CN111635864A - Liquid culture medium for culturing Cordyceps sinensis and its preparation method - Google Patents
Liquid culture medium for culturing Cordyceps sinensis and its preparation method Download PDFInfo
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Abstract
The invention relates to a liquid culture medium for culturing cordyceps sinensis. The culture medium contains carbon source, nitrogen source, and one or two of vitamin B complex and rapeseed oil. The invention also relates to a preparation method of the liquid culture medium and a method for culturing cordyceps sinensis by using the liquid culture medium. The culture medium of the invention is used for culturing the conidiospore of the cordyceps sinensis bacterial liquid and has the obvious advantages of short culture period, high concentration of the produced conidiospore, less possibility of polluting other microorganisms and the like.
Description
Technical Field
The invention belongs to the field of microbial culture, relates to a liquid culture medium for culturing cordyceps sinensis, and particularly relates to a liquid culture medium for promoting cordyceps sinensis to generate liquid conidia and a preparation method thereof.
Background
The Cordyceps sinensis (Ophiocerdyceps sinensis (Berk.) G.H.Sung, J.M.Sung, Hywel-Jones & Spataforda (Berk.) Sacc.) is a specific rare medicinal bacterium in Qinghai-Tibet plateau, and scientific research proves that the Cordyceps sinensis has wide pharmacological effects of immunoregulation, antibiosis, tumor resistance, antioxidation, anti-aging, blood sugar reduction, blood fat reduction and the like. Due to the unique medicinal value and good application prospect, and the limitation of distribution regions, wild resources are relatively limited, and artificial cultivation of the wild resources becomes a research hotspot and difficulty in recent decades.
As an entomogenous fungus, the cordyceps sinensis belongs to Ascomycota, and the life history of the cordyceps sinensis comprises two stages of sexual type and non-sexual type. Both morphological studies and molecular characterization confirm that Hirsutella sinensis (Hirsutella sinensis) is an anamorph of cordyceps sinensis. The sexual stage is mainly completed by the larval stage of dozens of insects mainly including Cordyceps sinensis obligate parasitic Hepialus (Thitarodes, Lepidoptera), wherein the larval insect body is equivalent to the culture medium for the growth of fungi.
During the liquid fermentation of fungi, there may be two forms of propagules produced, depending on the culture conditions: blastospores (blastospore) and sporozoites of liquid origin (subdivided conia). The shape and size of the liquid conidiophores are similar to those of the aerial conidiophores, the liquid conidiophores are usually produced by the spore production of the blastospores through microcirculation, the liquid conidiophores can also be directly produced by hyphae, and the embedded conidiophores are usually smaller than the aerial conidiophores on the solid culture medium. The culture and mass preparation of conidia are an important link in the artificial cultivation process of cordyceps sinensis. At present, the main difficulties in the research of cordyceps sinensis conidia are that the conidia have long production period, the conidia are low in yield, and the cordyceps sinensis conidia are easily polluted by other microorganisms in the culture process. Compared with other common fungi, the cordyceps sinensis has very low spore yield. How to obtain conidia with larger concentration by improving the culture medium and optimizing the culture conditions is one of the problems to be solved urgently for successfully realizing industrialization of the cordyceps sinensis.
In the prior art, the conventional components of the cordyceps sinensis liquid culture medium are a carbon source, a nitrogen source and inorganic salts, and the research on the cordyceps sinensis liquid culture medium at present mainly focuses on the optimization research of the cordyceps sinensis liquid fermentation culture medium (Liuxin, Zhang Zong Hao, Liyuling, and the like) on improving the mycelium yield of the cordyceps sinensis]Chinese grassland journal, 2010,32(s1):14-16.) the conidia of Cordyceps sinensis have been reported to be basically conidia cultured in solid state, the yield is low and the culture period is long, the conidia yield obtained by culturing is generally less than 1 × 105Per mL (Liyuling, research on molecular spores produced by Cordyceps sinensis fungus by different factors [ J)]Edible fungi, 2002,24(5): 8-8.). Therefore, there is an urgent need for a liquid culture medium for Cordyceps sinensis capable of obtaining conidia at a relatively high concentration.
