CN113717865A - Ganoderma lucidum mutant strain and application thereof - Google Patents
Ganoderma lucidum mutant strain and application thereof Download PDFInfo
- Publication number
- CN113717865A CN113717865A CN202111093569.3A CN202111093569A CN113717865A CN 113717865 A CN113717865 A CN 113717865A CN 202111093569 A CN202111093569 A CN 202111093569A CN 113717865 A CN113717865 A CN 113717865A
- Authority
- CN
- China
- Prior art keywords
- strain
- ganoderma lucidum
- culture medium
- mutant strain
- plate
- 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.)
- Granted
Links
Images
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
- C12N1/02—Separating microorganisms from their culture media
-
- 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
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Botany (AREA)
- Mycology (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a Ganoderma lucidum mutant strain and application thereof, wherein the Ganoderma lucidum SBL-LZ-KJYB-5 has the preservation number of CGMCC NO. 23055. The application of the mutant strain is that the ganoderma lucidum mutant strain is fermented to produce extracellular crude polysaccharide and extracellular polysaccharide. The Ganoderma colony is round, and has dense, regular, white mycelium, rapid growth, dryness, and easy picking. The physiological and biochemical characteristics of the strain are as follows: the growth temperature range is wider than that of the original strain, the high temperature resistance is realized, the strain can still grow at 45 ℃, and the optimal growth temperature is 28-32 ℃; the optimal rotating speed of liquid culture is 180 rpm; the natural pH and salinity tolerance of the strain are stronger than those of the strain before mutagenesis, and the strain still grows well in a PDA culture medium containing 1% NaCl.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a ganoderma lucidum mutant strain and a mutagenesis and screening method thereof.
Background
Ganoderma is fungus of Ganoderma of Ganodermataceae of Polyporales of Agaricales of Basidiomycota. The Han Dynasty's book of Shennong Ben Cao Jing considers that ganoderma lucidum can benefit heart qi, calm essence, nourish liver, tonify qi, strengthen tendons and bones, and has high medicinal value. Ganoderma lucidum contains various chemical components such as polysaccharides, triterpenes, nucleosides, proteins, fatty acids, etc., wherein the polysaccharides are considered as one of the main active components of Ganoderma lucidum. Modern researches have proved that ganoderan has various biological activities of resisting tumor, enhancing immunity, resisting aging, etc.
Therefore, the polysaccharide produced by the ganoderma lucidum mutant strain obtained by the mutagenesis technology in fermentation is remarkably improved.
Disclosure of Invention
In view of the above-mentioned drawbacks or deficiencies in the prior art, it would be desirable to provide a mutant strain of Ganoderma lucidum and a method for mutagenesis and screening thereof.
According to the technical scheme provided by the embodiment of the application, the Ganoderma lucidum mutant strain, namely the Ganoderma lucidum SBL-LZ-KJYB-5, has the preservation number of CGMCC NO. 23055.
The application of the ganoderma lucidum mutant strain comprises the step of fermenting the ganoderma lucidum mutant strain to produce extracellular crude polysaccharide and extracellular polysaccharide, wherein the ganoderma lucidum mutant strain is subjected to liquid fermentation for 10 days in a conventional ganoderma lucidum fermentation culture medium potato glucose broth culture medium, the extracellular crude polysaccharide content is 3.10 g/L-3.56g/L, and is improved by 49% -52.79% compared with the original strain. The ganoderma lucidum mutant strain is subjected to liquid fermentation for 10 days in a potato glucose broth culture medium of a conventional ganoderma lucidum fermentation culture medium, the content of extracellular polysaccharide reaches 18g/L-20.77g/L, and is improved by 55% -59.52% compared with the original strain. The Ganoderma lucidum mutant strain belongs to a mutant variety of Ganoderma lucidum, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms at 21.07.21.2021 (address: No. 3 of the national institute of microbiology, Japan institute of academy of sciences, Japan, No. 1 of the North Kyoho, Beijing), is classified as Ganoderma lucidum (Ganoderma lucidum) and has the preservation number of CGMCC NO. 23055.
