A strain of myceliophthora thermophila producing a biogenic component and its use

Abstract

The application discloses a Cordyceps farinosa strain for producing beneficial components and application thereof, and belongs to the technical field of microorganisms. The Cordyceps farinosa strain Gsxcf1 is preserved in the China General Microbiological Culture Collection Center, has a preservation number of CGMCC NO.41894, a preservation date of April 21, 2025, and a preservation address of No.3, Xili, Beichen West Road, Chaoyang District, Beijing. The Cordyceps farinosa strain Gsxcf1 provided by the application is separated from wild Cordyceps sinensis, and after liquid fermentation or solid fermentation, the active components such as polysaccharide, ergosterol and cordycepin have high content, and have great potential in the development of Cordyceps sinensis fruiting body or mycelium related food.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a powdery Cordyceps strain that produces beneficial components and its applications. Background Technology

[0002] Cordyceps militaris is a fungus of the genus Cordyceps with important medicinal and scientific research value. It possesses unique biological characteristics and medicinal components, and is used as a substitute for Cordyceps sinensis. Studies have shown that Cordyceps militaris contains adenosine, amino acids, cordycepin, and other chemical components, which have effects such as enhancing immunity, calming the nerves, and combating fatigue, with relatively low toxicity. It has significant medicinal value and broad development prospects. However, Cordyceps militaris has a limited distribution in nature and a long growth cycle, leading to its scarcity. Furthermore, over-harvesting puts pressure on natural resources. Therefore, the development, utilization, and protection of Cordyceps militaris biological resources are crucial. With the rapid increase in demand for Cordyceps fungi and the depletion of wild Cordyceps sinensis resources, the development value of Cordyceps militaris is becoming increasingly prominent. The isolation and purification of strains from wild Cordyceps militaris and the screening of superior strains with active ingredients are of great significance for the resource utilization of Cordyceps militaris.

[0003] Cordycepin, ergosterol, and cordyceps polysaccharides are all major components of cordyceps. Cordycepin, also known as cordycepin or cordyceps militaris, is the main active ingredient in cordyceps (especially nucleoside-based), belonging to the nucleoside class of new drugs and purine alkaloids. Due to its special effects in repairing cells and protecting the genetic code of living organisms, it has become a leader in modern biomedicine. Cordycepin, a naturally derived drug, possesses various pharmacological effects such as anti-tumor, antibacterial, antiviral, immunomodulatory, and free radical scavenging, and has promising clinical application prospects. Ergosterol is also one of the main chemical components of cordyceps. Numerous studies have shown that it possesses various physiological activities, such as antiviral, immune-enhancing, anti-inflammatory, cell-growth-inhibiting, and blood sugar-regulating effects, showing great application potential. Cordyceps polysaccharides have attracted attention due to their antioxidant, anti-inflammatory, anti-tumor, anti-hyperlipidemia, anti-diabetic, and immunomodulatory effects. Cordyceps polysaccharides can be used as prebiotics to promote the growth of beneficial bacteria and have good application prospects in functional foods or natural medicines.

[0004] Cordyceps militaris is an important entomopathogenic fungus widely used in the biological control of agricultural and forestry pests. Furthermore, as a type of entomopathogenic fungus with medicinal value, its abundant secondary metabolites show broad development prospects. Pharmacological activity studies have shown that Cordyceps militaris promotes neuronal differentiation, inhibits leukemia cell growth, lowers blood sugar, has anti-tumor effects, and enhances immunity. These research results indicate that Cordyceps militaris has the potential to be developed into a drug. By isolating and identifying high-quality strains from Cordyceps militaris, screening for high-yield polysaccharide and cordycepin-producing strains, and developing efficient fermentation processes, the technical barriers to the large-scale production of active ingredients have been overcome. This provides scientific data support for using Cordyceps militaris isolated strains as artificially cultivated alternatives to wild Cordyceps militaris or Cordyceps sinensis, alleviating pressure on wild resources and promoting the sustainable utilization of Cordyceps fungal resources.

