CN108359613A - Aweto antifreeze and the ultralow temperature preservation based on the antifreeze and method for resuscitation - Google Patents
Aweto antifreeze and the ultralow temperature preservation based on the antifreeze and method for resuscitation Download PDFInfo
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- CN108359613A CN108359613A CN201810458998.8A CN201810458998A CN108359613A CN 108359613 A CN108359613 A CN 108359613A CN 201810458998 A CN201810458998 A CN 201810458998A CN 108359613 A CN108359613 A CN 108359613A
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Abstract
The present invention relates to a kind of aweto antifreeze and the ultralow temperature preservation based on the antifreeze and method for resuscitation; the aweto antifreeze is raw material by using natural polymer, polyalcohol, surfactant, carbohydrate, pH buffer solutions, purified water and carries out suitable weight proportion; each ingredient plays each self-applying; protect aweto strain preservation under condition of ultralow temperature without damage; the preservation time is longer; genetic stability is more preferable; growth situation and no significant difference before preservation; survival rate is high, and each raw material is cheap and easy to get.Data are shown, aweto strain after preservation be averaged anabiosis rate be 95%, mycelia bacterium colony center is prominent and fold is apparent, metabolic capability is normal after bacterium cell recovery, 30d and 60d average colony sizes have respectively reached 16.3~17.2mm and 28.1~31.1mm, and culture medium melanin deposition generates normal.
Description
Technical field
The invention belongs to the technical fields that thermophilic cold fungi ultralow temperature preservation and low temperature are recovered, and in particular to a kind of cordyceps sinensis
Bacterium antifreeze and ultralow temperature method for preserving based on the antifreeze.
Background technology
Cordyceps sinensis also known as cordyceps sinensis belong to the dark dark worm of cordyceps sinensis Cordycepps of Ascomycota excrement shell Gammaproteobacteria meat seat bacterium subclass Hypocreales
Careless Pseudomonas, be refer in particular to infect the larva of Hepialidae insect by aweto and the larva body that is formed and fungi stroma it is compound
Body is always treated as in preciousness, Tibetan medicine material with a variety of pharmacological functions, the whole world is enjoyed great prestige, especially in for a long time
State, south east asia influence extensively, are a kind of important medicinal fungis, and are collectively referred to as " Chinese medicine three is big precious " with ginseng, pilose antler.
Since cordyceps sinensis has complete shape (also referred to as sexual generation) and Incomplete Shepe (also referred to as asexual generation) two completely
Different form, means fall behind in terms of strain idenfication before 10 years, and Molecular Identification is not carried out also, has very long one period mostly only
Accidentally Paecilomyces hepiali chen is cultivated as aweto strain with separation hypha form, has walked many detours.In cordyceps sinensis
In the determination problem of phorozoon, academia debates endlessly always.Currently, phorozoon strain related with cordyceps sinensis it has been reported that
22 scientific names are related to as many as 13 categories, and the multiple registered sequences for " cordyceps sinensis " retrieved from ncbi database
Difference between row is very notable.
Culture presevation is to allow strain to be in complete dormant state with physics, biological means, so as to for a long time
Keep strain vitality and original character, ensure strain safely, prevent from degenerating.Mainly have about big culture collection process at present
Atoleine preserving process, secondary culture preserving process, sand tube preservation method, ultralow temperature preserving process and liquid nitrogen cryogenics preserving process etc..It passes
Being commissioned to train, the foster preserving process preservation time is short, and cumbersome, heavy workload, strain stability is bad, with the increase of passage number, bacterium
Kind easily morphs or degenerates；Atoleine preserving process can be because paraffin evaporation be reduced, and slant culture is caused to expose, if
It adds not in time, spawn degeneration can be caused even dead；Husky, soil needs washing, removal of impurities, scrap iron removing in sand tube preservation method；Sand
The ratio of sand is not fixed in native pipe, is influenced by sand quality and strain properties, is generally required by depending on experiment, survival rate
It is low, aberration rate is high；The liquid nitrogen cryogenics preserving process preservation time is long, but maintains cost higher, and only a small number of scientific research institutions make
With.
Invention content
In order to solve the above problem of the existing technology, it is long suitable for aweto strain that the present invention provides one kind
The antifreeze of phase ultralow temperature preservation, further also provide a kind of method based on the antifreeze preservation aweto strain and
The method of low temperature recovery.