The mutation of the ganoderma lucidum mutant strain comprises the following steps:
1) ground-based simulated mutagenesis
Preparation of A strain:
inoculating the starting strain with the colony size of 0.2cm x 0.2cm on a PDA plate culture medium, culturing at 25-35 deg.C, and keeping when the colony diameter on the plate exceeds 3 cm;
b, ground simulation space mutagenesis experiment:
the strains on the plates were subjected to the following three sets of combined mutagenesis experiments:
simulating a vibration test of an aircraft during flying: fixing a strain plate with the diameter of more than 3cm on a double-vibration table, and vibrating for 1h under 0-400kN, wherein the first 0.5 h is a low-frequency sinusoidal vibration test, and the last 0.5 h is a random vibration test;
vacuum low energy particle irradiation test: placing the strain plate with diameter more than 3cm into space low-energy comprehensive irradiation experimental equipment at 10 deg.C-3-10-6Recovering after 30-180 min of irradiation under Pa vacuum degree with radiation dose of 1 × 1014eV-4×1014eV；
And (3) gravity acceleration test: selecting 4g of gravity acceleration, and carrying out an overweight test for 30 min;
c, culturing:
taking a ground simulated space mutagenesis sample to culture under the aseptic condition, and comprising the following steps: taking a simulated space mutation sample with the culture area of 0.2cm x 0.2cm under the aseptic condition, eluting bacteria into an aseptic centrifuge tube by using 5 ml of aseptic water, sucking and beating the uniform bacteria eluent by using a pipette, putting 0.5 ml of bacteria liquid into 4.5 ml of aseptic water, sucking and beating uniformly, repeating the steps of diluting by analogy, coating 10-4 and 10-5 diluents onto a flat plate containing a solid culture medium, coating 50-250 microliters of diluted bacteria liquid on each flat plate, coating a plurality of flat plates on each dilution gradient, after the bacteria liquid is absorbed by the culture medium, inversely culturing for 5-20 days at the temperature of 25-35 ℃ in an incubator, selecting a strain which grows rapidly and is large in a single colony, continuously culturing and storing, and carrying out a next screening test;
2) strain screening after ground-based simulated mutagenesis
A, adding a high-concentration metabolite beta-glucan in a PDA solid culture medium to form a screening culture medium, wherein the preparation method comprises the following steps: weighing 39g of Potato Dextrose Agar (PDA) culture medium powder and 50-150g of beta-glucan, diluting to a constant volume of 1L with distilled water, naturally adjusting pH, sterilizing at 121 ℃ for 30min, pouring into a sterile culture dish, cooling and solidifying to obtain a solid culture medium;
b, sterilizing the screening culture medium, pouring the screening culture medium into a flat plate, and condensing to obtain a screening flat plate;
c, uniformly coating, marking and dibbling the strain subjected to the ground simulation space mutagenesis on a screening plate;
d, placing the inoculated flat plate in a mould incubator, culturing for 3-10 days at a constant temperature of 25-35 ℃, screening strains which grow faster and have larger changes in colony morphology as alternative strains according to two indexes of growth speed and colony size, carrying out space mutagenesis test when a return aircraft exists,
after the screened strains are subjected to passage work of 3-10 generations, inoculating the strains on a PDA slant culture medium, storing in a refrigerator at 4 ℃, and rejuvenating and carrying out space mutagenesis after an emission plan is determined;
3) space mutagenesis assay
The space mutagenesis is to transfer a strain screened in ground simulation mutagenesis to a carrying pipe, place the strain in a space capsule, and then send the space capsule into a space mutagenesis environment, wherein the space environment is as follows: microgravity 10-3-10-6g, the vibration in the launching and recovering stage is 0-400 kN; the radiation dose in the space capsule is 0.1mGy-0.9 mGy.
