CN110643518A - Method for culturing hirsutella sinensis - Google Patents
Method for culturing hirsutella sinensis Download PDFInfo
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Abstract
The invention relates to a hirsutella sinensis inoculation culture method, which comprises the following steps: and inoculating the hirsutella sinensis conidium suspension into a culture medium, and culturing in a dark room at a constant temperature of 16-20 ℃ for 7-10 days. The preparation of the hirsutella sinensis conidium suspension comprises the following steps: adding hirsutella sinensis conidium slant hypha into a Tween 80 solution, shaking uniformly, filtering and purifying by using medium-speed filter paper, and diluting a glucose solution to obtain a suspension for later use. The method of the invention omits the liquid strain culture process, directly skips the liquid strain culture process, and greatly shortens the culture period because the germination period of the conidia is more revived than the hyphae and the activity of the conidia is higher than the hyphae.
Description
Technical Field
The invention relates to the field of cordyceps sinensis, and in particular relates to a hirsutella sinensis culture method.
Background
Hirsutella sinensis, a material published in the isolation and identification of Cordyceps sinensis in the asexual stage, is Cordyceps sinensis in the anamorphic stage.
The method has the advantages of high inoculation efficiency, fast hypha growth and low pollution. However, the biggest defects of hirsutella sinensis are slow recovery, slow growth and long growth period of hyphae, and the characteristics are determined by hirsutella sinensis per se, and the long period and the low efficiency can be caused by adopting liquid strains for later culture and inoculation.
The university of Ludong filed an invention patent of a culture method of Cordyceps militaris (patent No. CN105733957A) for effectively replacing liquid strains with slant solid strains of Cordyceps militaris, wherein the patent mentions that the solid strains are directly used for inoculation instead of liquid strains, and the method is to mix the solid strains with glucose solution and then directly inoculate, so that the method can actually shorten the period. But the solid culture medium is adopted and is directly mixed with the glucose solution for inoculation, the slant strain and the glucose injection are required to be extruded out from a needle after being mixed, impurities are easily mixed in a mode of mixing for many times, the inoculated strain not only comprises conidia, but also comprises impurities or mixed bacteria, and the production efficiency is reduced; there is no description in the patent of reducing the degeneration of the properties of cordyceps militaris.
Disclosure of Invention
Technical problem to be solved
In order to solve the problems in the prior art, the invention provides a method for inoculating and culturing hirsutella sinensis, which shortens the culture period and improves the culture efficiency by a suspension culture method.
(II) technical scheme
In order to achieve the purpose, the invention adopts the main technical scheme that:
a hirsutella sinensis inoculation culture method comprises the following steps: inoculating the hirsutella sinensis conidium suspension into a culture medium, and carrying out indoor shading culture at a constant temperature of 16-20 ℃ for 7-10 days.
Further, the preparation of the hirsutella sinensis conidium suspension comprises the following steps: adding hirsutella sinensis conidium slant mycelium into Tween 80 solution, and shaking to make the number of conidia in Tween 80 solution be 106～5×107one/mL, after purification by filtration through medium speed filter paper, the glucose solution was diluted to give a suspension for use.
Further, the concentration of the tween 80 solution is 0.01-0.05%, and the volume of the tween 80 solution is 6-10 mL;
furthermore, the mass concentration of the glucose solution is 2% -5%, and the dilution times of the glucose solution are 5-10 times.
Further, the culture medium comprises the following components in parts by weight: 1.5-3.5% of rice, 0.3-0.7% of peptone, 1-3% of sucrose, and MgSO40.5 to 1.3 percent of potassium dihydrogen phosphate, 0.1 to 0.4 percent of vitamin B1 and the balance of sterile distilled water in percentage by weight.
Further, the culture medium comprises the following components in percentage by weight/volume: 15-25% of potato, 2-5% of glucose, 0.5-1.5% of yeast powder, 1.5-2.5% of milk powder, CaCl2·2H2O is 0.02-0.05%, MgSO4·7H20.03-0.07% of O and KH2PO40.05-0.15%, NaCl 0.07-0.15%, and the balance of sterile distilled water.
Further, the hirsutella sinensis conidium suspension and the culture medium are inoculated according to the volume percentage of 0.5-1%.
The principle of the invention is as follows:
the key technology of the invention is that conidium is adopted to replace hypha for inoculation, and the conidium suspension of hirsutella sinensis is used for carrying out the inoculation culture of the hirsutella sinensis; the key point is that conidia are collected from hirsutella sinensis strains and purified to prepare pure conidia suspension.
