Culture medium for artificially culturing cordyceps sinensis sporocarp as well as preparation method and application of culture medium

Abstract

The invention relates to the technical field of microbial cultivation, in particular to a culture medium for artificially cultivating cordyceps sinensis sporocarp and a preparation method and application of the culture medium. The culture medium comprises the following components in parts by mass: 40-50 parts of rice, 10-20 parts of cavings, 10-20 parts of bran, 8-16 parts of silkworm chrysalis meal, 5-10 parts of tenebrio molitor powder, 5-15 parts of a nutrient solution, 0.1-0.3 part of a preservative and 30-60 parts of water. The cordyceps sinensis sporocarp with the microbiological contamination rate lower than 6.7%, the sporocarp induction rate higher than 80%, the cordycepin content up to 0.8 mg / g and the cordyceps polysaccharide up to 25% or above can be cultured according to the culture medium. And a basis is provided for large-scale artificial cultivation of cordyceps sinensis.

Description

Technical Field

[0001] This invention relates to the field of microbial cultivation technology, and in particular to a culture medium for artificially cultivating Cordyceps sinensis fruiting bodies, its preparation method, and its application. Background Technology

[0002] Cordyceps sinensis (scientific name: *Ophiocordyceps sinensis*) is a fungus belonging to the family Nematodae and the genus *Ophiocordyceps*. It is a valuable medicinal fungus containing active ingredients such as cordycepin, adenosine, and cordyceps polysaccharides, and has extremely high application value in the pharmaceutical and health product fields. However, natural Cordyceps sinensis relies on specific alpine meadow ecosystems (3000–5000 meters above sea level) and host insects (hepialus moth larvae), with a growth cycle of 3–5 years. Due to over-harvesting and the impact on the ecological environment, wild Cordyceps sinensis resources are facing challenges and cannot meet market demand.

[0003] Artificial cultivation has become a key approach to solving the shortage of Cordyceps sinensis resources, and the design of the culture medium formulation is a crucial factor affecting cultivation efficiency. Current technologies for cultivating Cordyceps sinensis primarily use grains (rice, wheat), but these culture media suffer from nutritional imbalances, low levels of active ingredients, and high contamination rates.

[0004] Therefore, developing a nutritionally balanced, pollution-resistant culture medium that can meet the growth requirements of Cordyceps sinensis has become an urgent problem to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a culture medium with scientifically formulated nutrients, high cultivation efficiency, and strong resistance to pollution, enabling large-scale, high-quality cultivation of Cordyceps sinensis mycelium and fruiting bodies. In particular, it relates to a culture medium for the artificial cultivation of Cordyceps sinensis fruiting bodies, its preparation method, and its applications.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a culture medium for artificially cultivating Cordyceps sinensis fruiting bodies, comprising the following components in parts by weight:

[0008] Rice 40-50 parts, rice bran 10-20 parts, wheat bran 10-20 parts, silkworm pupa powder 8-16 parts, yellow mealworm powder 5-10 parts, nutrient solution 5-15 parts, preservative 0.1-0.3 parts, water 30-60 parts.

[0009] Preferably, the rice, rice bran, wheat bran, silkworm pupa powder, and mealworm powder have an individual particle size of 80-100 mesh.

[0010] Preferably, the nutrient solution is prepared from components in the following mass ratio:

[0011] Sucrose: Yeast extract: Methionine: Lysine: Sodium selenite: Zinc sulfate: Vitamin B1: Water = 3-5: 2-3: 0.3-0.5: 0.3-0.8: 0.01-0.03: 0.01-0.03: 0.005-0.01: 5-10;

[0012] The yeast extract is brewer's yeast extract.

[0013] Preferably, the preservative is potassium sorbate and / or tea polyphenols;

[0014] When the preservatives are potassium sorbate and tea polyphenols, the mass ratio of potassium sorbate to tea polyphenols is 1 to 3:1.