The technical solution adopted in the present invention is：
A kind of aweto antifreeze, raw material components include：
5~10 parts by weight of natural polymer, 10~20 parts by weight of polyalcohol, 1~5 parts by weight of surfactant,
5~10 parts by weight of carbohydrate, 5~10 parts by weight of pH buffer solutions, 45~74 parts by weight of purified water.
The raw material components of the further preferred aweto antifreeze include：
5~8 parts by weight of natural polymer, 10~15 parts by weight of polyalcohol, 2~3 parts by weight of surfactant, sugar
6~7 parts by weight of class, 5~8 parts by weight of pH buffer solutions, 59~72 parts by weight of purified water.
The natural polymer is in corn dextrin, gelatin sodium alginate, guar gum, chitosan and xanthans
One or more of mixtures；
The polyalcohol is one kind or several in ethylene glycol, propylene glycol, glycerine, butanediol, xylitol and pentaerythrite
The mixture of kind；
The surfactant is one kind in Tween-80, lauryl sodium sulfate, glycine betaine, polyvinylpyrrolidone
Or several mixture；
The carbohydrate is the mixture of one or more of glucose, lactose, fructose, sucrose, trehalose；
The pH buffer solutions are Na2HPO4/NaH2PO4、K2HPO4/KH2PO4、Na2HPO4/K2HPO4、Na2HPO4/KH2PO4、
NaH2PO4/K2HPO4、NaH2PO4/KH2PO4One or more of mixture.
A method of based on the antifreeze preservation aweto strain, include the following steps：
(1) PPDA slant tube culture mediums are made, after sterilized, the parent species of aweto are inoculated into the culture medium
On, 40~60d of light culture is carried out under the conditions of 15~18 DEG C, and the diameter of aweto bacterium colony is made to grow to 2~3cm；
(2) natural polymer, polyalcohol, surfactant, carbohydrate, pH buffer solutions and purified water are taken respectively, are filled
Divide and is uniformly mixed to get antifreeze；
The antifreeze is fitted into cryopreservation tube, the volume of antifreeze, which accounts for, freezes the 1/2~4/5 of pipe volume, after sterilized
It is cooling, for use；
(3) the aweto bacterium colony in step (1) PPDA slant tube culture mediums is cut into several mycelia blocks, by bacterium
Silk block is transferred in the cryopreservation tube containing antifreeze described in step (2), and mycelia block is completely submerged in the antifreeze；
(4) after 24~48h being pre-chilled at -15~-25 DEG C in the cryopreservation tube, then be quickly transferred to -75~-85 DEG C it is ultralow
Preservation under the conditions of temperature.
In step (1), the PPDA slant tubes culture medium at being grouped into：
Potato, 100~200 parts by weight；
Glucose, 10~20 parts by weight；
Peptone, 5~15 parts by weight；
MgSO4, 1~2 parts by weight；
KH2PO4, 1~2 parts by weight；
Agar powder, 18~25 parts by weight；
Purified water, 736~865 parts by weight.
The pH of the PPDA slant tubes culture medium is 6~6.5.
In step (1), the temperature for carrying out the sterilizing is 121 DEG C, and the time for carrying out the sterilizing is 30min；
The temperature for carrying out the light culture is 15~18 DEG C, and the time for carrying out the light culture is 40~60d.
In step (2), the temperature for carrying out the sterilizing is 121 DEG C, and the time for carrying out the sterilizing is 30min.
In step (3), the size of the mycelia block is 2mm × 2mm.
A method of low temperature recovery is carried out to the aweto strain after preservation, is as follows：
The cryopreservation tube of preservation under condition of ultralow temperature is taken out, and oscillation defrosting is carried out under the conditions of being immediately placed in 15~20 DEG C,
Until antifreeze melts completely, it is inoculated into PPDA slant tube culture mediums later and is cultivated.
Beneficial effects of the present invention are：
(1) aweto antifreeze provided by the invention, by using natural polymer, polyalcohol, surface
Activating agent, carbohydrate, pH buffer solutions, purified water are raw material and carry out suitable weight proportion, and each ingredient plays each self-applying, protection
Aweto strain preservation under condition of ultralow temperature is without damage, and the preservation time is longer, and genetic stability is more preferable, growth shape
Condition and no significant difference before preservation, survival rate is high, and each raw material is cheap and easy to get.Data show, the aweto strain after preservation
Average anabiosis rate is 95%, and mycelia bacterium colony center is prominent and fold is apparent, and metabolic capability is normal after bacterium cell recovery, 30d and 60d
Average colony size has respectively reached 16.3~17.2mm and 28.1~31.1mm, and culture medium melanin deposition generates normal.
