A strain of Glechospora tetraspinata and a method for preparing cordycepin using the strain

Abstract

The present invention discloses a method for using a tetraspora strain ( Emericella quadrilineata, Also known as Aspergillus four-lined ) A method for producing cordycepin by fermentation. The NCPC1875 strain provided by the present invention is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, and the deposit number is CGMCC No.40757. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The present invention uses a solid static culture method, and the unit of cordycepin produced by fermentation can reach more than 12 mg / g. The culture medium components used in this method are simple and easy to obtain, the fermentation process is simple, the cycle is short, the separation and purification process is simple and easy, the yield is more than 60%, and the product purity reaches more than 98%, which can be used for the efficient preparation of cordycepin.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Gymnosporium tetraspinata strain and a method for preparing cordycepin by using the strain. Background Art

[0002] Cordycepin is a unique active ingredient in the traditional Chinese medicine Cordyceps sinensis, also known as cordycepin and cordycepin. Modern pharmacological studies have shown that cordycepin has multiple functions: anti-tumor, antibacterial, antiviral, immune regulation, lowering blood sugar, lowering blood lipids, and treating leukemia. It has significant and potential application space in the clinical treatment of the top ten malignant diseases.

[0003] Cordycepin was first detected and isolated from Cordyceps militaris. In addition to Cordyceps militaris fruiting bodies and their asexual forms, the fermented mycelium of Paecilomyces militaris, Taishan Cordyceps, Kyushu Cordyceps, Cicadae and its asexual forms, Paecilomyces cicadae, Cordyceps sinensis, Cordyceps sinensis, Gunni Cordyceps, Acropyrum nidulans, Mortierella bat moth, Pseudomonas nigricans, Mortierella bat moth, and Mortierella clethni can also produce cordycepin; cordycepin has also been detected in Cordyceps militaris obtained by inoculating Cordyceps militaris with the larvae of the bean moth as the host. In addition to the natural strains reported above, genetically engineered bacteria have been constructed for obtaining cordycepin. For example, CN114075515A constructed an engineered Aspergillus nidulans bacterium, CN116004697A constructed an engineered Aspergillus oryzae bacterium that heterologously expresses cordycepin, and CN115975828A reported an engineered yeast bacterium that produces cordycepin. The fermentation units of the above-mentioned engineered bacteria ranged from 4.9 mg / L to 4362.54 mg / L.

[0004] There are three ways to obtain cordycepin: deep liquid fermentation, shallow liquid fermentation, and solid fermentation. Zheng Wei et al. analyzed the effects of different deep liquid culture conditions on cordycepin synthesis, and the highest cordycepin mass concentration reached 1.465 g / L (Biotechnology Progress, 2017, 7 (6): 625-631). Mina Masuda used the Cordyceps militaris mutant G81-3 as a fermentation strain, and the liquid shallow fermentation cordycepin fermentation unit reached 14.7 g / L. However, the fermentation cycle was very long, reaching 45 days, and the production efficiency was low (Process Biochemistry, 2014, 49: 181-187). Sun Cui et al. used the Cordyceps militaris C1-7 strain and optimized the solid fermentation medium. The fermentation cycle was 28 days, and the highest cordycepin content reached 9.595 g / kg (Journal of Biology, 2019, 36 (2): 103-108). Liu Xiwen et al. used oil-containing loofah as the fermentation medium matrix for semi-solid fermentation. The fermentation cycle was 28 days and the cordycepin content reached 10.13 g / L (Food Industry Science and Technology, 2021, 42 (6): 118-125).

[0005] The components of the liquid deep fermentation medium reported so far are relatively complex or require the addition of a large amount of precursors such as adenine and glycine; although the fermentation unit of liquid shallow fermentation is relatively high, the components of the medium are also relatively large, and the fermentation cycle is usually very long. The solid fermentation reported so far also has the problems of complex medium components and long fermentation cycle. It can be seen that finding a high-efficiency strain of cordycepin, increasing the fermentation yield of cordycepin, and shortening the fermentation time of cordycepin have become problems that need to be solved urgently. Summary of the invention

[0006] The purpose of the present invention is to provide an efficient strain for high-yield cordycepin fermentation, and at the same time to provide an application method of the strain in preparing cordycepin, so as to obtain cordycepin efficiently.