[0005] The content of ergosterol and cordycepin, two active substances, in natural powdery cordyceps is low, and there are very few research reports on the cultivation of fruiting bodies. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0008] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a strain of Cordyceps farinosa, Gsxcf1, which is deposited at the China General Microbiological Culture Collection Center (CGMCC), with the suggested classification and name: Cordyceps farinosa, accession number CGMCC No. 41894, deposit date of April 21, 2025, and deposit address at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: Cordyceps farinosa strain Gsxcf1, deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.41894, deposit date of April 21, 2025, and deposit address at No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing.

[0010] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of Cordyceps farinosa strain Gsxcf1 in the preparation of food, health products and pharmaceuticals.

[0011] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing fermented mycelium of the powdery Cordyceps strain Gsxcf1, comprising,

[0012] The Cordyceps farinosa strain Gsxcf1 was activated to obtain an activated Cordyceps farinosa strain, which was then inoculated into a seed culture medium to obtain a seed culture solution.

[0013] The seed culture solution was inoculated into the liquid fermentation medium and placed in a constant temperature shaker at 25℃ and 150 rpm for shaking fermentation.

[0014] The Cordyceps fermentation broth after fermentation culture was filtered to obtain fermented mycelium;

[0015] Wash the mycelium three times with distilled water, filter it again, dry it in a 60℃ oven, grind and pulverize it, and pass it through a 60-mesh sieve to obtain fermented mycelium.

[0016] In a preferred embodiment of the preparation method described in this invention, the method for preparing the seed culture medium includes:

[0017] Take 20 g of sucrose, 20 g of peptone, 0.15 g of magnesium sulfate, and 0.3 g of potassium dihydrogen phosphate. Dissolve them completely in distilled water and bring the volume to 1 L. The pH should be natural.

[0018] As a preferred embodiment of the preparation method described in this invention, the liquid fermentation culture medium comprises: 20 g of glucose, 10 g of peptone, 1.5 g of magnesium sulfate, 2 g of potassium dihydrogen phosphate, and 20 mg of vitamin B1, which are completely dissolved in distilled water and then brought to a final volume of 1 L, with a natural pH.

[0019] Another objective of this invention is to overcome the shortcomings of the prior art and provide a powdery Cordyceps strain Gsxcf1 fermentation mycelium, wherein the powdery Cordyceps strain Gsxcf1 fermentation mycelium contains polysaccharides, ergosterol and cordycepin.

[0020] The polysaccharide content is greater than 18%, the ergosterol content is greater than 7.10 mg / g, and the cordycepin content is greater than 220 ug / g.

[0021] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of the fermented mycelium of the powdery Cordyceps strain Gsxcf1 in the preparation of food, health products and pharmaceuticals.

[0022] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing the fruiting body of the powdery Cordyceps strain Gsxcf1, comprising,

[0023] Preparation of solid-state fermentation medium: Peptone, sucrose, MgSO4·7H2O, KH2PO4, and water are mixed evenly, and the pH is kept natural to obtain the nutrient solution;

[0024] Dry wheat was mixed with nutrient solution to prepare a solid fermentation culture medium.

[0025] Artificial culture conditions: Mycelial growth period: 20℃, culture in the dark, for 0-7 days;

[0026] Color change and primordium induction differentiation period: provide 14 hours of light per day, 1500 lx, 16℃, and 9℃ at night for 8-20 days;

[0027] Fruiting body formation and growth period: After the mycelium completes color change and forms primordia, continue to cultivate under the conditions of 1500 Lx light per day, 11 h / d, temperature of 16℃ and humidity of 80% until the fruiting body emerges, which takes 21-55 days.

[0028] The fruiting bodies are harvested over a period of 3 months. They are dried in an oven at 40°C to obtain the fruiting bodies of the powdery Cordyceps strain Gsxcf1.

[0029] Another objective of this invention is to overcome the shortcomings of the prior art and provide a fruiting body of a powdery Cordyceps strain Gsxcf1, wherein the fruiting body of the powdery Cordyceps strain Gsxcf1 contains polysaccharides, ergosterol and cordycepin, wherein the polysaccharide content is 10.53%, the ergosterol content is 7.78 mg / g, and the cordycepin content is 162.97 ug / g.