(2) the present invention also provides a kind of methods based on the antifreeze preservation aweto strain, first by the worm summer in winter
The parent species of careless bacterium are inoculated on PPDA slant tube culture mediums, and culture obtains bacterium colony；Bacterium colony is cut into mycelia block later, by bacterium
Silk block is transferred in the cryopreservation tube containing antifreeze, the cryopreservation tube is first pre-chilled, then be quickly transferred to condition of ultralow temperature
Lower preservation；The method for preserving can protect aweto strain without damage under condition of ultralow temperature, and the preservation time is longer, lose
It is more preferable to pass stability, growth situation and no significant difference before preservation, survival rate are high.
Specific implementation mode
To make the object, technical solutions and advantages of the present invention clearer, technical scheme of the present invention will be carried out below
Detailed description.Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Base
Embodiment in the present invention, those of ordinary skill in the art are obtained all without making creative work
Other embodiment belongs to the range that the present invention is protected.
In following example 1g is represented with 1 parts by weight.
Embodiment 1
The present embodiment provides a kind of aweto antifreeze, raw material components include：
5 parts by weight of corn dextrin, 20 parts by weight of glycerine, Tween-80 are 1 parts by weight, 10 parts by weight of trehalose, K2HPO4/
KH2PO45 parts by weight of buffer solution, 74 parts by weight of purified water.
Based on the method for the antifreeze preservation aweto strain, include the following steps：
(1) PPDA slant tube culture mediums are made, through 121 DEG C of sterilizing 30min, the water after placement a period of time on test tube wall
Pearl and moisture evaporation disappear, and have no living contaminants；The wherein pH of PPDA slant tubes culture medium is 6, at being grouped into：
Potato, 100 parts by weight；
Glucose, 20 parts by weight；
Peptone, 5 parts by weight；
MgSO4, 2 parts by weight；
KH2PO4, 1 parts by weight；
Agar powder, 25 parts by weight；
Purified water, 847 parts by weight.
The parent species of aweto are inoculated on the PPDA slant tubes culture medium, are secretly trained under the conditions of 15 DEG C
60d is supported, the diameter of aweto bacterium colony is made to grow to 2cm, bacterium colony shows as dark brown, and bacterium colony is solid, and center protrusion protuberance has
Apparent fold, and there is melanin to penetrate into culture medium；
(2) corn dextrin, glycerine, Tween-80, trehalose, K are taken respectively2HPO4/KH2PO4Buffer solution and purified water, fill
Divide and is uniformly mixed to get antifreeze；
The antifreeze is fitted into the high-low temperature resistant cryopreservation tube of 1.8ml, the volume of antifreeze, which accounts for, freezes the 1/ of pipe volume
2, through 121 DEG C of sterilizing 30min postcoolings, placement a period of time ensures sterile rear for use；
(3) in superclean bench, in an aseptic environment, by the worm summer in winter in step (1) PPDA slant tube culture mediums
Careless bacterium bacterium colony cuts into the mycelia block of several 2mm × 2mm, and mycelia block is transferred to freezing containing antifreeze described in step (2)
Guan Zhong, mycelia block are completely submerged in the antifreeze, and it is 3 pieces that every cryopreservation tube, which is put into mycelia number of blocks, covers cryopreservation tube lid,
It is used in combination sealed membrane to be sealed；
(4) after being marked on cryopreservation tube, after the cryopreservation tube is pre-chilled for 24 hours at -15 DEG C, then be quickly transferred to -
Long term storage under 75 DEG C of condition of ultralow temperature.
Embodiment 2
The present embodiment provides a kind of aweto antifreeze, raw material components include：
10 parts by weight of gelatin sodium alginate, 10 parts by weight of ethylene glycol, 5 parts by weight of lauryl sodium sulfate, 5 weight of glucose
Part, Na2HPO4/NaH2PO410 parts by weight of buffer solution, 45 parts by weight of purified water.
