CN108359613B - Cordyceps sinensis antifreeze agent and ultralow-temperature preservation and recovery method based on antifreeze agent - Google Patents
Cordyceps sinensis antifreeze agent and ultralow-temperature preservation and recovery method based on antifreeze agent Download PDFInfo
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- 241001248610 Ophiocordyceps sinensis Species 0.000 title claims abstract description 69
- 239000007798 antifreeze agent Substances 0.000 title claims abstract description 57
- 238000000034 method Methods 0.000 title claims abstract description 35
- 238000004321 preservation Methods 0.000 title claims abstract description 34
- 238000011084 recovery Methods 0.000 title abstract description 9
- 239000001963 growth medium Substances 0.000 claims abstract description 48
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 43
- 239000008213 purified water Substances 0.000 claims abstract description 27
- 239000007853 buffer solution Substances 0.000 claims abstract description 20
- 238000007710 freezing Methods 0.000 claims description 52
- 230000008014 freezing Effects 0.000 claims description 45
- 238000012360 testing method Methods 0.000 claims description 41
- CBCKQZAAMUWICA-UHFFFAOYSA-N 1,4-phenylenediamine Chemical compound NC1=CC=C(N)C=C1 CBCKQZAAMUWICA-UHFFFAOYSA-N 0.000 claims description 38
- 230000001954 sterilising effect Effects 0.000 claims description 22
- 230000001580 bacterial effect Effects 0.000 claims description 17
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 14
- 239000007836 KH2PO4 Substances 0.000 claims description 14
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 14
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 13
- 239000008103 glucose Substances 0.000 claims description 13
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 11
- 238000001816 cooling Methods 0.000 claims description 10
- 229920001817 Agar Polymers 0.000 claims description 8
- 239000001888 Peptone Substances 0.000 claims description 8
- 108010080698 Peptones Proteins 0.000 claims description 8
- 244000061456 Solanum tuberosum Species 0.000 claims description 8
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 8
- 239000008272 agar Substances 0.000 claims description 8
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 8
- 235000019319 peptone Nutrition 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 7
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 7
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 6
- 229940083575 sodium dodecyl sulfate Drugs 0.000 claims description 6
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 5
- 108010010803 Gelatin Proteins 0.000 claims description 5
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 5
- 239000008273 gelatin Substances 0.000 claims description 5
- 229920000159 gelatin Polymers 0.000 claims description 5
- 235000019322 gelatine Nutrition 0.000 claims description 5
- 235000011852 gelatine desserts Nutrition 0.000 claims description 5
- 235000010413 sodium alginate Nutrition 0.000 claims description 5
- 239000000661 sodium alginate Substances 0.000 claims description 5
- 229940005550 sodium alginate Drugs 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 2
- 235000012015 potatoes Nutrition 0.000 claims description 2
- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 claims 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims 1
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 abstract description 18
- 239000002994 raw material Substances 0.000 abstract description 12
- 241000190633 Cordyceps Species 0.000 abstract description 6
- 150000001720 carbohydrates Chemical class 0.000 abstract description 6
- 150000001875 compounds Chemical class 0.000 abstract description 6
- 150000005846 sugar alcohols Polymers 0.000 abstract description 6
- 239000004094 surface-active agent Substances 0.000 abstract description 6
- 230000004083 survival effect Effects 0.000 abstract description 4
- 230000002068 genetic effect Effects 0.000 abstract description 3
- 230000002503 metabolic effect Effects 0.000 abstract description 3
- 239000002244 precipitate Substances 0.000 abstract description 3
- 241000894006 Bacteria Species 0.000 abstract description 2
- 238000005138 cryopreservation Methods 0.000 description 18
- 230000002528 anti-freeze Effects 0.000 description 14
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- 238000007789 sealing Methods 0.000 description 12
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 9
- 239000003795 chemical substances by application Substances 0.000 description 7
- 238000001704 evaporation Methods 0.000 description 7
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 7
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 6
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 6
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 6
- 238000011109 contamination Methods 0.000 description 6
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 6
- 239000012466 permeate Substances 0.000 description 6
- 230000037303 wrinkles Effects 0.000 description 6
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 5
- 229920000053 polysorbate 80 Polymers 0.000 description 5
- 239000004576 sand Substances 0.000 description 5
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000000230 xanthan gum Substances 0.000 description 5
- 235000010493 xanthan gum Nutrition 0.000 description 5
- 229920001285 xanthan gum Polymers 0.000 description 5
- 229940082509 xanthan gum Drugs 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 4
- 229930006000 Sucrose Natural products 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 235000011187 glycerol Nutrition 0.000 description 4
- 230000036512 infertility Effects 0.000 description 4
