CN110616148A - Application of trehalose as protective agent in liquid nitrogen cryopreservation of edible fungi - Google Patents
Application of trehalose as protective agent in liquid nitrogen cryopreservation of edible fungi Download PDFInfo
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 title claims abstract description 94
- 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 title claims abstract description 52
- 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 title claims abstract description 52
- 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 title claims abstract description 52
- 239000007788 liquid Substances 0.000 title claims abstract description 48
- 239000003223 protective agent Substances 0.000 title claims abstract description 48
- 229910052757 nitrogen Inorganic materials 0.000 title claims abstract description 47
- 241000233866 Fungi Species 0.000 title claims abstract description 29
- 238000005138 cryopreservation Methods 0.000 title claims abstract description 27
- 240000000588 Hericium erinaceus Species 0.000 claims abstract description 8
- 235000007328 Hericium erinaceus Nutrition 0.000 claims abstract description 8
- 241000221638 Morchella Species 0.000 claims abstract description 4
- 240000001462 Pleurotus ostreatus Species 0.000 claims description 18
- 235000001603 Pleurotus ostreatus Nutrition 0.000 claims description 18
- 240000000599 Lentinula edodes Species 0.000 claims description 10
- 240000001080 Grifola frondosa Species 0.000 claims description 9
- 235000007710 Grifola frondosa Nutrition 0.000 claims description 9
- 241000318836 Pleurotus nebrodensis Species 0.000 claims description 7
- 240000006794 Volvariella volvacea Species 0.000 claims description 7
- 241000222336 Ganoderma Species 0.000 claims description 5
- 235000001715 Lentinula edodes Nutrition 0.000 claims description 5
- 241000221377 Auricularia Species 0.000 claims description 3
- 235000004501 Volvariella volvacea Nutrition 0.000 claims description 3
- 241000222350 Pleurotus Species 0.000 claims 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 abstract description 55
- 235000001674 Agaricus brunnescens Nutrition 0.000 abstract description 6
- 210000004027 cell Anatomy 0.000 abstract description 3
- 230000001681 protective effect Effects 0.000 abstract description 3
- 238000007710 freezing Methods 0.000 description 51
- 230000008014 freezing Effects 0.000 description 51
- 238000000034 method Methods 0.000 description 20
- 238000004321 preservation Methods 0.000 description 19
- 235000011187 glycerol Nutrition 0.000 description 18
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- 239000001963 growth medium Substances 0.000 description 9
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- 244000307697 Agrimonia eupatoria Species 0.000 description 6
- 235000000125 common agrimony Nutrition 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 235000007685 Pleurotus columbinus Nutrition 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 238000005057 refrigeration Methods 0.000 description 5
- 241000222532 Agrocybe Species 0.000 description 4
- 240000002769 Morchella esculenta Species 0.000 description 4
- 235000002779 Morchella esculenta Nutrition 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 240000006499 Flammulina velutipes Species 0.000 description 3
- 235000016640 Flammulina velutipes Nutrition 0.000 description 3
- 240000008397 Ganoderma lucidum Species 0.000 description 3
- 235000001637 Ganoderma lucidum Nutrition 0.000 description 3
- 241000219238 Pleurotus abalonus Species 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 235000014102 seafood Nutrition 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
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- 241000143060 Americamysis bahia Species 0.000 description 1
- 244000028550 Auricularia auricula Species 0.000 description 1
- 235000000023 Auricularia auricula Nutrition 0.000 description 1
- 241001474374 Blennius Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 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 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000228347 Monascus <ascomycete fungus> 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
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000254105 Tenebrio Species 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 235000013405 beer Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002826 coolant Substances 0.000 description 1
- 239000002577 cryoprotective agent Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 235000021107 fermented food Nutrition 0.000 description 1
- 238000009920 food preservation Methods 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
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- 239000003381 stabilizer Substances 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
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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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Abstract
The invention discloses an application of trehalose as a protective agent in liquid nitrogen cryopreservation of edible fungi, in particular an application of 5-10% of trehalose as a protective agent in liquid nitrogen cryopreservation of edible fungi, and in particular an application of 10% of trehalose as a protective agent in liquid nitrogen cryopreservation of morchella strains, hericium erinaceus strains, abalone mushroom strains and agaric strains. The invention has the advantages that the characteristic that trehalose can form a protective film on the cell surface is fully utilized, the trehalose is used as a protective agent for liquid nitrogen cryopreservation of edible fungus strains, the strain types which can be cryopreserved by taking the trehalose as the protective agent are far more than those of the traditional glycerol protective agent, and the invention is suitable for various strains.
