CN114276934A - Preservation method of flammulina velutipes strains - Google Patents
Preservation method of flammulina velutipes strains Download PDFInfo
- Publication number
- CN114276934A CN114276934A CN202111579296.3A CN202111579296A CN114276934A CN 114276934 A CN114276934 A CN 114276934A CN 202111579296 A CN202111579296 A CN 202111579296A CN 114276934 A CN114276934 A CN 114276934A
- Authority
- CN
- China
- Prior art keywords
- flammulina velutipes
- strains
- percent
- freezing
- tube
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of strain preservation, and particularly relates to a method for preserving flammulina velutipes strains. The preservation method of the flammulina velutipes strains comprises the following steps: (1) preparing strains: perforating plate strains, scraping aerial hyphae on the surface of a plate, putting fungus blocks into a liquid culture medium for culturing to obtain needle mushroom hypha balls; (2) preparing strains of the frozen tube: sealing the flammulina velutipes mycelium pellets in a freezing tube with a protective agent, pre-freezing the freezing tube, and finally storing in a liquid nitrogen tank. The strain preserved by the method can be directly used for inoculating the liquid strain in the triangular flask, and has the advantages of good stability, high uniformity, simple operation, consistent strain character and long preservation period of 5-10 years.
Description
Technical Field
The invention belongs to the technical field of strain preservation, and particularly relates to a method for preserving flammulina velutipes strains.
Background
The golden mushroom has high economic and practical value, and the key for planting the golden mushroom is the strain with excellent characters. The current method for strain preservation comprises the following steps: conventional low-temperature subculture preservation, liquid nitrogen ultralow-temperature preservation, freeze-drying preservation, paraffin wax sealing preservation, host preservation, soil preservation, silica gel preservation, filter paper strip preservation, distilled water preservation and the like.
For example, in patent No. cn202010315370.x, the wood chip preservation method is adopted to preserve the strain of needle mushroom, and the culture medium is optimized to reduce the degradation of the strain of needle mushroom during long-term preservation, so that the strain of needle mushroom can maintain excellent characters and yield after long-term preservation.
Patent CN201710882239.X provides a preservation method of flammulina velutipes mycelium pellets, which has the principle that no nutrient substances are provided for the flammulina velutipes mycelium pellets in the preservation process, the preservation temperature of the flammulina velutipes mycelium pellets is lower, the growth of the flammulina velutipes mycelium pellets is greatly reduced or stopped in a low-temperature environment, and the physiological activity and the cell morphology of strains of the flammulina velutipes mycelium pellets are kept; the culture medium is cultured by adopting a liquid culture medium, and the culture medium is gently and orderly shaken, so that the propagation of the flammulina velutipes strains is more uniform, the concentrated propagation of the flammulina velutipes strains in a certain space is avoided, and the propagation efficiency and the propagation effect of the flammulina velutipes strains are improved.
At present, the strain is preserved in a refrigerator at 4 ℃ by domestic flammulina velutipes enterprises, and is preserved in a test tube inclined plane and subcultured at regular intervals. It has the following disadvantages: (1) the preservation time is short, and the degradation is easy; (2) the strain properties can not be maintained for a long time, so that the production is unstable; (3) the consistency of strains is poor; (4) the technician has a large workload of operating test tube passage; (5) frequent outsourcing or strain selection is too costly.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the preserved flammulina velutipes strain preservation method is provided, the preserved strain can be directly used for inoculation of liquid strains in triangular flasks, the stability is good, the uniformity is high, the operation is simple, the strain characters are consistent, and the preservation period is as long as 5 to 10 years.
The preservation method of the flammulina velutipes strains comprises the following steps:
(1) preparing strains: perforating plate strains, scraping aerial hyphae on the surface of a plate, putting fungus blocks into a liquid culture medium for culturing to obtain needle mushroom hypha balls;
(2) preparing strains of the frozen tube: sealing the flammulina velutipes mycelium pellets in a freezing tube with a protective agent, pre-freezing the freezing tube, and finally storing in a liquid nitrogen tank.
