CN115067152A - Culture medium and culture method for artificially culturing cordyceps sinensis sporocarp - Google Patents
Culture medium and culture method for artificially culturing cordyceps sinensis sporocarp Download PDFInfo
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- CN115067152A CN115067152A CN202210749999.4A CN202210749999A CN115067152A CN 115067152 A CN115067152 A CN 115067152A CN 202210749999 A CN202210749999 A CN 202210749999A CN 115067152 A CN115067152 A CN 115067152A
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a culture medium and a culture method for artificially culturing cordyceps sinensis sporocarp. The culture medium contains 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, and the solvent is water. The culture medium and the culture method for artificially culturing the cordyceps sinensis sporocarp provided by the invention shorten the culture time of the cordyceps sinensis sporocarp, remarkably improve the yield of the cordyceps sinensis sporocarp, can be used for quickly screening whether the cordyceps sinensis bacterial strain is degraded and cannot form a sporocarp primordium so as to avoid resource waste and economic risk caused by large-scale culture of cordyceps sinensis, and can also be used for culturing the cordyceps sinensis sporocarp in a large scale.
Description
Technical Field
The invention relates to the technical field of microbial culture, in particular to a culture medium and a culture method for artificially culturing cordyceps sinensis sporocarp.
Background
Cordyceps sinensis is a complex of stiff insects and fungal stroma formed by infecting hepialdae larvae with Cordyceps sinensis Ophiococcineps sinensis (Berk), has important medicinal and economic values, and is known as Chinese medicine with ginseng and pilose antler. Because of its special habitat (natural reproduction only in alpine regions), slow growth speed, long period and extremely limited natural resources, relevant departments have classified Cordyceps as a national second-level protective species.
In recent years, due to the rapid increase of the demand for cordyceps sinensis at home and abroad, resources are being exhausted. The artificial cultivation of the cordyceps sinensis can not only meet the national demand and market demand, but also protect biological resources and ecological environment. After more than 30 years of research, the artificial culture technology of Cordyceps sinensis has been greatly developed, and the metabolic components of the fruiting body of Cordyceps sinensis obtained by artificial culture and wild Cordyceps sinensis have no significant difference, suggesting that the fruiting body and wild Cordyceps sinensis have the same pharmacodynamic function (Tang et al, Stage-and reading-Dependent metabolism Profiling of ocular dynamics research and Its Pipeline products, instruments 2021,12: 666). Therefore, with the mature technology of artificially culturing the cordyceps sinensis sporocarp and the deep knowledge of the efficacy and function of the cordyceps sinensis, the artificially cultured cordyceps sinensis becomes an ideal substitute of wild cordyceps sinensis, and can well meet the national demand and relieve the exhaustion of cordyceps sinensis resources. At present, the cordyceps sinensis sporocarp is artificially cultured mainly by using a rice wheat culture medium (haohiping, Cao Li, Han Ri Domain. sugar and plant growth regulator influence the cordyceps sinensis sporocarp artificial culture. environmental insects' proceedings, 2020,42(2):274 plus 281.), the sporocarp culture time is long, the cost is higher, if the inoculated strain is degenerated, the sporocarp or the yield of the sporocarp cannot be formed, and the industrialization of the cordyceps sinensis artificial culture 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 sporocarp, which shorten the culture time of the cordyceps sinensis sporocarp and obviously improve the yield of the cordyceps sinensis sporocarp.
The culture medium for artificially culturing the cordyceps sinensis sporocarp 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 a solid medium, 15 to 20g of agar powder per liter of medium is contained.
Preferably, the culture medium for artificially culturing the cordyceps sinensis sporocarp contains 200g of potatoes, 10g of tryptone, 20-40 g of maltose, 0.5g of magnesium sulfate, 1.5g of monopotassium 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 grinding fluid is galleria mellonella grinding fluid.
The second purpose of the invention is to provide a method for artificially culturing cordyceps sinensis fruiting bodies, which comprises the following steps:
A. preparation of an inoculated spore liquid: inoculating small pieces of Cordyceps sinensis fruiting body to the culture medium for artificially culturing Cordyceps sinensis fruiting body, fermenting, and collecting spore liquid obtained by liquid fermentation as mother seed for inoculating and culturing fruiting body;
B. and (3) fruiting body induction culture: inoculating spore liquid to a solid culture medium for artificially culturing the cordyceps sinensis sporocarp, culturing until a colony is formed, then stimulating and inducing the primordium of the sporocarp to differentiate at low temperature, and growing the primordium of the sporocarp until the sporocarp is mature.