Based on the method for the antifreeze preservation aweto strain, include the following steps：
(1) PPDA slant tube culture mediums are made, through 121 DEG C of sterilizing 30min, the water after placement a period of time on test tube wall
Pearl and moisture evaporation disappear, and have no living contaminants；The wherein pH of PPDA slant tubes culture medium is 6.5, at being grouped into：
Potato, 200 parts by weight；
Glucose, 10 parts by weight；
Peptone, 15 parts by weight；
MgSO4, 1 parts by weight；
KH2PO4, 2 parts by weight；
Agar powder, 18 parts by weight；
Purified water, 754 parts by weight.
The parent species of aweto are inoculated on the PPDA slant tubes culture medium, are secretly trained under the conditions of 18 DEG C
40d is supported, the diameter of aweto bacterium colony is made to grow to 3cm, bacterium colony shows as dark brown, and bacterium colony is solid, and center protrusion protuberance has
Apparent fold, and there is melanin to penetrate into culture medium；
(2) gelatin sodium alginate, ethylene glycol, lauryl sodium sulfate, glucose, Na are taken respectively2HPO4/NaH2PO4Buffering
Liquid and purified water are sufficiently mixed uniformly to get antifreeze；
The antifreeze is fitted into the high-low temperature resistant cryopreservation tube of 1.8ml, the volume of antifreeze, which accounts for, freezes the 4/ of pipe volume
5, through 121 DEG C of sterilizing 30min postcoolings, placement a period of time ensures sterile rear for use；
(3) in superclean bench, in an aseptic environment, by the worm summer in winter in step (1) PPDA slant tube culture mediums
Careless bacterium bacterium colony cuts into the mycelia block of several 2mm × 2mm, and mycelia block is transferred to freezing containing antifreeze described in step (2)
Guan Zhong, mycelia block are completely submerged in the antifreeze, and it is 5 pieces that every cryopreservation tube, which is put into mycelia number of blocks, covers cryopreservation tube lid,
It is used in combination sealed membrane to be sealed；
(4) after being marked on cryopreservation tube, after 48h is pre-chilled at -25 DEG C in the cryopreservation tube, then be quickly transferred to -
Preservation under 85 DEG C of condition of ultralow temperature.
Embodiment 3
The present embodiment provides a kind of aweto antifreeze, raw material components include：
8 parts by weight of guar gum, 15 parts by weight of propylene glycol, 2 parts by weight of glycine betaine, 6 parts by weight of lactose, Na2HPO4/K2HPO4
8 parts by weight of buffer solution, 59 parts by weight of purified water.
Based on the method for the antifreeze preservation aweto strain, include the following steps：
(1) PPDA slant tube culture mediums are made, through 121 DEG C of sterilizing 30min, the water after placement a period of time on test tube wall
Pearl and moisture evaporation disappear, and have no living contaminants；The wherein pH of PPDA slant tubes culture medium is 6~6.5, at being grouped into：
Potato, 150 parts by weight；
Glucose, 15 parts by weight；
Peptone, 10 parts by weight；
MgSO4, 1.5 parts by weight；
KH2PO4, 1.5 parts by weight；
Agar powder, 22 parts by weight；
Purified water, 800 parts by weight；
The parent species of aweto are inoculated on the PPDA slant tubes culture medium, are carried out under the conditions of 15~18 DEG C
Light culture 50d makes the diameter of aweto bacterium colony grow to 3cm, and bacterium colony shows as dark brown, and bacterium colony is solid, and center protrusion is grand
It rises, has apparent fold, and there is melanin to penetrate into culture medium；
(2) guar gum, propylene glycol, glycine betaine, lactose, Na are taken respectively2HPO4/K2HPO4Buffer solution and purified water, it is fully mixed
It closes uniformly to get antifreeze；
The antifreeze is fitted into the high-low temperature resistant cryopreservation tube of 1.8ml, the volume of antifreeze, which accounts for, freezes the 3/ of pipe volume
5, through 121 DEG C of sterilizing 30min postcoolings, placement a period of time ensures sterile rear for use；
(3) in superclean bench, in an aseptic environment, by the worm summer in winter in step (1) PPDA slant tube culture mediums
Careless bacterium bacterium colony cuts into the mycelia block of several 2mm × 2mm, and mycelia block is transferred to freezing containing antifreeze described in step (2)
Guan Zhong, mycelia block are completely submerged in the antifreeze, and it is 1 piece that every cryopreservation tube, which is put into mycelia number of blocks, covers cryopreservation tube lid,
It is used in combination sealed membrane to be sealed；
(4) after being marked on cryopreservation tube, after 36h is pre-chilled at -20 DEG C in the cryopreservation tube, then be quickly transferred to -
Preservation under 80 DEG C of condition of ultralow temperature.