[0007] The present invention provides a strain for preparing cordycepin by fermentation, the strain Gymnosporium tetraspinata ( Emericella quadrilineata )NCPC1875, the depositor is the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit date is August 21, 2023, the deposit number is CGMCC No.40757, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0008] The strain NCPC1875 provided by the present invention was identified as Gymnosporium tetraspinosum ( Emericella quadrilineata , also known as Aspergillus quadrilineatus ).

[0009] The identification method of the four-ridged naked spore shell strain NCPC1875 described in the present invention is to spread the slant culture of NCPC1875 on a slide for microscopic morphological observation. The spore liquid of the strain NCPC1875 is inoculated on CYA, CY20S, MEA and G25N culture media, cultured at 25°C for 7-14 days, and macroscopic morphological observation is performed, wherein CYA is cultured at 25°C and 37°C at the same time. The genome of the strain NCPC1875 is extracted and ITS sequence amplification, sequencing and NCBI comparison are performed. Through macroscopic and microscopic morphological observations and ITS sequence analysis, NCPC1875 is identified as a four-ridged naked spore shell strain.

[0010] The Gnomela tetraspora strain NCPC1875 of the present invention is isolated from the soil of Meihuashan National Nature Reserve in Fujian Province.

[0011] The soil collection method of the strain NCPC1875 of the present invention is to use a sterile shovel to scoop up the soil close to the plant roots and 5-10 cm from the ground surface and store it in a sterile bag.

[0012] The isolation method of the soil sample of the strain NCPC1875 of the present invention is a soil gradient dilution separation method. A fungus strain is obtained by this method and is named NCPC1875.

[0013] The invention discloses application of the Gnomela tetraspora strain in fermentation production of cordycepin.

[0014] The present invention provides a method for preparing cordycepin, which comprises using a strain of Gymnosporium tetraspinata ( Emericella quadrilineata ) NCPC1875 fermentation culture of Gymnospora tetraspinata, collecting mycelium to extract cordycepin may include the following steps:

[0015] a. Preparation of seed solution of Gymnosporium tetraspinosum strain NCPC1875;

[0016] b. Preparation of solid fermentation culture of G. tetraspora strain NCPC1875.

[0017] Specifically, the following steps may be included:

[0018] a. Preparation of seed solution of four-ridged Gymnosporium strain NCPC1875:

[0019] The slant culture or spore liquid of the Gymnospora tetraspinata strain NCPC1875 was inoculated into the seed culture medium at a temperature of 22-37°C, a rotation speed of 100-220 rpm, and a culture time of 48-72 h to obtain the seed liquid;

[0020] b. Preparation of fermentation culture of four-ridged naked spore shell strain NCPC1875:

[0021] Inoculate the seed liquid in step a into a solid fermentation medium at an inoculation rate of 3-10%, at a fermentation temperature of 22° C.-30° C., for 7-15 days, to obtain a fermentation culture;

[0022] c. Soaking the fermentation culture with an organic solvent to obtain a filtrate, concentrating the filtrate into an extract, removing oil with an organic solvent, and concentrating and drying under reduced pressure to obtain a crude extract;

[0023] d. The crude extract was dissolved in hot water, filtered, and the filtrate was introduced into a PS microsphere column, eluted with 20%-40% organic solvent water, and the eluate was concentrated and dried under reduced pressure to obtain a crude product;

[0024] e. The crude product was dissolved in hot water and prepared using a preparative chromatography column to collect the cordycepin chromatographic peak, concentrated under reduced pressure and then dissolved in an organic solvent while warm, filtered while hot, and the organic solvent was evaporated under reduced pressure to obtain a cordycepin solid.