[0015] This invention also provides a method for preparing a culture medium for artificially cultivating Cordyceps sinensis fruiting bodies, comprising the following steps:

[0016] (1) Weigh each component according to the mass fractions of the culture medium described above;

[0017] (2) Mix rice, rice bran, wheat bran, silkworm pupa powder, yellow mealworm powder and water to obtain mixture 1;

[0018] (3) Mix the mixture 1 with nutrient solution and preservative, sterilize, and obtain culture medium.

[0019] Preferably, the mixing speed in step (2) is 150-200 rpm; the mixing time in step (2) is 30-40 min.

[0020] Preferably, the mixing speed in step (3) is 150-200 rpm, and the mixing time in step (3) is 1-2 hours.

[0021] Preferably, the sterilization temperature in step (3) is 115-125°C and the sterilization time is 1-2 hours.

[0022] The present invention also provides the application of the culture medium described herein or the culture medium prepared by the preparation method described herein in the artificial cultivation of high-quality Cordyceps sinensis fruiting bodies.

[0023] This invention also provides a method for artificially cultivating Cordyceps sinensis fruiting bodies, comprising the following steps:

[0024] 1) Inoculate the Cordyceps sinensis suspension into the culture medium and culture at 10-12℃ for 45-55 days to obtain Cordyceps sinensis mycelium;

[0025] 2) Cordyceps sinensis mycelium was cultured at 2-6℃ for 65-75 days to obtain Cordyceps sinensis fruiting body primordia;

[0026] 3) The fruiting body primordia of Cordyceps sinensis are cultured at 10-12℃ for 30-38 days to obtain the fruiting body of Cordyceps sinensis;

[0027] The culture medium is the culture medium described above or the culture medium prepared by the preparation method described above.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The culture medium of the present invention provides balanced nutrition and significantly increases biomass: rice, bran, and wheat bran in the culture medium provide carbohydrates, while silkworm pupa powder and mealworms provide protein; it also supplements essential amino acids (methionine and lysine), trace elements (selenium and zinc), and vitamins; thus meeting the nutritional needs of Cordyceps sinensis at different growth stages. The induction rate of Cordyceps sinensis fruiting bodies cultured according to the culture medium of the present invention can reach more than 80%.

[0030] (2) The addition of sodium selenite and zinc sulfate to the culture medium of this invention not only meets the trace element requirements of Cordyceps sinensis but also promotes the synthesis of cordycepin and cordyceps polysaccharide in the fruiting body. The Cordyceps sinensis fruiting bodies cultured using the culture medium of this invention have a cordycepin content ≥0.8mg / g and a cordyceps polysaccharide content ≥25%, which is significantly higher than that of Cordyceps sinensis fruiting bodies cultured using traditional rice culture medium.

[0031] (3) Low contamination rate: The culture medium of this invention contains potassium sorbate and / or tea polyphenols, which gives it the property of inhibiting the growth of penicillin, yeast and other miscellaneous bacteria. Tea polyphenols also have antioxidant function, protecting other components in the culture medium from oxidative damage. The contamination rate of Cordyceps sinensis fruiting bodies cultured using the culture medium of this invention is ≤6.7%, which is significantly lower than the contamination rate of traditional culture medium culture. Detailed Implementation

[0032] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] The Cordyceps sinensis suspension in this embodiment of the invention was prepared using Cordyceps sinensis purchased from Jinri Jianli (Zhangzhou) Biotechnology Co., Ltd.

[0034] The preparation method of the Cordyceps sinensis suspension of the present invention is as follows: Cordyceps sinensis is inoculated into PDA solid culture medium and cultured upside down at 10-12℃ for 50 days to obtain solid strain; the solid strain is inoculated into PDA liquid culture medium and cultured at 10-12℃ and 150rpm for 50 days, and mycelial balls of uniform size with a diameter of 2-3mm are selected as liquid strain; the liquid strain is diluted 5 times with sterile water to obtain Cordyceps sinensis suspension.

[0035] The brewer's yeast extract described in this embodiment of the invention was purchased from Xi'an An'ao Biotechnology Co., Ltd.

