Cordyceps militaris strain with high yield of cordycepin as well as sporocarp cultivation method and application of cordyceps militaris strain

Abstract

The invention provides a cordyceps militaris strain with high yield of cordycepin as well as a sporocarp cultivation method and application of the cordyceps militaris strain, and relates to the technical field of microorganisms. The cordyceps militaris strain KN1.6. 778 provided by the invention is a mutant strain obtained in a conventional breeding process, and the preservation number is CGMCC No.40465. The cordyceps militaris strain is cultured and cultivated under proper conditions, and after harvesting, the content of cordycepin in a cordyceps militaris sporocarp reaches 35000mg / Kg or above, the highest content can reach 148000mg / Kg and is far beyond the level of the prior art, and an unexpected technical effect is generated.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Cordyceps militaris strain with high cordycepin production, a cultivation method for its fruiting body and applications thereof. Background Art

[0002] Cordyceps militaris belongs to the phylum Ascomycota, class Sordariomycetes, order Hypocreales, family Cordycipitaceae, genus Cordyceps ( Cordyceps ). It has been more than 150 years since it was first domesticated in 1867. Domestication in my country began in the 1980s, and it also took several decades. It can be seen that its commercial cultivation is difficult. However, after the joint efforts of practitioners in this field and continuous multi-faceted joint efforts, large-scale and commercial cultivation has been achieved. It is one of the more successful purely artificially cultivated Cordyceps.

[0004] In recent years, the influence of the selection of nutrients in the culture medium, the optimization of culture conditions (such as different light and temperature treatments), and the addition of exogenous substances (amino acids, cordycepin synthesis precursors, plant growth hormones, plant growth regulators NAA, salt, vegetable oils) on the biosynthesis of cordycepin has been studied. To date, the cordycepin content in the fruiting bodies of Cordyceps militaris that are available on the market or have been publicly reported is basically between 100-20000 mg / Kg, and there are few reports of better technical effects. For example, patent application number CN201810119734.X provides a high-yield cordycepin mutant strain of Cordyceps militaris and its application. After application, the mutant strain can produce up to 11777.094 μg / g of cordycepin in its fruiting body, that is, 11777.094 mg / Kg; for example, patent application number CN202310148047.1 provides a solid fermentation medium and a method for culturing Cordyceps militaris fruiting bodies. In the form of an excellent solid fermentation medium, the cordycepin content in the fruiting body of Cordyceps militaris is increased to a maximum of 7580 mg / Kg; patent application number CN202111270696.6 provides a method for culturing high-yield cordycepin silkworm pupae Cordyceps militaris using dry silkworm pupae after reeling as hosts, using CGMCC The No.13179 strain was cultured in a specific liquid culture medium, and then the mycelium was transferred into the dry silkworm pupae after reeling, and cultivated under specific temperature and light conditions. Finally, the cordycepin content of Cordyceps militaris fruiting bodies reached 2.8%, or 28,000 mg / Kg. In 2022, Li Jian et al. reported "A Preliminary Study on the Cultivation of a High-yield Cordyceps Militaris". Excluding commercial value, the cordycepin content reached 21,670 mg / Kg.

[0005] Although the synthetic metabolic pathway of cordycepin has been analyzed by many scientific researchers, obtaining excellent varieties or high-yield Cordyceps militaris strains according to breeding requirements is still the primary problem in this field. This is also the primary problem faced by the edible fungus industry in promoting the construction of a seed industry innovation system that is compatible with food development under the current background of practicing the big food concept and building a diversified food supply system. Therefore, based on the positioning of characteristic Cordyceps militaris to enrich large-scale agriculture, aiming at the practical technical problem of increasing the cordycepin content level in Cordyceps militaris fruiting bodies and leading the industry to implement the Cordyceps militaris seed industry revitalization action, this technical solution provides a Cordyceps militaris strain with high cordycepin production. After application, the cordycepin content in its fruiting body can reach 35000mg / Kg to 148000mg / Kg. Compared with the existing technical status, it has produced unexpected technical effects, far exceeding the existing technical level. Therefore, the application of Cordyceps militaris strain KN1.6.778 in this technical solution has outstanding substantive characteristics and significant progress. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a Cordyceps militaris strain with high cordycepin production and a cultivation method and application of its fruiting body.

[0007] On the one hand, the present invention provides a Cordyceps militaris strain KN1.6.778 with high cordycepin production, the original wild strain of which was collected in Tieling, Liaoning. The strain described in the present invention was obtained accidentally (by chance) through natural mutation during tissue separation after cultivation and domestication by the applicant.