The space mutagenesis test method comprises the following steps:
a space mutagenesis
Inoculating a mutant strain which is selected from a ground simulation experiment and grows more rapidly on a solid culture medium, putting the inoculated inclined plane into an incubator, and culturing for 3-7 days at the temperature of 25-35 ℃ so as to carry out a space mutagenesis test;
under the aseptic condition, scraping a cultured inclined plane, adding the inclined plane into 2mL of carrying small tubes, sealing by a sealing film, then filling the carrying small tubes into a carrying large tube with 30-50mL of solvent, delivering the carrying large tube to relevant personnel of a launching base, installing the carrying large tube on a Shenzhou No. eleven airship, carrying out a space mutagenesis test along with the airship, putting the carrying tube into a Shenzhou No. eleven airship, and recovering the carrying tube after flying along with the airship for 33 days in space;
b recovery carrying pipe
Taking a carried recovered sample with a culture area of 0.2cm x 0.2cm under aseptic condition, eluting thallus with 5 ml of sterile water into a sterile centrifuge tube, sucking and beating the uniform thallus eluent by a pipettor, taking 0.5 ml of the bacteria liquid into 4.5 ml of sterile water, sucking and beating uniformly, repeating the steps, diluting by multiple ratios, taking 10-4And 10-5Coating the diluted solution on a plate culture medium, coating 50-250 microliters of diluted bacteria solution on each plate, coating a plurality of plates on each dilution gradient, and inversely culturing the bacteria solution in an incubator at 25-35 ℃ for 3-10 days after the bacteria solution is absorbed by the culture medium;
4) screening of strains after spatial mutagenesis
Selecting single colony which grows rapidly after space mutagenesis, inoculating the single colony on a pre-prepared screening plate by a line drawing method, placing the inoculated plate in a mould incubator, culturing at constant temperature of 25-35 ℃ for 3-10 days, and picking out the single colony which grows rapidly on the plate.
In the invention, the preparation method of the PDB culture medium comprises the following steps: weighing 35 g of potato dextrose broth culture medium, adding into 1000ml of distilled water, naturally adjusting pH, shaking up, and autoclaving at 121 ℃ for 30 minutes for later use.
In the invention, a strain is activated on a flat PDA culture medium, the strain is cultured for 5-10 days at 25-35 ℃, after mycelium grows over the flat plate, a bacterium block with the size of about 4mm2 is selected and inoculated into 250mL PDB culture medium, after shaking culture is carried out for 5-10 days at 120r/min-200r/min and at 25-35 ℃, fermentation liquor is collected to measure the biomass of the mycelium, the content of extracellular crude polysaccharide and the content of extracellular polysaccharide.
In the invention, the starting strain of Ganoderma lucidum (Ganoderma lucidum) SBL-LZ-6 (laboratory number) is obtained by natural collection of the inventor, and the initial strain has higher mycelium biomass and capability of producing extracellular crude polysaccharide and extracellular polysaccharide compared with the wild strain after being cultured, but still does not reach a very ideal state.
In the invention, a starting strain, namely Ganoderma lucidum (Ganoderma lucidum) SBL-LZ-6, is subjected to a ground simulation space mutagenesis test on the ground, a mutagenesis material is recovered, a screening culture medium plate is used for screening a plurality of strains (with laboratory number SBL-LZ-MNYB 9-SBL-LZ-MNYB 21) with higher growth speed from a recovered sample, space mutagenesis is carried out by using Shenzhou No. eleven, and after the mutagenesis material is recovered, a strain capable of producing more mycelia, extracellular crude polysaccharide and extracellular polysaccharide, namely Ganoderma lucidum (Ganoderma lucidum) SBL-LZ-KJYB-5, is screened and finally determined by a fermentation method to be preserved, so that the aim of the invention is fulfilled.
