CN114591842A - Method for preserving aspergillus flavus strain by using aspergillus flavus sclerotium - Google Patents
Method for preserving aspergillus flavus strain by using aspergillus flavus sclerotium Download PDFInfo
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- CN114591842A CN114591842A CN202210348298.XA CN202210348298A CN114591842A CN 114591842 A CN114591842 A CN 114591842A CN 202210348298 A CN202210348298 A CN 202210348298A CN 114591842 A CN114591842 A CN 114591842A
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- 241000228197 Aspergillus flavus Species 0.000 title claims abstract description 55
- 241001558929 Sclerotium <basidiomycota> Species 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 19
- 241000576755 Sclerotia Species 0.000 claims abstract description 17
- 238000004321 preservation Methods 0.000 claims abstract description 13
- 238000001035 drying Methods 0.000 claims abstract description 6
- 230000004936 stimulating effect Effects 0.000 claims abstract description 5
- 238000003860 storage Methods 0.000 claims abstract description 4
- 238000005406 washing Methods 0.000 claims abstract description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 14
- 239000002609 medium Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 238000007789 sealing Methods 0.000 claims description 5
- 239000002655 kraft paper Substances 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 239000000123 paper Substances 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 2
- 230000002068 genetic effect Effects 0.000 abstract description 3
- 238000011081 inoculation Methods 0.000 abstract description 3
- 235000011187 glycerol Nutrition 0.000 description 4
- 239000005662 Paraffin oil Substances 0.000 description 3
- 239000004576 sand Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 239000003708 ampul Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005086 pumping Methods 0.000 description 2
- DLYUQMMRRRQYAE-UHFFFAOYSA-N tetraphosphorus decaoxide Chemical compound O1P(O2)(=O)OP3(=O)OP1(=O)OP2(=O)O3 DLYUQMMRRRQYAE-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 235000011868 grain product Nutrition 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012546 transfer 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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- 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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/85—Food storage or conservation, e.g. cooling or drying
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- General Engineering & Computer Science (AREA)
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Abstract
The invention discloses a method for preserving aspergillus flavus strain by using aspergillus flavus sclerotium, which mainly comprises the following steps: s1: stimulating the generation of aspergillus flavus sclerotia; s2: collecting, washing and drying aspergillus flavus sclerotium; s3: preserving aspergillus flavus sclerotium; the aspergillus flavus sclerotium has stronger stress resistance and stable genetic background, is suitable for long-time storage and is not easy to mutate; by preserving the sclerotium of the aspergillus flavus, the problems that the strain is easy to mutate and the environment pollution is easily caused in the preservation and inoculation processes are effectively solved, so that the preservation method of the aspergillus flavus is simple, convenient and effective, and the genetic stability of the strain is realized.
Description
Technical Field
The invention belongs to the technical field of aspergillus flavus preservation, and particularly relates to a method for preserving aspergillus flavus strains by using aspergillus flavus sclerotium.
