CN115067152B - Culture medium and culture method for artificially culturing cordyceps sinensis fruiting bodies - Google Patents
Culture medium and culture method for artificially culturing cordyceps sinensis fruiting bodies Download PDFInfo
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- CN115067152B CN115067152B CN202210749999.4A CN202210749999A CN115067152B CN 115067152 B CN115067152 B CN 115067152B CN 202210749999 A CN202210749999 A CN 202210749999A CN 115067152 B CN115067152 B CN 115067152B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/20—Culture media, e.g. compost
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
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Abstract
The invention discloses a culture medium and a culture method for artificially culturing cordyceps sinensis fruiting bodies. The culture medium contains 150-300 g of potato, 10-30 g of tryptone, 20-40 g of maltose, 0.5-1.5 g of magnesium sulfate, 1.5-3 g of monopotassium phosphate, 20-30mg of vitamin B1, 3-10 g of sodium glutamate and 5-10 g of living insect grinding fluid, and the solvent is water. The culture medium and the culture method for artificially culturing the cordyceps sinensis fruiting body, provided by the invention, shorten the culture time of the cordyceps sinensis fruiting body, remarkably improve the yield of the cordyceps sinensis fruiting body, not only can be used for rapidly screening whether the cordyceps sinensis strain is degenerated and can not form a fruiting body primordium so as to avoid resource waste and economic risks caused by large-scale culturing of the cordyceps sinensis, but also can be used for large-scale culturing of the cordyceps sinensis fruiting body, and the formula of the culture medium is lower in price and convenient to operate compared with a rice-wheat culture medium, so that the artificial culture cost is reduced.
Description
Technical Field
The invention relates to the technical field of microorganism culture, in particular to a culture medium for artificially culturing cordyceps sinensis fruiting bodies and a culture method.
Background
Cordyceps sinensis is a stiff worm and fungus sub-seat complex formed by infecting Hepialidae larva by cordyceps sinensis Ophiocordyceps sinensis (Berk), has important medicinal and economic values, and is known as three treasures in China with ginseng and pilose antler. Because of special habitat (natural reproduction only in alpine regions), the growth speed is slow, the period is long, the natural resources are extremely limited, and related departments already list the cordyceps sinensis as a national secondary protection species.
In recent years, the demands for cordyceps sinensis at home and abroad are rapidly increased, and resources are exhausted. The artificial cultivation of Cordyceps not only can meet the national demands and market demands, but also can protect biological resources and ecological environment. Through more than 30 years of research, the artificial culture technology of the cordyceps sinensis is greatly developed, and the metabolic components of the cordyceps sinensis fruiting body obtained through artificial culture are not remarkably different from that of the wild cordyceps sinensis, which implies that the cordyceps sinensis fruiting body and the wild cordyceps sinensis have the same drug effect function (Tang et al, stage-and-return-Dependent Metabolomics Profiling of Ophiocordyceps sinensis and Its Pipeline products.instruments 2021, 12:666). Therefore, with the maturation of the technology for artificially culturing the fruiting body of the cordyceps sinensis and the deep understanding of people on the drug effect function of the cordyceps sinensis, the artificially cultured cordyceps sinensis becomes an ideal substitute for the wild cordyceps sinensis, and can well meet the national demand and simultaneously relieve the condition of the exhaustion of cordyceps sinensis resources. At present, the artificial culture of the cordyceps sinensis fruiting body mainly uses a rice-wheat culture medium (Tao Haiping, cao Li, korean domain, sugar and plant growth regulator to influence the artificial culture of the cordyceps sinensis fruiting body, environmental insect school, 2020,42 (2): 274-281.) and has long fruiting body culture time and high cost, and if the inoculated strain is degraded, the fruiting body or fruiting body yield is not obviously reduced, so that the industrialization of the artificial culture of the cordyceps sinensis and the diversification of cordyceps sinensis products are restricted.
Disclosure of Invention
The invention aims to provide a culture medium and a culture method for artificially culturing cordyceps sinensis fruiting bodies, which shorten the culture time of cordyceps sinensis fruiting bodies and remarkably improve the yield of cordyceps sinensis fruiting bodies.