(III) advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
1. the method is feasible through experimental verification, and the fact that white hyphae can be seen on the 7 th day of inoculation and grow more uniformly is found, compared with the fact that the growth of star point hyphae is seen only in the 15 th day of the traditional culture method, the culture period is shorter.
2. The invention omits the liquid strain culture process, directly skips the liquid strain culture process, and greatly shortens the culture period because the germination period of the conidium is more revived than the hypha and the activity of the conidium is higher than the hypha.
Detailed Description
For a better understanding of the present invention, reference will now be made in detail to the present invention by way of specific embodiments thereof.
A hirsutella sinensis inoculation culture method comprises the following steps: and inoculating the hirsutella sinensis conidium suspension into a culture medium, and culturing in a dark room at a constant temperature of 16-20 ℃ for 7-10 days.
Further, the preparation of the hirsutella sinensis conidium suspension comprises the following steps: adding hirsutella sinensis conidium slant hypha into Tween 80 solution, and shaking to make the number of conidia in Tween 80 solution be 106～5×107The cells/mL are filtered and purified by medium-speed filter paper, and then the glucose solution is diluted to obtain a suspension for later use.
The medium-speed filter paper is adopted for filtering before the glucose solution is diluted so as to achieve the purpose of purification, and compared with the purification after the glucose solution is diluted, the purification effect is better. Experiments show that the direct purification effect is better after mycelium is dissolved by adopting the Tween 80 solution. The tween 80 solution may also be another emulsifier, such as calcium stearoyl lactylate, which is purified after dilution.
Further, the concentration of the Tween 80 solution is 0.01-0.05%, and the volume of the Tween 80 solution is 6-10 mL;
furthermore, the mass concentration of the glucose solution is 2% -5%, and the dilution times of the glucose solution are 5-10 times.
Further, the culture medium comprises the following components in parts by weight: 1.5-3.5% of rice, 0.3-0.7% of peptone, 1-3% of sucrose, and MgSO40.5 to 1.3 percent of potassium dihydrogen phosphate, 0.1 to 0.4 percent of vitamin B1 and the balance of sterile distilled water in percentage by weight.
Further, the culture medium comprises the following components in percentage by weight/volume: 15-25% of potato, 2-5% of glucose, 0.5-1.5% of yeast powder, 1.5-2.5% of milk powder, CaCl2·2H2O is 0.02-0.05%, MgSO4·7H20.03-0.07% of O and KH2PO4Is 0.05 &0.15%, NaCl 0.07-0.15%, and the balance of sterile distilled water. Wherein the potato is fresh potato.
Further, the hirsutella sinensis conidium suspension and the culture medium are inoculated according to the volume percentage of 0.5-1%.
Compared with the liquid strain culture adopted in the prior art, the liquid strain culture needs equipment and instruments, the liquid strain culture needs time, the growth period of hirsutella sinensis mycelia is very long, if the link of the liquid strain can be omitted, the cost can be reduced (the equipment cost of the culture instrument), the whole period can be shortened, and the production efficiency is improved. The invention has the advantages of saving the link of liquid strains, simplifying the culture steps, shortening the period, reducing the cost and improving the production efficiency on the premise of achieving the same culture effect. The invention adopts conidium to replace hypha for inoculation, because the germination period of the conidium is shorter than the total period of the hypha recovery and the growth, and the activity of the conidium is higher than that of the hypha, thereby achieving the effect of shortening the period.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
Example 1
A hirsutella sinensis inoculation culture method comprises the following steps:
s1 preparation of hirsutella sinensis conidia suspension
Adding the conidium slant hypha of hirsutella sinensis with good growth vigor into 6mL of Tween 80 solution with the mass concentration of 0.01%, and vibrating uniformly to ensure that the number of the conidia in the Tween 80 solution is 106The suspension is prepared by filtering and purifying the solution by using medium-speed filter paper, and diluting the solution by 5 times by using a glucose solution with the mass concentration of 2 percent.
Preparation of S2 rice culture medium
Mixing rice 15g, peptone 3g, sucrose 10g, and MgSO41.0g, 1g of monopotassium phosphate and 0.5g of vitamin B1, adding sterile distilled water, uniformly dissolving, adding sterile distilled water to a constant volume of 1000mL, and shaking uniformly to obtain the rice culture medium.
S3 inoculation: the conidium suspension is directly inoculated into the rice culture medium by using a pipette gun, and the inoculation amount is 5 mL.
S4 culture: placing the inoculated culture medium into a 16 ℃ constant temperature chamber for shading culture;
in this example, the growth of white hairy hyphae was observed on the surface of the medium at 7.5 days of culture, with a culture period of 30 days.