Disclosure of Invention
The invention aims to overcome the limitation of the prior art and provide a liquid culture medium for promoting cordyceps sinensis to generate liquid conidium.
The first purpose of the invention is to provide a liquid culture medium for culturing cordyceps sinensis, which contains a carbon source, a nitrogen source and one or two of vitamin B complex and rapeseed oil. Preferably, the carbon source is selected from one or more of potato, corn grit and sucrose and the nitrogen source is selected from one or both of peptone and yeast extract. More preferably, the weight ratio of the carbon source to the nitrogen source of the culture medium is 100: 1-20, and preferably, the culture medium comprises 100-200 parts by weight of potatoes, 20-50 parts by weight of corn residues, 2.5-10 parts by weight of peptone, 1-10 parts by weight of yeast extract, 10-20 parts by weight of sucrose, and one or two of 0.1-1.5 parts by weight of vitamin B complex and 1-5 parts by weight of rapeseed oil.
Preferably, each liter of the culture medium contains 100-200 g of potatoes, 20-50 g of corn residues, 2.5-10 g of peptone, 1-10 g of yeast extract, 10-20 g of cane sugar and one or two of 0.1-1.5 g of vitamin B complex and 1-5 g of rapeseed oil. Further preferably, the culture medium contains 150g of potato, 30g of corn grit, 5g of peptone, 2g of yeast extract, and 20g of sucrose, and one or both of 0.25g to 0.5g of vitamin B complex and 1.5g to 3g of rapeseed oil per liter of the culture medium. The liquid medium further comprises the balance water.
The second object of the present invention is to provide a method for preparing a liquid medium for culturing cordyceps sinensis, the method comprising: adding carbon source, nitrogen source, and one or two of vitamin B complex and rapeseed oil into water, adding water, stirring, and dissolving completely. Preferably, the method comprises: (1) weighing corn dregs, and boiling in boiling water for 10-20 minutes; (2) adding potatoes into the product obtained in the step (1), boiling for 20-30 minutes, cooling and filtering to obtain filtrate; (3) weighing peptone, yeast extract, sucrose and one or two of vitamin B complex and rapeseed oil, adding into the filtrate obtained in step (2), adding water, and stirring to dissolve completely; (4) adding water into the product obtained in the step (3) to a constant volume, packaging and sterilizing.
The third purpose of the invention is to provide a culture method for culturing cordyceps sinensis by using the liquid culture medium so as to obtain high-concentration cordyceps sinensis bacterial liquid conidium, which comprises the following steps: inoculating the cordyceps sinensis strain to the liquid culture medium, and performing shake culture on a shaking table at the temperature of 14-18 ℃ and the rpm of 90-120 for 15-30 days to obtain the cordyceps sinensis strain for inoculation. Inoculating the cordyceps sinensis liquid strain into a new liquid culture medium according to the inoculation amount of 2-5% of the volume ratio, and performing shake culture on a shaker for 15-30 days at 14-18 ℃ and 90-120 rpm to obtain the cordyceps sinensis liquid strain.
The addition of the vitamin B complex and the colza oil can respectively promote the generation of the conidia of the cordyceps sinensis bacterial liquid, and the conidia production concentration can reach 106The addition of the two components can obtain the highest concentration of the liquid biogenetic sporozoites with the concentration of 2.5 × 106Control group without addition of either promoter had a yield of lyotropic sporozoites of less than 1 × 104one/mL, which cannot be directly counted with a hemacytometer.
The culture medium of the invention is used for culturing the conidiospore of the cordyceps sinensis bacterial liquid and has the obvious advantages of short culture period, high concentration of the produced conidiospore, less possibility of polluting other microorganisms and the like.
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Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a microscopic photograph (400X) of a conidia of Cordyceps sinensis cultured according to the method of example 3.
FIG. 2 is a scanning electron microscope (20 μm) of conidia of Cordyceps sinensis bacterial liquid cultured according to the method of example 3.
Detailed Description
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the following examples, glass flasks used for liquid culture of Cordyceps sinensis were all of the same size and had a volume of 1000 mL.
For a better understanding of the present invention, the present invention is illustrated below by the example of Cordyceps sinensis bacterial strain IOZ-07, which is intended to be illustrative and not limiting.