Description
Technical Field
The invention relates to the field of edible fungus liquid nitrogen preservation, in particular to application of trehalose as a protective agent in edible fungus liquid nitrogen cryopreservation.
Background
In the field of microorganisms, both basic scientific research and application research of biotechnology need to ensure the quality and activity of strains, and the application value of microorganisms is continuously increased by researching and mining the application value of the microorganisms. The edible fungi are large-scale fungi which can be eaten by human beings, the variety is more than 2000, and the edible fungi which can be artificially cultivated in a large area have more than 60. Because edible fungi are easy to pollute, mutate and even die in the using and passage processes, strains are degenerated and even good strains are lost, the strains are required to be preserved frequently to keep the original characters and stable activity of the strains as much as possible, ensure that the strains are not dead, mutated and polluted, keep the good characters of the strains not degenerated and keep the good strains alive so as to meet the requirements of various aspects of research, exchange, use and the like.
At present, two preservation methods of edible fungus strains mainly comprise a hypha preservation method and a spore preservation method, and the hypha preservation method is commonly used in actual production due to the fact that the spore preservation method is complex. The hypha preservation method comprises a periodical transplantation method, a mineral oil sealing preservation method, a monascus hypha liquid preservation method, a glycerin low-temperature preservation method, a freezing vacuum drying method, a liquid nitrogen ultralow-temperature freezing method and the like. The liquid nitrogen ultra-low temperature freezing method is that the preserved strain is sealed in a freezing tube, a freezing protective agent is added, the freezing tube is pre-cooled to-40 ℃ and then is put into a liquid nitrogen tank at-196 ℃ to realize the long-term preservation of the strain.
The liquid nitrogen ultra-low temperature freezing method can be applied to various microorganisms, so the method is adopted by domestic and foreign preservation institutions. For the liquid nitrogen low temperature freezing method, the survival rate of each strain after freezing preservation is required to be more than or equal to 80 percent. The cryoprotectant is an important factor influencing the freezing survival rate of the strain, glycerol is a commonly used protectant for freezing edible strain liquid nitrogen, the strain can be placed in a liquid nitrogen tank after the temperature is reduced by a program during the preservation, otherwise the survival rate of the strain cannot be ensured at all. Meanwhile, the glycerol protective agent is suitable for a few strains, and the liquid nitrogen cryopreservation requirements of different strains are difficult to meet.
Trehalose is a non-reducing sugar composed of two glucose molecules by 1, 1-glycosidic bonds, is a safe and reliable natural sugar, widely exists in animals and plants and microorganisms, and has a uniform and high content in mushrooms, seaweed, beans, dried shrimps, beer and yeast fermented food which are eaten by people in daily life. Trehalose can form a unique protective film on the cell surface under the severe environmental conditions of high temperature, high osmotic pressure, drying and water loss and the like, and effectively protects protein molecules from being inactivated without denaturation, thereby maintaining the vital characteristics of organisms, so that the trehalose is widely used in the fields of food preservation technology and medical treatment, for example, trehalose solution is sprayed on seafood for freezing and storing at the temperature of-16 ℃, the hardness and the flexibility of the seafood can be ensured, the taste of the seafood can be improved, and for example, trehalose is used as a stabilizer of biological reagents such as diagnostic tool enzyme and the like, and the diagnostic tool enzyme can be dried and stored under the normal temperature condition or the high temperature condition (70 ℃). However, trehalose as a protective agent for preserving edible fungus liquid nitrogen in a freezing way is not reported at home and abroad.
Disclosure of Invention
The invention aims to provide a new application of trehalose aiming at the defects in the prior art: namely the application of trehalose as a protective agent in the aspect of liquid nitrogen cryopreservation of edible fungi.
Further, the present invention also provides:
the application of 5-10% trehalose as a protective agent in the liquid nitrogen cryopreservation of edible fungi.
The application of 5% trehalose and 10% trehalose as protective agents in liquid nitrogen cryopreservation of Pleurotus Ostreatus strains.
