Cordyceps militaris domestication culture medium, culture method and application

Abstract

The invention discloses a cordyceps militaris domestication culture medium, a culture method and application, and belongs to the technical field of cordyceps militaris. The domestication culture method of cordyceps militaris comprises the following steps: (1) performing first domestication culture: inoculating a cordyceps militaris strain into a domestication culture medium, and performing domestication culture at 17-20 DEG C in a dark place for 7 days to obtain a fresh slant culture of an experimental strain; (2) second-time domestication culture: aseptically selecting fungus blocks on a fresh slant culture medium, placing the fungus blocks on a domestication plate culture medium, and culturing the fungus blocks in a dark place at 17-20 DEG C until the plate is full of strains; and (3) carrying out third domestication culture, namely punching a plate culture of the strains into 1cm < 2 > strain blocks by adopting a sterilized puncher, inoculating the strain blocks into a liquid culture medium in a sterile manner, inoculating 3-4 strain blocks into each bottle, and culturing for 90-96 hours in a dark place at the temperature of 17-20 DEG C and the rotating speed of 140-160r / min. The cordyceps militaris with high cordycepin content and good domestication effect is obtained by limiting the component composition of a domestication culture medium and a domestication culture method and limiting first domestication, second domestication and third domestication.

Description

Technical Field

[0001] The present application relates to a Cordyceps militaris acclimation culture medium, a culturing method and an application thereof, and belongs to the technical field of Cordyceps militaris. Background Art

[0002] Cordyceps militaris, also known as northern cordyceps and northern winter cordyceps, is an insect-fungus complex formed by fungi parasitizing on the bodies of insects such as Lepidoptera. It is a complex composed of two parts: the fruiting body (i.e. the grass part) and the sclerotium (i.e. the body part of the insect). In winter, the larvae hibernate in the soil, and the fungi parasitize in them, absorbing nutrients, and the larvae die of mycelium. In summer, seedlings grow from the bodies of the larvae, which are grass-like and collected around the summer solstice. It is a nutritious product with a tonic effect. Cordyceps militaris contains a variety of biologically active substances, including cordycepin, adenosine and other nucleoside substances, as well as cordyceps polysaccharides, superoxide dismutase and amino acids. The most important active ingredient is cordycepin. Studies have shown that cordycepin has the effects of regulating immunity, anti-tumor, anti-inflammatory, improving metabolism, and scavenging free radicals. At this stage, cordycepin is becoming an extremely active field in pharmaceutical chemistry, anti-aging, beauty, and health products. Cordyceps militaris is distributed worldwide. The number of natural resources is very small, and the market demand is very large.

[0003] Therefore, how to realize the domestication and cultivation of new varieties of Cordyceps militaris and obtain Cordyceps militaris with high cordycepin content and good domestication effect to solve the shortage of natural resources of Cordyceps militaris and meet the strong market demand is a technical problem that needs to be solved urgently. Summary of the invention

[0004] In order to solve the above problems, a Cordyceps militaris acclimation culture medium, a culture method and an application are provided. By limiting the component composition of the acclimation culture medium and the acclimation culture method, and by limiting the first acclimation, the second acclimation and the third acclimation, a Cordyceps militaris with high cordycepin content and good acclimation effect can be obtained.

[0005] According to one aspect of the present application, a method for acclimating and cultivating Cordyceps militaris is provided, comprising the following steps:

[0006] (1) First acclimatization culture: Inoculate the Cordyceps militaris strain into the acclimatization medium, acclimatize and culture at 17-20°C in the dark for 7 days to obtain a fresh slant culture of the experimental strain;

[0007] (2) Second acclimatization culture: Aseptically pick the bacterial blocks on the fresh slant culture medium onto the acclimatization plate culture medium and culture them at 17-20°C in the dark until the bacteria fill the plate;

[0008] (3) The third acclimatization culture: Use a sterile puncher to punch the plate culture of the strain into 1 cm 2The bacterial blocks were aseptically inoculated into liquid culture medium, 3 to 4 bacterial blocks were inoculated into each bottle, and cultured at 17 to 20°C, at a rotation speed of 140 to 160 r / min in the dark for 90 to 96 hours.

