CN105505778A - Ultralow temperature preservation method for pleurotus eryngii original mother strains - Google Patents
Ultralow temperature preservation method for pleurotus eryngii original mother strains Download PDFInfo
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- CN105505778A CN105505778A CN201610081333.0A CN201610081333A CN105505778A CN 105505778 A CN105505778 A CN 105505778A CN 201610081333 A CN201610081333 A CN 201610081333A CN 105505778 A CN105505778 A CN 105505778A
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Abstract
The invention discloses an ultralow temperature preservation method for pleurotus eryngii original mother strains, which comprises: A, PDA (potato dextrose agar) slant tubes are made, and water beads on tube walls and moisture are evaporated; B, original mother strains are transferred into PDA culture mediums and put into a thermostat at 22+/-1 DEG C for cultivating in darkness for 16-18 days, and the mediums are full of hyphae on the whole; C, glycerite is prepared from glycerinum, tween-80 and purified water at a volumetric proportion of (15%-30%):(0-5%):(80%-65%), separately loaded into high/low-temperature resistant plastic tubes to reach 1/2-4/5 of total volume, subjected to high-pressure sterilization and then cooled; D, the plurality of culture mediums with hyphae are transferred into the tubes, and the strain culture mediums are submerged in glycerite and sealed; E, the plastic tubes are precooled for 1-2 days at subzero 18+/-2 DEG C, and then transferred to a refrigerator at subzero 80+/-5 DEG C for long-term preservation. The ultralow temperature preservation method can be used for preserving pleurotus eryngii original mother strains for 8 years, and can ensure the vitality of the strains and the heredity stability; the possibility of variation is low and the cost is low.
Description
Technical field
The present invention relates to the method for preserving field that the original mother of Pleurotus eryngii plants, particularly the very low temperature method for preserving of the original mother's kind of a kind of Pleurotus eryngii.
Background technology
In order to keep the good character of edible fungus species constant, need to carry out preservation to its female kind.Liquid nitrogen cryogenics method for preserving, the regular transfer methods in inclined-plane etc. are mainly used in current edible fungus species preservation, liquid nitrogen cryogenics method for preserving, preservation cost very high, the preservation times such as the regular transfer methods in inclined-plane are short, bacterial classification stability is bad, easily morph or degeneration etc., it is 10-40% aqueous trehalose that the low temperature conservation method for Chinese (20061003531.7) of a kind of straw mushroom bacterial classification of the prior art adds massfraction in the test tube slant of straw mushroom strain inoculation, test tube slant can be made 1-4 DEG C of preservation 8 months, and the time is short.Method for preserving stropharia rugoso-annulata strain (200810017694.4) adopts mycelium vacuum freeze-drying method to improve the low temperature frost resistance of bacterial classification, then carries out preservation with freeze pipe, complicated operation.
Summary of the invention
The object of this invention is to provide the very low temperature method for preserving of the original mother's kind of Pleurotus eryngii that a kind of applicable batch production is used, energy long term storage, and the activity that original mother plants can be ensured.
The present invention proposes the very low temperature method for preserving that the original mother of a kind of Pleurotus eryngii plants, and comprises the following steps:
A, making PDA slant tube, through Sterility testing, disappear the globule on test tube wall and moisture evaporation;
B, original mother planted be transferred to above above-mentioned PDA substratum qualified after testing, in thermostat container 22 ± 1 DEG C, dark culturing 16-18 days, makes mycelia substantially cover with substratum;
C, by glycerine: tween-80: the volumetric ratio preparation glycerin of pure water=15%-30%:0-5%:80-65%, then is dispensed in the plastic test tube of high-and low-temperature resistance; Each plastic test tube dress glycerin is to the total volume of its 1/2-4/5, and cool after carrying out autoclaving, sterile preservation is for subsequent use;
D, in Bechtop, under aseptic condition, the substratum in PDA slant tube is cut into culture block, then will several culture block of band mycelia, transfer to inside aseptic plastic test tube, do not have bacterial classification block for standard with glycerin, cover lid, seals with sealed membrane;
E, by plastic test tube at-18 ± 2 DEG C after first precooling 1-2 days, then long term storage in the refrigerator transferring to-80 ± 5 DEG C.