The 10% trehalose is used as protective agent in liquid nitrogen cryopreservation of Morchella esculenta strains, Hericium erinaceus strains, Pleurotus abalonus strains and Auricularia auricular strains.
The 10% trehalose is used as protective agent in liquid nitrogen cryopreservation of Lentinus Edodes strains, Volvariella volvacea strains, Pleurotus nebrodensis strains, Ganoderma strains and Grifola frondosa strains.
When in use, the edible fungus strain to be preserved is put into a freezing tube, trehalose is added into the freezing tube, and the freezing tube is directly put into a liquid nitrogen tank at the temperature of 196 ℃ below zero for freezing preservation after being sealed by a cover.
The invention has the advantages that the characteristic that trehalose can form a protective film on the cell surface is fully utilized, the trehalose is used as a protective agent for liquid nitrogen cryopreservation of edible fungus strains, the strain types which can be cryopreserved by taking the trehalose as the protective agent are far more than those of the traditional glycerol protective agent, and the invention is suitable for various strains. Specifically, the trehalose as a protective agent is not subjected to program cooling, can be suitable for strains such as oyster mushrooms, agaric, needle mushrooms, hericium erinaceus, morchella and the like, and omits the program cooling step compared with the traditional glycerol protective agent; the trehalose as a protective agent is particularly suitable for the cryopreservation of strains such as shiitake mushroom, straw mushroom, ganoderma lucidum, grifola frondosa and the like after programmed cooling and freezing.
Detailed Description
The present invention is described in detail below with reference to specific examples, wherein trehalose and glycerol are commercially available, and 10% glycerol and trehalose at different concentrations are formulated with water.
Example 1 application of trehalose as protectant to cryopreservation of Pleurotus Ostreatus hyphae
The invention takes Heiping 16-1 (provided by edible fungus research and development center of agricultural institute of Henan province), Xin831 (provided by edible fungus research and development center of agricultural institute of Henan province), Heiji 650 (provided by edible fungus research and development center of agricultural institute of Henan province), Su quote No. 6 (provided by edible fungus research and development center of agricultural institute of Henan province) and No. 4 (provided by edible fungus research and development center of agricultural institute of Henan province) five oyster mushroom strains as reference strains, and examines the influence of trehalose as a protective agent on the survival rate of the oyster mushroom strains after liquid nitrogen cryopreservation, and comprises the following steps:
step one, water is used as a contrast, three protective agents of 10% glycerol, 5% trehalose and 10% trehalose are prepared respectively, the prepared protective agents are subjected to high-pressure sterilization, and then the protective agents are cooled to room temperature for standby;
secondly, respectively inoculating hypha of each oyster mushroom strain into a PDA culture medium, culturing at 25 ℃ until the hypha is full of the dish, taking 100 mother seed hypha blocks on the PDA culture medium by using a hole puncher (the diameter is 0.5 cm), respectively putting the mother seed hypha blocks of each oyster mushroom strain into 20 freezing tubes (5 mother seed hypha blocks are put into each freezing tube), setting 5 freezing tubes as a group, respectively adding a protective agent in the first step into each group, covering and sealing the groups, and respectively adding 850uL of the protective agent;
thirdly, directly placing the sealed freezing pipe into a liquid nitrogen tank at the temperature of 196 ℃ below zero for low-temperature refrigeration for 6 months;
and fourthly, after refrigeration, taking out the freezing tubes from the liquid nitrogen tank, thawing the freezing tubes in a water bath at 37 ℃ for 10min, standing the freezing tubes at 25 ℃ for 24h, respectively inoculating hyphae in each freezing tube into a PDA culture medium, inoculating 5 dishes into each freezing tube, culturing the freezing tubes at 25 ℃, observing the growth vigor of the pleurotus ostreatus hyphae, wherein the growth vigor of the hyphae is represented by "+", the white growth vigor of the hyphae is better, the "+" hyphae is good, the "+" hyphae is poor in growth vigor, and the survival rate of each pleurotus ostreatus strain is shown in table 1.