[0025] Further, the seed culture medium formula: corn starch 10.0-40.0 g, glucose 10.0-40.0 g, hot pressed soybean cake powder 4.0-20.0 g, malt powder 1.0-6.0 g, yeast powder 1.0-3.0 g, sodium chloride 0.5-2.0 g, magnesium sulfate heptahydrate 0.5-2.0 g, add water to make up to 1000 mL, pH 6.5-7.0;

[0026] The solid fermentation medium comprises a solid granular matrix and water, and a powdered matrix, an inorganic salt, and trace elements are optionally added or not added; the solid granular matrix is ​​any one of rice, millet, bran, and corn residues or a combination thereof, the powdered matrix is ​​any one of hot-pressed soybean cake powder, cottonseed powder, and corn protein powder or a combination thereof, the inorganic salt component is any one of phosphate, magnesium sulfate, sodium chloride, and sodium nitrate or a combination thereof, and the trace element is any one of cobalt chloride, zinc sulfate, and manganese chloride or a combination thereof.

[0027] In the step c, the soaking is preferably performed 2-4 times, each time for 2-4 hours; the soaking organic solvent is preferably methanol or ethanol, and the degreasing organic solvent is preferably petroleum ether.

[0028] In the step d, the temperature of the hot water is preferably 40-50° C., the volume is 100 times that of the crude extract, and the organic solvent is preferably a 30% methanol-water solution.

[0029] In the step e, the temperature of the hot water is preferably 40-50° C., the preparative chromatographic column is preferably an ODS column, and the mobile phase is methanol water (containing KH2PO4).

[0030] In the step e, the organic solvent is preferably methanol, and the warming temperature is preferably 40-50°C.

[0031] The method for preparing cordycepin in the present invention may preferably be as follows:

[0032] The strain G. tetraspora ( Emericella quadrilineata ) The slant culture or spore liquid of NCPC1875 is inoculated into the seed culture medium, the culture temperature is 22-30°C, the rotation speed is 100-220 rpm, and the culture time is 48-72 h to obtain the seed liquid; wherein the seed culture medium is prepared according to the following method: 10.0-40.0 g corn starch, 10.0-40.0 g glucose, 4.0-20.0 g hot pressed soybean cake powder, 1.0-6.0 g malt powder, 1.0-3.0 g yeast powder, 0.5-2.0 g sodium chloride, 0.5-2.0 g magnesium sulfate heptahydrate, add water to make up to 1000 mL, pH 6.5-7.0; sterilize at 121°C for 30 min.

[0033] The seed liquid is inoculated into a solid fermentation medium at an inoculation rate of 3-10%, the fermentation temperature is 22°C-30°C, and the fermentation time is 7-14 days to obtain a fermentation culture; the solid fermentation medium includes a solid granular matrix and water, and a powdered matrix, an inorganic salt, and trace elements are optionally added or not added; the solid granular matrix is ​​any one or a combination of rice, millet, bran, and corn residue, the powdered matrix is ​​any one or a combination of hot-pressed soybean cake powder, cottonseed powder, and corn protein powder, the inorganic salt component is any one or a combination of phosphate, magnesium sulfate, sodium chloride, and sodium nitrate, and the trace element is any one or a combination of cobalt chloride, zinc sulfate, and manganese chloride. The culture medium is sterilized at 121°C for 30 minutes.

[0034] The fermentation culture is soaked in industrial ethanol for 2-4 times, each time for 2-4 hours, filtered, and the filtrate is concentrated under reduced pressure (30-50°C) to obtain an extract, which is deoiled with petroleum ether and concentrated under reduced pressure (30-50°C) to obtain a crude extract. The crude extract is dissolved in about 100 times hot water (40-50°C), filtered, and the filtrate is adsorbed on a PS microsphere column (spherical), eluted with 30% methanol-water (v / v), and the eluent is concentrated under reduced pressure (30-50°C) to obtain a crude product. The crude product is dissolved in hot water (40-50°C), prepared by HPLC (ODS column), and the cordycepin chromatographic peak is collected and concentrated under reduced pressure (30-50°C). Methanol is added to dissolve warm (40-50°C), filtered while hot, KH2PO4 is removed, and the methanol solution is evaporated under reduced pressure (30-50°C) to obtain cordycepin solid.