[0030] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of the fruiting body of the powdery Cordyceps strain Gsxcf1 in the preparation of food, health products and pharmaceuticals.

[0031] Beneficial effects of this invention:

[0032] (1) The Cordyceps farinosa strain Gsxcf1 provided by the present invention was isolated from wild Cordyceps militaris. After liquid or solid fermentation, it has high content of active ingredients such as polysaccharides, ergosterol, and cordycepin. The polysaccharide content of the fruiting body of the strain of the present invention is 10.53%, and the polysaccharide content of the mycelium in liquid fermentation reaches 14.03%. The ergosterol content of the fruiting body and the fermented mycelium is 7.78 mg / g and 7.18 mg / g, respectively, and the cordycepin content of the fruiting body and the fermented mycelium is 162.97 μg / g and 220.23 μg / g, respectively. The ergosterol content of the strain of the present invention is significantly higher than that of its close relative Cordyceps militaris. In liquid fermentation culture, the mycelium is easy to obtain and suitable for large-scale culture. High yields of polysaccharides, ergosterol, and cordycepin can be achieved in a short time. Ergosterol, polysaccharides, and cordycepin can be efficiently produced through liquid or solid-state cultivation. The extracts exhibit significant antitumor and immunomodulatory activities, making them suitable for pharmaceutical or functional food development. Compared to wild cordyceps, which relies on insect hosts and specific ecological environments, making harvesting difficult and highly seasonal, artificial cultivation allows for continuous production year-round, unaffected by natural conditions. The growth cycle of artificially cultivated powdery cordyceps fruiting bodies is 3 months, significantly improving production efficiency. Artificially cultivated fruiting bodies can serve as alternative raw materials to natural cordyceps for the research and development of antitumor and immunomodulatory drugs.

[0033] (2) In the fermented mycelium and fruiting body of the powdery Cordyceps strain Gsxcf1 of the present invention, the contents of ergosterol and cordycepin are significantly higher than those of different parts of fresh Cordyceps militaris. In the liquid fermented mycelium, the contents of ergosterol are 5.16 and 7.33 times that of Cordyceps militaris sclerotia and stroma, respectively, and the contents of cordycepin are 1.7 and 15.24 times that of Cordyceps militaris sclerotia and stroma, respectively. In the solid fermented fruiting body, the contents of ergosterol are 5.59 and 8.19 times that of Cordyceps militaris sclerotia and stroma, respectively, and the contents of cordycepin are 1.26 and 12.28 times that of Cordyceps militaris sclerotia and stroma, respectively. The contents of ergosterol in the fruiting body are significantly higher than those in the mycelium, increasing by 1.08 times, and the contents of cordycepin in the fermented mycelium are 1.35 times higher than those in the fermented fruiting body.

[0034] It is evident that the powdery Cordyceps strain Gsxcf1 of this invention has high ergosterol and cordycepin content after fermentation, and thus high medicinal value. Attached Figure Description

[0035] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0036] Figure 1This is an image showing the colony morphology of the strain Gsxcf1 of the present invention on PDA medium. Detailed Implementation

[0037] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0038] The Cordyceps farinosa strain Gsxcf1 of this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC), with the suggested classification and name: Cordyceps farinosa, accession number CGMCC No. 41894, deposit date April 21, 2025, and deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0039] Example 1

[0040] Strain acquisition and identification:

[0041] 1.1 Strain Isolation

[0042] Strain isolation: Select fresh Cordyceps sinensis with robust stroma, slowly rinse with running water to remove surface dirt and impurities, then transfer to a clean bench and immerse the sample in 75% ethanol solution for 1 min to achieve disinfection.

[0043] The sample was then rinsed with sterile water to remove residual ethanol.

[0044] After rinsing, use sterile filter paper to blot dry the sample surface.

[0045] Using a sterile scalpel, the sample was cut open from the mandible of the insect's head, separating it into two parts: the stroma and the sclerotium. The stroma and the internal sclerotium were further cut into 3 mm × 3 mm tissue blocks, and the cut surfaces were inoculated into PDA medium, Bengal red agar medium, and modified PDA medium, with 5 tissue blocks evenly inoculated into each dish.