[0008] Specifically, the Cordyceps militaris strain KN1.6.778 has a deposit number of CGMCC No.40465, and was deposited in the General Microbiology Center of the China Culture Collection Administration on December 26, 2022.

[0009] More specifically, the 18S coding sequence of the Cordyceps militaris strain KN1.6.778 is shown as SEQ ID NO.1, and the ITS coding sequence is shown as SEQ ID NO.2.

[0010] SEQ ID NO.1:

[0011] SEQ ID NO.2: .

[0012] More specifically, the Cordyceps militaris strain KN1.6.778 is a single-mating type Cordyceps militaris strain, and its fruiting body is orange-yellow in color and has a smooth surface.

[0013] In a preferred embodiment of the present invention, after 18-30 days of cultivation, the fruiting bodies of KN1.6.778 are finely differentiated and dense, brush-shaped, and pointed; some fruiting bodies begin to have multiple branches after 35-40 days of growth, and the fruiting body diameter is about 0.15cm-0.55cm, and the fruiting body density is 4-8 roots / cm 2 , there is no swelling on the head of the fruiting body.

[0014] In a preferred embodiment of the present invention, the cultivation box or cultivation container can be different according to the height of the desired commercial fruiting body. Usually, the height of the fruiting body cultivated in a bottle can reach 3-8cm, and the height of the fruiting body cultivated in a box can reach 5-12cm. Using transparent glass bottles and plastic boxes for cultivation can produce a large amount of side fur grass.

[0015] In a preferred embodiment of the present invention, some fruiting bodies of KN1.6.778 can still maintain their original shape and color from light yellow to orange-red during the harvest period (80 days to 98 days).

[0016] On the other hand, the present invention provides a method for cultivating the fruiting body of Cordyceps militaris strain KN1.6.778.

[0017] Specifically, the method comprises the following steps: (1) Preparation of primary culture: Take the thawed KN1.6.778 strain and inoculate it into sterilized liquid culture medium at a volume ratio of 1:200-1500 for cultivation; (2) Preparation of solid culture medium: Weigh grain solid materials, including one or more of rice, wheat, oats, and brown rice, place them in a cultivation container, add water or nutrient solution at a solid-liquid mass ratio of 1:1.8-2.5, and stir evenly; (3) Sterilization and cooling: Place the prepared culture medium in a sterilization device and sterilize it at 120-135°C for 5-30 minutes. After the temperature drops to 95-115°C, sterilize it again for 10-30 minutes. After the two-stage sterilization treatment, take it out and cool it; (4) Inoculation: dilute the primary seed at a volume ratio of 1:6-80 and spray evenly on the surface of the Cordyceps militaris solid culture medium; (5) Cultivation and cultivation: The inoculated solid culture medium of Cordyceps militaris is placed in dark culture at a temperature of 18-30°C, a humidity of 30-90%RH, and a CO2 concentration of 0.07-0.12% until the surface of the culture medium of Cordyceps militaris is completely covered with mycelium. After the dark culture is completed, light stimulation is given to induce color change and primordium differentiation, and grass growth or cultivation management is carried out.

[0018] (6) Harvesting.

[0019] More specifically, in the preparation of the primary seed in step (1), the liquid culture medium formula is: 8-25 g potato flour, 0.5-2 g fish scale powder, 6-12 g dried fish intestine powder, 8-12 g glucose, 8-15 g beef extract, 0.5-2 g KH2PO4, 2-5 g MgSO4, 0.3-1 g vitamin B complex, and 1000 mL water.

[0020] More specifically, in step (2) of preparing the solid culture medium, the nutrient solution comprises: 1000 mL of water, 8-15 g of glucose, 0.05-0.1 g of KH2PO4, and 0.02-0.05 g of MgSO4. More specifically, in step (5) of culturing and cultivating, the lighting conditions are: the light intensity is 150-5000 Lux, and the distance between the light source and the top of the cultivation container is 10-70 cm.

[0021] In yet another aspect, the present invention provides the use of Cordyceps militaris strain KN1.6.778 in the production of cordycepin.

[0022] Compared with the prior art, the present invention has the following advantages: The present invention provides a Cordyceps militaris strain KN1.6.778 with high cordycepin production. After application, the cordycepin content in the fruiting bodies of Cordyceps militaris after harvest produces an unexpected technical effect, reaching 35000 mg / Kg to 148000 mg / Kg, while still maintaining a good commercial appearance. It is at a significantly leading level in the prior art, has outstanding substantive characteristics and significant progress.

[0023] Collection Instructions Name of biological material: KN1.6.778.