TABLE 1 Effect of various protectants on the survival of Pleurotus ostreatus strains
The result shows that 10% of trehalose is used as a protective agent, and is directly frozen and preserved by liquid nitrogen without program cooling, the survival rate of hyphae of different oyster mushroom strains is more than or equal to 85%, the requirement of the liquid nitrogen frozen preservation of the oyster mushroom strains is met, the program cooling step is omitted, and the preservation step is simplified; 5% trehalose as a protective agent can be used for liquid nitrogen cryopreservation of four oyster mushroom strains except for Heijia nobilis 650 without programmed cooling; the glycerol is used as a protective agent, the oyster mushroom strains are directly frozen and preserved by liquid nitrogen without a temperature reduction procedure, and the survival rate of oyster mushroom hyphae is less than or equal to 50 percent, so the glycerol used as the protective agent cannot be used for directly freezing and preserving the oyster mushroom strains; while the survival rate of five oyster mushroom strains only added with water is zero.
Example 2 nine strains of Auricularia (Auricularia auricula 1, provided by Agrimony and sciences of Heilongjiang province), Ganoderma (Huangshan No. 8, provided by Agrimony and sciences research and development center of Henan province), Lentinus edodes (Lentinus edodes 856, provided by Agrimony and sciences research and development center of Henan province), Flammulina velutipes (gold 19, provided by Agrimony and sciences research and development center of Henan province), Morchella esculenta (Morchella esculenta 1, provided by Agrimony and sciences of Sichuan province), Pleurotus abalonus (Pleurotus abalonus 02, provided by Agrocybe and research and development center of Henan province), Grifola frondosa (Grifola frondosa 1, provided by Agrocybe research and development center of Henan province), Pleurotus nebrodensis (Tenebrio velutipes (Teng 2, provided by Agrocybe research and development center of Henan province and sciences) and Hericium erinaceus (Henan provinc, the method for investigating the influence of 10% trehalose as a protective agent on the survival rate of the nine strains after liquid nitrogen cryopreservation comprises the following steps:
step one, 10% of glycerol and 10% of trehalose are prepared respectively, the protective agent is sterilized under high pressure after the preparation is finished, and then the protective agent is cooled to room temperature for standby;
secondly, respectively inoculating agaric, ganoderma lucidum, shiitake mushroom, flammulina velutipes, morchella esculenta, abalone mushroom, grifola frondosa, pleurotus nebrodensis and hericium erinaceus into a PDA culture medium, culturing at 25 ℃ until mycelia grow over a culture dish, taking mother strain mycelia blocks of each strain from the culture medium by using a 0.5cm puncher, respectively subpackaging the mother strain mycelia blocks of each strain into 20 aseptic freezing tubes (5 mother strain mycelia blocks are filled in each freezing tube), grouping every 10 aseptic freezing tubes, adding 850uL of 10% glycerol into one group of freezing tubes and 850uL of 10% trehalose into the other group of freezing tubes, and sealing by covering;
thirdly, directly placing the sealed freezing pipe into a liquid nitrogen tank at the temperature of 196 ℃ below zero for low-temperature refrigeration for 4-12 months;
fourthly, after refrigeration, the freezing tubes are taken out of the liquid nitrogen tank, water bath at 37 ℃ is carried out for thawing for 10min, then standing is carried out for 24h at 25 ℃, hyphae in each freezing tube are respectively inoculated in a PDA culture medium, each freezing tube is inoculated with 5 dishes and cultured at 25 ℃, the hypha growth vigor of each strain is observed, the hypha growth vigor is represented by "+", the "+" indicates that the hyphae have good white growth vigor, the "+" hyphae have good growth vigor, and the "+" hyphae have poor growth vigor, and the survival rate of each strain is shown in table 2.
TABLE 2 influence of trehalose on the survival of different edible bacterial strains
The result shows that the 10% trehalose serving as the protective agent can be used for liquid nitrogen cryopreservation of agaric, hericium erinaceus, abalone mushroom, flammulina velutipes and morchella without program cooling, and the survival rate of the mycelia after the liquid nitrogen cryopreservation is 100%; the 10% glycerol protective agent can be directly used for liquid nitrogen cryopreservation of three strains of hericium erinaceus, abalone mushroom and agaric without programmed cooling, and is less in applicable strains.
Example 3 the present invention uses ganoderma lucidum (agrimony 1-1, provided by the academy of agricultural sciences of the Henan province; Huangshan 8, Hongling, provided by the academy of agricultural sciences edible fungi research and development center of the Henan province, respectively), shiitake mushroom (shiitake 856, Xiang087, Yuhua 3, and L808, provided by the academy of agricultural sciences of the Henan province, and the same is provided by the academy of agricultural sciences, respectively), pleurotus nebrodensis (Pleurotus nebrodensis 2, and Tianshan 2, provided by the academy of agricultural sciences edible fungi research and development center of the Henan province, and Zhongnong 1, provided by the academy of agricultural sciences), volvariella volvacea (V23, V34, Nongcao 1, Agrocybe Volvaria virginiana, and V58, provided by the academy of agricultural sciences edible fungi research and development center of the Henan, respectively) as ginseng test strains, and investigate the effects of temperature reduction on the, the method specifically comprises the following steps:
step one, respectively preparing 10% of glycerol (contrast), 10% of trehalose and a mixed protective agent (volume ratio is 1: 1) of 10% of glycerol and 10% of trehalose, carrying out high-pressure sterilization on the three protective agents after the preparation is finished, and then cooling to room temperature for later use;
secondly, respectively inoculating hypha of three strains of shiitake mushroom, straw mushroom, lucid ganoderma and grifola frondosa into a PDA culture medium, culturing at 25 ℃ until the hypha grows over a culture dish, taking a mother strain hypha block of each strain from the culture medium by using a 0.5cm puncher, respectively subpackaging the mother strain hypha block of each strain into 30 sterile freezing tubes (5 mother strain hypha blocks are filled in each freezing tube), wherein each 10 sterile freezing tubes form a group, and each group of freezing tubes are respectively added with a protective agent in the first step;
thirdly, cooling the sealed freezing pipe by using a program cooling instrument, taking liquid nitrogen as a cooling medium, cooling the freezing pipe to 1 ℃ per minute, cooling the freezing pipe to-40 ℃, then putting the freezing pipe into a liquid nitrogen tank at-196 ℃, and freezing, storing and refrigerating for 6 months;
fourthly, after refrigeration, the freezing tubes are taken out of the liquid nitrogen tank, unfreezing is carried out for 10min in a water bath at 37 ℃, standing is carried out for 24h at 25 ℃, hyphae in each freezing tube are respectively inoculated into a PDA culture medium, the hyphae in each freezing tube are inoculated into 5 dishes, culture is carried out at 25 ℃, the hyphae growth vigor of each strain is observed, the hyphae growth vigor is represented by "+", the hyphae growth vigor is better in pure white, the "+" hyphae growth vigor is better, the "+" hypha growth vigor is poor, and the survival rate of each strain is shown in a table 3.
TABLE 3 Effect of temperature programmed reduction on the survival of various strains
The result shows that the 10% trehalose is used as a protective agent and is combined with program cooling, five strains of the shiitake mushroom, the straw mushroom, the lucid ganoderma, the grifola frondosa and the pleurotus nebrodensis can be cold-preserved, and the 10% glycerin is used as the protective agent and still cannot be used for liquid nitrogen freezing preservation of the straw mushroom and the grifola frondosa after the program cooling.
As can be seen from examples 1-3 of the present invention, when the edible fungus strain is cryopreserved by using liquid nitrogen, the strain types suitable for trehalose as a protective agent are significantly higher than those of glycerol, and the preservation effect of trehalose as a protective agent for cryopreserving the edible fungus strain is significantly better than that of glycerol.
Claims (5)
1. The trehalose is used as a protective agent in the liquid nitrogen cryopreservation of edible fungi.
2.5 to 10 percent of trehalose is used as a protective agent in the liquid nitrogen cryopreservation of edible fungi.
The application of 3.5% trehalose and 10% trehalose as protective agents in the liquid nitrogen cryopreservation of oyster mushroom strains.
The application of 4.10% trehalose as protectant in liquid nitrogen cryopreservation of Morchella strains, Hericium Erinaceus strains, Pleurotus Haliotidis strains and Auricularia auricular strains.
The application of 5.10% trehalose as protectant in liquid nitrogen cryopreservation of Lentinus Edodes strains, Volvariella volvacea strains, Pleurotus nebrodensis strains, Ganoderma strains and Grifola frondosa strains.
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| CN111982629A (en) * | 2020-08-25 | 2020-11-24 | 北京市农林科学院 | Ultrathin freezing slicing method for edible mushroom tissue cells |
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