To sum up, the beneficial effect of this application:
(1) the Ganoderma colony is round, and has dense, regular, white mycelium, rapid growth, dryness, and easy picking. The physiological and biochemical characteristics of the strain are as follows: the growth temperature range is wider than that of the original strain, the high temperature resistance is realized, the strain can still grow at 45 ℃, and the optimal growth temperature is 28-32 ℃; the optimal rotating speed of liquid culture is 180 rpm; the natural pH and salinity tolerance of the strain are stronger than those of the strain before mutagenesis, and the strain still grows well in a PDA culture medium containing 1% NaCl.
(2) The method of combining ground simulation and space mutagenesis is adopted as a novel mutagenesis means of the ganoderma lucidum, and the method has the remarkable characteristic of high mutagenesis beneficial mutation rate;
(3) the screening method is based on the metabolite feedback inhibition principle in the microbial metabolic pathway, high-concentration metabolite beta-glucan is added into a PDA culture plate, strains with good growth conditions in the plate are generally strains with faster growth and higher metabolic efficiency, so that only a single colony with good growth on the plate is selected, and the single colony is generally required in production, so that the screening method is an efficient, simple and convenient excellent strain plate screening method;
(4) the method realizes the screening of the single culture plate of the high-yield strain, reduces the workload by more than times compared with the traditional screening method, and greatly reduces the screening blindness.
(4) The method has simple operation process, and the required reagents and materials are common reagents in laboratories.
Drawings
Other features, objects and advantages of the present application will become more apparent upon reading of the following detailed description of non-limiting embodiments thereof, made with reference to the accompanying drawings in which:
FIG. 1 shows the growth of the strain on 1% NaCl PDA plates before and after mutagenesis;
FIG. 2 is a histogram of differential protein subcellular localization;
reference numbers in the figures:
Detailed Description
The present application will be described in further detail with reference to the following drawings and examples. It is to be understood that the specific embodiments described herein are merely illustrative of the relevant invention and not restrictive of the invention. It should be noted that, for convenience of description, only the portions related to the present invention are shown in the drawings.
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments with reference to the attached drawings.
Example 1
(1) Ground simulation space mutagenesis experiment
1) Strain preparation
Inoculating a starting strain of Ganoderma lucidum (Ganoderma lucidum) SBL-LZ-KJYB-5 (laboratory number) on a flat plate containing a PDA culture medium for culturing, and preparing for a ground simulated space mutagenesis test when the diameter of the strain exceeds 3 cm. The plate preparation method of the PDA culture medium is as follows: 1) weighing 39g of Potato Dextrose Agar (PDA) culture medium powder, diluting to constant volume to 1L with distilled water, naturally p H, sterilizing at 121 deg.C for 30min, pouring into a sterile culture dish, cooling and solidifying to obtain solid culture medium; 2) pouring 20-30ml of culture medium into each plate, and culturing at 33-37 ℃ for 24 hours to be used in an aseptic manner; 4) after inoculating the original strain, the strain was cultured at 30 ℃ for 5 days.
2) The following mutagenesis experiments were performed on the strain sequences on the plates:
vacuum low energy particle mutagenesis: placing the cultured Ganoderma strain plate (phi 9cm) into space low-energy comprehensive irradiation experimental equipment for vacuum low-energy particle irradiation test at 10 deg.C-5Recovering after 120 minutes of irradiation under Pa vacuum degree, the radiation dose is 2.6 multiplied by 1014eV。
Simulating a vibration test of an aircraft during flying: fixing the cultured Ganoderma strain plate (phi 9cm) on 400kN double vibration table, testing for 1h, starting 0.5 h as low frequency sinusoidal vibration test, and then 0.5 h as random vibration test. The test simulates the vibration experienced by the aircraft during launch and return.
And (3) gravity acceleration test: the cultured strain is put into a 10mL centrifuge tube, and an overweight test is carried out for 30min by selecting a gravity acceleration of 4 g.