Embodiment 4
The present embodiment provides a kind of aweto antifreeze, raw material components include：
8 parts by weight of chitosan, 15 parts by weight of butanediol, 3 parts by weight of polyvinylpyrrolidone, 7 parts by weight of fructose,
Na2HPO4/KH2PO48 parts by weight of buffer solution, 72 parts by weight of purified water.
Based on the method for the antifreeze preservation aweto strain, include the following steps：
(1) PPDA slant tube culture mediums are made, through 121 DEG C of sterilizing 30min, the water after placement a period of time on test tube wall
Pearl and moisture evaporation disappear, and have no living contaminants；The wherein pH of PPDA slant tubes culture medium is 6, at being grouped into：
Potato, 200 parts by weight；
Glucose, 20 parts by weight；
Peptone, 15 parts by weight；
MgSO4, 2 parts by weight；
KH2PO4, 2 parts by weight；
Agar powder, 25 parts by weight；
Purified water, 736 parts by weight；
The parent species of aweto are inoculated on the PPDA slant tubes culture medium, are secretly trained under the conditions of 16 DEG C
50d is supported, the diameter of aweto bacterium colony is made to grow to 3cm, bacterium colony shows as dark brown, and bacterium colony is solid, and center protrusion protuberance has
Apparent fold, and there is melanin to penetrate into culture medium；
(2) chitosan, butanediol, polyvinylpyrrolidone, fructose, Na are taken respectively2HPO4/KH2PO4Buffer solution and purifying
Water is sufficiently mixed uniformly to get antifreeze；
The antifreeze is fitted into the high-low temperature resistant cryopreservation tube of 1.8ml, the volume of antifreeze, which accounts for, freezes the 1/ of pipe volume
2, through 121 DEG C of sterilizing 30min postcoolings, placement a period of time ensures sterile rear for use；
(3) in superclean bench, in an aseptic environment, by the worm summer in winter in step (1) PPDA slant tube culture mediums
Careless bacterium bacterium colony cuts into the mycelia block of several 2mm × 2mm, and mycelia block is transferred to freezing containing antifreeze described in step (2)
Guan Zhong, mycelia block are completely submerged in the antifreeze, and it is 4 pieces that every cryopreservation tube, which is put into mycelia number of blocks, covers cryopreservation tube lid,
It is used in combination sealed membrane to be sealed；
(4) after being marked on cryopreservation tube, after 36h is pre-chilled at -20 DEG C in the cryopreservation tube, then be quickly transferred to -
Preservation under 80 DEG C of condition of ultralow temperature.
Embodiment 5
The present embodiment provides a kind of aweto antifreeze, raw material components include：
8 parts by weight of xanthans, 15 parts by weight of xylitol, 2 parts by weight of lauryl sodium sulfate, 6 parts by weight of sucrose,
NaH2PO4/K2HPO46 parts by weight of buffer solution, 65 parts by weight of purified water.