Preferably, the fermentation culture in the step A is 110rpm and dark culture at 15 +/-3 ℃.
Preferably, the solid culture medium inoculated with the spore liquid in the step B is cultured in a dark condition in a culture room at the temperature of 15 +/-3 ℃ until a colony is formed, then is transferred to an incubator at the temperature of 4 +/-2 ℃ for dark culture, and is used for stimulating the formation of the sporophore primordium, and after the sporophore primordium is formed to be 1cm long, the solid culture medium is transferred to the culture room at the temperature of 15 +/-3 ℃ for continuous culture in the dark condition until the sporophore is mature.
The culture medium and the culture method for artificially culturing the cordyceps sinensis sporocarp provided by the invention shorten the culture time of the cordyceps sinensis sporocarp, remarkably improve the yield of the cordyceps sinensis sporocarp, can be used for quickly screening whether the cordyceps sinensis bacterial strain is degraded and cannot form a sporocarp primordium so as to avoid resource waste and economic risk caused by large-scale culture of cordyceps sinensis, and can also be used for culturing the cordyceps sinensis sporocarp in a large scale.
Description of the drawings:
FIG. 1 shows a culture solution for culturing Cordyceps sinensis by liquid fermentation.
FIG. 2 shows the liquid conidiophores and blastospore morphologies in the spore liquid obtained by liquid fermentation culture according to the present invention, black arrows indicate blastospores, and white arrows indicate sporozoites.
FIG. 3 shows the fruiting body of Cordyceps sinensis obtained by artificial culture according to the present invention.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Solid and liquid media were prepared according to the media formula (table 1), wherein 200g of peeled potatoes was 200g of potato filtrate, and was prepared by: 200g of shredded potatoes are boiled by water and then filtered, and the filtrate is taken, namely 200g of boiled potato filtrate. The wax moth is obtained by grinding living wax moth. The components are mixed in water according to the formula of the table 1, then the water is used for fixing the volume to 1L, and 18g of agar powder is added into the solid culture medium. The liquid medium was dispensed into 250mL triangular flasks, each containing 120mL of liquid medium. The culture medium is sterilized by autoclaving at 121 deg.C for 25min, and the liquid culture medium is pre-cooled in 13 + -2 deg.C culture chamber. And (3) cooling the solid culture medium to 60 ℃, pouring 25mL of culture medium into each culture dish, drying the culture medium with moisture, bagging, and placing in a 13 +/-2 ℃ culture room for precooling for later use.
Culturing and preparing inoculated spore liquid, shearing Cordyceps fruiting body aseptically cultured in laboratory into small pieces with length of about 0.5cm on clean bench with sterile scissors, clamping 2-3 pieces of fruiting body with sterile forceps, inoculating to the liquid culture medium, and inoculatingThe culture bottle is placed on a shaker at 110rpm and cultured for 15d at the temperature of 13 +/-2 ℃. Passing the culture solution (figure 1) through 3 layers of mirror paper, centrifuging the filtrate at 8000rpm and 4 deg.C for 15min to obtain spore solution, and counting spores with a blood counting plate under microscope to obtain spore solution with a spore concentration of 1 × 10 9 The ratio of the sporophyte to the blastospore in the spore liquid is 100:1, and the shapes of the sporophyte and the blastospore are shown in figure 2.
Inoculating 10uL spore liquid into the center of the solid culture medium plate, sealing the plate with a sealing film, placing into a sterile bag, culturing in a 15 + -3 deg.C artificial climate box in the dark for 40 days until colony formation, transferring to a 4 + -2 deg.C refrigerator for dark culture, stimulating the formation of sporophore primordium, transferring to a 15 + -3 deg.C artificial climate box when the sporophore primordium is formed to 1cm length, and culturing in the dark for 5 months until the sporophore is mature (FIG. 3). The harvested fruiting bodies were placed in an oven, dried at 80 ℃ for about 4h to constant weight, and the dry weight of the fruiting bodies in each dish was weighed, the results are shown in table 2.