[0009] Optionally, in step (1) and step (2), the acclimation temperature is 18°C.

[0010] Optionally, in step (3), the acclimation temperature is 18° C., the rotation speed is 140 r / min, and the light-proof culture time is 96 h.

[0011] Optionally, the Cordyceps militaris species is a Cordyceps militaris strain, with a preservation number of CICC14014, CICC14015 and / or CICC14013.

[0012] Optionally, the liquid culture medium comprises the following components: 180-200 g of potato, 18-20 g of glucose, 8-11 g of peptone, 1.5-2.5 g of glycine, 2-3.5 g of KH2PO4, 1-1.5 g of MgSO4, 10-15 mg of vitamin B1, natural pH, and 1000 mL of water.

[0013] According to another aspect of the present application, a Cordyceps militaris acclimation culture medium is also provided, characterized in that it comprises the following components: 190-200 g of potato, 18-20 g of glucose, 8-10 g of peptone, 2-3 g of glycine, 3-4 g of KH2PO4, 1.5-2 g of MgSO4, 10-12 mg of vitamin B1, 12-18 g of agar, and 1,000 mL of water.

[0014] Optionally, 0.5 to 2.5 g of seaweed polysaccharide extract is also included.

[0015] Specifically, the mass ratio of seaweed polysaccharide extract to glycine is (0.15-1.25):1.

[0016] Preferably, the mass ratio of seaweed polysaccharide extract to glycine is (0.5-1.2):1.

[0017] Optionally, the following components are included: 200 g potato, 20 g glucose, 10 g peptone, 2 g glycine, 3.0 g KH2PO4, 1.5 g MgSO4, 10 mg vitamin B1, 15 g agar, 1 000 mL water and 1.3 g seaweed polysaccharide extract.

[0018] According to another aspect of the present application, there is provided an application of the domestication and cultivation method described above in the domestication of Cordyceps militaris.

[0019] According to another aspect of the present application, there is provided application of the above-mentioned acclimation medium in acclimation of Cordyceps militaris.

[0020] The beneficial effects of this application include but are not limited to:

[0021] 1. According to the domestication and cultivation method of Cordyceps militaris of the present application, by limiting the components of the domestication medium and the domestication and cultivation method, and by limiting the first domestication, the second domestication and the third domestication, Cordyceps militaris with high cordycepin content and good domestication effect can be obtained.

[0022] 2. According to the domestication medium of Cordyceps militaris of the present application, specific components are limited and compounded in specific proportions to form the domestication medium, and the domestication medium is used in conjunction with the domestication and culture method of the present application to promote mycelium growth, increase the cordycepin content, and have good application stability.

[0023] 3. According to the acclimation medium of Cordyceps militaris of the present application, by adding seaweed polysaccharide extract, on the one hand, the seaweed polysaccharide extract is compounded with potatoes, glucose, etc. to provide Cordyceps militaris with rich carbon sources and nutrients, and provide Cordyceps militaris with a new source of nutrition; on the other hand, the seaweed polysaccharide extract can promote the growth of Cordyceps militaris to a certain extent. The glycerol and amino acids rich in the seaweed polysaccharide extract act as signal molecules synergistically with glycine to stimulate the expression of genes related to strain growth and enzyme reactions, thereby promoting strain growth and metabolism.

[0024] 4. The domestication culture medium and domestication culture method of Cordyceps militaris provided in this application have simple components, simple process steps and equipment, are easy to operate, and have low cost, and are suitable for use in general strain factories. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0026] Figure 1 The plate colony images before (a) and after (b) acclimation involved in Example 3 of the present application;

[0027] Figure 2 The mycelium of the liquid culture medium involved in Example 3 of the present application;

[0028] Figure 3 This is a microscope image of the mycelium involved in Example 3 of the present application. DETAILED DESCRIPTION

[0029] The present application is described in detail below with reference to embodiments, but the present application is not limited to these embodiments.