Preferably, in described E step, plastic test tube should uprightly disperse to place, and prevents test tube from outwelling or extrudes.
In described E step, within being limited in year 8 years of long term storage.
In described step A, the PDA substratum in PDA slant tube is containing food grade salts 0.8%-1.4%.
The invention has the beneficial effects as follows: adding mass content in PDA substratum is 0.8%-1.4% salt, at vegetative stage, owing to containing the metal ions such as sodium, magnesium, calcium in salt, healthy and strong mycelia can be cultivated; Between very low temperature preservation term, mycelia can be made to absorb glycerine small molecules faster, better play a protective role; At mycelia Restoration stage because the flow of water of bacterial classification block is poor, the bacterial classification block mycelium germination surviving rate that easily absorbs water is high.Original mother will be planted and be put into thermostat container dark culturing 16-18 days, this is the key of cultivating the healthy and strong nourishing body of mycelia, is just beneficial to very low temperature refrigeration on this basis, because mycelia grows under light illumination, it slowly easily changes to reproduction direction, is unfavorable for cultivating healthy and strong vegetative mycelium; The effect of precooling is exactly lower the temperature stage by stage, reduces because sharply lowering the temperature to the destruction of Pleurotus eryngii hyphal cell, ensures the integrity of Pleurotus eryngii mycelia nourishing body to greatest extent.
Tween-80 is a kind of tensio-active agent, can freeze with dehydration in reduce the freezing denaturation of hyphal cell caused by ice-water interfacial tension, in reconstitution process, Surfactant component can play the effect of wetting agent and refolded fold agent again, be conventional non-ionic surfactant.Glycerine has the micromolecular compound protective material of excellent permeability; cell interior can be infiltrated into; hydrogen bond is formed with macromole in hydroxyl and born of the same parents; partly replace the hydrogen bond formed by water molecules; make the protein in organism, carbohydrate, fat and other macromolecular substance still can keep its original structure under water deficit conditions, maintain organism vigor.But the too high sample that easily causes of glycerol concentration is clamminess, not easily dry.The hydroxyl that glycerine has can connect with cell surface free radical, avoids thalline to expose in media as well, also can form hydrogen bond with protein and replace water, ensure protein stability.Glycerine has very strong retentiveness, cell moisture in drying process can be made to be unlikely to decline too fast, destroy cellular protein structures and make thalline impaired.But when glycerol concentration is increased to a certain degree, can reach the limit to protein stabilization ability, it is rotten that excessive concentrations can make protein in freezing dry process occur.Following change will occur: the concentration of the middle solute salt that dilutes the solution after adding glycerine time freezing, cellular uptake salt amount reduces, and is substituted by protective material; Protective material enters cell, changes supercooled state in born of the same parents, makes born of the same parents' internal pressure close to born of the same parents' external pressure, reduces cell dehydration shrinkage degree and speed; Protective material turnover cell is easy, the damage that when alleviating rehydration, perviousness swelling causes.Glycerin can avoid mycelium icing generation ice crystal damage mycelial cell, makes the original mother of Pleurotus eryngii plant the very low temperature of ability-80 DEG C, with the enzymic activity of very low temperature reduction Pleurotus eryngii mycelia, keeps its biological activity simultaneously, reach long period preservation object.
Present method can be planted original mother reach the very low temperature long term storage of 8 years, and survival rate is high, resurrection rate more than 90%, the kind of viable bacteria and function-stable, its genetic stability, secondary metabolite, existence are active all without changing; After refrigeration, bacterial classification is after twice tube activation, and the speed of growth, robustness, bright degree etc. of its bacterial classification are all consistent with incipient original strain; Bacterial classification can not be polluted and be degenerated, in the production process of original seed, cultivar, production bottle, and the test tube kind that very low temperature is preserved for a long time, its infection rate and the speed of growth are all planted consistent with original mother; Do not need tube, simple to operate; Bacterial classification inheritance stability is good, in mushroom type, the bacterial classification of long term storage is the same with the original female bacterium before preservation, yield and quality is indifference also, and the present invention can keep genotype and the morphotype of original female bacterium for a long time, for the constant product quality of suitability for industrialized production provides guarantee.