- 239000005720 sucrose Substances 0.000 description 4
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 3
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 3
- 229920001661 Chitosan Polymers 0.000 description 3
- 229930091371 Fructose Natural products 0.000 description 3
- 239000005715 Fructose Substances 0.000 description 3
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 3
- 229920002907 Guar gum Polymers 0.000 description 3
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 3
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 description 3
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 3
- 229960003237 betaine Drugs 0.000 description 3
- CDQSJQSWAWPGKG-UHFFFAOYSA-N butane-1,1-diol Chemical compound CCCC(O)O CDQSJQSWAWPGKG-UHFFFAOYSA-N 0.000 description 3
- 229940045110 chitosan Drugs 0.000 description 3
- FYGDTMLNYKFZSV-MRCIVHHJSA-N dextrin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)OC1O[C@@H]1[C@@H](CO)OC(O[C@@H]2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-MRCIVHHJSA-N 0.000 description 3
- 239000000665 guar gum Substances 0.000 description 3
- 235000010417 guar gum Nutrition 0.000 description 3
- 229960002154 guar gum Drugs 0.000 description 3
- 239000008101 lactose Substances 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 3
- WXZMFSXDPGVJKK-UHFFFAOYSA-N pentaerythritol Chemical compound OCC(CO)(CO)CO WXZMFSXDPGVJKK-UHFFFAOYSA-N 0.000 description 3
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 3
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 3
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- 239000000811 xylitol Substances 0.000 description 3
- 235000010447 xylitol Nutrition 0.000 description 3
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 3
- 229960002675 xylitol Drugs 0.000 description 3
- 241000233866 Fungi Species 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229940057995 liquid paraffin Drugs 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 241000235349 Ascomycota Species 0.000 description 1
- 241000130660 Hepialidae Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000221781 Hypocreaceae Species 0.000 description 1
- 241000221775 Hypocreales Species 0.000 description 1
- 241001416980 Paecilomyces hepiali Species 0.000 description 1
- 244000131316 Panax pseudoginseng Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 210000003056 antler Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 229960001375 lactose Drugs 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229960004063 propylene glycol Drugs 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 229960004793 sucrose Drugs 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
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- Organic Chemistry (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
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Abstract
The invention relates to a cordyceps fungus antifreeze agent and an ultra-low temperature preservation and recovery method based on the antifreeze agent, wherein the antifreeze agent takes natural high molecular compound, polyalcohol, surfactant, saccharide, pH buffer solution and purified water as raw materials and carries out proper weight proportion, each component plays a role respectively, the cordyceps fungus strain is protected from being damaged during preservation under the ultra-low temperature condition, the preservation time is longer, the genetic stability is better, the strain growth condition has no obvious difference with that before preservation, the survival rate is high, and each raw material is cheap and easy to obtain. The data show that the average recovery rate of the preserved cordyceps sinensis strains is 95%, the center of hypha colonies protrudes and folds are obvious, the metabolic capacity of the bacteria cells is normal after recovery, the average colony sizes of 30d and 60d reach 16.3-17.2 mm and 28.1-31.1 mm respectively, and the melanin precipitates in the culture medium are normal.
Description
Technical Field
The invention belongs to the technical field of cryopreservation and low-temperature resuscitation of psychrophilic fungi, and particularly relates to an antifreeze agent for cordyceps sinensis and an cryopreservation method based on the antifreeze agent.
Background
Cordyceps sinensis is also called cordyceps sinensis, belongs to the genus Cordyceps sinensis belonging to the family of Hypocreaceae of Hypocreales of Ascomycota, and particularly relates to a complex of larva bodies and fungal stroma formed by infecting larvae of insects of Hepialidae with Cordyceps sinensis, which has various pharmacological functions, is long-term considered as a precious medium and Tibetan medicine material, is widely influenced in the world, particularly in China and southeast Asia regions, is an important medicinal fungus, and is called as 'three treasures of traditional Chinese medicine' in combination with ginseng and pilose antler.
Because the cordyceps sinensis has two completely different forms of complete form (also called sexual generation) and incomplete form (also called asexual generation), the method for identifying the strain is lagged behind before 10 years, molecular identification is not developed, and paecilomyces hepiali is wrongly cultured as the cordyceps sinensis strain only by separating the form of the hypha for a long period of time, so that a lot of tortuosity is caused. In the problem of determining the anamorph of Cordyceps sinensis, the academic world has been controversial. At present, 22 anamorph strains related to Cordyceps sinensis have been reported, and there are 13 genera, and many sequences registered as "Cordyceps sinensis" retrieved from NCBI database are significantly different from each other.