Based on the method for the antifreeze preservation aweto strain, include the following steps：
(1) PPDA slant tube culture mediums are made, through 121 DEG C of sterilizing 30min, the water after placement a period of time on test tube wall
Pearl and moisture evaporation disappear, and have no living contaminants；The wherein pH of PPDA slant tubes culture medium is 6, at being grouped into：
Potato, 100 parts by weight；
Glucose, 10 parts by weight；
Peptone, 5 parts by weight；
MgSO4, 1 parts by weight；
KH2PO4, 1 parts by weight；
Agar powder, 18 parts by weight；
Purified water, 865 parts by weight；
The parent species of aweto are inoculated on the PPDA slant tubes culture medium, are secretly trained under the conditions of 17 DEG C
50d is supported, the diameter of aweto bacterium colony is made to grow to 2cm, bacterium colony shows as dark brown, and bacterium colony is solid, and center protrusion protuberance has
Apparent fold, and there is melanin to penetrate into culture medium；
(2) xanthans, xylitol, lauryl sodium sulfate, sucrose, NaH are taken respectively2PO4/K2HPO4Buffer solution and purifying
Water is sufficiently mixed uniformly to get antifreeze；
The antifreeze is fitted into cryopreservation tube, the volume of antifreeze, which accounts for, freezes the 7/10 of pipe volume, sterilized postcooling,
For use；
(3) the aweto bacterium colony in step (1) PPDA slant tube culture mediums is cut into several 2mm × 2mm's
Mycelia block is transferred in the cryopreservation tube containing antifreeze described in step (2) by mycelia block, and mycelia block is completely submerged in described freeze proof
In agent, it is 4 pieces that every cryopreservation tube, which is put into mycelia number of blocks, covers cryopreservation tube lid, sealed membrane is used in combination to be sealed；
(4) after being marked on cryopreservation tube, after 36h is pre-chilled at -20 DEG C in the cryopreservation tube, then be quickly transferred to -
Preservation under 80 DEG C of condition of ultralow temperature.
Embodiment 6
The present embodiment provides a kind of aweto antifreeze, raw material components include：
8 parts by weight of xanthans, 15 parts by weight of pentaerythrite, Tween-80 are 2 parts by weight, 6 parts by weight of trehalose, NaH2PO4/
KH2PO46 parts by weight of buffer solution, 65 parts by weight of purified water.
Based on the method for the antifreeze preservation aweto strain, include the following steps：
(1) PPDA slant tube culture mediums are made, through 121 DEG C of sterilizing 30min, the water after placement a period of time on test tube wall
Pearl and moisture evaporation disappear, and have no living contaminants；The wherein pH of PPDA slant tubes culture medium is 6, at being grouped into：
Potato, 100 parts by weight；
Glucose, 10 parts by weight；
Peptone, 5 parts by weight；
MgSO4, 1 parts by weight；
KH2PO4, 1 parts by weight；
Agar powder, 18 parts by weight；
Purified water, 865 parts by weight；
The parent species of aweto are inoculated on the PPDA slant tubes culture medium, are secretly trained under the conditions of 18 DEG C
50d is supported, the diameter of aweto bacterium colony is made to grow to 2cm, bacterium colony shows as dark brown, and bacterium colony is solid, and center protrusion protuberance has
Apparent fold, and there is melanin to penetrate into culture medium；
(2) xanthans, pentaerythrite, Tween-80, trehalose, NaH are taken respectively2PO4/KH2PO4Buffer solution and purified water,
It is sufficiently mixed uniformly to get antifreeze；
The antifreeze is fitted into cryopreservation tube, the volume of antifreeze, which accounts for, freezes the 7/10 of pipe volume, sterilized postcooling,
For use；
(3) the aweto bacterium colony in step (1) PPDA slant tube culture mediums is cut into several 2mm × 2mm's
Mycelia block is transferred in the cryopreservation tube containing antifreeze described in step (2) by mycelia block, and mycelia block is completely submerged in described freeze proof
In agent, it is 4 pieces that every cryopreservation tube, which is put into mycelia number of blocks, covers cryopreservation tube lid, sealed membrane is used in combination to be sealed；
(4) after being marked on cryopreservation tube, after 48h is pre-chilled at -20 DEG C in the cryopreservation tube, then be quickly transferred to -
Preservation under 80 DEG C of condition of ultralow temperature.
Comparative example 1
It is small pre- in -20 DEG C of conditions by aweto culture presevation in the glycerin solution that weight percent is 15%
Then cold 36h is put into rapidly preservation 18 months in -80 DEG C of refrigerators.
Comparative example 2
By aweto culture presevation in the aqueous trehalose that weight percent is 7%, in the small precooling of -20 DEG C of conditions
Then 36h is put into rapidly preservation 18 months in -80 DEG C of refrigerators.
Comparative example 3
By aweto culture presevation in the sucrose solution that weight percent is 20%, in the small precooling of -20 DEG C of conditions
Then 36h is put into rapidly preservation 18 months in -80 DEG C of refrigerators.
Comparative example 4
Without the aweto strain of preservation process, by PPDA slant tubes culture medium, per 60d, switching is primary, and
It is cultivated under the conditions of 18 DEG C, was continuously forwarded to for the tenth generation.