The culture medium for artificially culturing cordyceps sinensis fruiting bodies comprises 150-300 g of potatoes, 10-30 g of tryptone, 20-40 g of maltose, 0.5-1.5 g of magnesium sulfate, 1.5-3 g of monopotassium phosphate, 20-30mg of vitamin B1, 3-10 g of sodium glutamate and 5-10 g of living insect grinding fluid per liter of culture medium, and the solvent is water.
Preferably, in the case of solid medium, 15-20g of agar powder is contained per liter of medium.
Preferably, the culture medium for artificially culturing cordyceps sinensis fruiting bodies contains 200g of potatoes, 10g of tryptone, 20-40 g of maltose, 0.5g of magnesium sulfate, 1.5g of potassium dihydrogen phosphate, 20mg of vitamin B1, 3-10 g of sodium glutamate and 5-10 g of living insect grinding fluid per liter of culture medium, and the solvent is water.
Preferably, the living insect polishing liquid is a wax moth polishing liquid.
The second object of the present invention is to provide a cultivation method for artificially cultivating cordyceps sinensis fruiting bodies, comprising the steps of:
A. and (3) preparing inoculated spore liquid: inoculating small blocks of Cordyceps sinensis fruiting body to culture medium for artificially culturing Cordyceps sinensis fruiting body, fermenting, and collecting spore liquid obtained by liquid fermentation as mother strain for fruiting body culture inoculation;
B. induction culture of fruiting bodies: inoculating spore liquid onto solid culture medium for artificially culturing Cordyceps fruiting body, culturing until colony is formed, and inducing fruiting body primordium differentiation at low temperature to grow until fruiting body is mature.
Preferably, the fermentation culture in the step A is 110rpm, 15+/-3 ℃ dark culture.
Preferably, the solid culture medium inoculated with spore liquid in the step B is cultured in a dark condition of a culture room with the temperature of 15+/-3 ℃ until colonies are formed, then transferred to a culture box with the temperature of 4+/-2 ℃ for dark culture, the formation of primordia of the fruiting body is stimulated, and after the primordia of the fruiting body is formed to be 1cm long, the solid culture medium is transferred to the culture room with the temperature of 15+/-3 ℃ for continuous culture in the dark condition until the fruiting body is mature.
The culture medium and the culture method for artificially culturing the cordyceps sinensis fruiting body, provided by the invention, shorten the culture time of the cordyceps sinensis fruiting body, remarkably improve the yield of the cordyceps sinensis fruiting body, not only can be used for rapidly screening whether the cordyceps sinensis strain is degenerated and can not form a fruiting body primordium so as to avoid resource waste and economic risks caused by large-scale culturing of the cordyceps sinensis, but also can be used for large-scale culturing of the cordyceps sinensis fruiting body, and the formula of the culture medium is lower in price and convenient to operate compared with a rice-wheat culture medium, so that the artificial culture cost is reduced.
Description of the drawings:
FIG. 1 shows a liquid fermentation culture of Cordyceps sinensis.
FIG. 2 shows liquid conidia and blastospore morphology in spore liquid obtained by liquid fermentation culture according to the invention, black arrows indicate blastospores, and white arrows indicate liquid conidia.
FIG. 3 shows the fruiting body of Cordyceps sinensis obtained by artificial culture in accordance with the present invention.
The specific embodiment is as follows:
the following examples are further illustrative of the invention and are not intended to be limiting thereof.
Solid and liquid media were prepared according to the media formulation (table 1), wherein 200g peeled potatoes were 200g potato filtrate, and the preparation method was: 200g of shredded potatoes are cooked by water and then filtered, and the filtrate is taken to be 200g of cooked potato filtrate. The Chilo suppressalis is obtained by grinding living Chilo suppressalis. The ingredients were mixed in water according to the formulation of Table 1, then the volume was set to 1L with water and 18g of agar powder was added to the solid medium. The liquid culture medium was dispensed into 250mL triangular flasks, each flask containing 120mL of liquid culture medium. Sterilizing the culture medium at 121deg.C for 25min, and pre-cooling the liquid culture medium in a culture chamber at 13+ -2deg.C. The solid culture medium is cooled to 60 ℃, poured into plates, each culture dish is poured into 25mL of culture medium, and the culture medium is packaged after the water content of the culture medium is dried, and is placed in a 13+/-2 ℃ culture room for precooling for standby.