[0035] The four-ridged Gymnosporium strain NCPC1875 in the present invention is a wild strain. The present invention uses a solid static culture method, and the unit of cordycepin produced by fermentation can reach more than 12 mg / g. The culture medium components used in the present invention are simple and easy to obtain, the fermentation process is simple, the cycle is short, the separation and purification process is simple and easy, the yield is more than 60%, and the product purity reaches more than 98%, which is suitable for the efficient preparation of cordycepin. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Colony characteristics of the strain cultured in CYA medium at 37°C for 14 days

[0037] Figure 2 Colony characteristics of the strain cultured in CYA medium at 25°C for 14 days

[0038] Figure 3 Colony characteristics of the strain cultured in CY20S medium at 25°C for 14 days

[0039] Figure 4 Colony characteristics of the strain cultured in MEA medium at 25°C for 14 days

[0040] Figure 5Colony characteristics of strain G25N cultured at 25℃ for 14 days

[0041] Figure 6 Ascocarp and pericarp cells of strains

[0042] Figure 7 Ascus

[0043] Figure 8 Shell cells of strain

[0044] Fig. 9 Phylogenetic tree of ITS sequences of strains

[0045] Fig.10 Cordycepin 1 H-NMR spectrum

[0046] Fig.11 Cordycepin 13 C-NMR spectrum DETAILED DESCRIPTION

[0047] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. The experimental materials used in the following examples are all industrial grade raw materials unless otherwise specified.

[0048] Cordycepin sample preparation and detection method: After the fermentation culture is crushed, methanol is added at a ratio of fermentation culture: methanol of 1:10 (M / V) and soaked for 2-3 hours. The extract is centrifuged at 10000 r / min to obtain the cordycepin sample solution.

[0049] Detection method: chromatographic column nanometer (150 mm × 4.6 mm, 10 μm); detection wavelength 260 nm, flow rate 1.0 mL / min; mobile phase ratio of pure water: methanol (85:15), column temperature 30 °C.

[0050] Example 1. Isolation and identification of strain NCPC1875

[0051] (1) Isolation of strain NCPC1875

[0052] The culture medium for the isolation of the fungal strain NCPC1875 was PDA (Beijing Shuangxuan). The isolation method was to take 1 gram of soil sample (collected from the soil of Meihuashan National Nature Reserve in Fujian Province, China), put it into a test tube containing 10 mL of sterile water, and perform soil gradient dilution separation. Take 0.2 mL of each gradient and spread it on a plate, which was incubated at 26°C, and a fungal strain was obtained from it, which was named NCPC1875.

[0053] (2) Identification of strain NCPC1875

[0054] The slant culture of strain NCPC1875 was spread on a glass slide for microscopic morphology observation. The spore liquid of strain NCPC1875 was spotted in 4 identification media (CYA, CY20S, MEA, G25N), cultured at 25°C for 7-14 days, and macroscopic morphology observation was performed, among which CYA was cultured at both 25°C and 37°C.

[0055] The morphological characteristics of strain NCPC1875 in the four identification media are as follows:

[0056] The strain was cultured on CYA at 37℃ for 14 days, and the colony diameter reached 38-55mm; the main body of the colony was off-white, the central part was dark purple-red, and there were granular ascocarps in the center. The colony was dense, with radial grooves, a central bulge, and a relatively neat edge of the colony, with dark purple-red ascocarps (see Figure 1 ).

[0057] The colony was cultured on CYA at 25℃ for 14 days, and the colony diameter reached 32-42mm. The center of the colony was dark iron red, and the rest was grayish white. The center of the colony was bulging, dense, and granular, and the rest was velvety and loose. The edge of the colony was very irregular. There was a large amount of colorless exudate on the surface of the colony (see Figure 2 ).

[0058] The colony was cultured on CY20S at 25℃ for 14 days, and the colony diameter reached 35-55mm. The colony was yellowish brown. The colony was granular, with a raised center and densely distributed ascocarp particles, while the rest of the ascocarp particles were loosely distributed. The colony edge was neat (see Figure 3 ).

[0059] The colony was cultured on MEA at 25℃ for 14 days, and the colony diameter reached 35-46mm. The colony was light yellowish brown. The colony was relatively flat and granular, with a large number of ascocarps. The ascocarps were densely distributed in the center of the colony, and sparsely distributed in the rest of the colony. The colony edge was relatively neat (see Figure 4 ).