Example 1
In the present embodiment, each liter of the liquid culture medium of cordyceps sinensis is prepared by the following method: weighing 30g of corn grit, boiling in boiling water for 15 minutes, adding 150g of sliced peeled potatoes, boiling with soft fire for 25 minutes, cooling and filtering. Weighing 5g of peptone, 2g of yeast extract and 20g of sucrose, adding into the filtrate, adding water, stirring until the peptone, the yeast extract and the sucrose are fully dissolved, adding water to a constant volume of 1000mL, subpackaging into a triangular flask, sterilizing for 20 minutes by high-pressure steam at 121 ℃, and placing in a 15 ℃ culture chamber for precooling for later use.
Inoculating a cordyceps sinensis strain IOZ-07 stored in a laboratory into the liquid culture medium, performing shaking culture on a shaking table at 16 ℃ and 110rpm for 18 days to obtain an inoculated cordyceps sinensis strain, inoculating the cordyceps sinensis liquid strain into a new liquid culture medium according to an inoculation amount of 2.5% by volume ratio, performing shaking culture on the shaking table at 16 ℃ and 110rpm for about 30 days to obtain a strain, counting the fermentation liquor by using a blood counting plate, filtering the mycelium by using a filter membrane because a large amount of mycelium exists in the fermentation liquor and interferes counting, then counting conidia, taking a proper amount of the filtrate on the blood counting plate, observing and counting the number of the conidia under a microscope, concentrating the fermentation liquor by 100 times by using the blood counting plate, then counting, and counting the concentration of the liquid conidia by a centrifugal method, wherein the concentration of the liquid conidia is 3.5 × 103one/mL.
Example 2
In the present embodiment, each liter of the liquid culture medium of cordyceps sinensis is prepared by the following method: weighing 30g of corn grit, boiling in boiling water for 15 minutes, adding 150g of sliced peeled potatoes, boiling with soft fire for 25 minutes, cooling and filtering. Weighing 5g of peptone (purchased from Oxoid company, the same below), 2g of yeast extract (purchased from Oxoid company, the same below), 20g of sucrose, 0.5g of vitamin B complex (purchased from Tangchen Beijia company, the same below), grinding into powder in advance, fully dissolving in water, adding 3g of rapeseed oil (purchased from Luhua company, the same below) into the filtrate, adding water, stirring until fully dissolving, adding water to a constant volume of 1000mL, subpackaging into triangular flasks, sterilizing with high-pressure steam at 121 ℃ for 20 minutes, and placing in a 15 ℃ culture room for precooling for later use.
Inoculating the Cordyceps strain IOZ-07 stored in laboratory to the liquid culture medium, and shake culturing at 16 deg.C and 110rpm for 18 days to obtain inoculated Cordyceps strain. Inoculating Cordyceps liquid strain into new liquid according to the inoculation amount of 2.5 vol%In the culture medium, shaking culture is carried out on a shaking table at 16 ℃ and 110rpm for about 30 days to obtain the fermentation liquor, the fermentation liquor is counted by a blood counting plate, because the fermentation liquor contains a large amount of mycelia and has interference on counting, a filter membrane is used for filtering the mycelia, conidia are counted, a proper amount of the filtrate is taken on the blood counting plate, observation and counting are carried out under a microscope, the number of the conidia is counted, and the concentration of the conidia generated by the counting liquid is 2.5 × 106one/mL.
Example 3
In the present embodiment, each liter of the liquid culture medium of cordyceps sinensis is prepared by the following method: weighing 30g of corn grit, boiling in boiling water for 15 minutes, adding 150g of sliced peeled potatoes, boiling with soft fire for 25 minutes, cooling and filtering. Weighing 5g of peptone, 2g of yeast extract, 20g of sucrose and 3g of rapeseed oil, adding water, stirring until the peptone, the yeast extract and the rapeseed oil are fully dissolved, adding water to a constant volume of 1000mL, subpackaging the obtained mixture into a triangular flask, sterilizing the obtained product for 20 minutes by using high-pressure steam at 121 ℃, and placing the obtained product in a 15 ℃ culture chamber for precooling for later use.
