Powdery cordyceps sinensis strain capable of producing probiotic components and application of powdery cordyceps sinensis strain

Abstract

The invention discloses a Cordyceps sinensis powder strain capable of producing beneficial components and application thereof, and belongs to the technical field of microorganisms, the Cordyceps sinensis powder strain Gsxcf1 is preserved in China General Microbiological Culture Collection Center, the preservation number is CGMCC NO.41894, the preservation date is April 21, 2025, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The Cordyceps farinosa strain Gsxcf1 provided by the invention is separated from wild cordyceps sinensis, after liquid fermentation or solid fermentation, the content of active ingredients such as polysaccharide, ergosterol and cordycepin is high, and the Cordyceps farinosa strain Gsxcf1 has great potential to be applied to development of cordyceps sinensis sporocarp or mycelium related food.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Cordyceps sinensis strain producing beneficial ingredients and application thereof. Background Art

[0002] Cordyceps sinensis is a Cordyceps fungus with important medicinal and scientific value. It has unique biological properties and medicinal ingredients and is used as a substitute for Cordyceps sinensis. Research has shown that Cordyceps sinensis contains chemical components such as adenosine, amino acids, and cordycepin, which have effects such as enhancing immunity, calming the nerves, and resisting fatigue. It is also relatively low in toxicity, and has significant medicinal value and broad development prospects. Cordyceps sinensis has a limited distribution in nature and a long growth cycle, resulting in its resource scarcity. In addition, its overharvesting has put certain pressures on natural resources. Therefore, the development, utilization, and protection of Cordyceps sinensis biological resources are of great importance. With the rapid growth in demand for Cordyceps sinensis resources and the depletion of wild Cordyceps sinensis resources, the development value of Cordyceps sinensis has become increasingly prominent. The isolation and purification of strains from wild Cordyceps sinensis and the screening of excellent strains with active ingredients are of great significance for the resource utilization of Cordyceps sinensis.

[0003] Cordycepin, ergosterol, and cordyceps polysaccharides are all major components of Cordyceps. Cordycepin, also known as cordycepin and cordycepin, is the main active ingredient in Cordyceps (especially nucleosides). It is a new nucleoside drug and a purine alkaloid. Due to its unique ability to repair cells and protect the genetic code of living organisms, it has become a leader in modern biomedicine. Cordycepin, a naturally derived drug, has multiple pharmacological effects, including anti-tumor, antibacterial and antiviral, immunomodulatory, and free radical scavenging, and has promising clinical application prospects. Ergosterol is also a major chemical component of Cordyceps. Numerous studies have shown that it possesses a variety of physiological activities, such as antiviral, immune-enhancing, anti-inflammatory, cell growth inhibition, and blood sugar regulation, and has great application potential. Cordyceps polysaccharides have attracted widespread attention due to their antioxidant, anti-inflammatory, anti-tumor, anti-hyperlipidemic, anti-diabetic, and immunomodulatory effects. Cordyceps polysaccharides can be used as prebiotics to promote the growth of beneficial bacteria and have promising application prospects in functional foods or natural medicines.

[0004] Cordyceps sinensis is an important entomogenous fungus widely used in the biological control of agricultural and forestry pests. Furthermore, as a medicinal entomogenous fungus, its rich secondary metabolites show broad development prospects. Pharmacological activity studies have shown that Cordyceps sinensis has the potential to promote neuronal differentiation, inhibit leukemia cell growth, lower blood sugar, fight tumors, and enhance immunity. These research results indicate that Cordyceps sinensis has the potential to be developed into a pharmaceutical. By isolating and identifying high-quality strains of Cordyceps sinensis, screening for functional strains that produce high polysaccharides and cordycepin, and developing efficient fermentation processes, we have overcome the technical barriers to large-scale production of active ingredients. This provides scientific data support for the use of isolated Cordyceps sinensis strains as alternatives to wild Cordyceps sinensis or artificially cultivated Cordyceps sinensis, alleviating pressure on wild resources and promoting the sustainable utilization of Cordyceps sinensis resources.

[0005] The contents of ergosterol and cordycepin, two active substances, in natural powdered Cordyceps are low, and there are very few research reports on fruiting body culture. Summary of the Invention

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

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

[0008] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a Cordyceps farinose strain Gsxcf1, which is deposited in the General Microbiology Center of the China Culture Collection of Microorganisms, with the recommended classification name: Cordyceps farinose, the deposit number is CGMCC No.41894, the deposit date is April 21, 2025, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

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

[0010] Another object of the present invention is to overcome the deficiencies in the prior art and provide a use of the Cordyceps farinose strain Gsxcf1 in the preparation of foods, health products and medicines.