Example 2:
a hirsutella sinensis inoculation culture method comprises the following steps:
s1 preparation of hirsutella sinensis conidia suspension
Adding the conidium slant hypha of hirsutella sinensis with good growth vigor into 8mL of Tween 80 solution with the mass concentration of 0.03%, and shaking uniformly to ensure that the number of conidia in the Tween 80 solution is 5 multiplied by 106The cells/mL of the cells were purified by filtration through a medium-speed filter paper, and then diluted 7-fold with a 3% glucose solution to obtain a suspension for use.
Preparation of S2 rice culture medium
Mixing rice 30g, peptone 5g, sucrose 20g, and MgSO41g of rice culture medium, 2g of monopotassium phosphate and 1g of vitamin B1 are mixed, then sterile distilled water is added for uniform dissolution, water is added for constant volume to 1000mL, and the mixture is shaken uniformly to obtain the rice culture medium.
S3 inoculation: the conidium suspension is directly inoculated into the rice culture medium by using a pipette gun, and the inoculation amount is 7 mL.
S4 culture: placing the inoculated culture medium into a constant temperature chamber at 19 ℃ for shading culture;
in this example, white hairy hyphae growth was observed on the surface of the medium at the time of culture at 8 d. The culture period was 33 d.
Example 3
A hirsutella sinensis inoculation culture method comprises the following steps:
s1 preparation of hirsutella sinensis conidia suspension
Adding the conidium slant hypha of hirsutella sinensis with good growth vigor into 10mL of Tween 80 solution with the mass concentration of 0.05%, and vibrating uniformly to ensure that the number of conidia in the Tween 80 solution is 5 multiplied by 107The extract was filtered and purified by medium speed filter paper, and then purified by 5% by mass of glucoseThe glucose solution is diluted by 10 times to obtain a suspension for later use.
Preparation of S2 rice culture medium
Mixing rice 35g, peptone 7g, sucrose 30g, and MgSO41.0g, 4g of monopotassium phosphate and 1.0g of vitamin B1, adding sterile distilled water, uniformly dissolving, adding sterile distilled water to a constant volume of 1000mL, and shaking uniformly to obtain the rice culture medium.
S3 inoculation: a liquid transfer gun is used for sucking the conidium suspension liquid to be directly inoculated into the rice culture medium, and the inoculation amount is 10 mL.
S4 culture: placing the inoculated culture medium into a 20 ℃ constant temperature chamber for shading culture;
in this example, white hairy hyphae growth was observed on the surface of the medium at 8.5 days of culture. The culture period was 38 d.
Example 4
A method for inoculating hirsutella sinensis cultured by adopting a liquid culture medium comprises the following steps:
s1 preparation of hirsutella sinensis conidia suspension
Adding the conidium slant hypha of hirsutella sinensis with good growth vigor into 8mL of Tween 80 solution with the mass concentration of 0.01%, and shaking uniformly to ensure that the number of conidia in the Tween 80 solution is 3 multiplied by 106The suspension was diluted 7-fold with a 5% glucose solution after being purified by filtration through a medium-speed filter paper.
Preparing an S2 liquid culture medium: 200g of fresh potatoes, 30g of glucose, 10g of yeast powder and CaCl2·2H2O is 0.3g, MgSO4·7H2O is 0.5g, KH2PO41.0g of NaCl, 1.0g of NaCl and 20g of milk powder are mixed, then sterile distilled water is added for uniform dissolution, the sterile distilled water is added for constant volume till 1000mL is shaken up, and the constant volume is 1000 mL.
S3 inoculation: the conidium suspension is directly inoculated into the liquid culture medium by using a pipette gun, and the inoculation amount is 6 mL.
S4 culture: placing the inoculated culture medium into a constant-temperature shaking incubator at 18 ℃ for shading culture.
And (4) observing results: in this example, when the liquid medium inoculated with the conidium suspension was cultured at 9d, there was observed a sign of hyphal growth in the medium; the culture period was 39 d. And the growth sign of star point hypha can be seen only after the rice culture medium inoculated with the liquid strain is cultured for 15 days.
Example 5
A method for inoculating hirsutella sinensis cultured by adopting a liquid culture medium comprises the following steps:
s1 preparation of hirsutella sinensis conidia suspension
Adding the conidium slant hypha of hirsutella sinensis with good growth vigor into 10mL of Tween 80 solution with the mass concentration of 0.04%, and vibrating uniformly to ensure that the number of conidia in the Tween 80 solution is 1 multiplied by 107The cells/mL of the cells were purified by filtration through a medium-speed filter paper, and then diluted 10-fold with a 2% glucose solution to obtain a suspension for use.