Inoculating the Cordyceps strain IOZ-07 stored in laboratory to the liquid culture medium, shake culturing at 16 deg.C and 110rpm for 18 days to obtain inoculated Cordyceps strain, inoculating the Cordyceps strain to new liquid culture medium at 16 deg.C and 110rpm, shake culturing at 16 deg.C and 110rpm for 30 days, counting the fermentation liquid with blood counting plate, filtering the mycelium with filter membrane, counting conidia, taking appropriate amount of the filtrate on the blood counting plate, observing and counting under microscope, counting conidia, and determining the concentration of conidia to be 1.5 × 106one/mL. The microscope and scanning electron microscope images of cultured Cordyceps sinensis liquid conidium are shown in FIG. 1 and FIG. 2.
Example 4
In the present embodiment, each liter of the liquid culture medium of cordyceps sinensis is prepared by the following method: weighing 30g of corn grit, boiling in boiling water for 15 minutes, adding 150g of sliced peeled potatoes, boiling with soft fire for 25 minutes, cooling and filtering. Weighing 5g of peptone, 2g of yeast extract, 20g of sucrose and 0.5g of vitamin B complex (which are ground into powder in advance and fully dissolved in water), adding into the filtrate, adding water, stirring until the solution is fully dissolved, adding water to a constant volume of 1000mL, subpackaging into triangular flasks, sterilizing for 20 minutes by high-pressure steam at 121 ℃, and placing in a 15 ℃ culture chamber for precooling for later use.
Inoculating the Cordyceps strain IOZ-07 stored in laboratory to the liquid culture medium, shake culturing at 16 deg.C and 110rpm for 18 days to obtain inoculated Cordyceps strain, inoculating the Cordyceps strain to new liquid culture medium at 16 deg.C and 110rpm, shake culturing at 16 deg.C and 110rpm for 30 days, counting the fermentation liquid with blood counting plate, filtering the mycelium with filter membrane, counting conidia, taking appropriate amount of the filtrate on the blood counting plate, observing and counting under microscope, counting conidia, and measuring the conidia concentration with counting liquid to 2.0 × 106one/mL.
Example 5
In the present embodiment, each liter of the liquid culture medium of cordyceps sinensis is prepared by the following method: weighing 20g of corn grit, boiling in boiling water for 15 minutes, adding 100 g of cut peeled potatoes, boiling with soft fire for 25 minutes, cooling and filtering. Weighing 5g of peptone, 2g of yeast extract, 20g of sucrose and 0.5g of vitamin B complex (which are ground into powder in advance and fully dissolved in water), adding 3g of rapeseed oil into the filtrate, adding water, stirring until the solution is fully dissolved, adding water to a constant volume of 1000mL, subpackaging the obtained mixture into triangular flasks, sterilizing the obtained mixture for 20 minutes by using high-pressure steam at 121 ℃, and placing the obtained product in a 15 ℃ culture chamber for precooling for later use.
Inoculating the Cordyceps strain IOZ-07 stored in laboratory to the liquid culture medium, and shake culturing at 16 deg.C and 110rpm for 18 days to obtain inoculated Cordyceps strain. Inoculating Cordyceps liquid strain into new liquid culture medium according to the inoculation amount of 2.5% by volume, performing shake culture at 16 deg.C and 110rpm for about 30 days, and counting the fermentation liquid with blood counting plate. Due to the large amount of fermentation liquorThe mycelium has interference to counting, so the mycelium is filtered by a filter membrane, and then conidium counting is carried out, a proper amount of the filtrate is taken on a blood counting chamber, observation and counting are carried out under a microscope, the number of conidia is counted, and the concentration of the conidia generated by the counting solution is 2.4 × 105one/mL.
Example 6
In the present embodiment, each liter of the liquid culture medium of cordyceps sinensis is prepared by the following method: weighing 30g of corn grit, boiling in boiling water for 15 minutes, adding 150g of sliced peeled potatoes, boiling with soft fire for 25 minutes, cooling and filtering. Weighing 2.5 g of peptone, 1g of yeast extract, 20g of sucrose, 0.5g of vitamin B complex (which needs to be ground into powder in advance and fully dissolved in water), 3g of rapeseed oil, adding water into the filtrate, stirring until the vitamin B complex is fully dissolved, adding water to a constant volume of 1000mL, subpackaging into triangular flasks, sterilizing for 20 minutes by high-pressure steam at 121 ℃, and placing in a 15 ℃ culture chamber for precooling for later use.