[0036] Example 1

[0037] Crush the rice, rice bran, and wheat bran separately through a 100-mesh sieve and set aside.

[0038] The oil in silkworm pupae is removed by pressing to obtain defatted silkworm pupae. The defatted silkworm pupae are dried at 50℃ until the moisture content is 6%, crushed, and passed through a 100-mesh sieve to obtain silkworm pupa powder for later use.

[0039] The oil in mealworms is removed by pressing to obtain defatted mealworms. The defatted mealworms are dried at 50°C until the moisture content is 6%, then pulverized and passed through a 100-mesh sieve to obtain mealworm powder for later use.

[0040] Dissolve 40g sucrose, 30g brewer's yeast extract, 3g methionine, 5g lysine, 0.3g sodium selenite, 0.2g zinc sulfate, and 0.05g vitamin B1 in 80g water and stir to obtain a nutrient solution.

[0041] A preservative is obtained by mixing 2g of potassium sorbate with 1g of tea polyphenols.

[0042] Add 40g of rice, 20g of rice bran, 15g of wheat bran, 12g of silkworm pupa powder, and 8g of yellow mealworm powder to 50g of water, and stir at 180rpm for 30min to obtain mixture 1.

[0043] Add 15g of nutrient solution and 0.1g of preservative to mixture 1, and stir at 180rpm for 1.5h to ensure all substances are evenly mixed to obtain the culture medium. Pour the culture medium into glass culture flasks (500mL), filling each flask to half its volume. Cap the flasks and sterilize at 121℃ and 0.12MPa for 1.5h. Cool to room temperature to obtain the culture medium.

[0044] In a sterile laminar flow hood, 200 μL of Cordyceps sinensis mycelium suspension was inoculated into the culture medium. After incubation at 10°C for 50 days, Cordyceps sinensis mycelium was obtained.

[0045] Adjust the temperature of the incubator to 2℃ and the humidity to 75%. Place the Cordyceps sinensis mycelium under these conditions and culture it. Control the light time to 12h / d. Induce the primordia of the fruiting bodies at low temperature. After 68 days of culture, the Cordyceps sinensis fruiting body primordia are obtained. Place the Cordyceps sinensis fruiting body primordia at 10℃ and culture for 36 days to obtain Cordyceps sinensis fruiting bodies with an average length of 6.3cm.

[0046] Example 2

[0047] Crush the rice, rice bran, and wheat bran separately through an 80-mesh sieve and set aside.

[0048] The oil in silkworm pupae is removed by pressing to obtain defatted silkworm pupae. The defatted silkworm pupae are dried at 50°C until the moisture content is 6%, then crushed and passed through an 80-mesh sieve to obtain silkworm pupa powder for later use.

[0049] The oil in mealworms is removed by pressing to obtain defatted mealworms. The defatted mealworms are dried at 50°C until the moisture content is 6%, then pulverized and passed through an 80-mesh sieve to obtain mealworm powder for later use.

[0050] Dissolve 30g sucrose, 35g brewer's yeast extract, 5g methionine, 8g lysine, 0.1g sodium selenite, 0.3g zinc sulfate, and 0.08g vitamin B1 in 50g water and stir to obtain a nutrient solution.

[0051] A preservative is obtained by mixing 2g of potassium sorbate with 1g of tea polyphenols.

[0052] Add 50g of rice, 10g of rice bran, 20g of wheat bran, 8g of silkworm pupa powder, and 10g of yellow mealworm powder to 60g of water, and stir at 180rpm for 30min to obtain mixture 1.

[0053] Add 5g of nutrient solution and 0.3g of preservative to mixture 1, and stir at 180rpm for 2 hours to ensure all substances are evenly mixed to obtain the culture medium. Pour the culture medium into glass culture flasks (500mL), filling each flask to half its volume. Cap the flasks and sterilize at 121℃ and 0.12MPa for 1.5 hours. Cool to room temperature to obtain the culture medium.