[0046] The PDA medium and Bengal red medium were both purchased from Beijing Luqiao Technology Co., Ltd.

[0047] PDA medium preparation: Weigh 40.0 g of ready-to-use potato dextrose agar medium, dissolve it completely in distilled water, and bring the volume to 1 L. The pH should be at its natural setting.

[0048] Preparation of Bengal Red Agar Medium: Weigh 36.6 g of ready-to-use Bengal Red Agar Medium, dissolve it completely in distilled water, and bring the volume to 1 L. The pH should be at its natural setting.

[0049] Preparation of modified PDA medium: Add 10 g of peptone, 0.5 g of magnesium sulfate, 1.0 g of potassium dihydrogen phosphate, and 50 mg of vitamin B1 to each liter of PDA medium. Dissolve completely in distilled water and bring the volume to 1 L. The pH should be natural.

[0050] After inoculation, the cells were incubated upside down at 22°C, 75% humidity, and in complete darkness.

[0051] Perform 3 parallel scans for each tissue site;

[0052] After colony formation, select single colonies with clear edges and independent structures. Use a sterile inoculation loop to pick up a small amount of hyphae or spores and transfer them to fresh PDA medium for purification. After multiple purification cycles, store the obtained pure colonies at 4°C for later use.

[0053] The culture characteristics of this strain are as follows: After about one week of cultivation on PDA medium (potato dextrose agar), the colonies completely covered the entire culture dish (90 mm in diameter). The colonies on the PDA plates were fluffy and white; the reverse side of the colonies was yellow. See [link to PDA medium colony morphology chart] for details. Figure 1 .

[0054] Furthermore, based on morphological identification, representative strains were screened from each morphological group and subjected to molecular biological identification to determine their phylogenetic position.

[0055] 1.2 Strain Identification

[0056] Take an appropriate amount of powdery Cordyceps mycelium and place it in a sterile centrifuge tube. Follow the operating procedures specified in the Solarbio Fungal DNA Extraction Kit to extract genomic DNA from the sample.

[0057] After extraction, the obtained DNA solution was stored at -20℃ for later use.

[0058] Using previously extracted DNA as a template, PCR amplification was performed using the universal primers ITS1 and ITS4 for the fungal ribosomal gene transcription spacer region (ITS).

[0059] All amplification primers were synthesized by Shanghai Bioengineering Technology Service Co., Ltd.

[0060] The PCR reaction system is as follows:

[0061] The reaction system used was 20 μL, with the following components: Mix 2.9 μL, ddH2O 14.8 μL, upstream ITS1 primer 0.4 μL, downstream ITS4 primer 0.4 μL, and template 1.5 μL.

[0062] The PCR amplification program settings are as follows:

[0063] First, pre-denaturation was performed at 94°C for 5 min. Then, the cycling phase was carried out, with each cycle consisting of three steps: denaturation (94°C, 30 s), annealing (55°C, 30 s), and extension (72°C, 1 min), for a total of 30 cycles. After the cycling phase, the final extension was performed at 72°C for 10 min. The amplified product was finally stored at 4°C.

[0064] The purification and sequencing of the PCR products were all performed by Shanghai Bioengineering Technology Co., Ltd.

[0065] The obtained sequences were compared with the NCBI database using BLAST analysis, and the taxonomic status of the target species was determined based on the comparison information.

[0066] Based on morphological characteristics and sequence alignment information, strain Gsxcf1 was transferred to the center of PDA plates and incubated at 25°C. Colony morphology and color were observed and recorded periodically. Colonies of strain Gsxcf1 on PDA plates were fluffy and white; the reverse side of the colonies was yellow. See attached figure for colony morphology on PDA medium. Figure 1 .

[0067] After sequencing, the obtained sequence was compared with the NCBI database using BLAST. The comparison results showed that the isolated strain Gsxcf1 was *Cordyceps farinosa*. The strain was identified as *Cordyceps farinosa*, named *Cordyceps farinosa* strain Gsxcf1, and deposited on April 21, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 41894; the address of the depository is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0068] Example 2

[0069] Preparation of fermented mycelium from Cordyceps militaris strain Gsxcf1:

[0070] The preserved powdery cordyceps slant culture was inoculated into PDA medium for activation and cultured in a constant temperature environment of 25℃.