Culturing and preparing inoculated spore liquid, cutting laboratory aseptic cultured Cordyceps fruiting body into small pieces of fruiting body of about 0.5cm with aseptic scissors on an ultra-clean workbench, inoculating 2-3 fruiting bodies to the liquid culture medium with aseptic forceps, and culturing in a culture flask at 110rpm shaking table and 13+ -2deg.C for 15d. Passing the culture solution (figure 1) through 3 layers of mirror cleaning paper, centrifuging the filtrate at a temperature of 4deg.C at 8000rpm for 15min to obtain spore solution, and counting spores with a blood cell counting plate under microscope to obtain spore concentration of 1×10 9 The ratio of the liquid conidium to the blastospore in the spore liquid is 100:1, and the liquid conidium and the blastospore are shown in figure 2.
Inoculating 10uL spore liquid at the center of the solid culture medium plate, sealing the plate with a sealing film, filling into a sterile bag, culturing under a dark condition of a climatic chamber at 15+ -3deg.C for 40 days until colony formation, transferring to a refrigerator at 4+ -2deg.C for dark culture, stimulating the formation of primordia of fruiting body, and culturing under a dark condition of a climatic chamber at 15+ -3deg.C for 5 months until primordia of fruiting body forms to 1cm long (FIG. 3). The harvested fruiting bodies were placed in an oven and dried at 80℃for about 4 hours to constant weight, and the fruiting bodies dry weight in each petri dish was weighed and the results are shown in Table 2.
Comparative example 1:
according to the comparison document (Tao Haiping, cao Li, korean domain. Influence of sugar and plant growth regulator on artificial culture of Cordyceps sinensis fruiting body. Environmental insect theory, 2020,42 (2): 274-281.) (adding rice 8g, wheat 8g, silkworm chrysalis powder 0.4g, and nutrient solution 22mL into 100mL culture flask, wherein the nutrient solution comprises glucose 20g, peptone 5g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g, ammonium citrate 1g, vitamin B120 mg, and 1L of 1-D glucose in 1L nutrient solution, and inoculating Cordyceps sinensis fruiting body (inoculating Cordyceps sinensis fungus solution of 12mL culture 30d per bottle culture flask), and containing conidium 3×10 per mL fungus solution) 9 4.5X10 of individual and bud spores 6 Culturing at 9-13 deg.c for 60d to cover the surface of the culture medium, inducing primordium differentiation at 4 deg.c and culturing until fruiting body ripens, and culturing in dark for 9 months to obtain Cordyceps fruiting body dry weight average value of 0.80+ -0.03 g/dish, which is significantly lower than that of the present invention in example 1 (Table 2).
Comparative example 2:
according to the comparison document (Tao Haiping, cao Li, korean domain. Influence of sugar and plant growth regulator on artificial culture of Cordyceps sinensis fruiting body, environmental insect theory report 2020,42 (2): 274-281.) the optimal culture medium of Cordyceps sinensis fruiting body (8 g of rice, 8g of wheat, 0.4g of silkworm chrysalis powder, 22mL of nutrient solution, 20g of glucose in every 1L of nutrient solution, 5g of peptone, 2g of potassium dihydrogen phosphate, 1g of magnesium sulfate, 1g of ammonium citrate, 120 mg of vitamin B) is added into a 100mL culture bottle, the Cordyceps sinensis fruiting body is cultured by the inoculation culture method according to the invention, namely, the inoculation spore liquid is cultured until the mycelium is covered with the culture medium under the dark condition of an artificial climate box with the temperature of 15+/-3 ℃, then the culture medium is transferred to a refrigerator with the temperature of 4+/-2 ℃, the fruiting body primordium is stimulated to form, after the fruiting body primordium is formed to be transferred to the artificial climate box with the temperature of 15+/-3 ℃ until the fruiting body is mature, the fruiting body is continued dark culture for 5 months, the dry weight average value of 1.04 dishes per 1.04 meter per 1 dry weight of fruiting body is obtained, and the inoculation culture method is remarkably higher than that according to the comparison example 1 (the invention is remarkably higher than that in the invention, 1 dry weight is obtained according to the invention).