[0060] The colony was cultured on G25N at 25℃ for 14 days, and the colony diameter reached 22-30mm. The center of the colony was light brown-red, with a circle of new green ring stripes outside the center, and the edge of the colony was white. The surface of the colony was velvety, with radial grooves, and the edge of the colony was relatively neat (see Figure 5 ).

[0061] The ascocarp is purple-red, with a large number of shell cells distributed in a periodic ring. The ascus is purple-red and contains purple-red ascospores (see Figure 6-8 ).

[0062] The ITS sequence was determined to be 546 bp (see the sequence table in the manual). Comparison with the type strain on NCBI identified NCPC1875 as Gymnosporium tetraspinosum (see Fig. 9 ).

[0063] Example 2. Preparation of Cordycepin-producing Bacteria Fermentation Culture

[0064] (a) Preparation of seed solution of G. tetraspora strain NCPC1875

[0065] The strain NCPC1875 of Gymnospora tetraspinata was inoculated into the seed culture medium and cultured at 26°C and 200 rpm for 72 hours to obtain the seed solution. The preparation method of the seed culture medium is as follows: 30.0 g corn starch, 10.0 g glucose, 4.0 g hot pressed soybean cake powder, 5.0 g malt powder, 3.0 g yeast powder, 2.0 g sodium chloride, 1.0 g magnesium sulfate heptahydrate, add tap water to dissolve to 1000 ml, pH value 6.5, and sterilize at 121°C for 30 min.

[0066] (b) Preparation of solid fermentation culture of G. tetraspora strain NCPC1875

[0067] The seed liquid was inoculated with 8% inoculum into a 750mL triangular flask containing 100 grams of solid rice fermentation medium, and cultured at 26°C for 15 days. The preparation method of the fermentation medium is as follows: rice and water are mixed evenly in a ratio of 4:1, and the soaking time is 3 hours. Then 5% hot-pressed soybean cake powder, 1% cottonseed powder, 0.2% potassium dihydrogen phosphate, 0.05% cobalt chloride, 0.01% manganese chloride, and 0.01% zinc sulfate are added, mixed evenly, and divided into 750mL triangular flasks, each bottle containing 100 grams, rubber stopper, and sterilized at 121°C for 30 minutes.

[0068] After fermentation was completed, the fermentation unit was measured to be 12.46 mg / g.

[0069] Example 3. Preparation of Cordycepin-producing Bacteria Fermentation Culture

[0070] (a) Preparation of seed solution of G. tetraspora strain NCPC1875

[0071] The strain NCPC1875 of Gymnospora tetraspinata was inoculated into the seed culture medium and cultured at 22 ℃ and 100 rpm for 72 h to obtain the seed solution. The preparation method of the seed culture medium is as follows: 10.0 g corn starch, 40.0 g glucose, 14.0 g hot pressed soybean cake powder, 6.0 g malt powder, 1.0 g yeast powder, 1.0 g sodium chloride, 0.5 g magnesium sulfate heptahydrate, add tap water to dissolve to 1000 ml, pH value 7.0, and sterilize at 121 ℃ for 30 min.

[0072] (b) Preparation of solid fermentation culture of G. tetraspora strain NCPC1875

[0073] The seed liquid was inoculated with 5% inoculum into a 750mL triangular flask containing 100g of solid corn residue fermentation medium, and cultured at 30°C for 14 days. The fermentation medium was prepared by mixing corn residue and water in a ratio of 3:1, adding 0.2% sodium nitrate by total weight, and soaking for 4 hours. Then, the mixture was divided into 750mL triangular flasks, each containing 100g, rubber stoppered, and sterilized at 121°C for 30min.

[0074] After fermentation was completed, the fermentation unit was measured to be 12.12 mg / g.

[0075] Example 4. Preparation of Cordycepin-producing Bacteria Fermentation Culture

[0076] (a) Preparation of seed solution of G. tetraspora strain NCPC1875

[0077] The strain NCPC1875 of Gymnospora tetraspinata was inoculated into the seed culture medium and cultured at 30℃ and 220 rpm for 48 h to obtain the seed solution. The preparation method of the seed culture medium is as follows: 40.0 g corn starch, 30.0 g glucose, 20.0 g hot pressed soybean cake powder, 1.0 g malt powder, 2.0 g yeast powder, 0.5 g sodium chloride, 2.0 g magnesium sulfate heptahydrate, add tap water to dissolve to 1000 ml, pH value 6.5, and sterilize at 121℃ for 30 min.