[0024] Taxonomic name: Cordyceps militaris Cordyceps militaris ).

[0025] Date of preservation: December 26, 2022.

[0026] Deposit number: CGMCC No.40465.

[0027] Depository: General Microbiology Center, China Culture Collection Administration.

[0028] Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a front view of the morphology comparison of the fruiting bodies cultivated for 35 days in Example 2, A is the KN1.6.778 strain, and B is the Cordyceps militaris strain of the control group.

[0030] Figure 2 This is a top view of the morphology comparison of the fruiting bodies cultivated for 35 days in Example 2, A is the KN1.6.778 strain, and B is the Cordyceps militaris strain of the control group.

[0031] Figure 3 The front view of the fruiting body morphology comparison after 48 days of cultivation in Example 2, A is the KN1.6.778 strain, and B is the Cordyceps militaris strain of the control group.

[0032] Figure 4 The top view of the fruiting body morphology comparison after 48 days of cultivation in Example 2, A is the KN1.6.778 strain, and B is the Cordyceps militaris strain of the control group.

[0033] Figure 5 The front view of the fruiting body morphology comparison after 64 days of cultivation in Example 2, A is the KN1.6.778 strain, and B is the Cordyceps militaris strain of the control group.

[0034] Figure 6 The top view of the fruiting body morphology comparison after 64 days of cultivation in Example 2, A is the KN1.6.778 strain, and B is the Cordyceps militaris strain of the control group. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0036] Some of the raw materials in the examples are from the following sources: Table 1 raw material factory Potato Flour Baoji Liupanyun Biotechnology Co., Ltd. Beef extract Sinopharm Chemical Reagent Co., Ltd. Vitamin B Complex Xi'an Fenghua Pharmaceutical Co., Ltd. The preparation method of the fish scale powder of the present invention comprises the following steps: drying and grinding fresh fish scales into powder; The preparation method of the dried fish intestine rice noodle of the present invention comprises the following steps: drying and grinding fresh fish intestine into powder.

[0037] Example 1 (1) Preparation of primary strain: Take 0.5 mL of thawed KN1.6.778 and control group (from Jiangsu Kangneng Biotechnology Co., Ltd.) glycerol-preserved strains and inoculate them into 400 mL of sterilized liquid culture medium. The liquid culture medium formula is 8 g potato flour, 2 g fish scale powder, 6 g dried fish intestine powder, 8 g glucose, 8 g beef extract, 0.8 g KH2PO4, 2 g MgSO4, 1 g complex vitamin B, and 1000 mL water. Seal the mouth and place it in a 19°C constant temperature rotary shaker with a speed of 130 rpm. Incubate in a dark and oscillating environment for 4 days. When the bacterial solution turns golden yellow, fine and uniform, and has no sterile balls, and is tested for no contamination by other bacteria, it can be used.

[0038] (2) Preparation of solid culture medium: Weigh 100 g of wheat and place it in a cultivation box. Add 250 mL of clean water and stir evenly.

[0039] (3) Sterilization and cooling: sterilize the culture carrier after loading in stages: sterilize at 120°C for 15 min, then reduce the temperature to 95°C and sterilize for 20 min; after sterilization, cool to room temperature for later use.

[0040] (4) Inoculation: Under sterile conditions, dilute the primary seed at a volume ratio of 1:80 and spray evenly on the surface of the Cordyceps militaris solid culture medium.

[0041] (5) Cultivation and cultivation: The inoculated Cordyceps militaris culture medium is placed in a culture room for dark culture at 20°C for 7 days until the surface of the Cordyceps militaris culture medium is completely covered with Cordyceps militaris mycelium. After the dark culture is completed, it is given 4500 Lux of light, with the light source 15 cm away from the top of the cultivation box. The conditions in the cultivation room are controlled at: humidity 30-70% RH, CO2 concentration 0.07-0.12%.

[0042] (6) Harvesting: Harvest on the 90th day after inoculation.

[0043] Example 2 (1) Preparation of primary strain: Take 0.5 mL of thawed KN1.6.778 and the control group glycerol-preserved strain and inoculate it into 400 mL of sterilized liquid culture medium. The liquid culture medium formula is 15 g potato flour, 1.5 g fish scale powder, 6 g dried fish intestine powder, 10 g glucose, 15 g beef extract, 2 g KH2PO4, 2 g MgSO4, 1 g vitamin B complex, and 1000 mL water. After inoculation, seal it with a breathable membrane, place it in a 19°C constant temperature rotary shaker, adjust the speed to 130 rpm, and culture it in a dark and oscillating manner for 4 days. When the bacterial solution turns golden yellow, fine and uniform, and has no sterile balls, and is tested to be free of contamination by other bacteria, it can be used.