3) Culturing
And (4) recovering the mutagenic material. Taking a simulated space mutation sample with the culture area of about 0.2cm x 0.2cm under the aseptic condition, eluting bacteria into an aseptic centrifuge tube by using 5 ml of aseptic water, sucking and beating the uniform bacteria eluent by using a pipette, taking 0.5 ml of the bacteria liquid into 4.5 ml of aseptic water, sucking and beating uniformly, and so on, and performing dilution by multiple ratios. And (3) taking 10-4 and 10-5 diluent solutions to coat on a PDA culture medium plate (the plate is prepared in the same way as the plate of the PDA solid culture medium prepared in the step (1)), coating 100 microliters of diluent solution on each plate, and coating a plurality of plates on each diluent gradient. After the bacterial liquid is absorbed by the culture medium, the bacterial liquid is inversely cultured for 5 days at the temperature of 30 ℃ in an incubator. And (4) selecting strains which grow rapidly and have large single colonies, continuously culturing and storing the strains, and carrying out the next screening test.
4) Screening
Making of screening plate
Weighing 39g of Potato Dextrose Agar (PDA) culture medium powder and 100g of beta-glucan, diluting to a constant volume of 1L with distilled water, naturally adjusting pH, sterilizing at 121 ℃ for 30min, pouring into a sterile culture dish, cooling and solidifying to obtain a solid culture medium;
b, taking sterilized culture dishes with the diameter of 90mm, pouring 30ml of culture medium into each culture dish, culturing for 24 hours at 32 ℃, selecting a sterile plate, entering the next working link, and sterilizing and destroying the contaminated plate.
② inoculation
Selecting single colony which grows rapidly after being mutagenized in ground simulation space, and inoculating the single colony on a screening plate prepared in advance by a scribing method.
(iii) cultivation
The inoculated plate is put into a mould incubator and cultivated for 5 days at the constant temperature of 30 ℃.
Bacterial colony selection
Picking out single colony with fast growth speed on the plate.
(2) Spatial mutagenesis
1) And (3) carrying out space mutagenesis. The growth of the selected strain from the ground simulation was more rapid (laboratory number SBL-LZ-MN21) and inoculated on a solid medium, and the inoculated slant was incubated at 30 ℃ for 3 days in an incubator to facilitate the space mutagenesis experiment. Under the aseptic condition, a cultured inclined plane is scraped and added into a 2mL carrying small tube, a sealing film is used for sealing, and then the carrying small tube is filled into a carrying large tube with 30-50mL of solvent. The large tube is carried to the personnel related to the launching base, and the flying boat is arranged on a Shenzhou No. eleven flying boat, and the space mutagenesis test is carried out along with the flying boat. The carrying pipe is put into a flying boat of 'Shenzhou No. eleven', and is recovered after flying with the flying boat in space for 33 days.
2) And recovering the carrying pipe. Taking a carried recovered sample with a culture area of about 0.2cm x 0.2cm under an aseptic condition, eluting bacteria into an aseptic centrifuge tube by using 5 ml of aseptic water, sucking and beating uniform bacteria eluent by using a pipette, taking 0.5 ml of the bacteria liquid into 4.5 ml of aseptic water, sucking and beating uniformly, and so on, and diluting by a multiple ratio. And (3) coating 10-4 and 10-5 times of the diluent on a plate culture medium, coating 50-250 microliters of diluent on each plate, and coating a plurality of plates on each dilution gradient. After the bacterial liquid is absorbed by the culture medium, the bacterial liquid is inversely cultured for 5 days at the temperature of 30 ℃ in an incubator.
3) Screening
39g of Potato Dextrose Agar (PDA) culture medium powder and 50-150g of beta-glucan are weighed, distilled water is used for fixing the volume to 1L, the mixture is naturally pH-stabilized, sterilized for 30min at 121 ℃, and poured into a sterile culture dish to be cooled and solidified into a solid culture medium. And (3) taking sterilized culture dishes with the diameter of 90mm, pouring 30ml of culture medium into each culture dish, culturing for 24 hours at 37 ℃, selecting a sterile plate, entering the next working link, and sterilizing and destroying the contaminated plate. The single colony growing rapidly after space mutagenesis is selected and inoculated on a screening plate prepared in advance by a line drawing method.