Experimental example
It transfers after the aweto of 1~3 preservation of Examples 1 to 3 and comparative example is recovered and in comparative example 4
Aweto strain progress shape to the tenth generation compares test, and concrete operations are：
18 months awetos of Examples 1 to 3 preservation are recovered, specially：It shakes under the conditions of 18 DEG C
Defrosting is swung, until antifreeze melts completely, is aseptically taken out from cryopreservation tube later, is gone with axenic purification washing
Antifreeze after being used in combination filter paper to be sufficiently absorbed through moisture, accesses on PPDA culture mediums, 60d is cultivated under the conditions of being placed in 18 DEG C.
The aweto of 1~3 preservation of comparative example is recovered, specially：It is carried out in 18 DEG C of thermostat water baths fast
Speed, concussion are thawed, and after being used in combination filter paper to be sufficiently absorbed through moisture, access on PPDA culture mediums, 60d is cultivated under the conditions of being placed in 18 DEG C.
The acquisition pattern that the tenth generation strain is forwarded in comparative example 4 is as follows：By the 9th generation strain transfer to PPDA culture mediums
In, cultivate 60d under the conditions of being placed in 18 DEG C.
The shape comparison sheet of aweto bacterium colony between table 1- different disposal groups
From table 1 it follows that the aweto strain of 1~3 method preservation of the embodiment of the present invention, the winter worm after preservation
The summer grass bacterium strain anabiosis rate that is averaged is 95%, and mycelia bacterium colony center is prominent and fold is apparent, and metabolic energy is metabolized after bacterium cell recovery
Power is normal, 30d and 60d average colony sizes have respectively reached 16.3~17.2mm and 28.1~31.1mm, culture medium melanin
Precipitation generates normal；There is the advantage of highly significant relative to other antifreezes (comparative example 1~3).
In addition, the preservation time limit of aweto strain is extended to 3 years, 5 years using antifreeze of the present invention, obtain
Result it is consistent with result in table 1.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any
Those familiar with the art in the technical scope disclosed by the present invention, can easily think of the change or the replacement, and should all contain
Lid is within protection scope of the present invention.Therefore, protection scope of the present invention should be based on the protection scope of the described claims.
Claims (10)
1. a kind of aweto antifreeze, which is characterized in that raw material components include：
5~10 parts by weight of natural polymer, 10~20 parts by weight of polyalcohol, 1~5 parts by weight of surfactant, carbohydrate 5
~10 parts by weight, 5~10 parts by weight of pH buffer solutions, 45~74 parts by weight of purified water.
2. aweto antifreeze according to claim 1, which is characterized in that raw material components include：
5~8 parts by weight of natural polymer, 10~15 parts by weight of polyalcohol, 2~3 parts by weight of surfactant, carbohydrate 6
~7 parts by weight, 5~8 parts by weight of pH buffer solutions, 59~72 parts by weight of purified water.
3. aweto antifreeze according to claim 1, which is characterized in that the natural polymer is jade
The mixture of one or more of rice dextrin, gelatin sodium alginate, guar gum, chitosan and xanthans；
The polyalcohol is one or more of ethylene glycol, propylene glycol, glycerine, butanediol, xylitol and pentaerythrite
Mixture；
The surfactant is Tween-80, lauryl sodium sulfate, glycine betaine, one kind in polyvinylpyrrolidone or several
The mixture of kind；
The carbohydrate is the mixture of one or more of glucose, lactose, fructose, sucrose, trehalose；
The pH buffer solutions are Na2HPO4/NaH2PO4、K2HPO4/KH2PO4、Na2HPO4/K2HPO4、Na2HPO4/KH2PO4、
NaH2PO4/K2HPO4、NaH2PO4/KH2PO4One or more of mixture.
4. a kind of method based on any one of claims 1 to 3 antifreeze preservation aweto strain, feature exist
In including the following steps：
(1) PPDA slant tube culture mediums are made, after sterilized, the parent species of aweto are inoculated on the culture medium,
40~60d of light culture is carried out under the conditions of 15~18 DEG C, and the diameter of aweto bacterium colony is made to grow to 2~3cm；
(2) natural polymer, polyalcohol, surfactant, carbohydrate, pH buffer solutions and purified water are taken respectively, it is fully mixed
It closes uniformly to get antifreeze；
The antifreeze is fitted into cryopreservation tube, the volume of antifreeze, which accounts for, freezes the 1/2~4/5 of pipe volume, sterilized postcooling,
For use；
(3) the aweto bacterium colony in step (1) PPDA slant tube culture mediums is cut into several mycelia blocks, by mycelia block
It is transferred in the cryopreservation tube containing antifreeze described in step (2), mycelia block is completely submerged in the antifreeze；
(4) after 24~48h being pre-chilled at -15~-25 DEG C in the cryopreservation tube, then -75~-85 DEG C of ultralow temperature items are quickly transferred to
Preservation under part.