Preparing an S2 liquid culture medium: 200g of fresh potatoes, 20g of glucose, 15g of yeast powder, 20g of milk powder and CaCl2·2H2O is 0.2g, MgSO4·7H2O is 0.7g, KH2PO41.1g and 0.7g of NaCl are mixed, then sterile distilled water is added for uniform dissolution, the sterile distilled water is added for constant volume till 1000mL is shaken up, and the constant volume till 1000mL is obtained, thus obtaining the liquid culture medium.
S3 inoculation: the conidium suspension is directly inoculated into the liquid culture medium by using a pipette gun, and the inoculation amount is 10 mL.
S4 culture: placing the inoculated culture medium into a constant-temperature shaking incubator at 17 ℃ for shading culture.
And (4) observing results: in this example, when the liquid medium inoculated with the conidium suspension was cultured at 9d, there was observed a sign of hyphal growth in the medium; the culture period was 40 days. And the growth sign of star point hypha can be seen only after the rice culture medium inoculated with the liquid strain is cultured for 15 days.
Example 6
A method for inoculating hirsutella sinensis cultured by adopting a liquid culture medium comprises the following steps:
s1 preparation of hirsutella sinensis conidia suspension
Adding the conidium slant hypha of hirsutella sinensis with good growth vigor into 6mL of Tween 80 solution with the mass concentration of 0.05%, and vibrating uniformly to ensure that the number of conidia in the Tween 80 solution is 5 multiplied by 107The suspension was diluted 5-fold with a 3% glucose solution after being purified by filtration through a medium-speed filter paper.
Preparing an S2 liquid culture medium: 200g of fresh potatoes, 30g of glucose, 6g of yeast powder, 25g of milk powder and CaCl2·2H2O is 0.3g, MgSO4·7H2O is 0.3g, KH2PO41.5g of NaCl and 1g of NaCl are mixed, then sterile distilled water is added for uniform dissolution, and the sterile distilled water is added for constant volume till 1000mL and is shaken up to obtain the liquid culture medium.
S3 inoculation: the conidium suspension is directly inoculated into the liquid culture medium by using a pipette gun, and the inoculation amount is 5 mL.
S4 culture: placing the inoculated culture medium into a constant-temperature shaking incubator at 18 ℃ for shading culture.
And (4) observing results: in this example, when the liquid medium inoculated with the conidium suspension was cultured at 8.5 days, the growth of hyphae was observed in the medium; the culture period was 35 d. And the growth sign of star point hypha can be seen only after the rice culture medium inoculated with the liquid strain is cultured for 15 days.
Example 7
A method for inoculating hirsutella sinensis cultured by adopting a liquid culture medium comprises the following steps:
s1 preparation of hirsutella sinensis conidia suspension
Adding the better-growing hirsutella sinensis conidia inclined plane hypha into 10mL of 0.05% Tween 80 solution by mass concentration, and vibrating uniformly to ensure that the number of conidia in the Tween 80 solution is 2 multiplied by 107The suspension was diluted 10-fold with a 5% glucose solution after being purified by filtration through a medium-speed filter paper.
Preparing an S2 liquid culture medium: 200g of fresh potatoes, 50g of glucose, 1g of yeast powder, 15g of milk powder and CaCl2·2H2O is 0.5g, MgSO4·7H2O is 0.5g, KH2PO40.5g of NaCl and 1.5g of NaCl are mixed, then sterile distilled water is added for uniform dissolution, and the sterile distilled water is added for constant volume to 1000mL and is shaken up to obtain the liquid culture medium.
S3 inoculation: the conidium suspension is directly inoculated into the liquid culture medium by using a pipette gun, and the inoculation amount is 10 mL.
S4 culture: placing the inoculated culture medium into a constant-temperature shaking incubator at 18 ℃ for shading culture.
And (4) observing results: in this example, when the liquid medium inoculated with the conidium suspension was cultured at 9.5 days, the growth of hyphae was observed in the medium; the culture period was 45 d. And the growth sign of star point hypha can be seen only after the rice culture medium inoculated with the liquid strain is cultured for 15 days.
Experimental data:
comparative example 1
The difference is that the liquid seed culture with the same number and concentration of conidia as the suspension is used in step S1 instead of the suspension, and the suspension is used in step S3 instead of the suspension in step S3, which is otherwise the same as that of example 2.
