and placing them in a pre-prepared optimal liquid culture medium for light yellow-brown Coleus XYS3, and then shaking and culturing in the dark at 28°C and 150 r / min for 7-8 days.

[0130] The optimal culture medium formula for the liquid culture of *Polyporus pachycarpa* XYS3 is as follows: 30 g mannitol, 10 g corn flour, 5 g yeast extract, 5 g wheat bran powder, 5 g peptone, 3 g poplar flower extract (10:1), 2 g potassium dihydrogen phosphate, 1 g magnesium sulfate, 10 mg vitamin B1, 1000 mL distilled water, pH 6.0.

[0131] The morphology of the cultured strains is as follows Figure 10 and Figure 11 As shown.

[0132] The above method was repeated for the culture experiment. The mycelial biomass of the three batches were 1.15 g / 100mL, 1.10 g / 100mL and 1.09 g / 100mL, with a relative deviation of 2.62%. The obtained process has good stability, which proves that the method can be used for large-scale production.

[0133] Example 4: Fruiting body culture Step 1: Mother culture and propagation 1. Preparation of mother culture slant culture medium Mother culture medium formula: 200 g potato, 20 g mannitol, 3 g yeast extract, 2 g potassium dihydrogen phosphate, 1 g magnesium sulfate, 10 mg vitamin B1, 15 g agar powder, 1000 mL distilled water, pH 6.0. Dissolve all components in water, dispense into test tubes (18*20 mm), sterilize at 121℃ for 30 min, and prepare test tube slant culture medium for later use.

[0134] 2. Propagation and cultivation of mother culture Under aseptic conditions, select fresh culture of *Inonotus obliquus* strain XYS3 and inoculate it into the middle of an agar slant. Incubate at 28°C in the dark for 10-12 days until the mycelium fully colonizes the agar slant. This will yield the mother culture of *Inonotus obliquus* strain XYS3, which can then be stored in a refrigerator (2°C-4°C) for later use.

[0135] Step 2: Liquid culture 1. Preparation of liquid culture medium Liquid culture medium formulation: 30 g mannitol, 10 g corn flour, 5 g yeast extract, 5 g wheat bran powder, 5 g peptone, 3 g poplar flower extract (10:1), 2 g potassium dihydrogen phosphate, 1 g magnesium sulfate, 10 mg vitamin B1, 1000 mL distilled water, pH 6.0. Dissolve all components in water and dispense into 1000 mL Erlenmeyer flasks (400 mL / flask). Sterilize at 121°C for 30 min.

[0136] 2. Liquid culture In a sterile environment, the slant culture block of the pale yellow-brown Flocculinus XYS3 strain, which was propagated in step one, was inoculated into a liquid culture medium and cultured in the dark at 28℃ and 150 r / min for 7-8 days. When the mycelial balls are abundant and the fermentation broth is thick, the culture can be stopped.

[0137] Step 3: Fruiting body cultivation 1. Culture medium preparation Cultivation substrate formula: 400 g sawdust, 400 g chestnut shells, 150 g wheat bran, 10 g soybean meal, 10 g quicklime, 10 g gypsum, 3 g poplar flower extract (10:1), 17 g mannitol; Dissolve quicklime, gypsum, and poplar flower extract in a small amount of water beforehand, then mix them with the other materials according to the ratio and stir evenly. Then add 800-1000 g of water and stir to make the moisture content of the cultivation substrate reach 55%-60%. Then put it into a 650 mL culture bottle, seal it with plastic film, and sterilize it at 121℃-125℃ for 2 hours.

[0138] 2. Vaccination After sterilization, cool the culture bottles in a sterile environment. When the temperature drops below 28°C, use a large-volume pipette to inoculate the cultured light yellow-brown Flocculinum liquid culture from the seal according to the aseptic operation procedure. Inoculate 10 mL of liquid culture into each culture bottle and seal the bottle after inoculation.

[0139] 3. Spawn Management After inoculation, place the culture bottle at a constant temperature of 28℃ and control the air humidity at 40%-60%. During the mycelium growth process, the carbon dioxide concentration should not exceed 4000 ppm. The mycelium will fully grow in the culture bottle in 25-30 days. Then, lower the temperature to 25℃ for post-ripening culture. After 10-15 days, when the mycelium turns brown, start fruiting management.