5. preparation method according to claim 4, which is characterized in that in step (1), the PPDA slant tubes culture medium
At being grouped into：
Potato, 100~200 parts by weight；
Glucose, 10~20 parts by weight；
Peptone, 5~15 parts by weight；
MgSO4, 1~2 parts by weight；
KH2PO4, 1~2 parts by weight；
Agar powder, 18~25 parts by weight；
Purified water, 736~865 parts by weight.
6. the method for preservation aweto strain according to claim 5, which is characterized in that the PPDA slant tubes training
The pH for supporting base is 6~6.5.
7. the method for preservation aweto strain according to claim 4, which is characterized in that in step (1), described in progress
The temperature of sterilizing is 121 DEG C, and the time for carrying out the sterilizing is 30min；
The temperature for carrying out the light culture is 15~18 DEG C, and the time for carrying out the light culture is 40~60d.
8. the method for preservation aweto strain according to claim 4, which is characterized in that in step (2), described in progress
The temperature of sterilizing is 121 DEG C, and the time for carrying out the sterilizing is 30min.
9. the method for preservation aweto strain according to claim 4, which is characterized in that in step (3), the mycelia
The size of block is 2mm × 2mm.
10. a kind of aweto strain to after the method preservation of any one of claim 4~9 carries out low temperature recovery
Method, which is characterized in that be as follows：
The cryopreservation tube of preservation under condition of ultralow temperature is taken out, and oscillation defrosting is carried out under the conditions of being immediately placed in 15~20 DEG C, until
Until antifreeze melts completely, it is inoculated into PPDA slant tube culture mediums later and is cultivated.
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| CN114223651A (en) * | 2021-12-29 | 2022-03-25 | 松山湖材料实验室 | Frozen stock solution, preparation method thereof and application thereof in human renal epithelial cells |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643749A (en) * | 2012-04-28 | 2012-08-22 | 中粮生物化学（安徽）股份有限公司 | Microorganism low-temperature preservation protective agent and microorganism low-temperature preservation method |
| CN103146580A (en) * | 2013-03-07 | 2013-06-12 | 西北民族大学 | Anaerobic ultralow temperature strain preserving agent |
| WO2017066454A2 (en) * | 2015-10-14 | 2017-04-20 | X-Therma, Inc. | Compositions and methods for reducing ice crystal formation |
| CN106754376A (en) * | 2016-12-07 | 2017-05-31 | 中国食品药品检定研究院 | A kind of microorganism low-temperature preservation protective agent |
-
2018
- 2018-05-14 CN CN201810458998.8A patent/CN108359613B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643749A (en) * | 2012-04-28 | 2012-08-22 | 中粮生物化学（安徽）股份有限公司 | Microorganism low-temperature preservation protective agent and microorganism low-temperature preservation method |
| CN103146580A (en) * | 2013-03-07 | 2013-06-12 | 西北民族大学 | Anaerobic ultralow temperature strain preserving agent |
| WO2017066454A2 (en) * | 2015-10-14 | 2017-04-20 | X-Therma, Inc. | Compositions and methods for reducing ice crystal formation |
| CN106754376A (en) * | 2016-12-07 | 2017-05-31 | 中国食品药品检定研究院 | A kind of microorganism low-temperature preservation protective agent |
Non-Patent Citations (2)
| Title |
|---|
| 张宏宇主编: "《药用昆虫养殖技术》", 30 September 2001, 广州：广东科技出版社 * |
| 李建国主编: "《奶牛饲养与繁殖技术指南》", 31 January 2003, 北京：北京农业大学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114223651A (en) * | 2021-12-29 | 2022-03-25 | 松山湖材料实验室 | Frozen stock solution, preparation method thereof and application thereof in human renal epithelial cells |
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