[0078] (b) Preparation of solid fermentation culture of G. tetraspora strain NCPC1875

[0079] The seed liquid was inoculated with 3% inoculum into a 750ml triangular flask containing 100g solid millet fermentation medium, and cultured at 30°C for 10 days. The preparation method of the fermentation medium is as follows: millet and water are mixed evenly in a ratio of 8:3, and the soaking time is 10 hours. Then corn protein powder is added at 2.5% of the total weight, and 0.2% potassium dihydrogen phosphate is added. After mixing evenly, the mixture is divided into 750ml triangular flasks, each containing 100g, rubber stoppered, and sterilized at 121°C for 30min.

[0080] After fermentation was completed, the fermentation unit was measured to be 12.38 mg / g.

[0081] Example 5. Preparation of Cordycepin-producing Bacteria Fermentation Culture

[0082] (a) Preparation of seed solution of G. tetraspora strain NCPC1875

[0083] The strain NCPC1875 of Gymnospora tetraspinata was inoculated into the seed culture medium and cultured at 30℃ and 220 rpm for 48 h to obtain the seed solution. The preparation method of the seed culture medium is as follows: 15.0 g corn starch, 10.0 g glucose, 10.0 g hot pressed soybean cake powder, 2.0 g malt powder, 1.5 g yeast powder, 0.5 g sodium chloride, 1.0 g magnesium sulfate heptahydrate, add tap water to dissolve to 1000 ml, pH value 7.0, and sterilize at 121℃ for 30 min.

[0084] (b) Preparation of solid fermentation culture of G. tetraspora strain NCPC1875

[0085] The seed liquid was inoculated with 5% inoculum into a 1000mL triangular flask containing 100g of solid fermentation medium and cultured at 26°C for 14 days. The fermentation medium was prepared by mixing hot pressed soybean cake powder, bran and water in a ratio of 4:3:5, and sterilized at 121°C for 30min after mixing evenly.

[0086] After fermentation was completed, the fermentation unit was measured to be 13.68 mg / g.

[0087] Example 6. Preparation of Cordycepin-producing Bacteria Fermentation Culture

[0088] (a) Preparation of seed solution of G. tetraspora strain NCPC1875

[0089] The strain NCPC1875 of Gymnospora tetraspinosa was inoculated into the seed culture medium and cultured at 26°C and 220 rpm for 48 h to obtain the seed solution. The preparation method of the seed culture medium is as follows: 20.0 g corn starch, 10.0 g glucose, 2.0 g hot pressed soybean cake powder, 6.0 g malt powder, 3.0 g yeast powder, 2.0 g sodium chloride, 1.0 g magnesium sulfate heptahydrate, add tap water to dissolve to 1000 ml, pH value 6.7, and sterilize at 121°C for 30 min.

[0090] (b) Preparation of solid fermentation culture of G. tetraspora strain NCPC1875

[0091] The seed liquid was inoculated with 10% inoculum into a shallow dish containing 1000g solid fermentation medium, the material thickness was 2-2.5cm, and the culture was static for 12 days at 26℃. The preparation method of the fermentation medium is as follows: the ratio of hot pressed soybean cake powder, bran and water is 5:3:6, and after mixing evenly, it is sterilized at 121℃ for 30min.

[0092] After fermentation was completed, the fermentation unit was measured to be 14.76 mg / g.