[0044] (2) Preparation of solid culture medium: Weigh 80 g of oats and place them in a culture box. Add 150 mL of water and stir evenly.

[0045] (3) Sterilization and cooling: sterilize the culture carrier after loading in stages: sterilize at 135°C for 5 minutes, then reduce the temperature to 95°C and sterilize for 25 minutes; after sterilization, cool to room temperature for later use.

[0046] (4) Inoculation: Under sterile conditions, dilute the primary seed at a volume ratio of 1:15 and spray evenly on the surface of the Cordyceps militaris solid culture medium.

[0047] (5) Cultivation and cultivation: The inoculated Cordyceps militaris culture medium is placed in a culture room for dark culture at 19°C for 4 days until the surface of the Cordyceps militaris culture medium is completely covered with Cordyceps militaris mycelium. After the dark culture is completed, 3500 Lux of light is applied, with the light source being 15 cm away from the top of the culture box. The conditions in the culture room are controlled at: humidity 30-70% RH, CO2 concentration 0.07-0.12%.

[0048] (6) Harvesting: Harvest on the 85th day after inoculation.

[0049] Example 3 (1) Preparation of primary strain: Take 0.5 mL of thawed KN1.6.778 and the control group glycerol-preserved strain, and inoculate into 400 mL of sterilized liquid culture medium. The liquid culture medium formula is 8 g potato flour, 1 g fish scale powder, 12 g dried fish intestine powder, 8 g glucose, 8 g beef extract, 0.5 g KH2PO4, 2 g MgSO4, 1 g complex vitamin B, and 1000 mL water. After inoculation, seal with a breathable membrane, place in a 19°C constant temperature rotary shaker, adjust the speed to 130 rpm, and culture in a dark and oscillating manner for 4 days. When the bacterial solution turns golden yellow, fine and uniform, and has no sterile balls, and is tested for no contamination by other bacteria, it can be used.

[0050] (2) Preparation of solid culture medium: Weigh 90 g of brown rice and place it in a culture box. Add 180 mL of nutrient solution (nutrient solution composition: 1000 mL of water, 10 g of glucose, 0.05 g of KH2PO4, and 0.1 g of MgSO4) and stir evenly.

[0051] (3) Sterilization and cooling: sterilize the culture carrier after loading as above in stages: sterilize at 120°C for 15 min, then reduce the temperature to 100°C and continue sterilizing for 10 min; after sterilization, cool to room temperature for later use.

[0052] (4) Inoculation: Under sterile conditions, dilute the primary seed at a volume ratio of 1:15 and spray evenly on the surface of the Cordyceps militaris solid culture medium.

[0053] (5) Cultivation and cultivation: The inoculated Cordyceps militaris culture medium is placed in a culture room for dark cultivation at 19°C for 4 days until the surface of the Cordyceps militaris culture medium is completely covered with Cordyceps militaris mycelium. After the dark cultivation is completed, 4500 Lux of light is applied, with the light source being 25 cm away from the top of the cultivation box. The conditions in the cultivation room are controlled at: humidity 30-70% RH, CO2 concentration 0.07-0.12%.

[0054] (6) Harvesting: Harvest on the 98th day after inoculation.

[0055] Example 4 (1) Preparation of primary strain: Take 0.5 mL of thawed KN1.6.778 and control group (purchased from Jiangsu Kangneng Biotechnology Co., Ltd.) glycerol-preserved strains and inoculate them into 400 mL of sterilized liquid culture medium. The liquid culture medium formula is 8 g potato flour, 2 g fish scale powder, 6 g dried fish intestine powder, 8 g glucose, 8 g beef extract, 0.8 g KH2PO4, 2 g MgSO4, 1 g complex vitamin B, and 1000 mL water. Seal the mouth and place it in a 19°C constant temperature rotary shaker with a speed of 130 rpm. Incubate in a dark and oscillating environment for 4 days. When the bacterial solution turns golden yellow, fine and uniform, and has no sterile balls, and is tested for no contamination by other bacteria, it can be used.

[0056] (2) Preparation of solid culture medium: Weigh 100 g of rice and place it in a cultivation box. Add 180 mL of water and stir evenly.

[0057] (3) Sterilization and cooling: sterilize the culture carrier after loading in stages: sterilize at 125°C for 10 min, then reduce the temperature to 95°C and sterilize for 15 min; after sterilization, cool to room temperature for later use.