② culturing: the inoculated plate is put into a mould incubator and cultivated for 5 days at the constant temperature of 30 ℃.
Thirdly, selecting bacterial colonies: picking out single colony with fast growth speed on the plate.
4) Fermentation of
Preparing a potato glucose broth culture medium (PDB culture medium): weighing 35 g of potato dextrose broth culture medium, adding into 1000ml of distilled water, naturally adjusting pH, shaking up, and autoclaving at 121 ℃ for 30 minutes for later use.
Activating the strain on a plate PDA culture medium, culturing at 30 deg.C for 10 days, and selecting about 4mm2The fungus pieces with the sizes are inoculated into 250mL PDB culture medium and cultured for 10d by shaking at the temperature of 28 ℃ at 120 r/min.
5) Measurement of
Mycelium biomass and extracellular crude polysaccharide content determination: culturing according to the above test method for several days, collecting fermentation liquid, centrifuging at 8000r/min for 20min, collecting precipitate to obtain mycelium, washing with distilled water for 3 times, freeze drying, and weighing to obtain mycelium biomass. The units are calculated in g/L.
Centrifuging the fermentation liquor, taking supernatant, measuring the volume of the supernatant, slowly adding 4 times of volume of absolute ethyl alcohol, uniformly stirring, standing at 4 ℃ for precipitating with ethanol for 12h, centrifuging at 8000r/min for 20min, collecting precipitate, washing the precipitate with ethanol of the same concentration for 2 times, adding distilled water to the precipitate for fully dissolving, heating to volatilize the ethanol, and freeze-drying to obtain the amount of extracellular crude polysaccharide. The results of the measurement of crude extracellular polysaccharides of the mutant strain and the original strain are shown in Table 1.
And (3) determining the content of the exopolysaccharide: and (3) carrying out alcohol precipitation on the fermentation liquor, taking a certain amount of supernatant, and measuring extracellular polysaccharide by adopting a phenol-sulfuric acid method. The units are calculated in g/L. The measurement results of exopolysaccharides of the mutant strain and the original strain are shown in Table 1.
6) Results
TABLE 1 Effect of spatial mutagenesis on the growth and exopolysaccharide content of mycelia of Ganoderma strains
Example 2
(1) Ground simulation space mutagenesis experiment
1) Strain preparation
Inoculating a starting strain Ganoderma lucidum (Ganoderma lucidum) SBL-LZ-6 (laboratory number) on a flat plate containing a PDA culture medium for culturing, and preparing for a ground simulated space mutagenesis test when the diameter of the strain exceeds 3 cm. The plate preparation method of the PDA culture medium is as follows: 1) weighing 39g of Potato Dextrose Agar (PDA) culture medium powder, diluting to constant volume with distilled water to 1L, naturally adjusting pH, sterilizing at 121 deg.C for 30min, pouring into a sterile culture dish, cooling and solidifying to obtain solid culture medium; 2) pouring 20-30ml of culture medium into each plate, and culturing at 33-37 ℃ for 24 hours to be used in an aseptic manner; 4) after inoculating the original strain, the strain was cultured at 30 ℃ for 5 days.
2) The following mutagenesis experiments were performed on the strain sequences on the plates:
vacuum low-energy particle mutagenesis: placing the cultured Ganoderma strain plate (phi 9cm) into space low-energy comprehensive irradiation experimental equipment for vacuum low-energy particle irradiation test at 10 deg.C-5Recovering after 120 minutes of irradiation under Pa vacuum degree, the radiation dose is 2.6 multiplied by 1014eV。
Simulating a vibration test of an aircraft during flying: fixing the cultured Ganoderma strain plate (phi 9cm) on 400kN double vibration table, testing for 1h, starting 0.5 h as low frequency sinusoidal vibration test, and then 0.5 h as random vibration test. The test simulates the vibration experienced by the aircraft during launch and return.