[0054] In a sterile laminar flow hood, 200 μL of Cordyceps sinensis mycelium suspension was inoculated into the culture medium. After incubation at 12°C for 49 days, Cordyceps sinensis mycelium was obtained.

[0055] Adjust the temperature of the incubator to 4℃ and the humidity to 75%. Place the Cordyceps sinensis mycelium under these conditions and culture it. Control the light time to 12h / d. Induce the primordia of the fruiting bodies at low temperature. After 70 days of culture, the primordia of the Cordyceps sinensis fruiting bodies are obtained. Place the primordia of the Cordyceps sinensis fruiting bodies at 12℃ and culture them for 32 days to obtain Cordyceps sinensis fruiting bodies with an average length of 6.8cm.

[0056] Example 3

[0057] Crush the rice, rice bran, and wheat bran separately through a 100-mesh sieve and set aside.

[0058] The oil in silkworm pupae is removed by pressing to obtain defatted silkworm pupae. The defatted silkworm pupae are dried at 50℃ until the moisture content is 6%, crushed, and passed through a 100-mesh sieve to obtain silkworm pupa powder for later use.

[0059] The oil in mealworms is removed by pressing to obtain defatted mealworms. The defatted mealworms are dried at 50°C until the moisture content is 6%, then pulverized and passed through a 100-mesh sieve to obtain mealworm powder for later use.

[0060] Dissolve 50g sucrose, 20g brewer's yeast extract, 4g methionine, 3g lysine, 0.2g sodium selenite, 0.1g zinc sulfate, and 0.1g vitamin B1 in 100g water and stir to obtain a nutrient solution.

[0061] A preservative is obtained by mixing 2g of potassium sorbate with 1g of tea polyphenols.

[0062] Add 45g of rice, 15g of rice bran, 10g of wheat bran, 16g of silkworm pupa powder, and 5g of yellow mealworm powder to 40g of water, and stir at 180rpm for 30min to obtain mixture 1.

[0063] Add 10g of nutrient solution and 0.1g of preservative to mixture 1, and stir at 180rpm for 2 hours to ensure all substances are evenly mixed to obtain the culture medium. Pour the culture medium into glass culture flasks (500mL), filling each flask to half its volume. Cap the flasks and sterilize at 121℃ and 0.12MPa for 1.5 hours. Cool to room temperature to obtain the culture medium.

[0064] In a sterile laminar flow hood, 200 μL of Cordyceps sinensis mycelium suspension was inoculated into the culture medium. After incubation at 11°C for 45 days, Cordyceps sinensis mycelium was obtained.

[0065] The temperature of the incubator was adjusted to 6℃ and the humidity to 75%. The Cordyceps sinensis mycelium was placed under these conditions for cultivation. The light time was controlled to be 12h / d. The fruiting body primordia were induced by low temperature and cultured for 75 days to obtain Cordyceps sinensis fruiting body primordia. The Cordyceps sinensis fruiting body primordia were placed at 11℃ and cultured for 31 days to obtain Cordyceps sinensis fruiting bodies with an average length of 6.5cm.

[0066] Comparative Example 1

[0067] Grind the rice into powder and pass it through a 100-mesh sieve. Set aside.

[0068] Add 100g of rice, 5g of sucrose, and 3g of brewer's yeast extract to 60g of water and stir at 180rpm for 2 hours to obtain the culture medium. Pour the culture medium into glass culture flasks (500mL), filling each flask to half its volume. Cap the flasks and sterilize at 121℃ and 0.12MPa for 1.5 hours. Cool to room temperature to obtain the culture medium.

[0069] In a sterile laminar flow hood, 200 μL of Cordyceps sinensis mycelium suspension was inoculated into the culture medium. After incubation at 10°C for 62 days, Cordyceps sinensis mycelium was obtained.

[0070] The temperature of the incubator was adjusted to 2℃ and the humidity to 75%. The Cordyceps sinensis mycelium was placed under these conditions for cultivation. The light time was controlled to be 12h / d. The fruiting body primordia were induced by low temperature and cultured for 86 days to obtain Cordyceps sinensis fruiting body primordia. The Cordyceps sinensis fruiting body primordia were placed at 10℃ and cultured for 47 days to obtain Cordyceps sinensis fruiting bodies with an average length of 5.8cm.