Comparative example 1:
according to a comparison document (Doochiping, Cao Li, Han Nippon domain. influence of sugar and plant growth regulator on the artificial culture of the cordyceps sinensis fruiting body. environmental insect report, 2020,42(2): 274) the optimal culture medium (8 g of rice, 8g of wheat, 0.4g of silkworm chrysalis powder and 22mL of nutrient solution are added into a 100mL culture bottle, the formula of the nutrient solution is that 20g of glucose, 5g of peptone, 2g of potassium dihydrogen phosphate, 1g of magnesium sulfate, 1g of ammonium citrate and 120 mg of vitamin B are added into 1L of nutrient solution, 12mL of cordyceps sinensis bacterial solution for culturing 30d is inoculated into each bottle of culture medium, and each milliliter of the bacterial solution contains 3 multiplied by 10 of conidia 9 Spore of seed and bud of 4.5 × 10 6 Culturing at 9-13 ℃ for 60 days until hyphae cover the surface of the culture medium, inducing primordium differentiation at 4 ℃ and culturing until the fruiting bodies are mature, and culturing for 9 months under a dark condition to obtain the cordyceps sinensis fruiting bodies with the average dry weight of 0.80 +/-0.03 g/dish, which is obviously lower than that of the cordyceps sinensis fruiting bodies in the embodiment 1 (table 2) of the invention.
Comparative example 2:
according to the contrast file (Taohiping, Cao Li, Han Nippon domain, sugar and plant growth regulator influence on the artificial culture of the cordyceps sinensis fruiting body. environmental insect report, 2020,42(2):274 plus 281.) the optimum culture medium (100mL culture bottle is added with 8g of rice, 8g of wheat, 0.4g of silkworm chrysalis powder and 22mL of nutrient solution, the formula of the nutrient solution is that 20g of glucose, 5g of peptone, 2g of potassium dihydrogen phosphate, 1g of magnesium sulfate, 1g of ammonium citrate and 120 mg of vitamin B in every 1L of nutrient solution is added to culture the cordyceps sinensis fruiting body by the inoculation culture method, namely, the inoculation spore solution is placed in an artificial climate box with the temperature of 15 plus or minus 3 ℃ for culture until the hypha is fully distributed with the culture medium, then is transferred to a refrigerator with the temperature of 4 plus or minus 2 ℃ for dark culture, the thorn fruiting body primordium is formed, and the fruiting body primordium is transferred to the artificial climate box with the temperature of 15 plus or minus 3 ℃ for continuous culture for 5 months until the fruiting body becomes finished The dry weight average of the fruiting bodies obtained by ripening was 1.04 + -0.04 g/dish, which is significantly lower than that of example 1 of the present invention, but the dry weight of the fruiting bodies obtained by the cultivation method according to the present invention was significantly higher than that of comparative example 1 (Table 2).
TABLE 1 composition of the culture media in the different examples (3 biological replicates per formulation, 15 dishes per replicate)
| Media Components | 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 |
| Potassium dihydrogen 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 |
| Greater wax moth | 10g | 10g | 5g | 5g | 5g | ||
| Vitamin B 1 | 20mg | 20mg | 20mg | 20mg | 20mg | 20mg | 20mg |
| Glutamic acid sodium salt | 3g | 10g | 3g | ||||
| Water (W) | 1L | 1L | 1L | 1L | 1L | 1L | 1L |
TABLE 2 different examples and comparative examples the dry weight (g/dish) of 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 tables, example 1 resulted in a mean dry weight of fruiting bodies of 1.89. + -. 0.10 g/dish, significantly higher than the dry weight of fruiting bodies in the comparative example (Table 2).
The sodium glutamate content of the medium formulation in example 2 is higher than that of example 1, and the rest of the components and the culture method are the same as those in example 1. Inoculating spore liquid, culturing in the dark until primordia of the sporocarp are formed, and then continuously culturing in the dark for 5 months to obtain the sporocarp dry weight average value of 1.82 +/-0.08 g/dish, wherein the dry weight of the sporocarp is not obviously different from that of the sporocarp obtained in the example 1 and is obviously higher than that of the sporocarp in a comparative example (Table 2).