In the step (1), each 1L of the liquid culture medium contains: 1.8 to 2.0 percent of white granulated sugar, 0.30 to 0.35 percent of bean flour, 0.15 to 0.20 percent of peptone, 0.28 to 0.30 percent of corn flour, 0.18 to 0.20 percent of yeast extract powder, 0.06 to 0.10 percent of monopotassium phosphate, 0.06 to 0.10 percent of sulfuric acid and the balance of water.
Preferably, a proper amount of antifoaming agent is also added to the liquid culture medium to eliminate foaming of the liquid culture medium.
In the step (1), the culture temperature is 19-22 ℃, and the culture time is 6-9 days.
In the step (2), the cryopreservation tube is a high-temperature-resistant cryopreservation tube sold in the market, generally comprises a tube cap and a tube body and is divided into an internal rotation cover cryopreservation tube and an external rotation cover cryopreservation tube, wherein the internal rotation cryopreservation tube is provided with a silica gel pad.
Preferably, the volume of the cryopreservation tube is 2-10 ml. Before use, the frozen tube needs to be sterilized at a high temperature of 121 ℃ before use.
In the step (2), the protective agent is glycerol or dimethyl sulfoxide water solution, and the content of glycerol or dimethyl sulfoxide is 10-15 wt%.
Preferably, the mass ratio of the protective agent to the flammulina velutipes mycelium pellets is 1-1.2: 1.
In the step (2), when the freezing tube is pre-frozen, the freezing speed is controlled to be reduced by 1-1.5 ℃ per minute, so that the sample is frozen to-35 ℃, is waited for 1-2 hours, and is then placed in a liquid nitrogen tank for quick freezing.
In the step (2), the preservation temperature of the liquid nitrogen tank is-150 to-196 ℃.
When the preserved strain needs to be recovered, the freezing tube is taken out from the liquid nitrogen tank, immediately placed into a water bath at 38-40 ℃ for rapid thawing until the strain is completely thawed, and then the freezing tube is opened, and the content is transferred into a proper culture medium for culture.
Compared with the prior art, the invention has the following beneficial effects:
(1) the cryopreservation tube strain preserved by the method can be directly used for inoculating the liquid strain in the triangular flask, and has the advantages of good stability, high uniformity and simple operation;
(2) the preservation method of the invention has the advantages that the consistency of the frozen tubes in the same batch is high, the characters are stable, the variation is less, the frozen tubes are not easy to degenerate, the frozen tubes can be preserved for a long time of 5 to 10 years, and the strain seed using safety is ensured.
Drawings
FIG. 1 is a view of a preservation apparatus for Flammulina velutipes strains according to the present invention;
in the figure: 1. a liquid nitrogen tank; 2. and (5) freezing and storing the tube.
Detailed Description
The present invention is further illustrated by, but is not limited to, the following examples.
Example 1
The preservation method of the invention is adopted to preserve the flammulina velutipes strains, and the method comprises the following steps:
(1) preparing a liquid culture medium: each 1L of liquid culture medium contains: 1.8 percent of white granulated sugar, 0.35 percent of bean flour, 0.20 percent of peptone, 0.28 percent of corn flour, 0.18 percent of yeast extract powder, 0.08 percent of monopotassium phosphate, 0.08 percent of sulfuric acid and the balance of water; uniformly mixing the raw materials according to the formula, sterilizing at high temperature, and cooling for later use;
(2) preparing strains: uniformly punching selected flat strains by using a puncher, selecting the optimal point, scraping aerial hyphae on the surface of the flat by using an inoculating knife, and putting the fungus blocks into a liquid culture medium for culturing at the culture temperature of 20 ℃ for 7 days to obtain needle mushroom hypha balls;
(3) preparing strains of the frozen tube: selecting needle mushroom mycelium pellets with uniform size, sealing the needle mushroom mycelium pellets in a cryopreservation tube with a protective agent through aseptic operation, wherein the specification of the cryopreservation tube is 10ml, sterilizing at the high temperature of 121 ℃, using a glycerol aqueous solution with the volume ratio of 10% as the protective agent, and the mass ratio of the protective agent to the needle mushroom mycelium pellets is 1: 1; then the freezing tube is prefrozen at the rate of reducing 1 ℃ per minute to freeze the sample to-35 ℃ for 1 hour, then the sample is placed in a liquid nitrogen tank at-150 ℃ to-196 ℃ for quick freezing, and liquid strains in a triangular flask are taken at any time or stored for a long time.