[0030] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents or raw materials used in the present invention can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in the present invention are used in a conventional manner in the art or in accordance with the product instructions. In addition, any method and material similar or equivalent to the described content can be applied to the method of the present invention. The preferred implementation methods and materials described in this patent are for demonstration purposes only.

[0031] The Cordyceps militaris strains used in this application have a deposit number of CICC14014, CICC14015 and / or CICC14013. The Latin name of Cordyceps militaris is Cordyceps militaris, and the strains CICC14014, CICC 14015 and / or CICC14013 are all purchased from the China Industrial Microbiological Culture Collection Administration Center.

[0032] Example 1

[0033] A method for taming and cultivating Cordyceps militaris:

[0034] (1) First acclimation culture: The Cordyceps militaris strain was inoculated into an acclimation medium, and acclimated and cultured at 17°C in the dark for 7 days to obtain a fresh slant culture of the experimental strain; the acclimation medium included: 190 g potato, 18 g glucose, 8 g peptone, 2 g glycine, 3 g KH2PO4, 1.5 g MgSO4, 10 mg vitamin B1, 12 g agar, and 1 000 mL water; the Cordyceps militaris strain was a Cordyceps militaris strain, with a deposit number of CICC14014;

[0035] (2) Second acclimation culture: Aseptically pick the bacterial blocks on the fresh slant culture medium and place them on the acclimation plate culture medium, and culture them at 17°C in the dark until the bacteria grow all over the plate; the acclimation plate culture medium includes: 190 g potato, 18 g glucose, 8 g peptone, 2 g glycine, 3 g KH2PO4, 1.5 g MgSO4, 10 mg vitamin B1, 12 g agar, and 1000 mL water;

[0036] (3) The third acclimatization culture: Use a sterile puncher to punch the plate culture of the strain into 1 cm 2 The bacterial blocks were aseptically inoculated into liquid culture medium, with 3 bacterial blocks inoculated in each bottle. The culture was carried out at 17°C, with a rotation speed of 140r / min and protected from light for 90h. The liquid culture medium included: 180g potato, 18g glucose, 8g peptone, 1.5g glycine, 2g KH2PO4, 1g MgSO4, 10mg vitamin B1, natural pH, and 1000mL water.

[0037] Example 2

[0038] A method for taming and cultivating Cordyceps militaris:

[0039] (1) First acclimation culture: The Cordyceps militaris strain was inoculated into an acclimation medium, and acclimated and cultured at 20°C in the dark for 7 days to obtain a fresh slant culture of the experimental strain; the acclimation medium included: 200 g potato, 20 g glucose, 10 g peptone, 3 g glycine, 4 g KH2PO4, 2 g MgSO4, 12 mg vitamin B1, 18 g agar, and 1 000 mL water; the Cordyceps militaris strain was a Cordyceps militaris strain, and the deposit number was CICC14013;

[0040] (2) Second acclimation culture: Aseptically pick the bacterial blocks on the fresh slant culture medium and place them on the acclimation plate culture medium, and culture them at 20°C in the dark until the bacteria grow all over the plate; the acclimation plate culture medium includes: 200 g potato, 20 g glucose, 10 g peptone, 3 g glycine, 4 g KH2PO4, 2 g MgSO4, 12 mg vitamin B1, 18 g agar, and 1000 mL water;

[0041] (3) The third acclimatization culture: Use a sterile puncher to punch the plate culture of the strain into 1 cm 2 The bacterial blocks were aseptically inoculated into liquid culture medium, with 4 bacterial blocks inoculated in each bottle. The culture time was 96 hours at 20°C, 160 r / min, and protected from light. The liquid culture medium included: 200 g potato, 20 g glucose, 11 g peptone, 2.5 g glycine, 3.5 g KH2PO4, 1.5 g MgSO4, 15 mg vitamin B1, natural pH, and 1000 mL water.