The strain preservation is to use physical and biological means to make the strain in a complete dormant state, thereby keeping the vitality and original characters of the strain for a long time, ensuring the safety of the strain and preventing degeneration. At present, the preservation methods of large strains mainly comprise a liquid paraffin preservation method, a subculture preservation method, a sand and soil tube preservation method, an ultra-low temperature preservation method, a liquid nitrogen low-temperature preservation method and the like. The subculture preservation method has short preservation time, complicated operation, large workload and poor strain stability, and strains are easy to change or degenerate along with the increase of the number of passages; the liquid paraffin preservation method can cause the slant culture to be exposed because the paraffin evaporation is reduced, and if the slant culture is not supplemented in time, the strain can be degraded and even die; in the sand-soil tube preservation method, sand and soil need to be washed, impurity removed and scrap iron removed; the proportion of the sand in the sand pipe is not fixed, is influenced by the quality of the sand and the characteristics of strains, is often determined by tests, and has low survival rate and high variation rate; the liquid nitrogen cryopreservation method has long preservation time, but has higher maintenance cost, and is only used by a few scientific research institutions.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an antifreeze agent suitable for long-term ultralow-temperature preservation of cordyceps sinensis strains, and further provides a method for preserving the cordyceps sinensis strains based on the antifreeze agent and a low-temperature resuscitation method.
The technical scheme adopted by the invention is as follows:
an antifreeze agent for cordyceps sinensis comprises the following raw material components:
5-10 parts of natural high molecular compound, 10-20 parts of polyhydric alcohol, 1-5 parts of surfactant, 5-10 parts of saccharide, 5-10 parts of pH buffer solution and 45-74 parts of purified water.
Further preferably, the cordyceps sinensis antifreeze comprises the following raw material components:
5-8 parts of natural high molecular compound, 10-15 parts of polyhydric alcohol, 2-3 parts of surfactant, 6-7 parts of saccharide, 5-8 parts of pH buffer solution and 59-72 parts of purified water.
The natural high molecular compound is one or a mixture of more of corn dextrin, gelatin sodium alginate, guar gum, chitosan and xanthan gum;
the polyalcohol is one or a mixture of more of ethylene glycol, propylene glycol, glycerol, butanediol, xylitol and pentaerythritol;
the surfactant is one or a mixture of more of tween-80, sodium dodecyl sulfate, betaine and polyvinylpyrrolidone;
the saccharide is one or a mixture of more of glucose, lactose, fructose, sucrose and trehalose;
the pH buffer solution is Na2HPO4/NaH2PO4、K2HPO4/KH2PO4、Na2HPO4/K2HPO4、Na2HPO4/KH2PO4、NaH2PO4/K2HPO4、NaH2PO4/KH2PO4One or a mixture of several of them.
A method for preserving cordyceps sinensis strains based on the antifreeze agent comprises the following steps:
(1) preparing a PPDA slant test tube culture medium, inoculating a mother strain of cordyceps sinensis to the culture medium after sterilization, and carrying out dark culture at 15-18 ℃ for 40-60 days to enable the diameter of a cordyceps sinensis colony to be 2-3 cm;
(2) respectively taking a natural high molecular compound, polyhydric alcohol, a surfactant, saccharides, a pH buffer solution and purified water, and fully and uniformly mixing to obtain an antifreeze agent;
filling the antifreeze agent into a freezing storage tube, wherein the volume of the antifreeze agent accounts for 1/2-4/5 of the volume of the freezing storage tube, and sterilizing and cooling the antifreeze agent for later use;
(3) cutting the cordyceps sinensis bacterial colony in the PPDA slant test tube culture medium in the step (1) into a plurality of hypha blocks, transferring the hypha blocks into the freezing storage tube containing the antifreeze agent in the step (2), and completely immersing the hypha blocks in the antifreeze agent;
(4) and pre-cooling the freezing tube at-15 to-25 ℃ for 24 to 48 hours, and then quickly transferring the tube to an ultralow temperature condition of-75 to-85 ℃ for preservation.
In the step (1), the PPDA slant test tube culture medium comprises the following components:
100-200 parts by weight of potatoes;
10-20 parts by weight of glucose;
5-15 parts of peptone;
MgSO41-2 parts by weight;
KH2PO41-2 parts by weight;
18-25 parts by weight of agar powder;
736-865 parts by weight of purified water.