(4) When the preserved strain needs to be recovered, the freezing tube is taken out from the liquid nitrogen tank, immediately placed into a water bath at 40 ℃ for rapid thawing until the strain is completely thawed, and then the freezing tube is opened, and the content is transferred into a proper culture medium for culture.
Example 2
The preservation method of the invention is adopted to preserve the flammulina velutipes strains, and the method comprises the following steps:
(1) preparing a liquid culture medium: each 1L of liquid culture medium contains: 2.0 percent of white granulated sugar, 0.30 percent of bean flour, 0.15 percent of peptone, 0.30 percent of corn flour, 0.20 percent of yeast extract powder, 0.06 percent of monopotassium phosphate, 0.06 percent of sulfuric acid and the balance of water; uniformly mixing the raw materials according to the formula, sterilizing at high temperature, and cooling for later use;
(2) preparing strains: uniformly punching selected flat strains by using a puncher, selecting the optimal point, scraping aerial hyphae on the surface of the flat by using an inoculating knife, and putting the fungus blocks into a liquid culture medium for culturing at the culture temperature of 19 ℃ for 9 days to obtain needle mushroom hypha balls;
(3) preparing strains of the frozen tube: selecting needle mushroom mycelium pellets with uniform size, sealing the needle mushroom mycelium pellets in a cryopreservation tube with a protective agent through aseptic operation, wherein the specification of the cryopreservation tube is 2ml, sterilizing at the high temperature of 121 ℃, using a glycerol aqueous solution with the volume ratio of 15% as the protective agent, and the mass ratio of the protective agent to the needle mushroom mycelium pellets is 1.2: 1; then the freezing tube is pre-frozen at the speed of reducing 1.5 ℃ per minute to freeze the sample to-35 ℃ for 2 hours, and then the sample is placed in a liquid nitrogen tank at-150 ℃ to-196 ℃ for quick freezing, and liquid strains in a triangular flask are taken at any time or stored for a long time.
(4) When the preserved strain needs to be recovered, the freezing tube is taken out from the liquid nitrogen tank, immediately placed into a water bath at 38 ℃ for rapid thawing until the strain is completely thawed, and then the freezing tube is opened, and the content is transferred into a proper culture medium for culture.
Example 3
The preservation method of the invention is adopted to preserve the flammulina velutipes strains, and the method comprises the following steps:
(1) preparing a liquid culture medium: each 1L of liquid culture medium contains: 1.9 percent of white granulated sugar, 0.32 percent of bean flour, 0.17 percent of peptone, 0.28 percent of corn flour, 0.20 percent of yeast extract powder, 0.10 percent of monopotassium phosphate, 0.06-0.10 percent of sulfuric acid and the balance of water; uniformly mixing the raw materials according to the formula, sterilizing at high temperature, and cooling for later use;
(2) preparing strains: uniformly punching selected flat strains by using a puncher, selecting the optimal point, scraping aerial hyphae on the surface of the flat by using an inoculating knife, and putting the fungus blocks into a liquid culture medium for culturing at the culture temperature of 22 ℃ for 6 days to obtain needle mushroom hypha balls;
(3) preparing strains of the frozen tube: selecting needle mushroom mycelium pellets with uniform size, sealing the needle mushroom mycelium pellets in a cryopreservation tube with a protective agent through aseptic operation, wherein the specification of the cryopreservation tube is 5ml, sterilizing at the high temperature of 121 ℃, and using a 10% dimethyl sulfoxide aqueous solution in volume ratio as the protective agent, wherein the mass ratio of the protective agent to the needle mushroom mycelium pellets is 1: 1; then the freezing tube is prefrozen at the rate of reducing 1 ℃ per minute to freeze the sample to-35 ℃ for 1 hour, then the sample is placed in a liquid nitrogen tank at-150 ℃ to-196 ℃ for quick freezing, and liquid strains in a triangular flask are taken at any time or stored for a long time.