Table 1 Medium composition in different examples (3 biological replicates per formulation, 15 dishes per replicate)
| Medium composition | Example 1 | Example 2 | Example 3 | Comparative example 3 | Comparative example 4 | Comparative example 5 | Comparative example 6 |
| Peeled potato | 200g | 200g | 200g | 200g | 200g | 200g | 200g |
| Glucose | 20g | 20g | |||||
| Maltose | 40g | 40g | 20g | 20g | 20g | ||
| Tryptone | 10g | 10g | 10g | 10g | 10g | 20g | 20g |
| Monopotassium phosphate | 1.5g | 1.5g | 1.5g | 1.5g | 1.5g | 1.5g | 1.5g |
| Magnesium sulfate | 0.5g | 0.5g | 0.5g | 0.5g | 0.5g | 0.5g | 0.5g |
| Chilo suppressalis (Chilo suppressalis) | 10g | 10g | 5g | 5g | 5g | ||
| Vitamin B 1 | 20mg | 20mg | 20mg | 20mg | 20mg | 20mg | 20mg |
| Sodium glutamate | 3g | 10g | 3g | ||||
| Water and its preparation method | 1L | 1L | 1L | 1L | 1L | 1L | 1L |
Table 2 different examples and comparative examples the dry weight (g/dish) of the fruiting body of Cordyceps sinensis was obtained
| Example 1 | Example 2 | Comparative example 1 | Comparative example 2 | Example 3 | Comparative example 3 | Comparative example 4 | Comparative example 5 | Comparative example 6 | |
| Dry weight of | 1.89±0.10a | 1.82±0.08a | 0.80±0.03b | 1.04±0.04b | 1.61±0.06a | 0.47±0.06c | 0.28±0.03c | 0.78±0.07b | 0.92±0.07b |
As can be seen from Table 1 and Table 1, example 1 gave a mean dry weight of fruiting bodies of 1.89.+ -. 0.10 g/dish, which is significantly higher than the dry weight of fruiting bodies of the comparative example (Table 2).
The sodium glutamate content of the medium formulation of example 2 was higher than that of example 1, and the remaining components and culture method were the same as in example 1. The spore liquid was inoculated and the dark culture was continued for 5 months after the formation of the primordium of the fruiting body, and the average dry weight of the fruiting body was 1.82.+ -. 0.08 g/dish, which was not significantly different from the dry weight of the fruiting body obtained in example 1, and was significantly higher than that of the fruiting body in comparative example (Table 2).
The content of maltose and the grinding fluid of the wax moth in the formula of the culture medium in the example 3 is 50% of that in the example 1, and the rest components and the culture method are the same as in the example 1. The spore liquid was inoculated and the dark culture was continued for 5 months after the formation of the primordium of the fruiting body, and the average dry weight of the fruiting body was 1.61.+ -. 0.06 g/dish, which was not significantly different from the dry weight of the fruiting body obtained in example 1, and was significantly higher than that of the fruiting body in comparative example (Table 2).
The content of maltose and wax moth grinding fluid in the culture medium formula in comparative example 3 is 50% of that in example 1, sodium glutamate is not added, and the rest components and the culture method are the same as in example 1. The spore liquid was inoculated and the dark culture was continued for 5 months after the formation of the primordium of the fruiting body, resulting in a fruiting body dry weight average of 0.47.+ -. 0.06 g/dish, significantly lower than that of example 1 and comparative example 1 (Table 2).
The maltose content in the medium formulation of comparative example 4 was 50% of that in example 1, without adding Chilo suppressalis polishing liquid and sodium glutamate, and the other components and the culture method were the same as in example 1. The spore liquid was inoculated and the dark culture was continued for 5 months after the formation of the primordium of the fruiting body, resulting in a fruiting body dry weight average of 0.28.+ -. 0.03 g/dish, significantly lower than that of example 1 and comparative example 1 (Table 2).
The sugar source in the culture medium formula in comparative example 5 is glucose, and the rest components and the culture method are the same as those in comparative example 4. The spore liquid was inoculated and the dark culture was continued for 5 months after the formation of the primordium of the fruiting body, resulting in a fruiting body dry weight average of 0.78.+ -. 0.07 g/dish, significantly lower than that obtained in example 1, no significant difference from comparative example 1, significantly higher than that of example 5 (Table 2).