[0071] The activated Cordyceps militaris spawn was inoculated into seed liquid culture medium and cultured in a constant temperature shaker at 25℃ and 200 rpm for 4 days to obtain the seed liquid (1.7×10⁻⁶). -7 (CFU / mL)

[0072] The seed culture medium consisted of 20 g sucrose, 20 g peptone, 0.15 g magnesium sulfate, and 0.3 g potassium dihydrogen phosphate, which were completely dissolved in distilled water and brought to a final volume of 1 L. The pH was left to stand at its natural setting.

[0073] Take 8% (v:w) seed culture solution and inoculate it into liquid fermentation medium, and place it in a constant temperature shaker at 25℃ and 150 rpm for 5 days for fermentation.

[0074] After 5 days of fermentation, the Cordyceps fermentation broth was filtered through a Buchner funnel to obtain fermented mycelium.

[0075] The mycelium was washed three times with distilled water, filtered again through a Buchner funnel, and finally dried in a 60°C oven, ground and pulverized, and passed through a 60-mesh sieve to obtain fermented mycelium.

[0076] The liquid fermentation medium consisted of 20 g glucose, 10 g peptone, 1.5 g magnesium sulfate, 2 g potassium dihydrogen phosphate, and 20 mg vitamin B1, all dissolved completely in distilled water and brought to a final volume of 1 L. The pH was left to stand at its natural setting.

[0077] Example 3

[0078] Culture of fruiting bodies of Cordyceps militaris strain Gsxcf1:

[0079] Inoculate with 10% (v:w) seed culture (1.7×10⁻⁶) under aseptic conditions. -7 Add (CFU / mL) to solid culture medium and incubate in a light incubator.

[0080] The above solid-state fermentation culture medium consists of 20g of dry wheat.

[0081] Nutrient solution: 2% (w / v) peptone, 2% (w / v) sucrose, 0.05% (w / v) MgSO4·7H2O, 0.1% (w / v) KH2PO4, 1000mL water, natural pH;

[0082] Dry feed: Nutrient solution = 1:1.5 (g / mL).

[0083] The above artificial culture conditions are as follows: mycelial growth period (0-7 days): after inoculation, culture at 20℃ in the dark until the mycelium completely covers the surface of the culture medium;

[0084] Color change and primordium induction differentiation period (8-20 days): Provide 14 hours of light per day, 1500 lx, 16℃, and 9℃ at night (diurnal temperature difference of 7-10℃).

[0085] Fruiting body formation and growth period (21-55 days): After the mycelium completes color change and forms primordia, continue to cultivate under the conditions of 1500 Lx light per day, 11 h / d, temperature of 16℃ and humidity of 80% until the fruiting body emerges.

[0086] The fruiting bodies are harvested in 3 months and dried in an oven at 40℃ for subsequent determination of active ingredients.

[0087] The stroma and sclerotia of fresh cordyceps are ground into powder using liquid nitrogen for subsequent determination of active ingredients.

[0088] Example 4

[0089] Determination of polysaccharide content in fermented mycelium and fruiting bodies of wild fresh Cordyceps and Cordyceps militaris strain Gsxcf1

[0090] Using the cultivation method of Example 2, mycelial dry powder of strain Gsxcf1 after fermentation was obtained.

[0091] Using the culture method of Example 3, dried fruiting bodies of strain Gsxcf1 were obtained after culturing.

[0092] The mycelium, fruiting bodies, and fresh Cordyceps militaris of strain Gsxcf1 were used as research objects. The fresh Cordyceps militaris used in the experiment were collected from Dajiao Town, Jiang County, Yuncheng City, Shanxi Province.

[0093] Fresh cordyceps fruiting bodies and sclerotia are ground into powder using liquid nitrogen.

[0094] The fruiting body of Cordyceps militaris consists of two parts: the stroma and the sclerotium.