Embodiment
Embodiment one
The present embodiment proposes the very low temperature method for preserving that the original mother of a kind of Pleurotus eryngii plants, and comprises the following steps:
A, making PDA slant tube, through Sterility testing, be evaporated the globule on test tube wall and moisture;
B, original mother planted be transferred to above above-mentioned PDA substratum qualified after testing, in thermostat container 22 ± 1 DEG C, dark culturing 16 days, makes mycelia substantially cover with substratum;
C, by glycerine: tween-80: the volumetric ratio preparation glycerin of pure water=20%:5%:75%, then is dispensed in the plastic test tube of resistance to low high temperature; Each plastic test tube dress glycerin is to the total volume of its 1/2-4/5, and cool after carrying out autoclaving, sterile preservation is for subsequent use;
D, in Bechtop, under aseptic condition, the substratum in PDA slant tube is cut into the culture block of 3mm × 3mm, again by 3-6 the culture block of band mycelia, inside the plastic test tube transferring to aseptic 5ml, bacterial classification block was not had for standard with glycerin, cover lid, seals with sealed membrane;
E, by plastic test tube at-18 ± 2 DEG C after first precooling 1-2 days, then long term storage in the refrigerator transferring to-80 DEG C, plastic test tube should uprightly be placed, and prevents test tube from outwelling or extrudes, within being limited in year 8 years of long term storage.
In described step A, the PDA substratum in PDA slant tube is containing food grade salts 0.9%, and be massfraction here, namely the quality of salt accounts for 0.9% of PDA substratum total mass.
Embodiment two
The present embodiment proposes the very low temperature method for preserving that the original mother of a kind of Pleurotus eryngii plants, and comprises the following steps:
A, making PDA slant tube, through Sterility testing, be evaporated the globule on test tube wall and moisture;
B, original mother planted be transferred to above above-mentioned PDA substratum qualified after testing, in thermostat container 22 ± 1 DEG C, dark culturing 17 days, makes mycelia substantially cover with substratum;
C, by glycerine: tween-80: the volumetric ratio preparation glycerin of pure water=25%:4%:71%, then is dispensed in the plastic test tube of resistance to low high temperature; Each plastic test tube dress glycerin is to the total volume of its 1/2-4/5, and cool after carrying out autoclaving, sterile preservation is for subsequent use;
D, in Bechtop, under aseptic condition, the substratum in PDA slant tube is cut into the culture block of 3mm × 3mm, again by 3-6 the culture block of band mycelia, inside the plastic test tube transferring to aseptic 5ml, bacterial classification block was not had for standard with glycerin, cover lid, seals with sealed membrane;
E, by plastic test tube at-18 ± 2 DEG C after first precooling 1-2 days, then long term storage in the refrigerator transferring to-78 DEG C, plastic test tube should uprightly be placed, and prevents test tube from outwelling or extrudes, within being limited in year 8 years of long term storage.
In described step A, the PDA substratum in PDA slant tube is containing food grade salts 1.2%, and be massfraction here, namely the quality of salt accounts for 1.2% of PDA substratum total mass.
Embodiment three
The present embodiment proposes the very low temperature method for preserving that the original mother of a kind of Pleurotus eryngii plants, and comprises the following steps:
A, making PDA slant tube, through Sterility testing, be evaporated the globule on test tube wall and moisture;
B, original mother planted be transferred to above above-mentioned PDA substratum qualified after testing, in thermostat container 22 ± 1 DEG C, dark culturing 18 days, makes mycelia substantially cover with substratum;
C, by glycerine: tween-80: the volumetric ratio preparation glycerin of pure water=30%:2%:68%, then is dispensed in the plastic test tube of resistance to low high temperature; Each plastic test tube dress glycerin is to the total volume of its 1/2-4/5, and cool after carrying out autoclaving, sterile preservation is for subsequent use;
D, in Bechtop, under aseptic condition, the substratum in PDA slant tube is cut into the culture block of 3mm × 3mm, again by 3-6 the culture block of band mycelia, inside the plastic test tube transferring to aseptic 5ml, bacterial classification block was not had for standard with glycerin, cover lid, seals with sealed membrane;
E, by plastic test tube at-18 ± 2 DEG C after first precooling 1-2 days, then long term storage in the refrigerator transferring to 85 DEG C, plastic test tube should uprightly be placed, and prevents test tube from outwelling or extrudes, within being limited in year 8 years of long term storage.