[0140] 4. Mushroom Management After the mycelium has finished incubating, transfer the culture bottles to the fruiting room, place them upright, remove the plastic film, and manage the fruiting and fruiting processes under 300-500 Lux diffused light, 800-1000 ppm carbon dioxide, and 80%-90% humidity. During the fruiting stage, control the culture temperature between 18℃ and 28℃. Once the mycelium has twisted and formed mycelial clusters, adjust the culture temperature to 25℃-28℃ for fruiting management. Harvest the fruiting bodies when the pale yellow growth ring disappears.

[0141] 5. Mushroom growth The growth of the pale yellowish-brown pleuromyotrophic lateral pore strain XYS3 on artificial culture medium is shown in the figure. Figure 12The results showed that *Inonotus pachychromis* can be produced as a fruiting body using agricultural methods. On an artificially synthesized culture medium with sawdust and chestnut shells as the main raw materials, the mycelium of *Inonotus pachychromis* was white, vigorous, and uniform, and could cover the cultivation medium in 28 days. After the mycelium matured, it produced a yellowish-brown pigment. The fruiting body had obvious growth rings, with a brown center and a dark yellow edge. The yield per bottle was 15-20 g.

[0142] Example 5: Preparation and Evaluation of Fruiting Body Lyophilized Powder Step 1: Sub-entity extraction 1. Preparation of water extracts from fruiting bodies The pale yellowish-brown pore-forming strain XYS3 fruiting bodies obtained by the method in Example 4 were dried at 60°C to a constant weight, pulverized using an electric grinder, and passed through an 80-mesh sieve for later use. 100 g of the fruiting body powder was accurately weighed into a 3000 mL ground-glass stoppered conical flask, 1500 mL of water was added, and the mixture was refluxed at 80°C for 3 h. After centrifugation at 5000 r / min, the supernatant was collected to obtain the water extract of the fruiting bodies.

[0143] 2. Preparation of ethanol extract from fruiting bodies The pale yellowish-brown fruiting bodies of *Inonotus obliquus* XYS3 obtained by the method in Example 4 were dried at 60°C to a constant weight, pulverized using an electric grinder, and passed through an 80-mesh sieve for later use. 100 g of the fruiting body powder was accurately weighed into a 3000 mL ground-glass stoppered conical flask, and 1500 mL of 65% ethanol was added. The mixture was refluxed at 80°C for 3 h, centrifuged at 5000 r / min, and the supernatant was collected to obtain the ethanol extract of the fruiting body.

[0144] Step 2: Evaluation of the bioactivity of the extract 1. Determination of active ingredient content: Polysaccharide content was determined using the phenol-sulfuric acid method. The standard curve equation was obtained by plotting glucose content on the x-axis and absorbance at 490 nm on the y-axis: y = 5.4529x + 0.0092 (R0). 2 =0.9990)(Reference: Zhao Wenwen, Sun Bolin, Chen Xijun, et al. Comparative analysis of polysaccharide content in different parts of Hericium erinaceus [J]. Edible Fungi, 2024, 46(4):72-73,75.); Polyphenols were determined by the Folin-Ciocalteu colorimetric method, with gallic acid content as the abscissa and absorbance at 760 nm wavelength as the ordinate. The standard curve equation obtained was y=18.671x-0.021 (R 2=0.9991)(Reference: Wang Jing, Ye Dianfeng, Deng Minggao, et al. Determination of polyphenol content and comparison of antioxidant activity of three edible and medicinal fungi[J]. Food Industry Technology, 2020, 41(4):51-57.); The flavonoids were determined by the aluminum nitrate method. The standard curve equation obtained by using rutin content as the abscissa and absorbance at 510 nm wavelength as the ordinate was y=2.874x-0.0081(R 2 =0.9991)(Reference: Ma Tengfei, Li Wenzhuo, Xue Zaoxun, et al. Study on the process of ultrasonic extraction of total flavonoids from Hericium erinaceus[J]. Shandong Chemical Industry, 2022, 51(16):32-34,38.). The absorbance values ​​of the test sample solutions prepared by different extraction methods were measured after reaction, and the concentrations in the test sample solutions were calculated by substituting them into the standard curve equations of polysaccharides, polyphenols and flavonoids respectively. Then the content in the fruiting bodies was calculated according to formula (2).