[0058] (4) Inoculation: Under sterile conditions, dilute the primary seed at a volume ratio of 1:80 and spray evenly on the surface of the Cordyceps militaris solid culture medium.

[0059] (5) Cultivation and cultivation: The inoculated Cordyceps militaris culture medium is placed in a culture room for dark culture at 19°C for 4 days until the surface of the Cordyceps militaris culture medium is completely covered with Cordyceps militaris mycelium. After the dark culture is completed, it is given 4500 Lux of light, with the light source 15 cm away from the top of the cultivation box. The conditions in the cultivation room are controlled at: humidity 30-70% RH, CO2 concentration 0.07-0.12%.

[0060] (6) Harvesting: Harvest on the 80th day after inoculation.

[0061] Experimental Example: Detection of cordycepin content in Cordyceps militaris fruiting bodies Drying: The Cordyceps militaris fruiting bodies obtained in Example 1-3 were dried at 55°C.

[0062] Detection method: The detection method of cordycepin refers to NY / T 2116-2012 Determination of cordycepin and adenosine in cordyceps products by high performance liquid chromatography.

[0063] Table 2

[0064] In order to further prove the authenticity of the content, the present invention also carried out an external inspection of the KN1.6.778 fruiting bodies obtained by culture in Example 3. The fruiting bodies obtained in Example 3 of the present invention were sent to the Guangzhou Institute of Analysis and Testing of China for testing according to the NY / T 2116-2012 testing method. The test results were: the cordycepin content was 1.48×10 5 mg / Kg, that is, the content of cordycepin in the fruiting body accounts for 14.8%.

[0065] The comparison data of the strain of the present invention with other strains in the prior art are as follows: Table 3

[0066] According to the above data, it can be seen that after application of the Cordyceps militaris strain KN1.6.778 provided in the present application, the content of cordycepin in its fruiting body has unexpectedly been significantly improved, which is much higher than the general level of the prior art.

[0067] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A Cordyceps militaris with high cordycepin production ( Cordyceps militaris ) strain KN1.6.778, characterized in that The deposit number of the Cordyceps militaris strain KN1.6.778 is CGMCC No.40465.

2. The fruiting body cultivation method of the Cordyceps militaris strain KN1.6.778 according to claim 1, characterized in that: The following steps are involved: (1) Preparation of primary culture: Take the thawed KN1.6.778 strain and inoculate it into sterilized liquid culture medium at a volume ratio of 1:200-1500 for cultivation; (2) Preparation of solid culture medium: Weigh grain solid materials, including one or more of rice, wheat, oats, and brown rice, place them in a cultivation container, add water or nutrient solution at a solid-liquid mass ratio of 1:1.8-2.5, and stir evenly; (3) Sterilization and cooling: Place the prepared culture medium in a sterilization device and sterilize it at 120-135°C for 5-30 minutes. After the temperature drops to 95-115°C, sterilize it again for 10-30 minutes. After the two-stage sterilization treatment, take it out and cool it; (4) Inoculation: dilute the primary seed at a volume ratio of 1:6-80 and spray evenly on the surface of the Cordyceps militaris solid culture medium; (5) Cultivation and cultivation: The inoculated solid culture medium of Cordyceps militaris is placed in dark culture under the conditions of temperature of 18-30°C, humidity of 30-90%RH, and CO2 concentration of 0.07-0.12% until the surface of the culture medium of Cordyceps militaris is completely covered with mycelium; after the dark culture is completed, light stimulation is given to induce color change and primordium differentiation, and grass growth or cultivation management is carried out; (6) Harvesting.

3. The fruiting body cultivation method according to claim 2, characterized in that: In the preparation of the primary seed in step (1), the liquid culture medium formula is: potato flour 8-25g, fish scale powder 0.5-2g, dried fish intestine powder 6-12g, glucose 8-12g, beef extract 8-15g, KH2PO4 0.5-2g, MgSO4 2-5g, complex vitamin B 0.3-1g, water 1000mL.

4. The fruiting body cultivation method according to claim 2, characterized in that: In step (2) of preparing the solid culture medium, the nutrient solution comprises: 1000 mL of water, 8-15 g of glucose, 0.05-0.1 g of KH2PO4, and 0.02-0.05 g of MgSO4.

5. The fruiting body cultivation method according to claim 2, characterized in that: During the cultivation and culturing of step (5), the lighting conditions are as follows: the light intensity is 150-5000 Lux, and the distance between the light source and the top of the cultivation container is 10-70 cm.

6. Use of the Cordyceps militaris strain KN1.6.778 according to claim 1 in the production of cordycepin.

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