Inoculating the Cordyceps strain IOZ-07 stored in laboratory to the liquid culture medium, shake culturing at 16 deg.C and 110rpm for 18 days to obtain inoculated Cordyceps strain, inoculating the Cordyceps strain to new liquid culture medium at 16 deg.C and 110rpm, shake culturing at 16 deg.C and 110rpm for 30 days, counting the fermentation liquid with blood counting plate, filtering the mycelium with filter membrane, counting conidia, taking appropriate amount of the filtrate on the blood counting plate, observing and counting under microscope, counting conidia, and measuring the conidia concentration with counting liquid to 2.8 × 105one/mL.
Example 7
In the present embodiment, each liter of the liquid culture medium of cordyceps sinensis is prepared by the following method: weighing 30g of corn grit, boiling in boiling water for 15 minutes, adding 150g of sliced peeled potatoes, boiling with soft fire for 25 minutes, cooling and filtering. Weighing 5g of peptone, 2g of yeast extract, 10g of sucrose, 0.5g of vitamin B complex (needing to be ground into powder in advance and fully dissolved in water), 3g of rapeseed oil, adding the mixture into the filtrate, adding water, stirring until the mixture is fully dissolved, adding water to a constant volume of 1000mL, subpackaging the mixture into triangular flasks, sterilizing the mixture for 20 minutes by using high-pressure steam at 121 ℃, and placing the mixture in a 15 ℃ culture chamber for precooling for later use.
Inoculating the Cordyceps strain IOZ-07 stored in laboratory to the liquid culture medium, shake culturing at 16 deg.C and 110rpm for 18 days to obtain inoculated Cordyceps strain, inoculating the Cordyceps strain to new liquid culture medium at 16 deg.C and 110rpm, shake culturing at 16 deg.C and 110rpm for 30 days, counting the fermentation liquid with blood counting plate, filtering the mycelium with filter membrane, counting conidia, taking appropriate amount of the filtrate on the blood counting plate, observing and counting under microscope, counting conidia, and determining the concentration of conidia to be 1.5 × 105one/mL.
Example 8
In the present embodiment, each liter of the liquid culture medium of cordyceps sinensis is prepared by the following method: weighing 30g of corn grit, boiling in boiling water for 15 minutes, adding 150g of sliced peeled potatoes, boiling with soft fire for 25 minutes, cooling and filtering. Weighing 5g of peptone, 2g of yeast extract, 10g of sucrose, one-half (0.25g) of vitamin B complex (needing to be ground into powder in advance and fully dissolved in water), 3g of rapeseed oil, adding water, stirring until fully dissolved, adding water to a constant volume of 1000mL, subpackaging into triangular flasks, sterilizing for 20 minutes by high-pressure steam at 121 ℃, and placing in a 15 ℃ culture chamber for precooling for later use.
Inoculating the Cordyceps strain IOZ-07 stored in laboratory to the liquid culture medium, and shake culturing at 16 deg.C and 110rpm for 18 days to obtain inoculated Cordyceps strain. Inoculating Cordyceps liquid strain into new liquid culture medium according to the inoculation amount of 2.5% by volume, performing shake culture at 16 deg.C and 110rpm for about 30 days, and counting the fermentation liquid with blood counting plate. Since the fermentation liquor contains a large amount of mycelia and interferes with counting, the conidia are counted after the mycelia are filtered by a filter membrane. Taking appropriate amount of the filtrateCounting the number of conidia by observing under a microscope on a blood counting plate, and counting the number of the conidia by using counting solution to obtain the conidia concentration of 2.2 × 106one/mL.
Example 9
In the present embodiment, each liter of the liquid culture medium of cordyceps sinensis is prepared by the following method: weighing 30g of corn grit, boiling in boiling water for 15 minutes, adding 150g of sliced peeled potatoes, boiling with soft fire for 25 minutes, cooling and filtering. Weighing 5g of peptone, 2g of yeast extract, 10g of sucrose, 0.5g of vitamin B complex (which needs to be ground into powder in advance and fully dissolved in water), 1.5g of rapeseed oil, adding water into the filtrate, stirring until the vitamin B complex is fully dissolved, adding water to a constant volume of 1000mL, subpackaging into triangular flasks, sterilizing for 20 minutes by high-pressure steam at 121 ℃, and placing in a 15 ℃ culture chamber for precooling for later use.