[0042] Example 3

[0043] A method for taming and cultivating Cordyceps militaris:

[0044] (1) First acclimation culture: The Cordyceps militaris strain was inoculated into an acclimation medium, and acclimated and cultured at 18°C ​​in the dark for 7 days to obtain a fresh slant culture of the experimental strain; the acclimation medium included: 200 g potato, 20 g glucose, 10 g peptone, 2 g glycine, 3.0 g KH2PO4, 1.5 g MgSO4, 10 mg vitamin B1, 15 g agar, 1 000 mL water, and 1.3 g seaweed polysaccharide extract; the Cordyceps militaris strain was a Cordyceps militaris strain, with a deposit number of CICC14015;

[0045] (2) Second acclimation culture: Aseptically pick the bacterial blocks on the fresh slant culture medium and place them on the acclimation plate culture medium, and culture them at 18°C ​​in the dark until the bacteria grow all over the plate; the acclimation plate culture medium includes: 200 g potato, 20 g glucose, 10 g peptone, 2 g glycine, 3.0 g KH2PO4, 1.5 g MgSO4, 10 mg vitamin B1, 15 g agar, 1 000 mL water and 1.3 g seaweed polysaccharide extract;

[0046] (3) The third acclimatization culture: Use a sterile puncher to punch the plate culture of the strain into 1 cm 2 The bacterial blocks were aseptically inoculated into liquid culture medium, with 4 bacterial blocks inoculated in each bottle. The culture time was 96 hours at 18°C, 140 r / min, and protected from light. The liquid culture medium included: 200 g potato, 20 g glucose, 11 g peptone, 2.5 g glycine, 3.5 g KH2PO4, 1.5 g MgSO4, 15 mg vitamin B1, natural pH, and 1000 mL water.

[0047] Example 4

[0048] The difference between Example 4 and Example 3 is that 3 g of glycine and 0.3 g of seaweed polysaccharide extract are contained in the acclimation plate culture medium, and the rest are the same.

[0049] Comparative Example 1

[0050] The difference between Comparative Example 1 and Example 3 is that the PDA comprehensive medium is used in the whole culture process of Comparative Example 1, and the PDA comprehensive medium includes 200g potato, 20g glucose, 0.7g peptone, 3g KH2PO4, 1.5g MgSO4, 10mg vitamin B1, 15g agar, natural pH, and 1000mL water. The temperature is kept at 121°C for 30min.

[0051] Experimental Example 1 Acclimation Stability Test

[0052] Taking Example 3 as an example, the liquid cultures obtained from the first acclimation culture, the second acclimation culture and the third acclimation culture of Example 3 were filtered through three layers of gauze, the mycelium was rinsed with clean water three times, and then the mycelium was collected and dried in an oven at 55° C. to constant weight. Comparative Example 1 was used as a control.

[0053] Accurately weigh 2.0g of the mycelium powder to be tested, place it in a 25mL volumetric flask, add 20mL of deionized water, place it in an ultrasonic instrument for ultrasonic extraction for 3h, take it out and make up to volume with deionized water, shake it well. Take 1mL of the sample solution and centrifuge it, then filter the supernatant through a 0.22μm microporous filter membrane, and determine the content of herbicide in the mycelium of the experimental strain according to the provisions of the agricultural industry standard NY / T2116-2012 of the People's Republic of China, and calculate the biomass according to the following formula:

[0054] Where: X is mycelium biomass (g / L); W is mycelium dry weight (g); V is sampling volume (mL). The test results are shown in Table 1.

[0055] Table 1 Cordyceps militaris strain deposit number CICC14015 acclimatization stability test

[0056]

[0057]

[0058] As can be seen from Table 1, the results of three domestication cultures show that on the optimized domestication culture medium, the colony morphology and mycelial growth of the Cordyceps militaris strain with the preservation number CICC14015 are better than those of the control group, and the cordycepin content in the mycelium is nearly 3 times higher than that of the control, indicating that the culture medium has a good domestication effect on the synthesis of cordycepin in the mycelium of the test strain and has good application stability.

[0059] Experimental Example 2

[0060] The liquid cultures obtained in Examples 1 to 4 and Comparative Example 1 were filtered through three layers of gauze, and the mycelia were washed with clean water three times, then the mycelia were collected and dried in an oven at 55°C to a constant weight. The content of mesoflavone in the mycelia was determined according to the method of Experimental Example 1, and the results are shown in Table 2.