The pH value of the PPDA slant test tube culture medium is 6-6.5.
In the step (1), the temperature for sterilizing is 121 ℃, and the time for sterilizing is 30 min;
the temperature for dark culture is 15-18 ℃, and the time for dark culture is 40-60 days.
In the step (2), the temperature for sterilizing is 121 ℃, and the time for sterilizing is 30 min.
In the step (3), the size of the mycelium block is 2mm multiplied by 2 mm.
A method for recovering preserved Cordyceps strains at low temperature comprises the following steps:
and taking out the cryopreservation tube preserved under the ultralow temperature condition, immediately putting the cryopreservation tube into a 15-20 ℃ condition for shaking and thawing until the antifreeze agent is completely melted, and then inoculating the cryopreservation tube to a PPDA slant test tube culture medium for culture.
The invention has the beneficial effects that:
(1) the anti-freezing agent for cordyceps sinensis provided by the invention takes natural high molecular compounds, polyhydric alcohols, surfactants, saccharides, pH buffer solution and purified water as raw materials and is prepared by proper weight proportion, each component plays a role respectively, the cordyceps sinensis strain is protected from being damaged during preservation under ultralow temperature conditions, the preservation time is longer, the genetic stability is better, the growth condition of the strain has no obvious difference with that before preservation, the survival rate is high, and each raw material is cheap and easy to obtain. The data show that the average recovery rate of the preserved cordyceps sinensis strains is 95%, the center of hypha colonies protrudes and folds are obvious, the metabolic capacity of the bacteria cells is normal after recovery, the average colony sizes of 30d and 60d reach 16.3-17.2 mm and 28.1-31.1 mm respectively, and the melanin precipitates in the culture medium are normal.
(2) The invention also provides a method for preserving the strain of the cordyceps sinensis based on the antifreeze, which comprises the steps of firstly inoculating the mother strain of the cordyceps sinensis to a PPDA slant test tube culture medium, and culturing to obtain a bacterial colony; then, the bacterial colonies are cut into hypha blocks, the hypha blocks are transferred to a freezing storage tube containing an antifreeze agent, the freezing storage tube is pre-cooled, and then the hypha blocks are rapidly transferred to an ultralow temperature condition for storage; the preservation method can protect the cordyceps sinensis strains from being damaged under the ultralow temperature condition, has longer preservation time and better genetic stability, has no obvious difference between the growth condition of the strains and the growth condition before preservation, and has high survival rate.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
In the following examples 1g is represented by 1 part by weight.
Example 1
The embodiment provides a cordyceps sinensis bacterial anti-freezing agent which comprises the following raw material components:
5 parts of corn dextrin, 20 parts of glycerol, 1 part of tween-80, 10 parts of trehalose and K2HPO4/KH2PO45 parts of buffer solution and 74 parts of purified water.
The method for preserving the cordyceps sinensis strain based on the antifreeze comprises the following steps:
(1) preparing PPDA slant test tube culture medium, sterilizing at 121 deg.C for 30min, standing for a period of time, evaporating water drop and water on the test tube wall, and removing contamination; wherein the pH of the PPDA slant test tube culture medium is 6, and the components comprise:
100 parts of potato;
20 parts of glucose;
peptone, 5 parts by weight;
MgSO42 parts by weight;
KH2PO41 part by weight;
25 parts by weight of agar powder;
purified water, 847 parts by weight.
Inoculating the mother strain of the cordyceps sinensis to the PPDA slant test tube culture medium, and performing dark culture at 15 ℃ for 60 days to ensure that the diameter of a cordyceps sinensis colony is as long as 2cm, the colony is black brown, the colony is firm, the center of the colony protrudes and bulges, obvious wrinkles are formed, and melanin permeates into the culture medium;
(2) respectively collecting corn dextrin, glycerol, tween-80, trehalose, and K2HPO4/KH2PO4Fully and uniformly mixing the buffer solution and the purified water to obtain the antifreeze agent;
filling the antifreeze agent into a 1.8ml high-low temperature resistant cryopreservation tube, wherein the volume of the antifreeze agent accounts for 1/2 of the volume of the cryopreservation tube, sterilizing at 121 ℃ for 30min, cooling, and standing for a period of time to ensure sterility for later use;
(3) in an ultra-clean workbench, under an aseptic environment, cutting the cordyceps sinensis bacterial colony in the PPDA slant test tube culture medium in the step (1) into a plurality of hypha blocks with the size of 2mm multiplied by 2mm, transferring the hypha blocks into the freezing storage tube containing the antifreeze in the step (2), completely immersing the hypha blocks in the antifreeze, putting 3 hypha blocks into each freezing storage tube, covering the freezing storage tube with a freezing storage tube cover, and sealing with a sealing film;
(4) marking on a freezing tube, precooling the freezing tube for 24 hours at-15 ℃, and then quickly transferring the freezing tube to the ultralow temperature condition of-75 ℃ for long-term storage.