Comparative example 2
The difference is that the liquid seed culture with the same number and concentration of conidia as those in the suspension is used instead of the suspension in step S1, and the suspension in step S3 is replaced, and the method is otherwise the same as that in example 5.
Hirsutella sinensis obtained in examples 1 to 7 and comparative examples 1 and 2 is equal in appearance, and the growth stability of examples 1 to 7 is higher than that of comparative examples 1 and 2, and the survival rate during cultivation is not lower than that of comparative examples 1 and 2. The inoculation and cultivation method of the present invention has unexpected positive and beneficial effects.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (7)
1. A hirsutella sinensis inoculation culture method is characterized by comprising the following steps: and inoculating the hirsutella sinensis conidium suspension into a culture medium, and carrying out indoor shading culture at a constant temperature of 16-20 ℃ for 7-10 d to obtain hirsutella sinensis.
2. The method for inoculating and culturing hirsutella sinensis according to claim 1, wherein the preparation of the hirsutella sinensis conidia suspension comprises the following steps: adding hirsutella sinensis conidium slant mycelium into Tween 80 solution, and shaking to make the number of conidia in Tween 80 solution be 106～5×107one/mL, after purification by filtration through medium speed filter paper, was diluted with glucose solution to give a suspension.
3. The method for the inoculation culture of hirsutella sinensis according to claim 2, wherein: the mass concentration of the Tween 80 solution is 0.01-0.05%.
4. The method for the inoculation culture of hirsutella sinensis according to claim 2, wherein: the mass concentration of the glucose solution is 2% -5%, and the dilution times of the glucose solution are 5-10 times.
5. The method for the inoculation culture of hirsutella sinensis according to claim 1, wherein: the culture medium comprises the following components in percentage by weight: 1.5-3.5% of rice, 0.3-0.7% of peptone, 1-3% of sucrose, and MgSO40.5 to 1.3 percent of potassium dihydrogen phosphate, 0.1 to 0.4 percent of monopotassium phosphate and 0.05 to 1.3 percent of vitamin B1.
6. The method for the inoculation culture of hirsutella sinensis according to claim 1, wherein: the culture medium comprises the following components in percentage by weight/volume: 15-25% of potato, 2-5% of glucose, 0.5-1.5% of yeast powder, 1.5-2.5% of milk powder, CaCl2·2H2O is 0.02-0.05%, MgSO4·7H2O is 0.03 &0.07％、KH2PO40.05-0.15% and 0.07-0.15% NaCl.
7. The method for the inoculation culture of hirsutella sinensis according to claim 1, wherein: the hirsutella sinensis conidium suspension is inoculated into a culture medium according to the volume percentage of 0.5-1%.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10117770A (en) * | 1996-10-24 | 1998-05-12 | Morikawa Kenkoudou Kk | Culture of mycelium of cordyceps |
| US20070004022A1 (en) * | 2004-05-31 | 2007-01-04 | Nanying Shen | Industrial fermenting production process of Hirsutella hepiali Chen & Shen of anamorphic fungi related to Chinese Cordyceps Sinensis |
| WO2015180519A1 (en) * | 2014-05-29 | 2015-12-03 | 熊艳 | Method for cultivating high-cordyceps-polysaccharide cordyceps militaris |
| CN105733957A (en) * | 2016-03-01 | 2016-07-06 | 鲁东大学 | Method for culturing cordyceps militaris with slant solid strains effectively instead of liquid strains |
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2019
- 2019-10-22 CN CN201911003617.8A patent/CN110643518A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10117770A (en) * | 1996-10-24 | 1998-05-12 | Morikawa Kenkoudou Kk | Culture of mycelium of cordyceps |
| US20070004022A1 (en) * | 2004-05-31 | 2007-01-04 | Nanying Shen | Industrial fermenting production process of Hirsutella hepiali Chen & Shen of anamorphic fungi related to Chinese Cordyceps Sinensis |
| WO2015180519A1 (en) * | 2014-05-29 | 2015-12-03 | 熊艳 | Method for cultivating high-cordyceps-polysaccharide cordyceps militaris |
| CN105733957A (en) * | 2016-03-01 | 2016-07-06 | 鲁东大学 | Method for culturing cordyceps militaris with slant solid strains effectively instead of liquid strains |
Non-Patent Citations (2)
| Title |
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| 张奇等: "蛹虫草液体发酵培养中菌丝体、分生孢子及虫草素变化规律初探", 《北京农业》 * |
| 王忠等: "甘肃冬虫夏草菌分离培养研究", 《甘肃农业科技》 * |
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