[0061] Table 2 Cordycepin content determination results

[0062] Group Mycelium dry weight (g / L) Cordycepin concentration (mg / g) Example 1 23 0.812 Example 2 22 0.805 Example 3 25 0.849 Example 4 22 0.801 Comparative Example 1 21 0.235

[0063] As shown in Table 2, compared with Examples 1 to 4, the cordycepin concentration of Comparative Example 1 is significantly reduced, and the cordycepin content of Cordyceps militaris obtained by domestication in Examples 1 to 3 is 3 to 4 times that of Comparative Example 1. It can be seen that the domestication medium and domestication culture method provided by the present application have good domestication effect and effectively improve the cordycepin content. Compared with Examples 1 to 2, the dry weight of mycelium and the cordycepin concentration of Example 3 are both improved. The analysis reason may be that the domestication medium in Example 3 adds seaweed polysaccharide extract, which effectively improves the domestication effect; compared with Example 3, Example 4 changes the addition amount and ratio of seaweed polysaccharide extract to glycine, and its mycelium dry weight and cordycepin concentration are lower than those of Example 3. The analysis reason is that the addition amount and ratio of seaweed polysaccharide extract to glycine change, affecting their synergistic effect.

[0064] The above is only the embodiment of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the technical ideas and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for acclimating and cultivating Cordyceps militaris, characterized in that: The following steps are involved: (1) First acclimatization culture: Inoculate the Cordyceps militaris strain into the acclimatization medium, acclimatize and culture at 17-20°C in the dark for 7 days to obtain a fresh slant culture of the experimental strain; (2) Second acclimatization culture: Aseptically pick the bacterial blocks on the fresh slant culture medium onto the acclimatization plate culture medium and culture them at 17-20°C in the dark until the bacteria fill the plate; (3) The third acclimatization culture: Use a sterile puncher to punch the plate culture of the strain into 1 cm 2 The bacterial blocks were aseptically inoculated into liquid culture medium, 3 to 4 bacterial blocks were inoculated into each bottle, and cultured at 17 to 20°C, at a rotation speed of 140 to 160 r / min in the dark for 90 to 96 hours.

2. The acclimation and cultivation method according to claim 1, characterized in that: In step (1) and step (2), the acclimatization temperature is 18°C.

3. The acclimation and cultivation method according to claim 1, characterized in that: In step (3), the acclimation temperature is 18° C., the rotation speed is 140 r / min, and the light-proof culture time is 96 h.

4. The acclimation and cultivation method according to claim 1, characterized in that: The Cordyceps militaris strain is a Cordyceps militaris strain, and the preservation number is CICC14014, CICC14015 and / or CICC14013.

5. The acclimation and cultivation method according to claim 1, characterized in that: The liquid culture medium comprises the following components: 180-200 g of potato, 18-20 g of glucose, 8-11 g of peptone, 1.5-2.5 g of glycine, 2-3.5 g of KH2PO4, 1-1.5 g of MgSO4, 10-15 mg of vitamin B1, natural pH, and 1000 mL of water.

6. A Cordyceps militaris acclimation culture medium, characterized in that: The invention comprises the following components: 190-200 g of potato, 18-20 g of glucose, 8-10 g of peptone, 2-3 g of glycine, 3-4 g of KH2PO4, 1.5-2 g of MgSO4, 10-12 mg of vitamin B1, 12-18 g of agar and 1,000 mL of water.

7. The acclimation medium according to claim 6, characterized in that It also includes 0.5 to 2.5 g of seaweed polysaccharide extract.

8. The acclimation medium according to claim 7, characterized in that The invention comprises the following components: 200 g potato, 20 g glucose, 10 g peptone, 2 g glycine, 3.0 g KH2PO4, 1.5 g MgSO4, 10 mg vitamin B1, 15 g agar, 1 000 mL water and 1.3 g seaweed polysaccharide extract.

9. Use of the domestication and cultivation method according to any one of claims 1 to 5 in the domestication of Cordyceps militaris.

10. Use of the acclimation medium according to any one of claims 6 to 8 in acclimation of Cordyceps militaris.

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