Background
Aspergillus flavus, a fungi imperfecti, is a common saprophytic fungus; the fertilizer is mostly found on mildewed grains, grain products and other mildewed organic matters; the aspergillus flavus colony has the advantages of fast growth and reproduction, loose structure, grayish green surface and colorless or slightly brown back surface; the method for preserving the aspergillus flavus mainly comprises the following steps: 1) a sand preservation method: mixing sand and soil at a ratio of 3:2, treating with dilute acid, cleaning, sieving, and placing into a small test tube with a height of 1 cm; sterilizing for 2-3 times, drying to obtain spore suspension, adding 2-2.5 ml of sterile distilled water into spore suspension, sucking a little, adding into sand tube, vacuum pumping to obtain loose appearance, and storing in refrigerator at 4 deg.C for 5-7 years; 2) freeze-drying, mixing spore suspension with protectant (generally skimmed milk or serum), and quick-freezing in ampoule in ethanol bath at-20 deg.C to-30 deg.C; then, pumping dry by a vacuum pump at low temperature, freezing water vapor by phosphorus pentoxide or dry ice, sealing the ampoule tube by melting, and storing at low temperature; the preservation period can be as long as 5 to 10 years; 3) the paraffin oil sealing method, pour the sterilized paraffin oil on the strain culture of the inclined plane, higher than the inclined plane by 1cm, preserve in the refrigerator or low-temperature dry place of 4 duC, this method is not suitable for the microorganism that can utilize the paraffin oil as carbon source, the retention period is more than one year; 4) preservation with filter paper sheets;
because the yield of the aspergillus flavus is large, the aspergillus flavus spores are easy to diffuse to pollute the environment during and after the inoculation in the preservation process; the sclerotium produced by the aspergillus flavus has stronger stress resistance and stable heredity, is suitable for long-time storage and is not easy to mutate.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for preserving aspergillus flavus strains by utilizing aspergillus flavus sclerotia, which comprises the steps of stimulating aspergillus flavus to generate sclerotia, processing the sclerotia, preserving the sclerotia of the aspergillus flavus independently, and culturing and propagating the sclerotia in the later period to generate the aspergillus flavus;
in order to achieve the technical purpose, the invention is realized by the following technical scheme: a method for preserving Aspergillus flavus strains by using Aspergillus flavus sclerotia is characterized by comprising the following steps:
s1: stimulating the production of aspergillus flavus sclerotia: inoculating the separated yellow mould on a PSA culture medium, and culturing for 12-15 days at 30-32 ℃ to induce and stimulate the yellow mould to generate sclerotia;
s2: collecting Aspergillus flavus sclerotium produced in S1, washing the sclerotium with sterile water until no large amount of spores are attached on the surface; absorbing the water on the surface of the dry sclerotium by using filter paper, and drying in a constant-temperature oven at 36-38 ℃ for 36-40 hours;
s3: putting the washed and dried aspergillus flavus sclerotium in the S2 into a storage medium for sealing and storing under the ultralow temperature condition;
preferably, the preservation medium is kraft paper or glycerol;
preferably, the ultra-low temperature is-80 ℃.
The invention has the beneficial effects that:
the invention stimulates aspergillus flavus to generate sclerotium by induction, the sclerotium is washed and dried, then is put into glycerol or sealed by kraft paper, and is preserved in an ultralow temperature environment; the method effectively solves the problems that the strain is easy to mutate and the environmental pollution is easily caused in the process of preservation and inoculation, so that the preservation method of the aspergillus flavus is simple, convenient and effective, and the genetic stability of the strain is realized.
Drawings
FIG. 1 is a sclerotium produced and grown by Aspergillus flavus of the present invention on a PSA medium;
FIG. 2 is a microscopic view of Aspergillus flavus sclerotia of the present invention;
FIG. 3 shows Aspergillus flavus sclerotium of the present invention preserved in glycerin and stored in a-80 deg.C ultra-low temperature refrigerator;
FIG. 4 shows the nuclear subculture of Aspergillus flavus of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
S1: stimulating the production of aspergillus flavus sclerotium: inoculating the separated yellow mould on a PSA culture medium, and culturing at 30 ℃ for 12-15 days to induce and stimulate the yellow mould to generate sclerotium;