[0145] The content of substances in the fruiting body (mg / g) = C × V × D / M………………………………(2) In the formula: C is the content of active ingredient in the test sample solution, mg / g; V is the total volume of sample extraction, mL; D is the dilution factor of the sample; M is the mass of the fruiting body, g.

[0146] 2. Assay for hypoglycemic activity (1) Determination of α-glucosidase inhibition rate: The 4-nitrophenyl-α-D-glucopyranoside (PNPG) colorimetric method was used for determination. Two 50 μL portions of the aqueous extract of Phytotrichum gloeosporioides XYS3 fruiting bodies and two equal volumes of 65% ethanol extract were taken. One portion was added with 100 μL of α-glucosidase solution (0.25 U / mL), and the other portion was added with 100 μL of phosphate buffer (0.2 mol / L, pH=7.0). The mixture was mixed and incubated in a water bath at 37℃ for 10 min. Then, 100 μL of pNPG (2.0 mmol / L) was added. After incubating in a water bath at 37℃ for 20 min, 1 mL of 1 mol / L sodium carbonate solution was added to terminate the reaction. The absorbance value was measured at 400 nm. Simultaneously, 50 μL of water and 65% ethanol were taken as controls. The inhibition rate of α-glucosidase by the test sample solution was calculated according to formula (3).

[0147] α-glucosidase inhibition rate (%) = [1 - (A1 - A2) / A0] × 100 ………………(3) In the formula: A0 is the absorbance of the control group; A1 is the absorbance of the test sample solution; A2 is the absorbance of the phosphate buffer (0.2 mol / L, pH=7.0) in place of the α-glucosidase solution; (2) Determination of α-amylase inhibition rate: Take two portions of 50 μL each of the aqueous extract of Phytotrichum gloeosporioides XYS3 fruiting body and the extract of 65% ethanol. Add 50 μL of α-amylase (1.0 U / mL) to one portion and 50 μL of phosphate buffer (0.2 mol / L, pH=7.0) to the other portion. Mix well and react at 37℃ for 10 min. Then add 100 μL of 1% soluble starch solution and incubate at 37℃ for 10 min. Add 1 mL of DNS reagent, incubate in boiling water for 5 min, cool to room temperature, add 1 mL of distilled water, and measure the absorbance at 520 nm. Simultaneously take 50 μL of water and 65% ethanol as controls. Calculate the inhibition rate of α-amylase on the test sample solution according to formula (4).

[0148] α-Amylase inhibition rate (%) = [1 - (A1 - A2) / A0] × 100 ………………(4) In the formula: A0 is the absorbance of the control group. A1 is the absorbance of the test sample solution; A2 is the absorbance of the phosphate buffer (0.2 mol / L, pH=7.0) instead of the α-amylase solution.

[0149] The measurement results are shown in Tables 10 and 11.

[0150] 3. Measurement and Analysis Results Table 10 shows that the fruiting bodies of strain XYS3 contain abundant polysaccharides, flavonoids, and polyphenols, and their contents vary significantly depending on the extraction solvent. Within the experimental range, the polysaccharide content in the water extract of the fruiting bodies of strain XYS3 was 47.87 ± 0.81 mg / g, which is 1.63 times that of the 65% ethanol extract. The contents of flavonoids and polyphenols in the 65% ethanol extract of the fruiting bodies were 5.29 and 2.59 times that of the water extract, respectively. This indicates that the polysaccharides in the fruiting bodies of strain XYS3 are mainly water-soluble, while ethanol solution is more conducive to the extraction of polyphenols and flavonoids.

[0151] Further analysis of the hypoglycemic activity (Table 11) showed that both the aqueous extract and the 65% ethanol extract of the XYS3 strain fruiting body possessed hypoglycemic activity, but with some differences. Specifically, the 65% ethanol extract of the XYS3 strain fruiting body exhibited strong inhibitory activity against α-glucosidase and α-amylase, with inhibition rates exceeding 97%; these were 2.17 and 1.16 times higher than those of the aqueous extract, respectively. Therefore, the extracts of the XYS3 strain fruiting body possess hypoglycemic activity, with the ethanol extract showing better activity than the aqueous extract.