(4) When the preserved strain needs to be recovered, the freezing tube is taken out from the liquid nitrogen tank, immediately placed into a water bath at 40 ℃ for rapid thawing until the strain is completely thawed, and then the freezing tube is opened, and the content is transferred into a proper culture medium for culture.
Comparative example 1
The conventional bevel preservation at 4 ℃ is adopted to preserve the flammulina velutipes strains, and the method comprises the following steps:
(1) preparing a liquid culture medium: each 1L of liquid culture medium contains: 1.9 percent of white granulated sugar, 0.32 percent of bean flour, 0.17 percent of peptone, 0.28 percent of corn flour, 0.20 percent of yeast extract powder, 0.10 percent of monopotassium phosphate, 0.06-0.10 percent of sulfuric acid and the balance of water; uniformly mixing the raw materials according to the formula, sterilizing at high temperature, and cooling for later use;
(2) preparing strains: selecting plate strains, uniformly punching holes by using a puncher, selecting an optimal point, scraping aerial hyphae on the surface of a plate by using an inoculating knife, inoculating a strain block into a culture container filled with the liquid culture medium, performing slant culture on the container at 22 ℃ by using a paraffin peak until the surface of a needle mushroom strain preservation culture medium in the culture container is full of hyphae;
(3)4 ℃ slant preservation: the culture vessel was kept at 4 ℃.
Comparative example 2
The conventional preservation method of distilled water at 4 ℃ is adopted to preserve the flammulina velutipes strains, and the method comprises the following steps:
(1) preparing a liquid culture medium: each 1L of liquid culture medium contains: 1.9 percent of white granulated sugar, 0.32 percent of bean flour, 0.17 percent of peptone, 0.28 percent of corn flour, 0.20 percent of yeast extract powder, 0.10 percent of monopotassium phosphate, 0.06-0.10 percent of sulfuric acid and the balance of water; uniformly mixing the raw materials according to the formula, sterilizing at high temperature, and cooling for later use;
(2) preparing strains: uniformly punching selected flat strains by using a puncher, selecting the optimal point, scraping aerial hyphae on the surface of the flat by using an inoculating knife, and putting the fungus blocks into a liquid culture medium for culturing at the culture temperature of 22 ℃ for 6 days to obtain needle mushroom hypha balls;
(3)4 ℃ distilled water preservation: selecting needle mushroom mycelium pellet with uniform size, aseptically inoculating needle mushroom mycelium pellet into test tube (sterilized at 121 deg.C) containing normal saline, and storing at 4 deg.C.
The needle mushroom strains preserved in examples 1 to 3 and comparative examples 1 to 2 were subjected to liquid culture fruiting experiments, and the results are shown in Table 1.
TABLE 1 liquid culture fruiting test results
As can be seen from Table 1, the hypha content and the yield per unit area preserved by the method for preserving flammulina velutipes strain of the present invention are better than the preservation effects of 4 ℃ slant preservation and 4 ℃ distilled water preservation.
Claims (9)
1. A preservation method of flammulina velutipes strains is characterized by comprising the following steps: the method comprises the following steps:
(1) preparing strains: perforating plate strains, scraping aerial hyphae on the surface of a plate, putting fungus blocks into a liquid culture medium for culturing to obtain needle mushroom hypha balls;
(2) preparing strains of the frozen tube: sealing the flammulina velutipes mycelium pellets in a freezing tube with a protective agent, pre-freezing the freezing tube, and finally storing in a liquid nitrogen tank.