[0093] Example 7. Extraction and purification of solid fermentation culture of G. tetrasporum strain NCPC1875

[0094] The solid culture described in Example 6 (b) was soaked in industrial ethanol for 3 times, each time for 2 hours, filtered, and the filtrate was concentrated under reduced pressure (40°C) to obtain a thick oily extract, which was deoiled with petroleum ether and concentrated under reduced pressure (40°C) to obtain a crude extract. The crude extract was dissolved in about 100 times hot water (45°C), filtered, and the filtrate was adsorbed on a PS microsphere column (spherical), eluted with 30% methanol-water (v / v), and the eluate was concentrated under reduced pressure (40°C) to obtain a yellowish brown powdery crude product. The crude product was dissolved in hot water (45°C), prepared by HPLC (ODS 20×250mm 10μm column, 20% methanol-water (containing 0.05% KH2PO4) (v / v), flow rate 18ml / min), and the cordycepin chromatographic peak was collected and concentrated under reduced pressure (30-50°C). Methanol was added and dissolved in warm water (40-50°C), and the mixture was filtered while hot to remove KH2PO4. The methanol solution was evaporated to dryness under reduced pressure (30-50°C) to obtain 8.9 g of white powdery cordycepin solid with HPLC purity of 98% and a total yield of 60%.

[0095] The molecular formula of cordycepin is: 10 H 13 N5O3, ESI-MS m / z 252.10 [M+H] + ; UVλmax(in MeOH):260nm.

[0096] 1 H-NMR (500MHz, CD3OD) δ (ppm) 8.41 (1H, s), 8.20 (1H, s), 5.95 (1H, d, J = 2.5Hz), 4.70 (1H, m), 4.52 (1H, m), 3.92 (1H, dd, J=12.5,2.5Hz), 3.66(1H,dd,J=12.5,3.5Hz), 2.37(1H,ddd,J=13.8,8.2,6.0Hz), 2.06(1H,ddd,J=13.2,6.7,3.8Hz), see Fig.10 .

[0097] 13 C-NMR (125MHz, CD3OD) δ (ppm) 157.5, 153.7, 150.0, 141.2, 120.7, 93.7, 82.6, 76.6, 64.3, 34.6, see Fig.11 .

[0098] Chemical structure of cordycepin

[0099]

Claims

1. A strain of Gymnosporum tetraspinata Emericella quadrilineata NCPC1875, the depository is the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit date is August 21, 2023, and the deposit number is CGMCCNo.40757.

2. Use of the Gymnosporium tetraspinosum strain NCPC1875 according to claim 1 in the preparation of cordycepin.

3. A method for preparing cordycepin using the Gnomela quadrangularis strain NCPC1875 according to claim 1, comprising: Fermenting and culturing Euspora tetraspora and collecting mycelium to extract cordycepin is characterized by comprising the following steps: a. Preparation of seed solution of Gymnosporum tetraspora strain NCPC1875; b. Preparation of solid fermentation culture of G. tetraspora strain NCPC1875.

4. The method according to claim 3, characterized in that It includes the following steps: a. Preparation of seed solution of four-ridged Gymnosporium strain NCPC1875: Inoculate the slant culture or spore liquid of the Gymnospora tetraspinata strain NCPC1875 into the seed medium to obtain the seed liquid; b. Preparation of fermentation culture of four-ridged naked spore shell strain NCPC1875: Inoculating the seed liquid in step a into a solid fermentation medium and fermenting to obtain a fermentation culture; c. Soaking the fermentation culture with an organic solvent to obtain a filtrate, concentrating the filtrate into an extract, removing oil with an organic solvent, and concentrating and drying under reduced pressure to obtain a crude extract; d. The crude extract was dissolved in hot water, filtered, and the filtrate was introduced into a PS microsphere column, eluted with a 20%-40% aqueous solution of an organic solvent, and the eluate was concentrated and dried under reduced pressure to obtain a crude product; e. The crude product was dissolved in hot water and prepared using a preparative chromatography column to collect the cordycepin chromatographic peak, concentrated under reduced pressure and then dissolved in an organic solvent while warm, filtered while hot, and the organic solvent was evaporated under reduced pressure to obtain a cordycepin solid.

5. The method according to claim 4, characterized in that Wherein in step a: the culture temperature is 22-37°C, the rotation speed is 100-220 rpm, and the culture time is 48-72h; the seed culture medium formula: 10.0-40.0 g corn starch, 10.0-40.0 g glucose, 4.0-20.0 g hot-pressed soybean cake powder, 1.0-6.0 g malt powder, 1.0-3.0 g yeast powder, 0.5-2.0 g sodium chloride, 0.5-2.0 g magnesium sulfate heptahydrate, add water to make the volume to 1000 mL, pH 6.5-7.