[0152] Table 10 Comparison of active ingredient content in different extracts of fruiting bodies

[0153] Table 11 Comparison of hypoglycemic activities of different extracts from fruiting bodies

[0154] Step 3: Post-processing and application of extracts The water extract and alcohol extract were combined and concentrated under reduced pressure at 0.8 MPa and 60℃. When the specific gravity of the concentrate was 1.1-1.2, it was freeze-dried at -40℃ to obtain light yellow-brown lyophilized powder of *Polygonum aviculare*. The yield of the freeze-dried powder reached more than 18%, which can be used as a raw material for the research and development of hypoglycemic products.

[0155] The above description is merely an 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 principle of this application should be included within the scope of the claims of this application.

Claims

1. A pale yellowish-brown inoculum ( Fuscoporia gilva XYS3, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 42288.

2. A method for culturing *Inonotus obliquus*, characterized in that, The method includes: Step 1: Inoculate the pale yellow-brown Flocculinus into the mother culture medium and culture it until the mycelium covers the plate. Stop the culture to obtain the mother culture. The culture conditions include: culture temperature 16℃-31℃, pH 3-9, and culture in the dark. Step 2: Inoculate the mother culture into a liquid culture medium for cultivation to obtain a liquid culture spawn; the cultivation conditions include: cultivation temperature 16℃-31℃, pH 3-9, 100-200 r / min, and cultivation in the dark for 5-20 days. Step 3: Inoculate the liquid culture medium with 1%-5% of the liquid culture medium to allow for mycelial growth and fruiting.

3. The cultivation method according to claim 2, characterized in that, The mother culture medium contains a carbon source and / or a nitrogen source; preferably, the carbon source contains one or more of glucose, sucrose, lactose, maltose, and mannitol; preferably, the nitrogen source contains one or more of peptone, beef extract, yeast extract, ammonium sulfate, and ammonium nitrate; preferably, the mother culture medium also contains one or more of potato, potassium dihydrogen phosphate, magnesium sulfate, and vitamin B1.

4. The cultivation method according to claim 2, characterized in that, The liquid culture medium contains plant extracts; preferably, the plant extracts include poplar flower extract and / or sea buckthorn extract; more preferably, poplar flower extract; even more preferably, the concentration of the poplar flower extract is 0.01%-1% by weight of the liquid culture medium; preferably, 0.3%; preferably, the liquid culture medium also includes one or more of mannitol, corn flour, yeast extract, wheat bran powder, peptone, potassium dihydrogen phosphate, magnesium sulfate, and vitamin B1.

5. The cultivation method according to claim 2, characterized in that, The bag cultivation medium contains plant extracts; preferably, the plant extracts include poplar flower extract and / or sea buckthorn extract; more preferably, poplar flower extract; even more preferably, the concentration of the poplar flower extract is 0.01%-1% by weight of the bag cultivation medium; preferably, 0.3%; preferably, the bag cultivation medium also includes one or more of the following: sawdust, chestnut shells, wheat bran, soybean meal, quicklime, gypsum, and mannitol.

6. The cultivation method according to claim 2, characterized in that, The pale yellow-brown porphyria is pale yellow-brown porphyria ( Fuscoporia gilva XYS3 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42288.

7. A light yellowish-brown culture medium for *Polyporus flocculationii*, characterized in that, The culture medium contains poplar flower extract and / or sea buckthorn extract; preferably, the culture medium contains poplar flower extract; more preferably, the concentration of the poplar flower extract is 0.01%-1% by weight of the culture medium; preferably, 0.3%.

8. The application of the *Paecilomyces pachycarpa* culture medium as described in claim 7 in the cultivation of *Paecilomyces pachycarpa*.

9. The application of the culture method according to any one of claims 2-6, the culture medium according to claim 7, or the *Paecilomyces paeonii* according to claim 1 in any one of the following A1)-A3): A1) Prepare products containing *Polyporus pachychromis*; A2) Prepare hypoglycemic products; A3) Prepare products that inhibit the activity of α-glucosidase and / or α-amylase.

10. A fungus containing the pale yellowish-brown porphyria as described in claim 1 ( Fuscoporia gilva XYS3 products.

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