[0011] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing fermented mycelium of the Cordyceps sinensis strain Gsxcf1, comprising:

[0012] activating the Cordyceps farinose strain Gsxcf1 to obtain an activated Cordyceps farinose strain, and inoculating the strain 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°C and 150 rpm for shaking culture and fermentation;

[0014] After the fermentation culture, the Cordyceps fermentation liquid is filtered to obtain fermentation mycelium;

[0015] The mycelia were washed with distilled water for three times, filtered again, dried in an oven at 60° C., ground and crushed, and passed through a 60-mesh sieve to obtain fermented mycelia.

[0016] As a preferred embodiment of the preparation method of the present invention, the preparation method of the seed culture medium comprises:

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

[0018] As a preferred embodiment of the preparation method of the present invention, the liquid fermentation 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 adjusted to 1 L with a natural pH.

[0019] Another object of the present invention is to overcome the deficiencies in the prior art and provide a fermentation mycelium of the powdery Cordyceps sinensis strain Gsxcf1, wherein the fermentation mycelium of the powdery Cordyceps sinensis strain Gsxcf1 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 object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing food, health products and medicines by fermenting mycelium of the Cordyceps sinensis strain Gsxcf1.

[0022] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing the fruiting body of Cordyceps militaris strain Gsxcf1, comprising:

[0023] Prepare solid-state fermentation medium: mix peptone, sucrose, MgSO4·7H2O, KH2PO4, and water evenly and adjust the pH to a natural level to prepare a nutrient solution;

[0024] Mixing dry wheat with nutrient solution to prepare solid fermentation medium;

[0025] Artificial culture conditions: Mycelial growth period: 20℃, shaded culture, time 0-7 days;

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

[0027] Fruiting body formation and growth period: After the mycelium has completed color change and formed primordia, continue to cultivate under the conditions of 1500Lx, 11h / d light, temperature 16℃, and humidity 80% to wait for grass to emerge, which takes 21-55 days;

[0028] The fruiting body is harvested in 3 months and dried in an oven at 40°C to obtain the fruiting body of the powdery Cordyceps sinensis strain Gsxcf1.

[0029] Another object of the present invention is to overcome the deficiencies in the prior art and provide a fruiting body of the powdery Cordyceps sinensis strain Gsxcf1, wherein the fruiting body of the powdery Cordyceps sinensis 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 object of the present invention is to overcome the deficiencies in the prior art and provide a use of the fruiting body of the Cordyceps sinensis strain Gsxcf1 in the preparation of foods, health products and medicines.

[0031] Beneficial effects of the present invention:

[0032] (1) The Cordyceps farinosa strain Gsxcf1 provided by the present invention is isolated from wild Cordyceps militaris. After liquid or solid fermentation, it has high contents 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 liquid fermentation mycelium is 14.03%. The ergosterol content of the fruiting body and fermented mycelium is 7.78 mg / g and 7.18 mg / g, respectively. The cordycepin content of the fruiting body and 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 closely related genus Cordyceps militaris. In liquid fermentation culture, the mycelium is simple and easy to obtain, suitable for large-scale culture, and high yields of polysaccharides, ergosterol, and cordycepin can be achieved in a short time. Ergosterol, polysaccharides and cordycepin are efficiently produced through liquid or solid culture. The extracts have significant anti-tumor and immunomodulatory activities and are suitable for the development of medicines or functional foods. Compared with wild Cordyceps, which relies on insect hosts and specific ecological environments, is difficult to collect and has strong seasonality, artificial cultivation can be continuously produced throughout the year and is not restricted by natural conditions. The growth cycle of artificially cultured powdered Cordyceps fruiting bodies is 3 months, which greatly improves production efficiency. Artificially cultured fruiting bodies can be used as an alternative raw material to natural Cordyceps for the research and development of anti-tumor and immunomodulatory drugs.

[0033] (2) The ergosterol content and cordycepin content in the fermented mycelium and fruiting body of the powdery Cordyceps sinensis strain Gsxcf1 of the present invention are significantly higher than those in different parts of fresh Cordyceps sinensis. In the liquid fermentation mycelium, the ergosterol content is 5.16 and 7.33 times that of the sclerotium and stroma of Cordyceps sinensis, respectively, and the cordycepin content is 1.7 and 15.24 times that of the sclerotium and stroma of Cordyceps sinensis, respectively; in the solid fermentation fruiting body, the ergosterol content is 5.59 and 8.19 times that of the sclerotium and stroma of Cordyceps sinensis, respectively, and the cordycepin content is 1.26 and 12.28 times that of the sclerotium and stroma of Cordyceps sinensis, respectively; the ergosterol content in the fruiting body is significantly increased compared with the mycelium, by 1.08 times, and the cordycepin content in the fermented mycelium is 1.35 times higher than that in the fermented fruiting body;

[0034] It can be seen that the powdery Cordyceps sinensis strain Gsxcf1 of the present invention has high ergosterol and cordycepin contents after fermentation, high medicinal value, antioxidant capacity and immunomodulatory effects, and can be used to prepare drugs with antioxidant, functional foods or health care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be derived from these drawings without inventive effort. Among them:

[0036] Figure 1 The image shows the colony morphology of the strain Gsxcf1 of the present invention on PDA culture medium. DETAILED DESCRIPTION

[0037] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0038] The Cordyceps farinose strain Gsxcf1 of the present invention is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, with the recommended classification name: Cordyceps farinose, the deposit number is CGMCC No. 41894, the deposit date is April 21, 2025, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0039] Example 1

[0040] Acquisition of strains and identification of strains:

[0041] 1.1 Strain Isolation

[0042] To isolate the fungus, select fresh Cordyceps sinensis with a thick stroma. Rinse slowly with running water to remove surface dirt and impurities. Then transfer the sample to a clean bench and soak it in 75% ethanol solution for 1 minute to achieve disinfection.

[0043] Subsequently, the samples were rinsed with sterile water to remove residual ethanol;

[0044] After rinsing, use sterile filter paper to absorb the moisture on the surface of the sample;

[0045] Use a sterile scalpel to cut open the insect head at the mandible, separating the sample into the stroma and sclerotia. Further, cut the stroma and internal sclerotia into 3 mm × 3 mm tissue blocks. Inoculate the blocks face-down onto PDA medium, Bengal rose agar medium, and modified PDA medium, evenly distributing five blocks per dish.

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

[0047] Preparation of PDA medium: Weigh 40.0 g of ready-to-use potato dextrose agar medium, dissolve it completely with distilled water, and dilute to 1 L. The pH was natural.

[0048] Preparation of Red Bengal agar medium: Weigh 36.6 g of ready-to-use Red Bengal agar medium, dissolve it completely with distilled water, and then dilute to 1 L. The pH is natural.

[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 150 mg of VB per liter of PDA medium, completely dissolve them with distilled water, and then adjust the volume to 1 L. The pH should be natural.

[0050] After inoculation, the culture was inverted and placed under 22°C, 75% humidity and no light conditions.

[0051] Three parallels were performed for each tissue site;

[0052] After colonies have formed, select single, clear-edged, independent colonies. Use a sterile inoculating loop to collect a small amount of hyphae or spores. Transfer the colonies to fresh PDA medium for purification. After multiple purifications, store the resulting pure colonies in a refrigerator at 4°C until further use.

[0053] The culture characteristics of this strain are: after about one week of culture on PDA medium (potato dextrose agar medium), it will grow all over the culture dish (diameter of the culture dish is 90mm). The colonies on the PDA plate are fluffy and white; the back of the colonies is yellow. Figure 1 .

[0054] Furthermore, based on morphological identification, representative strains were selected from each morphological group and molecular biological identification was performed to determine their phylogenetic status.

[0055] 1.2 Identification of bacterial species

[0056] Take an appropriate amount of powdery Cordyceps mycelium and place it in a sterile centrifuge tube. Extract the sample genomic DNA according to the operating procedures specified in the Solebaugh fungal DNA extraction kit.

[0057] After the extraction was completed, the obtained DNA solution was stored at -20°C for future use.

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

[0059] The above-mentioned amplification primers were synthesized by Shanghai Bioengineering Technology Service Co., Ltd.