The culture medium formula in the comparative example 6 is obtained by adding the grinding fluid of Chilo suppressalis on the basis of the culture medium formula in the comparative example 5. The spore liquid was inoculated and the dark culture was continued for 5 months after the formation of the primordium of the fruiting body, resulting in a fruiting body dry weight average of 0.92.+ -. 0.07 g/dish, significantly lower than that obtained in example 1, no significant difference from comparative example 1, significantly higher than that of examples 4 and 5 (Table 2).
Claims (5)
1. A culture method for artificially culturing cordyceps sinensis fruiting bodies, which is characterized by comprising the following steps:
A. and (3) preparing inoculated spore liquid: inoculating small blocks of Cordyceps sinensis fruiting body to culture medium for artificially culturing Cordyceps sinensis fruiting body, fermenting, and collecting spore liquid obtained by liquid fermentation as mother strain for fruiting body culture and inoculation;
B. induction culture of fruiting bodies: inoculating spore liquid onto solid culture medium for artificially culturing Cordyceps fruiting body, culturing until colony is formed, and then inducing fruiting body primordium differentiation by low temperature stimulation, and allowing fruiting body primordium to grow until fruiting body is mature;
the culture medium for artificially culturing cordyceps sinensis fruiting bodies comprises 150-300 g of potatoes, 10-30 g of tryptone, 20-40 g of maltose, 0.5-1.5 g of magnesium sulfate, 1.5-3 g of monopotassium phosphate, 20-30mg of vitamin B1, 3-10 g of sodium glutamate and 5-10 g of living insect grinding fluid per liter of culture medium, wherein the solvent is water;
the solid culture medium for artificially culturing the cordyceps sinensis fruiting body is a culture medium which is used for artificially culturing the cordyceps sinensis fruiting body and is added with 15-20g of agar powder per liter.
2. The method according to claim 1, wherein the culture medium for artificially culturing Cordyceps sinensis fruiting body contains 200g potato, 10g tryptone, 20-40 g maltose, 0.5g magnesium sulfate, 1.5g potassium dihydrogen phosphate, 20mg vitamin B1, 3-10 g sodium glutamate and 5-10 g live insect grinding fluid per liter of culture medium, and the solvent is water.
3. The method according to claim 1 or 2, wherein the living insect polishing liquid is a wax moth polishing liquid.
4. The method according to claim 1, wherein the fermentation culture in the step A is a dark culture at 110rpm, 15.+ -. 3 ℃.
5. The method according to claim 1, wherein the solid medium inoculated with the spore liquid in the step B is cultured in a dark condition in a culture room at 15+ -3deg.C until colony formation, then transferred to a 4+ -2deg.C incubator for dark culture, and the culture is continued until the fruiting body primordium is formed to a length of 1cm and transferred to a culture room at 15+ -3deg.C until the fruiting body is mature.
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| JP2676502B2 (en) * | 1995-08-03 | 1997-11-17 | 福島県 | Artificial cultivation method of fruiting body of Cordyceps sinensis |
| CN1362513A (en) * | 2001-01-04 | 2002-08-07 | 怀化市益康生物高科技有限公司 | Artificial cordyceps culturing medium |
| TWI460272B (en) * | 2012-05-23 | 2014-11-11 | Univ Nat Taiwan Ocean | Method for producing gamma-aminobutyric acid by fermentation with grain-based solid substrates used in producing cordyceps species and the application thereof |
| CN104082034B (en) * | 2014-06-25 | 2015-05-20 | 广东省昆虫研究所 | Ophiocordyceps sinensis sporocarp artificial cultivation method |
| CN108739050B (en) * | 2018-05-25 | 2020-12-01 | 广东省科学院动物研究所 | Cordyceps sinensis liquid culture medium and method for efficiently obtaining blastospores for Cordyceps sinensis host insect infection |
| CN112913572B (en) * | 2019-12-06 | 2022-08-02 | 陈衫润 | Method for culturing cordyceps militaris, culture product and pharmaceutical composition |
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| 不同蛹虫草杂粮培养基的生产性能及滋味物质分析;刘红等;食品工业科技;第第40卷卷(第第12期期);第77-84页 * |
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