2. The method for preserving flammulina velutipes strain according to claim 1, wherein: in the step (1), each 1L of the liquid culture medium contains: 1.8 to 2.0 percent of white granulated sugar, 0.30 to 0.35 percent of bean flour, 0.15 to 0.20 percent of peptone, 0.28 to 0.30 percent of corn flour, 0.18 to 0.20 percent of yeast extract powder, 0.06 to 0.10 percent of monopotassium phosphate, 0.06 to 0.10 percent of sulfuric acid and the balance of water.
3. The method for preserving flammulina velutipes strain according to claim 1, wherein: in the step (1), the culture temperature is 19-22 ℃, and the culture time is 6-9 days.
4. The method for preserving flammulina velutipes strain according to claim 1, wherein: in the step (2), the volume of the freezing storage tube is 2-10 ml.
5. The method for preserving flammulina velutipes strain according to claim 1, wherein: in the step (2), the protective agent is glycerol or dimethyl sulfoxide water solution, and the content of glycerol or dimethyl sulfoxide is 10-15 wt%.
6. The preservation method of enoki mushroom species according to claim 1 or 5, characterized in that: in the step (2), the mass ratio of the protective agent to the golden mushroom mycelium pellets is 1-1.2: 1.
7. The method for preserving flammulina velutipes strain according to claim 1, wherein: in the step (2), when the freezing storage tube is pre-frozen, the freezing speed is controlled to be reduced by 1-1.5 ℃ per minute, so that the sample is frozen to be below-35 ℃, is waited for 1-2 hours, and is then placed in a liquid nitrogen tank for quick freezing.
8. The method for preserving flammulina velutipes strain according to claim 1, wherein: in the step (2), the preservation temperature of the liquid nitrogen tank is-150 to-196 ℃.
9. The method for preserving flammulina velutipes strain according to claim 1, wherein: when the preserved strain needs to be recovered, the freezing tube is taken out from the liquid nitrogen tank, immediately placed into a water bath at 38-40 ℃ for rapid thawing until the strain is completely thawed, and then the freezing tube is opened, and the content is transferred into a proper culture medium for culture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111579296.3A CN114276934A (en) | 2021-12-22 | 2021-12-22 | Preservation method of flammulina velutipes strains |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111579296.3A CN114276934A (en) | 2021-12-22 | 2021-12-22 | Preservation method of flammulina velutipes strains |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114276934A true CN114276934A (en) | 2022-04-05 |
Family
ID=80874318
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111579296.3A Pending CN114276934A (en) | 2021-12-22 | 2021-12-22 | Preservation method of flammulina velutipes strains |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114276934A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1952108A (en) * | 2006-11-07 | 2007-04-25 | 华南农业大学 | Long term storage method for large-size fungus species |
| CN105950474A (en) * | 2016-05-11 | 2016-09-21 | 鲁东大学 | Tricholoma lobayense Heim strain low-temperature preservation and thawing method |
| CN107794224A (en) * | 2017-09-26 | 2018-03-13 | 广东香勤生物科技有限公司 | A kind of method for preserving of golden mushroom pompon |
| CN110741877A (en) * | 2019-10-29 | 2020-02-04 | 山东省农业科学院农业资源与环境研究所 | Method for ultralow-temperature cryopreservation and recovery of needle mushroom sterile fruiting bodies |
| CN111733108A (en) * | 2020-07-09 | 2020-10-02 | 江苏华绿生物科技股份有限公司 | Cooling method for liquid nitrogen preservation of needle mushroom strains |
| CN112961786A (en) * | 2021-02-26 | 2021-06-15 | 山东香育种业科技有限公司 | Freezing preservation method of edible fungi |
-
2021