[0060] The PCR reaction system is as follows:

[0061] A 20 μL reaction system was used, and the specific components were: 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 was set up as follows:

[0063] First, pre-denaturation was performed at 94°C for 5 minutes; then the cycling stage was entered, and each cycle included three steps: denaturation (94°C, 30 seconds), annealing (55°C, 30 seconds), and extension (72°C, 1 minute), for a total of 30 cycles; after the cycling was completed, a final extension was performed at 72°C for 10 minutes; the amplified products were finally stored in a 4°C environment.

[0064] The purification of PCR products and sequencing operations were completed by Shanghai Bioengineering Technology Co., Ltd.

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

[0066] Based on the morphological characteristics and sequence alignment information, the strain Gsxcf1 was transferred to the center of the PDA plate and cultured in a constant temperature incubator at 25°C. The morphology and color of the colonies were regularly observed and recorded. The colonies of the strain Gsxcf1 on the PDA plate were fluffy and white in texture; the back of the colonies was yellow. The colony morphology on the PDA medium is shown in the attached figure. Figure 1 .

[0067] After sequencing, the resulting sequence was compared with the NCBI database using a blast analysis. The alignment results confirmed that the isolated strain Gsxcf1 was Cordyceps farinosa. The strain was identified as Cordyceps farinosa and named Gsxcf1. It was deposited with the General Microbiology Center of the China Culture Collection of Microorganisms on April 21, 2025, under the accession number CGMCC No. 41894. The depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0068] Example 2

[0069] Preparation of fermentation mycelium of Cordyceps sinensis strain Gsxcf1:

[0070] The preserved powdered Cordyceps sinensis slant strain was inoculated into PDA medium for activation and cultured in a constant temperature environment of 25°C;

[0071] The activated powdered Cordyceps sinensis fungi were inoculated into the seed liquid culture medium and cultured in a constant temperature shaker at 25°C and 200 rpm for 4 days as the seed liquid (1.7×10 -7 CFU / mL);

[0072] The seed liquid culture medium is: 20 g sucrose, 20 g peptone, 0.15 g magnesium sulfate, and 0.3 g potassium dihydrogen phosphate. After completely dissolving with distilled water, the volume is adjusted to 1 L, and the pH is natural.

[0073] 8% (v:w) seed culture solution was inoculated into liquid fermentation medium and placed in a constant temperature shaker at 25°C and 150 rpm for 5 days;

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

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

[0076] The liquid fermentation medium is: 20 g glucose, 10 g peptone, 1.5 g magnesium sulfate, 2 g potassium dihydrogen phosphate, and 20 mg vitamin B1, which are completely dissolved in distilled water and then adjusted to 1 L. The pH is natural.

[0077] Example 3

[0078] Cultivation of fruiting bodies of Cordyceps sinensis strain Gsxcf1:

[0079] Inoculate 10% (v:w) seed solution (1.7×10 -7 CFU / mL) to solid culture medium and cultured in a light incubator.

[0080] The above solid-state fermentation medium: 20g dry wheat;

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

[0082] Dry material: nutrient solution = 1:1.5 (g / mL).

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

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

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

[0086] The fruiting body is harvested within 3 months and can be dried in an oven at 40°C for subsequent determination of active ingredients;

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

[0088] Example 4

[0089] Determination of polysaccharide content in fermentation mycelia and fruiting bodies of wild fresh Cordyceps and powdery Cordyceps strain Gsxcf1

[0090] The culture method of Example 2 was used to obtain dry powder of mycelium fermented by the strain Gsxcf1.

[0091] The culture method of Example 3 was used to obtain dried fruiting bodies of the strain Gsxcf1.

[0092] The mycelium, fruiting body and fresh Cordyceps sinensis fermented by strain Gsxcf1 were used as research objects. The fresh Cordyceps sinensis used in the experiment was collected from Dajiao Town, Jiangxian County, Yuncheng City, Shanxi Province.

[0093] The stroma and sclerotia of fresh Cordyceps are ground into powder using liquid nitrogen.

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

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

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

[0097] The obtained precipitate was completely dissolved in distilled water to prepare a crude polysaccharide solution of the sample;

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

[0099] Using glucose as the standard, a 0.1 mg / mL glucose standard solution was prepared for use. The concentrations of glucose standard solution in the standard curve were 0.0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL, respectively. The regression equation was calculated using glucose concentration (mg / mL) as the abscissa and absorbance (OD) as the ordinate: y = 0.5023x - 0.0038, with a correlation coefficient of R. 2 =0.9988.

[0100] The Cordyceps polysaccharide content was calculated according to the standard curve.