Example 2
The embodiment provides a cordyceps sinensis bacterial anti-freezing agent which comprises the following raw material components:
10 parts of gelatin sodium alginate, 10 parts of ethylene glycol, 5 parts of sodium dodecyl sulfate, 5 parts of glucose and Na2HPO4/NaH2PO410 parts of buffer solution and 45 parts of purified water.
The method for preserving the cordyceps sinensis strain based on the antifreeze comprises the following steps:
(1) preparing PPDA slant test tube culture medium, sterilizing at 121 deg.C for 30min, standing for a period of time, evaporating water drop and water on the test tube wall, and removing contamination; wherein the pH of the PPDA slant test tube culture medium is 6.5, and the components comprise:
200 parts of potato;
10 parts of glucose;
peptone, 15 parts by weight;
MgSO41 part by weight;
KH2PO42 parts by weight;
18 parts of agar powder;
754 parts by weight of purified water.
Inoculating the mother strain of the cordyceps sinensis to the PPDA slant test tube culture medium, and performing dark culture at 18 ℃ for 40 days to ensure that the diameter of a cordyceps sinensis colony is as long as 3cm, the colony is black brown, the colony is firm, the center of the colony protrudes and bulges, obvious wrinkles are formed, and melanin permeates into the culture medium;
(2) respectively taking gelatin sodium alginate, ethylene glycol, sodium dodecyl sulfate, glucose and Na2HPO4/NaH2PO4Fully and uniformly mixing the buffer solution and the purified water to obtain the antifreeze agent;
filling the antifreeze agent into a 1.8ml high-low temperature resistant cryopreservation tube, wherein the volume of the antifreeze agent accounts for 4/5 of the volume of the cryopreservation tube, sterilizing at 121 ℃ for 30min, cooling, and standing for a period of time to ensure sterility for later use;
(3) in an ultra-clean workbench, under an aseptic environment, cutting the cordyceps sinensis bacterial colony in the PPDA slant test tube culture medium in the step (1) into a plurality of hypha blocks with the size of 2mm multiplied by 2mm, transferring the hypha blocks into the freezing storage tube containing the antifreeze in the step (2), completely immersing the hypha blocks in the antifreeze, putting 5 hypha blocks into each freezing storage tube, covering the freezing storage tube with a freezing storage tube cover, and sealing with a sealing film;
(4) marking on a freezing tube, precooling the freezing tube at-25 ℃ for 48h, and then quickly transferring to the ultralow temperature condition of-85 ℃ for preservation.
Example 3
The embodiment provides a cordyceps sinensis bacterial anti-freezing agent which comprises the following raw material components:
8 parts of guar gum, 15 parts of propylene glycol, 2 parts of betaine, 6 parts of lactose and Na2HPO4/K2HPO48 parts of buffer solution and 59 parts of purified water.
The method for preserving the cordyceps sinensis strain based on the antifreeze comprises the following steps:
(1) preparing PPDA slant test tube culture medium, sterilizing at 121 deg.C for 30min, standing for a period of time, evaporating water drop and water on the test tube wall, and removing contamination; wherein the pH of the PPDA slant test tube culture medium is 6-6.5, and the components are as follows:
150 parts of potato;
15 parts of glucose;
peptone, 10 parts by weight;
MgSO41.5 parts by weight;
KH2PO41.5 parts by weight;
22 parts by weight of agar powder;
800 parts by weight of purified water;
inoculating a mother strain of cordyceps sinensis to the PPDA slant test tube culture medium, and carrying out dark culture for 50 days at 15-18 ℃ to ensure that the diameter of a cordyceps sinensis colony is as long as 3cm, the colony is black brown, the colony is firm, the center of the colony protrudes and bulges, obvious wrinkles are formed, and melanin permeates into the culture medium;
(2) respectively taking guar gum, propylene glycol, betaine, lactose and Na2HPO4/K2HPO4Fully and uniformly mixing the buffer solution and the purified water to obtain the antifreeze agent;
filling the antifreeze agent into a 1.8ml high-low temperature resistant cryopreservation tube, wherein the volume of the antifreeze agent accounts for 3/5 of the volume of the cryopreservation tube, sterilizing at 121 ℃ for 30min, cooling, and standing for a period of time to ensure sterility for later use;
(3) in an ultra-clean workbench, under an aseptic environment, cutting the cordyceps sinensis bacterial colony in the PPDA slant test tube culture medium in the step (1) into a plurality of hypha blocks with the size of 2mm multiplied by 2mm, transferring the hypha blocks into the cryopreservation tubes containing the antifreeze in the step (2), completely immersing the hypha blocks in the antifreeze, putting 1 hypha block in each cryopreservation tube, covering the cryopreservation tube with a tube cover, and sealing with a sealing film;
(4) marking on a freezing tube, precooling the freezing tube at-20 ℃ for 36h, and then quickly transferring to the ultralow temperature condition of-80 ℃ for preservation.