[0071] Experimental Example 1

[0072] Cordyceps mycelium was obtained on days 50, 49, and 45 of culture using the culture media described in Examples 1-3. Cordyceps fruiting bodies were obtained on days 154, 151, and 151 of culture.

[0073] Compared to the 62 days required to obtain Cordyceps sinensis mycelium using the traditional culture medium in Comparative Example 1, the time was 12–17 days earlier, and compared to the 62+86+47 days required to obtain Cordyceps sinensis fruiting bodies using the traditional culture medium, the time was 41–44 days earlier.

[0074] Furthermore, the average lengths of the Cordyceps sinensis fruiting bodies obtained using the culture media of Examples 1 to 3 of the present invention were 6.3 cm, 6.8 cm, and 6.5 cm, respectively, which were 0.5 to 1.0 cm longer than the average length of 5.8 cm of the fruiting bodies obtained in Comparative Example 1.

[0075] Experimental Example 2

[0076] The number of Cordyceps sinensis fruiting bodies obtained from the culture media of Examples 1-3 and Comparative Example 1 was observed, and the induction rate of Cordyceps sinensis fruiting bodies by different culture media was calculated. The results are shown in Table 1.

[0077] Cordyceps sinensis fruiting body induction rate = (number of culture bottles from which Cordyceps sinensis fruiting bodies were successfully induced / total number of culture bottles induced) × 100%.

[0078] Table 1. Induction of Cordyceps sinensis fruiting bodies by different culture media

[0079] Group Example 1 Example 2 Example 3 Comparative Example 1 Number of bottles containing child entities / bottle 12 13 13 6 Number of bottles co-cultured / bottle 15 15 15 15 Induction rate 80 86.7 86.7 40

[0080] Table 1 shows that the induction rate of Cordyceps sinensis fruiting bodies obtained by induction using the culture medium of Examples 1-3 of the present invention can reach more than 80%, which is significantly higher than the 40% of Comparative Example 1.

[0081] Experimental Example 3

[0082] The contamination status of the culture media used in Examples 1-3 and Comparative Example 1 during the cultivation of Cordyceps sinensis fruiting bodies was recorded, and the contamination rate was calculated. The results are shown in Table 2.

[0083] Contamination rate = (Number of bottles contaminated with miscellaneous bacteria / Number of bottles co-cultured) × 100%.

[0084] Table 2. Species contamination during the cultivation of Cordyceps sinensis fruiting bodies on different culture media.

[0085] Group Example 1 Example 2 Example 3 Comparative Example 1 Number of contaminated bottles / bottle 1 1 1 5 Number of bottles co-cultured / bottle 15 15 15 15 Contamination rate 6.7 6.7 6.7 33.3

[0086] Table 2 shows that when Cordyceps sinensis fruiting bodies were cultured using Examples 1-3, the contamination rate was much lower than that of the traditional comparative example 1 culture medium.

[0087] Experiment Example 4

[0088] Cordyceps sinensis fruiting bodies obtained from Examples 1-3 and Comparative Example 1 were collected. The contents of cordycepin and cordycepic acid in the fruiting bodies were determined by high performance liquid chromatography (HPLC), and the contents of polysaccharides were determined by the phenol-sulfuric acid method. The results are shown in Table 3.

[0089] Table 3. Nutrient composition of Cordyceps sinensis fruiting bodies cultured in different culture media

[0090] Element Example 1 Example 2 Example 3 Comparative Example 1 Cordycepin (calculated as adenosine) mg / g 0.86 0.92 0.83 0.42 Cordycepic acid (based on D-mannitol) % 9.68 9.87 9.56 4.16 Cordyceps polysaccharide % 26.42 26.85 25.94 16.74

[0091] As can be seen, the contents of cordycepin, cordycepic acid, and cordyceps polysaccharides in the Cordyceps sinensis fruiting bodies obtained in Examples 1-3 are much higher than those in Comparative Example 1. This indicates that the culture medium of the present invention can produce high-quality Cordyceps sinensis.