as shown in figure 1, after Aspergillus flavus is cultured in a PSA culture medium in a 30 ℃ constant temperature incubator for 3 days, sclerotia appears on the surface of the culture medium, the sclerotia is the growth stage of the Aspergillus flavus, mycelium is continuously differentiated and intertwined with each other to form a black brown and hard mycelium tissue which is a dormant body formed by pseudoparenchyma and mitochondrion tissue. The function of sclerotia is to store nutrients and help aspergillus flavus to go through adverse environmental conditions, etc. After 14 days of culture, the sclerotium is mature, the aspergillus flavus sclerotium is collected and washed, and the aspergillus flavus spores on the surface of the sclerotium are cleaned, so that the subsequent sclerotium preservation is facilitated;
s2: collecting Aspergillus flavus sclerotium produced in S1, washing and drying;
as shown in fig. 2, the cleaned sclerotium is placed in a constant temperature oven at 36 ℃ for drying for 36 hours to remove the moisture of the sclerotium, so as to be beneficial to the preservation of the sclerotium;
s3: placing the washed and dried aspergillus flavus sclerotium in the S2 into a centrifugal tube filled with 70% of glycerol for sealing and preserving at the ultralow temperature of-80 ℃; as shown in fig. 3
FIG. 4 is an example of culturing Aspergillus flavus stored at-80 ℃ for 4 days by nuclear transfer on a PSA medium.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Claims (3)
1. A method for preserving Aspergillus flavus strains by using Aspergillus flavus sclerotia is characterized by comprising the following steps:
s1: stimulating the production of aspergillus flavus sclerotium: inoculating the separated yellow mould on a PSA culture medium, and culturing for 12-15 days at 30-32 ℃ to induce and stimulate the yellow mould to generate sclerotia;
s2: collecting Aspergillus flavus sclerotium produced in S1, washing the sclerotium with sterile water until no large amount of spores are attached on the surface; absorbing the water on the surface of the dry sclerotium by using filter paper, and drying in a constant-temperature oven at 36-38 ℃ for 36-40 hours;
s3: and (4) putting the washed and dried aspergillus flavus sclerotium in the S2 into a storage medium for sealing and storing under the ultralow temperature condition.
2. The method for preserving Aspergillus flavus species using Aspergillus flavus sclerotia as claimed in claim 1, wherein the preservation medium is kraft paper or glycerol.
3. The method for preserving Aspergillus flavus species using Aspergillus flavus sclerotia according to claim 1, characterized in that the ultra-low temperature is 80 ℃ below zero.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210348298.XA CN114591842A (en) | 2022-04-01 | 2022-04-01 | Method for preserving aspergillus flavus strain by using aspergillus flavus sclerotium |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210348298.XA CN114591842A (en) | 2022-04-01 | 2022-04-01 | Method for preserving aspergillus flavus strain by using aspergillus flavus sclerotium |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003016545A2 (en) * | 2001-08-15 | 2003-02-27 | Ciba Specialty Chemicals Holding Inc. | Process for the production of scleroglucan |
| CN103396948A (en) * | 2013-08-13 | 2013-11-20 | 广西大学 | Method for preserving rhizoctonia solani strains for long time |
| CN105154343A (en) * | 2015-10-28 | 2015-12-16 | 四川省农业科学院植物保护研究所 | Simple method for separating and preserving ustilaginoidea virens |
| CN105754872A (en) * | 2016-03-30 | 2016-07-13 | 广东省农业科学院作物研究所 | Method for fast separating, identifying and storing sweet potato sclerotium rolfsii |
| CN112955015A (en) * | 2018-09-19 | 2021-06-11 | 拜耳作物科学生物制品有限责任公司 | Method for improving storage stability of fungal spores |
-
2022
- 2022-04-01 CN CN202210348298.XA patent/CN114591842A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003016545A2 (en) * | 2001-08-15 | 2003-02-27 | Ciba Specialty Chemicals Holding Inc. | Process for the production of scleroglucan |
| CN103396948A (en) * | 2013-08-13 | 2013-11-20 | 广西大学 | Method for preserving rhizoctonia solani strains for long time |
| CN105154343A (en) * | 2015-10-28 | 2015-12-16 | 四川省农业科学院植物保护研究所 | Simple method for separating and preserving ustilaginoidea virens |
| CN105754872A (en) * | 2016-03-30 | 2016-07-13 | 广东省农业科学院作物研究所 | Method for fast separating, identifying and storing sweet potato sclerotium rolfsii |
| CN112955015A (en) * | 2018-09-19 | 2021-06-11 | 拜耳作物科学生物制品有限责任公司 | Method for improving storage stability of fungal spores |
Non-Patent Citations (1)
| Title |
|---|
| 李玉伟等: "砂土管保藏菌种复活试验的研究", 《 中国热带医学》, vol. 6, no. 9, pages 1706 - 1707 * |
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Application publication date: 20220607 |