In described step A, the PDA substratum in PDA slant tube is containing food grade salts 1.4%, and be massfraction here, namely the quality of salt accounts for 1.4% of PDA substratum total mass.
The foregoing is only the preferred embodiments of the present invention; not thereby the scope of the claims of the present invention is limited; every equivalent structure transformation utilizing description of the present invention to do, or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.
Claims (4)
1. a very low temperature method for preserving for the original mother's kind of Pleurotus eryngii, is characterized in that, comprise the following steps:
A, making PDA slant tube, through Sterility testing after carrying out, disappear the globule on test tube wall and moisture evaporation;
B, original mother planted be transferred to above above-mentioned PDA substratum qualified after testing, in thermostat container 22 ± 1 DEG C, dark culturing 16-18 days, makes mycelia substantially cover with substratum;
C, by glycerine: tween-80: the volumetric ratio preparation glycerin of pure water=15%-30%:0-5%:65-80%, then is dispensed in the plastic test tube of high-and low-temperature resistance; Each plastic test tube dress glycerin is to the total volume of its 1/2-4/5, and cool after carrying out autoclaving, sterile preservation is for subsequent use;
D, in Bechtop, under aseptic condition, the substratum in PDA slant tube is cut into culture block, then will several culture block of band mycelia, transfer to inside aseptic plastic test tube, do not have bacterial classification block for standard with glycerin, cover lid, seals with sealed membrane;
E, by plastic test tube at-18 ± 2 DEG C after first precooling 1-2 days, then long term storage in the refrigerator transferring to-80 ± 5 DEG C.
2. the very low temperature method for preserving of the original mother's kind of Pleurotus eryngii according to claim 1, is characterized in that, in described E step, plastic test tube should uprightly disperse to place, and prevents test tube from outwelling or extrudes.
3. the Pleurotus eryngii according to claim 1 original mother very low temperature method for preserving of planting, is characterized in that, in described E step, within being limited in year 8 years of long term storage.
4. the very low temperature method for preserving of the original mother's kind of Pleurotus eryngii according to claim 1, it is characterized in that, in described step A, the PDA substratum in PDA slant tube is containing food grade salts 0.8%-1.4%.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110713934A (en) * | 2019-11-20 | 2020-01-21 | 常熟理工学院 | Edible fungus production strain preservation method and activation method thereof |
| CN111742785A (en) * | 2020-07-07 | 2020-10-09 | 辽东学院 | Long-term preservation method of cordyceps militaris strain with high cordycepin yield |
| CN113973651A (en) * | 2021-09-26 | 2022-01-28 | 洛阳农林科学院 | Method for starting mushroom preservation mother seeds |
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| CN1733899A (en) * | 2005-08-12 | 2006-02-15 | 中国农业大学 | Edible mushroom liquid bacteria preservation method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110713934A (en) * | 2019-11-20 | 2020-01-21 | 常熟理工学院 | Edible fungus production strain preservation method and activation method thereof |
| CN110713934B (en) * | 2019-11-20 | 2021-10-19 | 常熟理工学院 | Edible fungus production strain preservation method and activation method thereof |
| CN111742785A (en) * | 2020-07-07 | 2020-10-09 | 辽东学院 | Long-term preservation method of cordyceps militaris strain with high cordycepin yield |
| CN111742785B (en) * | 2020-07-07 | 2022-05-06 | 辽东学院 | Long-term preservation method of cordyceps militaris strain with high cordycepin yield |
| CN113973651A (en) * | 2021-09-26 | 2022-01-28 | 洛阳农林科学院 | Method for starting mushroom preservation mother seeds |
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Effective date of registration: 20190717 Address after: 425000 Food and Drug Industrial Park, Yongzhou National Agricultural Science and Technology Park, Yongzhou City, Hunan Province Patentee after: Hunan Guoxiu Food Co., Ltd. Address before: 425000 National Agricultural Science and Technology Park, Yitang Town, Lengshuitan District, Yongzhou City, Hunan Province Patentee before: HUNAN YUXIU BIOLOGICAL TECHNICAL CO., LTD. |