0.

6. The method according to claim 4, characterized in that Wherein, in step b: the seed liquid is inoculated at an inoculation rate of 3-10%, the fermentation temperature is 22°C-30°C, and the fermentation time is 7-15 days; the solid fermentation medium comprises a solid granular matrix and water, and a powdered matrix, an inorganic salt, and trace elements are optionally added or not added; the solid granular matrix is ​​any one or a combination of rice, millet, bran, and corn residues, the powdered matrix is ​​any one or a combination of hot-pressed soybean cake powder, cottonseed powder, and corn protein powder, the inorganic salt component is any one or a combination of phosphate, magnesium sulfate, sodium chloride, and sodium nitrate, and the trace element is any one or a combination of cobalt chloride, zinc sulfate, and manganese chloride.

7. The method according to claim 4, characterized in that Wherein step c is as follows: the soaking is performed for 2-4 times, each time for 2-4 hours; the soaking organic solvent is methanol or ethanol, and the degreasing organic solvent is petroleum ether.

8. The method according to claim 4, characterized in that Wherein step d is as follows: the hot water is at 40-50° C., the volume is 100 times that of the crude extract, and the organic solvent aqueous solution is a methanol aqueous solution.

9. The method according to claim 4, characterized in that Wherein step e is: the hot water is 40-50°C, the preparative chromatographic column is an ODS column, the mobile phase is a methanol aqueous solution containing KH2PO4; the organic solvent is methanol, and the warming temperature is 40-50°C.

10. The preparation method according to claim 4, characterized in that The following steps are involved: The slant culture or spore liquid of the four-ridged Gymnospora strain NCPC1875 is inoculated into a seed culture medium, the culture temperature is 22-37°C, the rotation speed is 100-220rpm, and the culture time is 48-72h to obtain a seed liquid; the seed culture medium formula: 10.0-40.0 grams of corn starch, 10.0-40.0 grams of glucose, 4.0-20.0 grams of hot-pressed soybean cake powder, 1.0-6.0 grams of malt powder, 1.0-3.0 grams of yeast powder, 0.5-2.0 grams of sodium chloride, 0.5-2.0 grams of magnesium sulfate heptahydrate, add water to make the volume to 1000mL, pH 6.5-7.0; The seed liquid is inoculated into a solid fermentation medium at an inoculation rate of 3-10%, the fermentation temperature is 22°C-30°C, and the fermentation time is 7-15 days to obtain a fermentation culture; the solid fermentation medium comprises a solid granular matrix and water, and a powdery matrix, an inorganic salt, and trace elements are optionally added or not added; the solid granular matrix is ​​any one or a combination of rice, millet, bran, and corn residues, the powdery matrix is ​​any one or a combination of hot-pressed soybean cake powder, cottonseed powder, and corn protein powder, the inorganic salt is any one or a combination of phosphate, magnesium sulfate, sodium chloride, and sodium nitrate, and the trace element is any one or a combination of cobalt chloride, zinc sulfate, and manganese chloride.

11. The fermentation culture is soaked in industrial ethanol for 2-4 times, each time for 2-4 hours, filtered, and the filtrate is concentrated under reduced pressure at 30-50°C to obtain an extract, which is deoiled with petroleum ether and concentrated under reduced pressure at 30-50°C to obtain a crude extract; the crude extract is dissolved in about 100 times 40-50°C hot water, filtered, and the filtrate is adsorbed on a PS microsphere column, eluted with a 30% volume ratio methanol aqueous solution, and the eluent is concentrated under reduced pressure at 30-50°C to obtain a crude product; the crude product is dissolved in 40-50°C hot water, prepared by ODS column HPLC, the mobile phase is a 20% volume ratio methanol aqueous solution containing 0.05% KH2PO4, the cordycepin chromatographic peak is collected, and concentrated under reduced pressure at 30-50°C; methanol is added to dissolve in a warm 40-50°C, filtered while hot, KH2PO4 is removed, and the methanol solution is evaporated under reduced pressure at 30-50°C to obtain cordycepin solid.

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