Inoculating the Cordyceps strain IOZ-07 stored in laboratory to the liquid culture medium, shake culturing at 16 deg.C and 110rpm for 18 days to obtain inoculated Cordyceps strain, inoculating the Cordyceps strain to new liquid culture medium at 16 deg.C and 110rpm, shake culturing at 16 deg.C and 110rpm for 30 days, counting the fermentation liquid with blood counting plate, filtering the mycelium with filter membrane, counting conidia, taking appropriate amount of the filtrate on the blood counting plate, observing and counting under microscope, counting conidia, and measuring the conidia concentration with counting liquid to 2.4 × 106one/mL.
Claims (10)
1. A liquid culture medium for culturing Cordyceps sinensis is characterized in that the culture medium contains a carbon source, a nitrogen source and one or two of vitamin B complex and rapeseed oil.
2. The liquid medium according to claim 1, wherein the carbon source is one or more selected from the group consisting of potato, corn grit and sucrose, and the nitrogen source is one or both selected from the group consisting of peptone and yeast extract.
3. The liquid medium of claim 2, wherein the weight ratio of the carbon source and the nitrogen source is 100: 1-20, and preferably the medium comprises 100-200 parts by weight of potato, 20-50 parts by weight of corn grit, 2.5-10 parts by weight of peptone, 1-10 parts by weight of yeast extract, and 10-20 parts by weight of sucrose, and one or both of 0.1-1.5 parts by weight of vitamin B complex and 1-5 parts by weight of rapeseed oil.
4. The liquid culture medium according to claim 3, wherein each liter of the liquid culture medium contains 100 to 200g of potato, 20 to 50g of corn grit, 2.5 to 10g of peptone, 1 to 10g of yeast extract, and 10 to 20g of sucrose, and one or both of 0.1g to 1.5g of B-complex vitamins and 1 to 5g of rapeseed oil.
5. The liquid culture medium according to any one of claims 1 to 4, characterized in that it contains 150g of potato, 30g of corn grit, 5g of peptone, 2g of yeast extract, and 20g of sucrose, and one or both of 0.25g to 0.5g of B-complex vitamin and 1.5g to 3g of rapeseed oil per liter of the culture medium.
6. The liquid culture medium according to any one of claims 1 to 5, characterized in that the liquid culture medium further comprises the balance water.
7. A method for producing a liquid culture medium according to any one of claims 1 to 6, characterized in that the method comprises: adding carbon source, nitrogen source, and one or two of vitamin B complex and rapeseed oil into water, adding water, stirring, and dissolving completely.
8. A method for producing the liquid medium according to claim 7, characterized in that the method comprises: (1) weighing corn dregs, and boiling in boiling water for 10-20 minutes; (2) adding potatoes into the product obtained in the step (1), boiling for 20-30 minutes, cooling and filtering to obtain filtrate; (3) weighing peptone, yeast extract, sucrose and one or two of vitamin B complex and rapeseed oil, adding into the filtrate obtained in step (2), adding water, and stirring to dissolve completely; (4) adding water into the product obtained in the step (3) to a constant volume, packaging and sterilizing.
9. Use of the liquid medium according to any one of claims 1 to 6 for culturing Cordyceps sinensis.
10. A method for culturing Cordyceps sinensis, comprising: inoculating a cordyceps sinensis strain to the liquid culture medium of any one of claims 1 to 6, and performing shake culture on a shaker at 14-18 ℃ and 90-120 rpm for 15-30 days to obtain a cordyceps sinensis liquid strain; then inoculating the cordyceps sinensis liquid strain into a new liquid culture medium according to the inoculation amount of 2-5% of the volume ratio, and performing shake culture on a shaker for 15-30 days at 14-18 ℃ and 90-120 rpm to obtain the cordyceps sinensis liquid strain.
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| CN113564097A (en) * | 2021-07-22 | 2021-10-29 | 重庆市中药研究院 | Method for producing conidia by liquid fermentation of cordyceps sinensis |
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