Example 4
The embodiment provides a cordyceps sinensis bacterial anti-freezing agent which comprises the following raw material components:
8 parts of chitosan, 15 parts of butanediol, 3 parts of polyvinylpyrrolidone, 7 parts of fructose and Na2HPO4/KH2PO48 parts of buffer solution and 72 parts of purified water.
The method for preserving the cordyceps sinensis strain based on the antifreeze comprises the following steps:
(1) preparing PPDA slant test tube culture medium, sterilizing at 121 deg.C for 30min, standing for a period of time, evaporating water drop and water on the test tube wall, and removing contamination; wherein the pH of the PPDA slant test tube culture medium is 6, and the components comprise:
200 parts of potato;
20 parts of glucose;
peptone, 15 parts by weight;
MgSO42 parts by weight;
KH2PO42 parts by weight;
25 parts by weight of agar powder;
736 parts by weight of purified water;
inoculating the mother strain of the cordyceps sinensis to the PPDA slant test tube culture medium, and performing dark culture for 50 days at 16 ℃ to ensure that the diameter of a cordyceps sinensis colony is as long as 3cm, the colony is black brown, the colony is firm, the center of the colony protrudes and bulges, obvious wrinkles are formed, and melanin permeates into the culture medium;
(2) respectively collecting chitosan, butanediol, polyvinylpyrrolidone, fructose, and Na2HPO4/KH2PO4Fully and uniformly mixing the buffer solution and the purified water to obtain the antifreeze agent;
filling the antifreeze agent into a 1.8ml high-low temperature resistant cryopreservation tube, wherein the volume of the antifreeze agent accounts for 1/2 of the volume of the cryopreservation tube, sterilizing at 121 ℃ for 30min, cooling, and standing for a period of time to ensure sterility for later use;
(3) in an ultra-clean workbench, under an aseptic environment, cutting the cordyceps sinensis bacterial colony in the PPDA slant test tube culture medium in the step (1) into a plurality of hypha blocks with the size of 2mm multiplied by 2mm, transferring the hypha blocks into the freezing storage tube containing the antifreeze agent in the step (2), completely immersing the hypha blocks in the antifreeze agent, putting 4 hypha blocks into each freezing storage tube, covering the freezing storage tube with a freezing storage tube cover, and sealing by using a sealing film;
(4) marking on a freezing tube, precooling the freezing tube at-20 ℃ for 36h, and then quickly transferring to the ultralow temperature condition of-80 ℃ for preservation.
Example 5
The embodiment provides a cordyceps sinensis bacterial anti-freezing agent which comprises the following raw material components:
8 parts of xanthan gum, 15 parts of xylitol, 2 parts of sodium dodecyl sulfate, 6 parts of sucrose and NaH2PO4/K2HPO46 parts of buffer solution and 65 parts of purified water.