[0092] As demonstrated by the above embodiments, this invention provides a scientifically formulated culture medium suitable for the growth of Cordyceps sinensis. Using this culture medium, Cordyceps sinensis fruiting bodies with a contamination rate of less than 6.7%, a fruiting body induction rate of over 80%, a cordycepin content as high as 0.8 mg / g, and a cordyceps polysaccharide content of over 25% can be obtained. Furthermore, this culture medium can shorten the cultivation time of Cordyceps sinensis fruiting bodies by 41-44 days compared to traditional culture media. This provides a basis for the large-scale artificial cultivation of Cordyceps sinensis.

[0093] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A culture medium for artificially cultivating Cordyceps sinensis fruiting bodies, characterized in that, The components include the following parts by mass: Rice 40-50 parts, rice bran 10-20 parts, wheat bran 10-20 parts, silkworm pupa powder 8-16 parts, yellow mealworm powder 5-10 parts, nutrient solution 5-15 parts, preservative 0.1-0.3 parts, water 30-60 parts.

2. The culture medium according to claim 1, characterized in that, The rice, rice bran, wheat bran, silkworm pupa powder, and yellow mealworm powder each have a particle size of 80–100 mesh.

3. The culture medium according to claim 1, characterized in that, The nutrient solution is prepared from the following components in the following mass ratio: Sucrose: Yeast extract: Methionine: Lysine: Sodium selenite: Zinc sulfate: Vitamin B1: Water = 3-5: 2-3: 0.3-0.5: 0.3-0.8: 0.01-0.03: 0.01-0.03: 0.005-0.01: 5-10; The yeast extract is brewer's yeast extract.

4. The culture medium according to claim 1, characterized in that, The preservative is potassium sorbate and / or tea polyphenols; When the preservatives are potassium sorbate and tea polyphenols, the mass ratio of potassium sorbate to tea polyphenols is 1 to 3:

1.

5. A method for preparing a culture medium for artificially cultivating Cordyceps sinensis fruiting bodies, characterized in that, Includes the following steps: (1) Weigh each component according to the mass fractions of the culture medium according to any one of claims 1 to 4; (2) Mix rice, rice bran, wheat bran, silkworm pupa powder, yellow mealworm powder and water to obtain mixture 1; (3) Mix the mixture 1 with nutrient solution and preservative, sterilize, and obtain culture medium.

6. The preparation method according to claim 5, characterized in that, The mixing speed in step (2) is 150-200 rpm; the mixing time in step (2) is 30-40 min.

7. The preparation method according to claim 5, characterized in that, The mixing speed in step (3) is 150-200 rpm, and the mixing time in step (3) is 1-2 hours.

8. The preparation method according to claim 5, characterized in that, The sterilization temperature in step (3) is 115-125℃, and the sterilization time is 1-2 hours.

9. The application of the culture medium according to any one of claims 1 to 4 or the culture medium prepared by the preparation method according to any one of claims 5 to 8 in the artificial cultivation of high-quality Cordyceps sinensis fruiting bodies.

10. A method for artificially cultivating Cordyceps sinensis fruiting bodies, characterized in that, Includes the following steps: 1) Inoculate the Cordyceps sinensis suspension into the culture medium and culture at 10-12℃ for 45-55 days to obtain Cordyceps sinensis mycelium; 2) Cordyceps sinensis mycelium was cultured at 2-6℃ for 65-75 days to obtain Cordyceps sinensis fruiting body primordia; 3) The fruiting body primordia of Cordyceps sinensis are cultured at 10-12℃ for 30-38 days to obtain the fruiting body of Cordyceps sinensis; The culture medium is the culture medium according to any one of claims 1 to 4 or the culture medium prepared by the preparation method according to any one of claims 5 to 8.