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

[0102] The polysaccharide content of the fermented mycelium of Cordyceps sinensis 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 represent significant differences, and the same letters represent no significant differences.

[0106] Example 5

[0107] Determination of ergosterol content in fermented mycelia and fruiting bodies of wild fresh Cordyceps sinensis and powdery Cordyceps sinensis strain Gsxcf1

[0108] The culture method of Example 2 was used to obtain dry powder of mycelium fermented by the strain Gsxcf1.

[0109] The culture method of Example 3 was used to obtain dried fruiting bodies of the strain Gsxcf1.

[0110] The mycelium, fruiting body and fresh Cordyceps sinensis of Gsxcf1 were used as the research objects. The fresh Cordyceps sinensis used in the experiment was collected from Dajiao Town, Jiangxian County, Yuncheng City, Shanxi Province.

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

[0112] The fruiting body of Cordyceps militaris is divided into 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. Ultrasonicate the stoppered conical flask in an ultrasonicator for 60 minutes. After cooling, weigh again and make up the loss with methanol. Shake well, filter, and set aside the filtrate.

[0114] Determination of ergosterol content: HPLC was used to determine the ergosterol content in the sample. Accurately pipette 1 mL of the prepared sample solution and filter it 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 ultrasonication for 30 min before analysis; flow rate: 1.0 mL / min; column temperature: 35°C; detection wavelength: 283 nm; injection volume: 10 μL. A 0.1 mg / mL ergosterol standard solution was prepared to prepare ergosterol standard solutions with mass concentrations of 10.00 μg / mL, 20.00 μg / mL, 40.00 μg / mL, 60.00 μg / mL, and 80.00 μg / mL, respectively. The ergosterol standard curve was plotted using peak area as the ordinate and ergosterol mass concentration as the abscissa according to the chromatographic conditions, and the linear regression equation was calculated: y = 7952033x + 86509, R 2 =0.9999.

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

[0117] Table 2 Ergosterol content of different Cordyceps samples

[0118]

[0119] Example 6 Determination of cordycepin content in fermented mycelia and fruiting bodies of wild fresh Cordyceps and powdery Cordyceps strain Gsxcf1

[0120] The dry powder of mycelium of the powdery strain Gsxcf1 fermented by the culture method of Example 2 was obtained. The dried fruiting body of the strain Gsxcf1 was obtained by the culture method of Example 3.

[0121] The mycelium, fruiting body and fresh Cordyceps sinensis fermented by strain Gsxcf1 were used as research objects. The fresh Cordyceps sinensis used in the experiment was collected from Dajiao Town, Jiangxian County, Yuncheng City, Shanxi Province.

[0122] The stroma and sclerotia of fresh Cordyceps are ground into powder using liquid nitrogen.

[0123] The fruiting body of Cordyceps militaris is divided into two parts: the stroma and the sclerotium.

[0124] 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 hours. After extraction, centrifuge at 4000 rpm for 10 minutes, collect the supernatant, and dilute to 50 mL for later use.

[0125] Determination of cordycepin content: Accurately pipette 1 mL of the prepared sample solution and filter it through a 0.45 μm microporous membrane. The filtrate is then used for sample analysis. Chromatographic conditions were used for determination. The column was a ZORBAX Eclipse XDB-C18 (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of acetonitrile:water (5:95, v / v), which was passed through a 0.45 μm filter and degassed by ultrasonication for 30 min before injection. The flow rate was 1.0 mL / min. The column temperature was 35°C. The detection wavelength was 260 nm. The injection volume was 10 μL. A 0.1 mg / mL cordycepin standard solution was prepared to prepare cordycepin standard solutions at 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, respectively. The chromatographic conditions were used for determination, and the cordycepin standard curve was drawn with the peak area as the ordinate and the cordycepin mass concentration as the abscissa, and the linear regression equation was calculated: y = 35479x - 6998.7, R 2 =0.9995.

[0126] As shown in Table 3, the cordycepin content in the liquid fermentation mycelium and solid fermentation fruiting body of Cordyceps sinensis Gsxcf1 was significantly higher than that in different parts of fresh Cordyceps sinensis.

[0127] Table 3 Cordycepin content of different Cordyceps samples

[0128]

[0129] The mycelia of the fermented Cordyceps sinensis Gsxcf1 strain of the present invention and the ergosterol and cordycepin contents of the solid-state fermented fruiting bodies are high, indicating that the Cordyceps sinensis Gsxcf1 strain of the present invention has high medicinal value and can be used to prepare drugs with antioxidant, functional foods or health care products with high added value and high economic benefits. The strain can be subjected to 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 the mycelia or fruiting bodies of Cordyceps sinensis.