The method for preserving the cordyceps sinensis strain based on the antifreeze comprises the following steps:
(1) preparing PPDA slant test tube culture medium, sterilizing at 121 deg.C for 30min, standing for a period of time, evaporating water drop and water on the test tube wall, and removing contamination; wherein the pH of the PPDA slant test tube culture medium is 6, and the components comprise:
100 parts of potato;
10 parts of glucose;
peptone, 5 parts by weight;
MgSO41 part by weight;
KH2PO41 part by weight;
18 parts of agar powder;
purified water, 865 parts by weight;
inoculating the mother strain of the cordyceps sinensis to the PPDA slant test tube culture medium, and performing dark culture for 50 days at 17 ℃ to ensure that the diameter of a cordyceps sinensis colony is as long as 2cm, the colony is black brown, the colony is firm, the center of the colony protrudes and bulges, obvious wrinkles are formed, and melanin permeates into the culture medium;
(2) respectively taking xanthan gum, xylitol, sodium dodecyl sulfate, sucrose and NaH2PO4/K2HPO4Fully and uniformly mixing the buffer solution and the purified water to obtain the antifreeze agent;
filling the antifreeze agent into a freezing storage tube, wherein the volume of the antifreeze agent accounts for 7/10 of the volume of the freezing storage tube, sterilizing and cooling for later use;
(3) cutting the cordyceps sinensis bacterial colony in the PPDA slant test tube culture medium in the step (1) into a plurality of hypha blocks with the size of 2mm multiplied by 2mm, transferring the hypha blocks into the freezing storage tube containing the antifreeze agent in the step (2), completely immersing the hypha blocks in the antifreeze agent, putting 4 hypha blocks into each freezing storage tube, covering a freezing storage tube cover, and sealing by using a sealing film;
(4) marking on a freezing tube, precooling the freezing tube at-20 ℃ for 36h, and then quickly transferring to the ultralow temperature condition of-80 ℃ for preservation.
Example 6
The embodiment provides a cordyceps sinensis bacterial anti-freezing agent which comprises the following raw material components:
8 parts of xanthan gum, 15 parts of pentaerythritol, 2 parts of tween-80, 6 parts of trehalose and NaH2PO4/KH2PO46 parts of buffer solution and 65 parts of purified water.
The method for preserving the cordyceps sinensis strain based on the antifreeze comprises the following steps:
(1) preparing PPDA slant test tube culture medium, sterilizing at 121 deg.C for 30min, standing for a period of time, evaporating water drop and water on the test tube wall, and removing contamination; wherein the pH of the PPDA slant test tube culture medium is 6, and the components comprise:
100 parts of potato;
10 parts of glucose;
peptone, 5 parts by weight;
MgSO41 part by weight;
KH2PO41 part by weight;
18 parts of agar powder;
purified water, 865 parts by weight;
inoculating the mother strain of the cordyceps sinensis to the PPDA slant test tube culture medium, and performing dark culture for 50 days at 18 ℃ to ensure that the diameter of a cordyceps sinensis colony is as long as 2cm, the colony is black brown, the colony is firm, the center of the colony protrudes and bulges, obvious wrinkles are formed, and melanin permeates into the culture medium;
(2) respectively taking xanthan gum, pentaerythritol, tween-80, trehalose and NaH2PO4/KH2PO4Fully and uniformly mixing the buffer solution and the purified water to obtain the antifreeze agent;
filling the antifreeze agent into a freezing storage tube, wherein the volume of the antifreeze agent accounts for 7/10 of the volume of the freezing storage tube, sterilizing and cooling for later use;
(3) cutting the cordyceps sinensis bacterial colony in the PPDA slant test tube culture medium in the step (1) into a plurality of hypha blocks with the size of 2mm multiplied by 2mm, transferring the hypha blocks into the freezing storage tube containing the antifreeze agent in the step (2), completely immersing the hypha blocks in the antifreeze agent, putting 4 hypha blocks into each freezing storage tube, covering a freezing storage tube cover, and sealing by using a sealing film;
(4) marking on a freezing tube, precooling the freezing tube at-20 ℃ for 48h, and then quickly transferring to the ultralow temperature condition of-80 ℃ for preservation.
Comparative example 1
Storing Cordyceps strain in 15 wt% glycerol solution, precooling for 36 hr at-20 deg.C, and rapidly storing in-80 deg.C refrigerator for 18 months.
Comparative example 2
Storing Cordyceps strain in 7 wt% trehalose solution, precooling for 36 hr at-20 deg.C, and rapidly storing in-80 deg.C refrigerator for 18 months.
Comparative example 3
Storing Cordyceps strain in 20 wt% sucrose solution, precooling for 36 hr at-20 deg.C, and rapidly storing in-80 deg.C refrigerator for 18 months.
Comparative example 4
The strains of the cordyceps sinensis which are not subjected to preservation treatment are transferred once every 60 days through a PPDA slant test tube culture medium, are cultured at the temperature of 18 ℃, and are continuously transferred to the tenth generation.