The contents of maltose and galleria mellonella grinding fluid in the formula of the culture medium in the example 3 are both 50% of those in the example 1, and the rest components and the culture method are the same as the example 1. Inoculating spore liquid, culturing in dark until sporophore primordium is formed, and then continuously culturing in dark for 5 months to obtain sporophore dry weight average value of 1.61 +/-0.06 g/dish, wherein the sporophore dry weight average value is not significantly different from the sporophore dry weight obtained in example 1 and is significantly higher than the sporophore dry weight in a comparative example (Table 2).
The content of maltose and galleria mellonella grinding fluid in the formula of the culture medium in the comparative example 3 is 50% of that in the example 1, no sodium glutamate is added, and the rest components and the culture method are the same as the example 1. Inoculating spore liquid, culturing in dark until primordia of the sporocarp are formed, and then continuously culturing in dark for 5 months to obtain the sporocarp with a dry weight average value of 0.47 +/-0.06 g/dish, which is obviously lower than that of the sporocarp in example 1 and comparative example 1 (Table 2).
Comparative example 4 the maltose content in the medium formulation was 50% of that in example 1, no galleria mellonella grinding fluid and sodium glutamate were added, and the remaining components and culture method were the same as in example 1. Inoculating spore liquid, culturing in dark until primordia of the sporocarp is formed, and then continuously culturing in dark for 5 months to obtain the sporocarp with a dry weight average value of 0.28 +/-0.03 g/dish, which is obviously lower than that of the sporocarp in example 1 and comparative example 1 (Table 2).
The sugar source in the medium formulation of comparative example 5 was glucose, and the remaining components and the culture method were the same as in comparative example 4. Inoculating spore liquid, culturing in the dark until primordia of the sporocarp are formed, and then continuously culturing in the dark for 5 months to obtain the sporocarp dry weight average value of 0.78 +/-0.07 g/dish, which is obviously lower than the sporocarp dry weight obtained in the example 1, has no obvious difference with the comparative example 1, and is obviously higher than the sporocarp dry weight in the example 5 (Table 2).
The formula of the culture medium in the comparative example 6 is that the galleria mellonella grinding fluid is added on the basis of the formula of the culture medium in the comparative example 5. Inoculating spore liquid, culturing in dark until primordia of the sporocarp is formed, and then continuously culturing in dark for 5 months to obtain the sporocarp dry weight average value of 0.92 +/-0.07 g/dish, which is obviously lower than the sporocarp dry weight obtained in the example 1, has no obvious difference with the comparative example 1, and is obviously higher than the sporocarp dry weight in the examples 4 and 5 (Table 2).
Claims (7)
1. A culture medium for artificially culturing cordyceps sinensis sporocarp is characterized in that each liter of culture medium contains 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, and the solvent is water.
2. The culture medium of claim 1, wherein the culture medium for artificially culturing fruiting bodies of Cordyceps sinensis comprises, per liter of the culture medium, 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, and the solvent is water.
3. The culture medium according to claim 1 or 2, wherein the living insect polishing solution is galleria mellonella polishing solution.
4. The culture medium according to claim 1, wherein the culture medium comprises 15 to 20g of agar powder per liter of the culture medium when the culture medium is a solid medium.
5. A culture method for artificially culturing cordyceps sinensis sporocarp is characterized by comprising the following steps:
A. preparation of an inoculated spore liquid: inoculating small pieces of Cordyceps sinensis fruiting body to the culture medium of artificially cultured Cordyceps sinensis fruiting body of claim 1, 2 or 3, fermenting, and collecting spore liquid obtained by liquid fermentation as mother seed for inoculating and culturing fruiting body;
B. and (3) fruiting body induction culture: inoculating spore liquid to the solid culture medium of the artificially cultured Cordyceps sinensis fruiting body of claim 4, culturing until colony formation, and then stimulating to induce differentiation of fruiting body primordia at low temperature, so that the fruiting body primordia grow until the fruiting body is mature.
6. The culture method according to claim 5, wherein the fermentation culture in step A is 110rpm and 15. + -. 3 ℃ dark culture.
7. The culture method according to claim 5, wherein the solid culture medium inoculated with the spore liquid in step B is cultured in the dark in a culture room at a temperature of 15 ± 3 ℃ until the colony is formed, and then transferred to a 4 ± 2 ℃ incubator for dark culture to stimulate the formation of sporophore primordium, and after the sporophore primordium is formed to a length of 1cm, transferred to a culture room at a temperature of 15 ± 3 ℃ for continuous culture in the dark until the sporophore is mature.
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