[0130] The Cordyceps farinosa strain Gsxcf1 provided by the present invention is isolated from wild Cordyceps militaris. After liquid or solid fermentation, the content of active ingredients such as polysaccharides, ergosterol, and cordycepin is high, which is higher than that of its closely related species Cordyceps militaris (polysaccharides 4% to 10%). [1] , ergosterol 1.18mg / g [1] , cordycepin content 0.297±0.016mg / g [2] ).

[0131] [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].

[0132] [2] Ji Wei, Liu Xiaomei, Wang Yipu, et al. Effects of different light qualities on the growth, development and metabolites of Cordyceps militaris [J]. Chinese Wild Plant Resources, 2024, 43(05): 68-76.

[0133] 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 the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, and all of these should be included in the scope of the present invention.

Claims

1. Powdery Cordyceps ( Cordyceps farinose ) strain Gsxcf1, deposited in the General Microbiology Center of China Culture Collection Administration, with the proposed taxonomic name: Cordyceps sinensis Cordyceps farinose The deposit number is CGMCC No. 41894, the deposit date is April 21, 2025, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The powdery Cordyceps sinensis according to claim 1 ( Cordyceps farinose ) Application of strain Gsxcf1 in the preparation of food, health products and medicines.

3. A method for preparing fermented mycelium of Cordyceps militaris strain Gsxcf1, characterized by: include, Powdery Cordyceps Cordyceps farinose ) activating the strain Gsxcf1 to obtain an activated powdery Cordyceps sinensis strain, and inoculating it into a seed culture medium to obtain a seed culture solution; The seed culture solution was inoculated into the liquid fermentation medium and placed in a constant temperature shaker at 25°C and 150 rpm for shaking culture and fermentation; After the fermentation culture, the Cordyceps fermentation liquid is filtered to obtain fermentation mycelium; The mycelia were washed with distilled water for three times, filtered again, dried in an oven at 60° C., ground and crushed, and passed through a 60-mesh sieve to obtain fermented mycelia.

4. The preparation method according to claim 2, wherein: The preparation method of the seed culture medium comprises: Take 20 g of sucrose, 20 g of peptone, 0.15 g of magnesium sulfate, and 0.3 g of potassium dihydrogen phosphate, completely dissolve them in distilled water, and then adjust the volume to 1 L. The pH is natural.

5. The preparation method according to claim 2 or 3, wherein: The liquid fermentation 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 fixed to 1 L with a natural pH.

6. The fermentation mycelium of the Cordyceps militaris strain Gsxcf1 obtained by the preparation method according to any one of claims 3 to 5, characterized in that: The fermented mycelium of the powdery Cordyceps sinensis 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. Use of the fermented mycelium of the Cordyceps sinensis strain Gsxcf1 according to claim 6 in the preparation of foods, health products and medicines.

8. A method for preparing fruiting bodies of Cordyceps militaris strain Gsxcf1, characterized by: include, Prepare solid-state fermentation medium: mix peptone, sucrose, MgSO4·7H2O, KH2PO4, and water evenly and adjust the pH to a natural level to prepare a nutrient solution; Mixing dry wheat with nutrient solution to prepare solid fermentation medium; Artificial culture conditions: Mycelial growth period: 20℃, shaded culture, time 0-7 days; Color change and primordium induction differentiation period: 14 hours of light per day, 1500 lx, 16 ° C, 9 ° C at night, for 8-20 days; Fruiting body formation and growth period: After the mycelium has completed color change and formed primordia, continue to cultivate under the conditions of 1500Lx, 11h / d light, temperature 16℃, and humidity 80% to wait for grass to emerge, which takes 21-55 days; The fruiting body is harvested in 3 months and dried in an oven at 40°C to obtain the fruiting body of the powdery Cordyceps militaris strain Gsxcf1.

9. The fruiting body of Cordyceps militaris strain Gsxcf1 obtained by the preparation method according to claim 8, characterized in that: The fruiting body of the powdery Cordyceps sinensis 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.

10. Use of the fruiting body of the Cordyceps sinensis strain Gsxcf1 according to claim 9 in the preparation of foods, health products and medicines.

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