Examples of the experiments
After the cordyceps sinensis preserved in the embodiments 1-3 and the comparative examples 1-3 are recovered, the shape comparison test is carried out on the cordyceps sinensis strain switched to the tenth generation in the comparative example 4, and the specific operation is as follows:
the method for recovering the cordyceps sinensis preserved for 18 months in the embodiment 1-3 specifically comprises the following steps: and (3) performing shaking thawing at 18 ℃ until the antifreeze agent is completely thawed, taking out from the cryopreservation tube under the aseptic condition, washing away the antifreeze agent by using sterile purified water, fully absorbing water by using filter paper, inoculating the filter paper to a PPDA culture medium, and culturing for 60 days at 18 ℃.
Recovering the cordyceps sinensis preserved in the comparative examples 1-3, which specifically comprises the following steps: thawing in 18 deg.C constant temperature water bath, quickly shaking, fully sucking water with filter paper, inoculating PPDA culture medium, and culturing at 18 deg.C for 60 d.
The transfer to the tenth generation strain in comparative example 4 was obtained as follows: transferring the ninth generation strain to PPDA culture medium, and culturing at 18 deg.C for 60 d.
TABLE 1 comparison of the shapes of the colonies of Cordyceps sinensis among different treatment groups
As can be seen from table 1, the average recovery rate of the cordyceps sinensis strains preserved by the methods of embodiments 1 to 3 of the present invention is 95%, the center of the hypha colony protrudes and folds are obvious, the metabolic capability of the bacterial cells after recovery is normal, the average colony sizes of 30d and 60d reach 16.3 to 17.2mm and 28.1 to 31.1mm, respectively, and the melanin precipitates in the culture medium are normal; compared with other antifreeze agents (comparative examples 1-3), the antifreeze agent has very remarkable advantages.
In addition, the antifreeze agent of the invention is adopted to prolong the preservation time of the cordyceps sinensis strains to 3 years and 5 years, and the obtained results are consistent with the results in the table 1.
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 (6)
1. A method for preserving cordyceps sinensis strains based on an antifreeze agent is characterized by comprising the following steps:
(1) preparing a PPDA slant test tube culture medium, inoculating a mother strain of cordyceps sinensis to the culture medium after sterilization, and carrying out dark culture at 15-18 ℃ for 40-60 days to enable the diameter of a cordyceps sinensis colony to be 2-3 cm;
(2) respectively taking gelatin sodium alginate, ethylene glycol, sodium dodecyl sulfate, glucose and Na2HPO4/NaH2PO4Fully and uniformly mixing the buffer solution and the purified water to obtain the antifreeze agent; wherein, 10 weight portions of gelatin sodium alginate, 10 weight portions of glycol, 5 weight portions of lauryl sodium sulfate, 5 weight portions of glucose and Na2HPO4/NaH2PO410 parts of buffer solution, 45 parts of purified water,
filling the antifreeze agent into a freezing storage tube, wherein the volume of the antifreeze agent accounts for 1/2-4/5 of the volume of the freezing storage tube, and sterilizing and cooling the antifreeze agent for later use;
(3) cutting the cordyceps sinensis bacterial colony in the PPDA slant test tube culture medium in the step (1) into a plurality of hypha blocks, transferring the hypha blocks into the freezing storage tube containing the antifreeze agent in the step (2), and completely immersing the hypha blocks in the antifreeze agent;
(4) and pre-cooling the freezing tube at-15 to-25 ℃ for 24 to 48 hours, and then quickly transferring to an ultralow temperature condition of-75 to-85 ℃ for preservation.
2. The method according to claim 1, wherein in step (1), the PPDA slant tube culture medium comprises the following components:
100-200 parts by weight of potatoes;
10-20 parts by weight of glucose;
5-15 parts of peptone;
MgSO41-2 parts by weight;
KH2PO41-2 parts by weight;
18-25 parts by weight of agar powder;
736-865 parts by weight of purified water.
3. The method according to claim 2, wherein the pH of the PPDA slant tube medium is 6-6.5.
4. The method according to claim 1, wherein the sterilization is performed at 121 ℃ for 30min in step (1).
5. The method according to claim 1, wherein in the step (2), the sterilization is performed at a temperature of 121 ℃ for a period of 30 min.
6. The method according to claim 1, wherein in step (3), the size of the mycelium block is 2mm x 2 mm.
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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 |
| 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 |
| WO2017066454A3 (en) * | 2015-10-14 | 2017-06-15 | X-Therma, Inc. | Compositions and methods for reducing ice crystal formation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| WO2017066454A3 (en) * | 2015-10-14 | 2017-06-15 | 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 |
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