[0095] The total polysaccharide content of Cordyceps was determined using the conventional method in this field: an appropriate amount of sample powder was weighed and placed in a glass test tube, and distilled water was added at a material-to-liquid ratio of 1:30. The mixture was extracted in an 80°C water bath for 2 hours, centrifuged for 10 minutes, and the precipitate was then extracted again with 30 times the amount of distilled water. This process was repeated 3 times, and the supernatants were combined.

[0096] Add 3 times the volume of anhydrous ethanol solution to the supernatant, let stand overnight at 4°C, after alcohol precipitation, centrifuge for 10 min, and collect the precipitate;

[0097] The precipitate was completely dissolved in distilled water to obtain a crude polysaccharide solution of the sample.

[0098] Take 1.0 mL of the diluted sample into a test tube, add 1.0 mL of 6% phenol solution and mix well, add 5.0 mL of concentrated sulfuric acid, mix well, let stand for 10 min, then boil in a water bath for 15 min, cool to room temperature, and measure the absorbance at 490 nm.

[0099] A glucose standard solution with a concentration of 0.1 mg / mL was prepared for use. The glucose standard solutions in the standard curve were prepared with concentrations of 0.0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL. The regression equation was calculated by plotting glucose concentration (mg / mL) on the x-axis and absorbance (OD) on the y-axis: y = 0.5023x - 0.0038, with a correlation coefficient R0. 2 =0.9988.

[0100] The content of cordyceps polysaccharides was calculated based on the standard curve.

[0101] Experimental results showed that the polysaccharide content of the mycelium of the powdery Cordyceps strain Gsxcf1 reached 18.05% after fermentation.

[0102] The polysaccharide content of the fermented mycelium of Cordyceps Gsxcf1 was significantly higher than that of different parts of fresh Cordyceps sinensis, and the polysaccharide production capacity of the fermented mycelium was higher than that of the fruiting body.

[0103] Table 1 Comparison of polysaccharide production capacity of different Cordyceps samples

[0104]

[0105] Note: Different letters indicate significant differences, while the same letter indicates no significant differences. The same applies to the following table.

[0106] Example 5

[0107] Determination of ergosterol content in fermented mycelium and fruiting bodies of wild fresh Cordyceps and Cordyceps militaris strain Gsxcf1

[0108] Using the cultivation method of Example 2, mycelial dry powder of strain Gsxcf1 after fermentation was obtained.

[0109] Using the culture method of Example 3, dried fruiting bodies of strain Gsxcf1 were obtained after culturing.

[0110] The research subjects were fermentation mycelium, fruiting body and fresh Cordyceps militaris of strain Gsxcf1. The fresh Cordyceps militaris used in the experiment were collected from Dajiao Town, Jiangxian County, Yuncheng City, Shanxi Province.

[0111] The stroma and sclerotia of fresh cordyceps are ground into powder using liquid nitrogen;

[0112] The fruiting body of Cordyceps militaris consists of two parts: the stroma and the sclerotium.

[0113] Extraction of ergosterol: Accurately weigh 0.25 g of sample into a stoppered glass test tube, add 15 mL of methanol, seal tightly, weigh and record the weight. Place the stoppered conical flask in an ultrasonicator for 60 min, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and collect the filtrate for later use.

[0114] Ergosterol content determination: The ergosterol content in the sample was determined by HPLC. 1 mL of the prepared sample solution was accurately transferred and filtered through a 0.45 μm microporous membrane. The filtrate was used for sample analysis.

[0115] Chromatographic column: ZORBAX Eclipse XDB-C18 (250 mm × 4.6 mm, 5 μm); mobile phase: 100% methanol, filtered through a 0.45 μm filter and degassed by sonication for 30 min before injection; flow rate: 1.0 mL / min; column temperature: 35 ℃; detection wavelength: 283 nm; injection volume: 10 μL. Ergosterol standard solutions of 0.1 mg / mL were prepared to concentrations of 10.00 μg / mL, 20.00 μg / mL, 40.00 μg / mL, 60.00 μg / mL, and 80.00 μg / mL. Determination was performed under the chromatographic conditions. A standard curve of ergosterol was plotted with peak area on the ordinate and ergosterol concentration on the abscissa. The linear regression equation was calculated as: y = 7952033x + 86509, R0 2 =0.9999.