- 2021-12-22 CN CN202111579296.3A patent/CN114276934A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1952108A (en) * | 2006-11-07 | 2007-04-25 | 华南农业大学 | Long term storage method for large-size fungus species |
| CN105950474A (en) * | 2016-05-11 | 2016-09-21 | 鲁东大学 | Tricholoma lobayense Heim strain low-temperature preservation and thawing method |
| CN107794224A (en) * | 2017-09-26 | 2018-03-13 | 广东香勤生物科技有限公司 | A kind of method for preserving of golden mushroom pompon |
| CN110741877A (en) * | 2019-10-29 | 2020-02-04 | 山东省农业科学院农业资源与环境研究所 | Method for ultralow-temperature cryopreservation and recovery of needle mushroom sterile fruiting bodies |
| CN111733108A (en) * | 2020-07-09 | 2020-10-02 | 江苏华绿生物科技股份有限公司 | Cooling method for liquid nitrogen preservation of needle mushroom strains |
| CN112961786A (en) * | 2021-02-26 | 2021-06-15 | 山东香育种业科技有限公司 | Freezing preservation method of edible fungi |
Non-Patent Citations (1)
| Title |
|---|
| 吕和平等: "食用菌菌株的长期保藏方法", 《中国食用菌》, vol. 26, no. 2, pages 29 - 30 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Carlyle et al. | Microbial thermogenesis in the decomposition of plant materials: Part II. Factors involved | |
| Latta | Preservation of suspension cultures of plant cells by freezing | |
| CN103782801A (en) | Agaricus Bisporus liquid spawn and preparation method thereof | |
| CN109082385B (en) | Preparation method of morchella liquid strain | |
| CN113215016B (en) | Bacillus amyloliquefaciens and application thereof | |
| CN112021068B (en) | Rejuvenation method of tremella aurantialba sporocarp strains | |
| CN110741877B (en) | Method for ultralow-temperature cryopreservation and recovery of needle mushroom sterile fruiting bodies | |
| CN115226571A (en) | Sparassis crispa liquid strain culture medium and culture method thereof | |
| CN110713934B (en) | Edible fungus production strain preservation method and activation method thereof | |
| CN117417840A (en) | Exotomycorrhizal fungi Yunnan sclerotium lappa ZSS01 and application thereof | |
| CN111235039A (en) | Culture medium for culturing metarhizium anisopliae with high toxicity, preparation and application thereof | |
| CN117652343B (en) | Mycorrhiza synthesis method of target plant | |
| CN103449914B (en) | Application of trehalose to prolonging storage life of pleurotus ostrcatus strain, and culture mediums and method for prolonging storage life of pleurotus ostrcatus strain | |
| CN113396934B (en) | Preparation method of bacillus preparation for promoting crop growth | |
| CN114276934A (en) | Preservation method of flammulina velutipes strains | |
| CN102533561B (en) | Protecting agent for freezing preservation of edible fungus strain and use method | |
| CN112680361A (en) | Trichoderma galnarum and application thereof | |
| CN107058110B (en) | Preservation and recovery method of haematococcus pluvialis cells | |
| Barnett et al. | Factors affecting the production of zygospores by Choanephora cucurbitarum | |
| CN103563651A (en) | Long preservation method of fertile production seed source of cordyceps militaris | |
| CN116114535A (en) | Use of retinol for promoting synthesis and/or growth of Pinus plant-Lactarius rupestris symbiotic system | |
| CN111184031A (en) | Liquid composite mycorrhizal inoculant for promoting growth of blueberry seedlings and preparation method thereof | |
| CN113583880A (en) | Culture medium suitable for preparing generalized cordyceps sinensis liquid fermentation seed liquid and preparation method and culture method thereof | |
| CN111394263A (en) | Special low-temperature freezing protective agent for cordyceps sinensis strain preservation and use method | |
| CN110713970A (en) | Continuous production method for suspension culture, preservation and recovery of BHK21 cells by using bioreactor and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |