CN111394263A - Special low-temperature freezing protective agent for cordyceps sinensis strain preservation and use method - Google Patents
Special low-temperature freezing protective agent for cordyceps sinensis strain preservation and use method Download PDFInfo
- Publication number
- CN111394263A CN111394263A CN202010464845.1A CN202010464845A CN111394263A CN 111394263 A CN111394263 A CN 111394263A CN 202010464845 A CN202010464845 A CN 202010464845A CN 111394263 A CN111394263 A CN 111394263A
- Authority
- CN
- China
- Prior art keywords
- cordyceps
- strain
- protective agent
- glycerol
- preservation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000004321 preservation Methods 0.000 title claims abstract description 45
- 239000003223 protective agent Substances 0.000 title claims abstract description 39
- 238000007710 freezing Methods 0.000 title claims abstract description 32
- 230000008014 freezing Effects 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 30
- 241001248610 Ophiocordyceps sinensis Species 0.000 title claims description 29
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerol group Chemical group OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 86
- 241000190633 Cordyceps Species 0.000 claims abstract description 27
- 239000000725 suspension Substances 0.000 claims abstract description 23
- 235000015097 nutrients Nutrition 0.000 claims abstract description 18
- 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 abstract description 10
- 239000008103 glucose Substances 0.000 claims abstract description 10
- 239000004570 mortar (masonry) Substances 0.000 claims abstract description 9
- 239000001888 Peptone Substances 0.000 claims abstract description 8
- 108010080698 Peptones Proteins 0.000 claims abstract description 8
- 229940041514 candida albicans extract Drugs 0.000 claims abstract description 8
- 239000000706 filtrate Substances 0.000 claims abstract description 8
- 235000019319 peptone Nutrition 0.000 claims abstract description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000012138 yeast extract Substances 0.000 claims abstract description 8
- 238000001914 filtration Methods 0.000 claims abstract description 7
- 238000005286 illumination Methods 0.000 claims abstract description 7
- 230000008569 process Effects 0.000 claims description 8
- 238000007789 sealing Methods 0.000 claims description 5
- 238000005138 cryopreservation Methods 0.000 claims description 3
- 239000002577 cryoprotective agent Substances 0.000 claims 4
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 230000001939 inductive effect Effects 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 230000001954 sterilising effect Effects 0.000 claims 1
- 241000233866 Fungi Species 0.000 abstract description 11
- 238000004519 manufacturing process Methods 0.000 abstract description 8
- 239000007788 liquid Substances 0.000 abstract description 7
- 241001264174 Cordyceps militaris Species 0.000 description 20
- 235000011187 glycerol Nutrition 0.000 description 20
- 239000001963 growth medium Substances 0.000 description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000009630 liquid culture Methods 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 241000357408 Ophiocordyceps sobolifera Species 0.000 description 3
- 230000007850 degeneration Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 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 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000426846 Carukia barnesi Species 0.000 description 1
- 241000931705 Cicada Species 0.000 description 1
- KQLDDLUWUFBQHP-UHFFFAOYSA-N Cordycepin Natural products C1=NC=2C(N)=NC=NC=2N1C1OCC(CO)C1O KQLDDLUWUFBQHP-UHFFFAOYSA-N 0.000 description 1
- 241000422920 Cordyceps gunnii Species 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 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 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 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 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 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 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- OFEZSBMBBKLLBJ-BAJZRUMYSA-N cordycepin Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)C[C@H]1O OFEZSBMBBKLLBJ-BAJZRUMYSA-N 0.000 description 1
- OFEZSBMBBKLLBJ-UHFFFAOYSA-N cordycepine Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)CC1O OFEZSBMBBKLLBJ-UHFFFAOYSA-N 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 235000012055 fruits and vegetables Nutrition 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 210000002686 mushroom body Anatomy 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 235000019640 taste Nutrition 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
Classifications
-
- 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
- C12N1/14—Fungi; Culture media therefor
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Botany (AREA)
- Mycology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of fungus strain preservation, and relates to a special low-temperature freezing protective agent for cordyceps strain preservation and a using method thereof.A strain is cultured, the strain is induced to form spores by illumination, hypha is collected to a sterilized mortar, a protective agent is added to the sterilized mortar for grinding, and suspension is prepared, the protective agent consists of two parts, one part is cordyceps nutrient solution, the two parts are glucose 10-20 g, peptone 4-8 g, yeast extract 1-2 g, water is added to a constant volume of 1000 m L, the other part is glycerol in nature pH., the two parts are sterilized respectively, 10% -50% glycerol solution is prepared by the cordyceps nutrient solution before use, a cell sieve is adopted for filtering, filtrate is collected, is subpackaged into a sterile freezing tube, is sealed and is placed in low-temperature freezing preservation, the strain preserved by the protective agent provided by the invention has the effective period of more than 3 years, and can be directly used for production of liquid strains, and the number of subculture is reduced.
Description
Technical Field
The invention belongs to the technical field of fungus strain preservation, and relates to a special low-temperature freezing protective agent for cordyceps strain preservation and a using method thereof.
Background
China is the biggest domestic fungus producing country and export country in the world, the domestic fungus industry has become the fifth major planting industry following grains, oil, fruits and vegetables, and the total yield of domestic fungus is nearly 4000 million tons in China in recent two years. Although the scale of edible fungi is continuously enlarged, the edible fungi in China have a plurality of problems, wherein the most prominent problem is the strain problem. The problems of insufficient provenance, serious strain degeneration, laggard seed production process and the like seriously restrict the development of the industry. Because the traditional seed production technology is carried out by the amplification culture of solid strains, the culture period is long, the dosage of the transferred strains is large, the starting strains can be put into cultivation production only by multiple times of the transfer amplification, and the mutation risk of the strains is also rapidly increased. The quality of the strain is degraded, so that the fresh mushroom tastes worse and worse, the special flavor is weakened, and even the yield of the edible mushroom is seriously influenced, so that the mushroom body is deformed or lost.
Cordyceps militaris (Cordyceps militaris) has high nutritional value, is rich in amino acids and vitamins, and is identified as a new resource food in 2009. The cordyceps militaris contains cordycepin which is a characteristic component of the cordyceps militaris and has a remarkable anti-tumor effect. According to statistics of the edible fungus association in China, the national yield of cordyceps militaris is increased by 400% in 2015, while the yield in 2016 is slightly reduced due to market influence, but the development trend is still very strong, and the yield in 2018 is increased by 13.57% compared with that in 2017. With the continuous development of the production scale of the cordyceps militaris sporocarp and the gradual stable production mode, the cordyceps militaris is accepted by the market and the wide consumers as a new edible strain and starts to be pursued, the cultivation enterprises are continuously increased and expanded, and the social and economic benefits are very obvious. The easy degeneration of cordyceps militaris strains is widely accepted, and a certain research is carried out on the mechanism of degeneration, but a better solution is not found yet. At present, the number of subcultures is mainly reduced as much as possible, and the risk of degradation is reduced. Therefore, whether the preservation method of the cordyceps militaris strain can meet the production requirement is very important.
Many kinds of cordyceps have extremely high utilization value, and the problem of biological resource strain preservation needs to be solved urgently. In addition to the full determination of the medicinal value of the rare Chinese medicinal herb Cordyceps (Cordyceps sinensis), modern scientific researches show that the Cordyceps also belongs to other types of large-scale fungi which have high application values and are homologous in medicine and food, such as cicada fungus, Guni Cordyceps (C. gunnii), Liangshang shanensis (C. liang shannensis), Baens Cordyceps (C. barnesis) and the like. At present, more than 500 kinds of cordyceps are known in the world, and more than 140 kinds of cordyceps exist in China. However, because the growth environment is hidden, the collection difficulty is high, and the restriction of factors such as germplasm resource collection is caused, only a very small proportion of species can be artificially cultured at present. Once the cordyceps species are obtained, the preservation conditions are important factors affecting the viability of the species. As resources increase, the amount of work brought by storage also increases. Due to insufficient hands, the conventional tube-rotating subculture preservation method has the problem that the culture medium is dried up to cause death of strains.
In conclusion, establishing a preservation technology special for cordyceps is an important content for developing the cordyceps industry. The existing strain preservation method mainly comprises the following steps: a periodical successive transfer tube method, a paraffin preservation method, a glycerol preservation method, a freeze-drying method, a liquid nitrogen ultra-low temperature preservation method and the like. The most widely applied method is a periodical subculture pipe transfer method, and the most excellent preservation effect is a liquid nitrogen ultralow temperature method. Each of these methods has advantages and disadvantages. At present, some reports are directed at preservation methods of cordyceps militaris strains, but no report is found on preservation of cordyceps fungus strains.
The invention aims to provide a special low-temperature freezing protective agent for preserving cordyceps sinensis strains and a using method thereof. The protective agent provided by the invention can effectively avoid the harm of low temperature to strains, and the matched use method can make the protective agent better play a role. The preserved strain has the validity period of more than 3 years, can be directly used for the production of liquid strains, does not need activation and propagation, and reduces the subculture times and workload. The innovation of the invention is the following three aspects: 1) adopts a compound protective agent. The low-temperature freezing preservation of the strain is usually performed by using a single protective agent, such as glycerol, glucose, mannitol, trehalose and the like. The invention adopts glycerin as the main material and glucose as the auxiliary material for protection, thereby preventing the ice crystals from damaging hypha cells. 2) The compound protective agent can also meet the requirement of the cordyceps on nutrition in the preservation process. Cordyceps sinensis is a large fungus with a unique life style taking insects as hosts. Both artificial domestication and culture research prove that the cordyceps sinensis has extremely high requirements on a nitrogen source, so that the necessary carbon source and the nitrogen source are added into the protective agent in the strain preservation process, the biological properties of the cordyceps sinensis can be maintained, and the loss of the biological properties of the cordyceps sinensis is avoided. 3) The cordyceps sinensis strain preserved by using the special protective agent can be directly used for culturing liquid strains without activating a rotating pipe again. The strain prepared by the invention contains a large amount of mycelium segments and spores, wherein the spores have strong stress resistance, can provide a large amount of germination points for the culture of liquid strains, and meets the culture requirements of the liquid strains.
The special cordyceps militaris protective agent provided by the invention is a creative labor achievement obtained through a large number of researches and experiments based on various considerations such as cordyceps militaris nutritional characteristics and strain preservation requirements, and has good universality. The invention is not only suitable for the preservation of the cordyceps militaris strains produced commercially, but also can be used for the preservation of scientific research wild cordyceps militaris strain resources.
Disclosure of Invention
The invention aims to provide a special low-temperature freezing protective agent for cordyceps sinensis strain preservation and a using method thereof.
The invention is realized by the following steps:
1. cordyceps strain culture
Cordyceps strains such as Cordyceps militaris, Cordyceps sobolifera, and Cordyceps Guni are inoculated on PDA slant culture medium or PDA plate culture medium, and dark culture is carried out at 15-25 deg.C. When the colony diameter is over 4 cm, the strain is subjected to illumination culture to induce the strain to turn color, reduce the formation of aerial hyphae and promote the formation of spores.
2. Preparation of protective agent
The protective agent consists of two parts, wherein one part is cordyceps militaris nutrient solution and consists of 10-20 g of glucose, 4-8 g of peptone, 1-2 g of yeast extract, water with constant volume of 1000 m L and the other part is glycerol, the two parts are sterilized respectively, 10-50% (m/v) glycerol solution is prepared from the cordyceps militaris nutrient solution under aseptic condition before use, and then the two parts are mixed uniformly.
3. Preparation of suspension of bacterial hypha and spore
In order to increase the effectiveness of the strains, the invention does not adopt the conventional method of strain block preservation, but scrapes all the hyphae of the cultured slant or flat strains as much as possible, collects the hyphae into a sterilized mortar, adds a protective agent, grinds the hyphae into paste, and gradually adds a proper amount of protective agent to prepare strain suspension. In order to improve the strain quality and reduce the number of blocky strains, a cell sieve is adopted for filtration, and filtrate, namely hypha and spore suspension, is collected. Subpackaging the mycelium and spore suspension into sterile cryovials, screwing caps, and sealing for storage. The process is finished in a sterile operation in equipment such as a super clean bench.
4. Low temperature freezing preservation
The freezing tube is frozen and preserved at low temperature by a stage cooling method, namely precooling for 30min at 4 ℃, freezing for 2 h at-20 ℃ and preserving for a long time at-80 ℃. If the temperature is not low at minus 80 ℃, the product can be stored for a long time at minus 20 ℃. The shelf life was 3 years.
Detailed Description
The present invention will be further illustrated with reference to specific embodiments, but the present invention is not limited to the following examples.
Example one
1. Cordyceps militaris strain culture
Inoculating Cordyceps militaris strain in PDA slant culture medium, and dark culturing at 25 deg.C. When the colony diameter is 4 cm, the strain is subjected to illumination culture for 4 d, the strain is induced to turn color, the formation of aerial hyphae is reduced, and the formation of spores is promoted.
2. Preparation of protective agent
The protective agent consists of two parts, namely a cordyceps sinensis liquid culture medium, and the cordyceps sinensis liquid culture medium consists of 10 g of glucose, 4 g of peptone, 1 g of yeast extract, water with constant volume of 1000 m L and glycerol as the other part of natural pH., wherein the cordyceps sinensis nutrient solution is sterilized at 115 ℃ for 30min, the glycerol is sterilized at 121 ℃ for 20 min, and the glycerol is prepared by the cordyceps sinensis nutrient solution under aseptic conditions before use, wherein the mass concentration of the glycerol is 30%.
3. Preparation of suspension of bacterial hypha and spore
The hypha of the cultured slant strain is lightly scraped by an inoculating needle, the hypha collected by two test tubes is transferred to a sterilized mortar, 5 m L protective agent is added, the hypha is ground into paste, 15 m L protective agent is added to prepare strain suspension, a pipettor is used for sucking the suspension to a cell sieve with the aperture of 100 um, the filtration is carried out, filtrate, namely hypha and spore suspension is collected, the hypha and spore suspension is subpackaged into a sterile 5 m L freezing tube, the cover is screwed, the sealing storage is carried out, and the process is finished by carrying out sterile operation in devices such as a clean bench and the like.
4. Low temperature freezing preservation
Freezing the freezing tube filled with the strain suspension at low temperature by stage cooling method, namely precooling at 4 deg.C for 30min, freezing at-20 deg.C for 2 h, and storing at-80 deg.C for a long time.
Example two
1. Culture of cordyceps sobolifera strain
The strain of Cordyceps sobolifera is inoculated on PDA plate culture medium, the diameter of plate is 9 cm, and dark culture is carried out at 25 deg.C. When the colony diameter is 3cm, the strain is subjected to illumination culture for 3 d to promote the formation of spores.
2. Preparation of protective agent
The protective agent consists of two parts, namely cordyceps sinensis nutrient solution, and the cordyceps sinensis nutrient solution consists of 20 g of glucose, 8 g of peptone and 2g of yeast extract, wherein the water volume is fixed to 1000 m L, the other part of natural pH. is glycerol, the two parts are respectively sterilized, the cordyceps sinensis nutrient solution is sterilized at 115 ℃ for 30min, the glycerol is sterilized at 121 ℃ for 20 min, the cordyceps sinensis nutrient solution is used for preparing the glycerol under the aseptic condition before use, and the mass concentration of the glycerol is 40%.
3. Preparation of suspension of bacterial hypha and spore
The hypha of the cultured plate strain is gently scraped by an inoculating needle, the obtained hypha is collected by a plate and is completely transferred to a sterilized mortar, 7 m L protective agent is added, the hypha is ground into paste, 13 m L protective agent is added to prepare strain suspension, a pipettor is used for sucking the suspension to a cell sieve with the aperture of 100 um, the filtration is carried out, the filtrate, namely the hypha and spore suspension, is collected, the hypha and spore suspension is subpackaged into a sterile 5 m L freezing tube, the cover is screwed, the sealing and the preservation are carried out, and the process is finished by carrying out aseptic operation in devices such as a clean bench and the like.
4. Low temperature freezing preservation
And (3) carrying out low-temperature freezing preservation on the freezing pipe according to a stage cooling method, namely precooling for 30min at 4 ℃ and carrying out long-term freezing preservation at-20 ℃.
Example three
1. Culture of Guni cordyceps sinensis
The Guni cordyceps strain is inoculated on a PDA plate culture medium, the diameter of the plate is 6 cm, and dark culture is carried out at 16 ℃. When the colony diameter is 3cm, the strain is subjected to illumination culture, and hyphae are induced to form spores.
2. Preparation of protective agent
The protective agent consists of two parts, namely a cordyceps sinensis liquid culture medium, and the cordyceps sinensis liquid culture medium consists of 18 g of glucose, 5 g of peptone and 1 g of yeast extract, wherein the water volume is fixed to 1000 m L, the other part of natural pH. is glycerol, the two parts are respectively sterilized, the cordyceps sinensis nutrient solution is sterilized at 115 ℃ for 30min, the glycerol is sterilized at 121 ℃ for 20 min, the cordyceps sinensis nutrient solution is used for preparing the glycerol under the aseptic condition before use, and the mass concentration of the glycerol is 35%.
3. Preparation of suspension of bacterial hypha and spore
The hypha of the cultured plate strain is lightly scraped by an inoculating needle, the hypha collected by two plates is completely transferred to a sterilized mortar, 5 m L protective agent is added, the hypha is ground into paste, 15 m L protective agent is added to prepare strain suspension, a pipettor is used for sucking the suspension to a cell sieve with the aperture of 100 um, the filtration is carried out, the filtrate, namely the hypha and spore suspension, is collected, the hypha and spore suspension is subpackaged into a sterile 5 m L freezing tube, the cover is screwed, the sealing storage is carried out, and the process is finished by carrying out aseptic operation in devices such as a clean bench and the like.
4. Low temperature freezing preservation
The freezing tube is frozen and preserved at low temperature by a stage cooling method, namely precooling for 30min at 4 ℃, freezing for 2 h at-20 ℃ and preserving for a long time at-80 ℃.
And (3) analyzing the effect of the protective agent on strain preservation:
comparison was made between two different preservation methods, cryopreservation at 4 ℃ and cryopreservation in glycerol. The test results show that the preservation effect of the compound protective agent containing the nutrient solution and the glycerol is better, and the yield of the cordyceps militaris fruiting body and the yield of the guli-ni cordyceps mycelium are higher than those of the strains preserved at the low temperature of 4 ℃ (shown in the table). Therefore, the effective method for preserving the cordyceps sinensis strains is to use the compound protective agent containing nutrient solution and glycerol to carry out freezing preservation.
Effect of surface protective agent on preservation of two kinds of Cordyceps
Claims (8)
1. A low-temperature freezing protectant special for cordyceps strain preservation is characterized by comprising the following steps of preparing a protectant from 10-20 g of glucose, 4-8 g of peptone and 1-2 g of yeast extract, fixing the volume of water to 1000 m L, keeping the pH value natural, and using glycerol as the other part, sterilizing the two parts respectively, preparing the glycerol into a 10-50% glycerol solution by using the cordyceps nutrient solution before use, inducing strains to form spores by utilizing illumination in the strain culture process, collecting the mycelia to a sterilized mortar, adding the protectant to grind the mycelia to prepare a suspension, filtering by using a cell sieve, collecting filtrate, subpackaging the filtrate into a sterile freezing tube, sealing and placing the sterile freezing tube for low-temperature freezing preservation.
2. The special low-temperature cryoprotectant for cordyceps strain preservation according to claim 1, wherein the cordyceps nutrient solution comprises 10-20 g of glucose, 4-8 g of peptone and 1-2 g of yeast extract, and the volume of water is up to 1000 m L, and the natural pH value is obtained.
3. The cordyceps sinensis nutrient solution composition according to claim 2, wherein the amount of glucose is 20 g, the amount of peptone is 8 g, the amount of yeast extract is 2g, and the water volume is 1000 m L, and the natural pH value is obtained.
4. The special low-temperature cryoprotectant for cordyceps sinensis strain preservation according to claim 1, wherein two components of the cryoprotectant are required to be sterilized respectively.
5. The special low-temperature freezing protective agent for preserving cordyceps sinensis strain according to claim 1, wherein the protective agent is a 10-50% glycerol solution prepared by using cordyceps sinensis nutrient solution and glycerol.
6. The glycerol solution according to claim 5, wherein the glycerol concentration is 30%.
7. The special low-temperature cryoprotectant for cordyceps strain preservation according to claim 1, wherein in the strain culture process, illumination is utilized to induce the strains to form spores, hyphae are collected to a sterilized mortar, a protectant is added to the mortar for grinding, and suspension is prepared.
8. The cryopreservation agent special for cordyceps sinensis strain preservation according to claim 1, wherein a cell sieve is adopted for filtration, and the filtrate is collected.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010464845.1A CN111394263A (en) | 2020-05-28 | 2020-05-28 | Special low-temperature freezing protective agent for cordyceps sinensis strain preservation and use method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010464845.1A CN111394263A (en) | 2020-05-28 | 2020-05-28 | Special low-temperature freezing protective agent for cordyceps sinensis strain preservation and use method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111394263A true CN111394263A (en) | 2020-07-10 |
Family
ID=71430028
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010464845.1A Pending CN111394263A (en) | 2020-05-28 | 2020-05-28 | Special low-temperature freezing protective agent for cordyceps sinensis strain preservation and use method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111394263A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115838635A (en) * | 2022-10-08 | 2023-03-24 | 江西国药有限责任公司 | Long-term preservation method of paecilomyces hepiali Cs-4 strain for fermenting cordyceps sinensis powder |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103283493A (en) * | 2013-06-28 | 2013-09-11 | 广东星河生物科技股份有限公司 | Large-scale cultivation method for Guangdong fruiting bodies of cordyceps military |
| CN106983134A (en) * | 2017-04-10 | 2017-07-28 | 北华大学 | The preparation method of northern Chinese caterpillar Fungus bacterium freeze-dried powder |
| CN107955794A (en) * | 2017-11-27 | 2018-04-24 | 沈阳农业大学 | The high-quality method for preserving of Cordyceps militaris spawn |
-
2020
- 2020-05-28 CN CN202010464845.1A patent/CN111394263A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103283493A (en) * | 2013-06-28 | 2013-09-11 | 广东星河生物科技股份有限公司 | Large-scale cultivation method for Guangdong fruiting bodies of cordyceps military |
| CN106983134A (en) * | 2017-04-10 | 2017-07-28 | 北华大学 | The preparation method of northern Chinese caterpillar Fungus bacterium freeze-dried powder |
| CN107955794A (en) * | 2017-11-27 | 2018-04-24 | 沈阳农业大学 | The high-quality method for preserving of Cordyceps militaris spawn |
Non-Patent Citations (1)
| Title |
|---|
| 方苏等: "冬虫夏草菌种保藏方法的比较", 《安徽农业科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115838635A (en) * | 2022-10-08 | 2023-03-24 | 江西国药有限责任公司 | Long-term preservation method of paecilomyces hepiali Cs-4 strain for fermenting cordyceps sinensis powder |
| CN115838635B (en) * | 2022-10-08 | 2023-08-22 | 江西国药有限责任公司 | Long-term preservation method of paecilomyces hepiali Cs-4 strain for fermented cordyceps sinensis powder |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111534439B (en) | Penicillium oxalicum SDF-25 strain and application thereof | |
| CN106399132A (en) | Irpex lacteus and application thereof | |
| CN113396934B (en) | Preparation method of bacillus preparation for promoting crop growth | |
| CN109699394B (en) | Method for producing xylaria longilineans sporocarp by using liquid culture medium | |
| CN110741877A (en) | Method for ultralow-temperature cryopreservation and recovery of needle mushroom sterile fruiting bodies | |
| CN111394263A (en) | Special low-temperature freezing protective agent for cordyceps sinensis strain preservation and use method | |
| CN111139206B (en) | Growth-promoting compound endophytic bacteria agent and application thereof | |
| CN110846262B (en) | Serratia marcescens SZ201 and application thereof | |
| CN111527989B (en) | Culture medium of Firmiana hirsuta, artificial cultivation method and application | |
| CN108148769A (en) | A kind of room temperature preserves culture medium of Arthrinium fungi strain and its preparation method and application | |
| CN112280730A (en) | Method for purifying and rejuvenating mushroom strains | |
| CN109266553A (en) | A kind of freeze drying protectant, the method and application that Pu'er tea direct putting type freeze-drying microbial inoculum is prepared with it | |
| CN109749940B (en) | Preparation method of fungal inoculant, product and application thereof | |
| CN114958613B (en) | Trichoderma spiral RS05 and application thereof in preventing and treating brown rot of hemp and bamboo | |
| CN110218657A (en) | One plant of long shoot trichoderma MD30 and its biological organic fertilizer of development | |
| CN116496909A (en) | Endophytic fungus sessile spore shell fungus and application thereof in dendrobium candidum cultivation | |
| CN102533561A (en) | Protecting agent for freezing preservation of edible fungus strain and use method | |
| CN112655561B (en) | Method for in vitro preservation of red bud taro test-tube plantlet | |
| CN110846234B (en) | Pleurotus cornucopiae culture medium | |
| CN101240241B (en) | Method for preserving stropharia rugoso-annulata strain | |
| CN103667062A (en) | Protective agent for low-temperature preservation of asexual spores of antrodia cinnamomea and protective agent using method | |
| CN110089388A (en) | A kind of Rhizoma Gastrodiae soilless culture substrate | |
| CN116491366B (en) | Ganoderma lucidum cultivation method capable of reducing heavy metal ion enrichment | |
| CN114907981B (en) | Preservation method of solid microbial inoculum of Pantoea ananatis | |
| CN113604362B (en) | Method for improving spore production of Piriformospora indica |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200710 |