[0116] As shown in Table 2, the ergosterol content of the liquid fermentation mycelium and solid fermentation fruiting body of powdery Cordyceps Gsxcf1 was significantly higher than that of different parts of fresh Cordyceps sinensis.

[0117] Table 2. Ergosterol content in different Cordyceps samples

[0118]

[0119] Example 6

[0120] Determination of Cordycepin Content in Fermented Mycelia and Fruiting Bodies of Wild Fresh Cordyceps and Powdery Cordyceps Strain Gsxcf1

[0121] Using the culture method of Example 2, the mycelial powder of the powdery strain Gsxcf1 after fermentation was obtained. Using the culture method of Example 3, the dried fruiting bodies of strain Gsxcf1 were obtained after cultivation.

[0122] The mycelium, fruiting bodies, and fresh Cordyceps militaris of strain Gsxcf1 were used as research objects. The fresh Cordyceps militaris used in the experiment were collected from Dajiao Town, Jiang County, Yuncheng City, Shanxi Province.

[0123] Fresh cordyceps fruiting bodies and sclerotia are ground into powder using liquid nitrogen.

[0124] The fruiting body of Cordyceps militaris consists of two parts: the stroma and the sclerotium.

[0125] Extraction of cordycepin: Accurately weigh 0.25 g of sample into a 50 mL volumetric flask, add 40 mL of distilled water, and sonicate for 3 h. After extraction, centrifuge at 4000 rpm for 10 min, collect the supernatant, and dilute to 50 mL for later use.

[0126] Determination of Cordycepin Content: Accurately transfer 1 mL of the diluted sample solution and filter it through a 0.45 μm microporous membrane. The filtrate is used for analysis. The determination is performed according to the chromatographic conditions. Chromatographic column: ZORBAX EclipseXDB-C18 (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile:water (5:95, V / V), degassed by sonication for 30 min before injection; flow rate: 1.0 mL / min; column temperature: 35 ℃; detection wavelength: 260 nm; injection volume: 10 μL. Prepare cordycepin standard solutions with mass concentrations of 1.00 μg / mL, 2.00 μg / mL, 5.00 μg / mL, 10.00 μg / mL, 20.00 μg / mL, and 50.00 μg / mL using a 0.1 mg / mL cordycepin standard solution. Chromatographic conditions were used for determination. A standard curve for cordycepin was plotted with peak area as the ordinate and cordycepin concentration as the abscissa. The linear regression equation was calculated as: y = 35479x - 6998.7, R0 2 =0.9995.

[0127] As shown in Table 3, the cordycepin content in the liquid fermentation mycelium and solid fermentation fruiting body of powdery Cordyceps Gsxcf1 was significantly higher than that in different parts of fresh Cordyceps sinensis.

[0128] Table 3 Cordycepin content in different Cordyceps samples

[0129]

[0130] The powdery Cordyceps Gsxcf1 strain of this invention has high polysaccharide content in its fermented mycelium and high ergosterol and cordycepin content in its solid-state fermented fruiting bodies, indicating that the powdery Cordyceps Gsxcf1 strain of this invention has high medicinal value. It can be used to prepare drugs with antioxidant, functional food or health product effects, with high added value and high economic benefits. This strain can carry out large-scale liquid fermentation culture in a short period of time, realizing the development and utilization of Cordyceps sinensis resources, and laying an important foundation for the development of functional foods related to powdery Cordyceps mycelium or fruiting bodies.

[0131] The Cordyceps farinosa strain Gsxcf1 provided by this invention was isolated from wild Cordyceps militaris. After liquid or solid-state fermentation, it exhibits high levels of active ingredients such as polysaccharides, ergosterol, and cordycepin, which are higher than those of its close relative Cordyceps militaris (4%–10% polysaccharides). [1] Ergosterol 1.18 mg / g [1] Cordycepin content: 0.297 ± 0.016 mg / g [2] ).

[0132] [1] Ren Sizhu, Song Rui, Yin Xiaotian, et al. Research progress on active ingredients and medicinal effects of Cordyceps militaris [J]. Anhui Agricultural Sciences, 1-6 [2025-06-10].

[0133] [2] Ji Wei, Liu Xiaomei, Wang Yipu, et al. Effects of different light qualities on growth, development and metabolites of Cordyceps militaris [J]. Chinese Wild Plant Resources, 2024, 43(05):68-76.

[0134] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. Cordyceps farinosa strain Gsxcf1, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41894, deposited on April 21, 2025, at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The use of the powdery Cordyceps strain Gsxcf1 according to claim 1 in the preparation of food, health products and pharmaceuticals.

3. A method for preparing fermented mycelium of the powdery Cordyceps strain Gsxcf1 as described in claim 1, characterized in that: include, The powdery Cordyceps strain Gsxcf1 was activated to obtain activated powdery Cordyceps strain, which was then inoculated into seed culture medium to obtain seed culture solution. The seed culture solution was inoculated into the liquid fermentation medium and placed in a constant temperature shaker at 25℃ and 150 rpm for shaking fermentation. The Cordyceps fermentation broth after fermentation culture was filtered to obtain fermented mycelium; Wash the mycelium three times with distilled water, filter it again, dry it in a 60℃ oven, grind and pulverize it, and pass it through a 60-mesh sieve to obtain fermented mycelium.

4. The preparation method according to claim 3, characterized in that: The method for preparing the seed culture medium includes, Take 20 g of sucrose, 20 g of peptone, 0.15 g of magnesium sulfate, and 0.3 g of potassium dihydrogen phosphate. Dissolve them completely in distilled water and bring the volume to 1 L. The pH should be natural.

5. The preparation method according to claim 3 or 4, characterized in that: The preparation method of the liquid fermentation medium includes taking 20 g of glucose, 10 g of peptone, 1.5 g of magnesium sulfate, 2 g of potassium dihydrogen phosphate, and 20 mg of vitamin B1, dissolving them completely in distilled water, and then adjusting the volume to 1 L. The pH is natural.

6. The powdery Cordyceps strain Gsxcf1 fermentation mycelium prepared by any one of claims 3 to 5, characterized in that: The fermented mycelium of the powdery Cordyceps strain Gsxcf1 contains polysaccharides, ergosterol, and cordycepin. The polysaccharide content is greater than 18%, the ergosterol content is greater than 7.10 mg / g, and the cordycepin content is greater than 220 ug / g.

7. The use of the powdery Cordyceps strain Gsxcf1 fermentation mycelium as described in claim 6 in the preparation of food, health products and pharmaceuticals.

8. A method for preparing the fruiting body of the powdery Cordyceps strain Gsxcf1 as described in claim 1, characterized in that: include, Preparation of solid-state fermentation medium: Peptone, sucrose, MgSO4·7H2O, KH2PO4, and water are mixed evenly, and the pH is kept natural to obtain the nutrient solution; Dry wheat was mixed with nutrient solution to prepare a solid fermentation culture medium. Artificial culture conditions: Mycelial growth period: 20℃, in the dark, for 0-7 days; Color change and primordium induction differentiation period: provide 14 hours of light per day, 1500 lx, 16℃, and 9℃ at night for 8-20 days; Fruiting body formation and growth period: After the mycelium completes color change and forms primordia, continue to cultivate under the conditions of 1500 Lx light per day, 11 h / d, temperature of 16℃ and humidity of 80% until the fruiting body emerges, which takes 21-55 days. The fruiting bodies are harvested over a period of 3 months. They are dried in an oven at 40°C to obtain the fruiting bodies of the powdery Cordyceps strain Gsxcf1.

9. The fruiting body of the powdery Cordyceps strain Gsxcf1 prepared by the method according to claim 8, characterized in that: The fruiting body of the powdery Cordyceps strain Gsxcf1 contains polysaccharides, ergosterol, and cordycepin, among which... The polysaccharide content is 10.53%, the ergosterol content is 7.78 mg / g, and the cordycepin content is 162.97 ug / g.

10. The use of the fruiting body of the powdery Cordyceps strain Gsxcf1 according to claim 9 in the preparation of food, health products and pharmaceuticals.

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