And (3) gravity acceleration test: the cultured strain is put into a 10mL centrifuge tube, and an overweight test is carried out for 30min by selecting a gravity acceleration of 4 g.
3) Culturing
And (4) recovering the mutagenic material. Taking a simulated space mutation sample with the culture area of about 0.2cm x 0.2cm under the aseptic condition, eluting bacteria into an aseptic centrifuge tube by using 5 ml of aseptic water, sucking and beating the uniform bacteria eluent by using a pipette, taking 0.5 ml of the bacteria liquid into 4.5 ml of aseptic water, sucking and beating uniformly, and so on, and performing dilution by multiple ratios. And (3) taking 10-4 and 10-5 diluent solutions to coat on a PDA culture medium plate (the plate is prepared in the same way as the plate of the PDA solid culture medium prepared in the step (1)), coating 100 microliters of diluent solution on each plate, and coating a plurality of plates on each diluent gradient. After the bacterial liquid is absorbed by the culture medium, the bacterial liquid is inversely cultured for 5 days at the temperature of 30 ℃ in an incubator. And (4) selecting strains which grow rapidly and have large single colonies, continuously culturing and storing the strains, and carrying out the next screening test.
4) Screening
Making of screening plate
Weighing 39g of Potato Dextrose Agar (PDA) culture medium powder and 100g of beta-glucan, diluting to a constant volume of 1L with distilled water, naturally adjusting pH, sterilizing at 121 ℃ for 30min, pouring into a sterile culture dish, cooling and solidifying to obtain a solid culture medium;
b, taking sterilized culture dishes with the diameter of 90mm, pouring 30ml of culture medium into each culture dish, culturing for 24 hours at 32 ℃, selecting a sterile plate, entering the next working link, and sterilizing and destroying the contaminated plate.
② inoculation
Selecting single colony which grows rapidly after being mutagenized in ground simulation space, and inoculating the single colony on a screening plate prepared in advance by a scribing method.
(iii) cultivation
The inoculated plate is put into a mould incubator and cultivated for 5 days at the constant temperature of 30 ℃.
Bacterial colony selection
Picking out single colony with fast growth speed on the plate.
(2) Spatial mutagenesis
1) And (3) carrying out space mutagenesis. The growth selected from the ground simulation experiment was more rapid, (laboratory number SBL-LZ-MN21) was inoculated on solid medium, and the inoculated slant was incubated at 30 ℃ for 3 days in an incubator to facilitate the spatial mutagenesis experiment. Under the aseptic condition, a cultured inclined plane is scraped and added into a 2mL carrying small tube, a sealing film is used for sealing, and then the carrying small tube is filled into a carrying large tube with 30-50mL of solvent. The large tube is carried to the personnel related to the launching base, and the flying boat is arranged on a Shenzhou No. eleven flying boat, and the space mutagenesis test is carried out along with the flying boat. The carrying pipe is put into a flying boat of 'Shenzhou No. eleven', and is recovered after flying with the flying boat in space for 33 days.
2) And recovering the carrying pipe. Taking a carried recovered sample with a culture area of about 0.2cm x 0.2cm under an aseptic condition, eluting bacteria into an aseptic centrifuge tube by using 5 ml of aseptic water, sucking and beating uniform bacteria eluent by using a pipette, taking 0.5 ml of the bacteria liquid into 4.5 ml of aseptic water, sucking and beating uniformly, and so on, and diluting by a multiple ratio. And (3) coating 10-4 and 10-5 times of the diluent on a plate culture medium, coating 50-250 microliters of diluent on each plate, and coating a plurality of plates on each dilution gradient. After the bacterial liquid is absorbed by the culture medium, the bacterial liquid is inversely cultured for 5 days at the temperature of 30 ℃ in an incubator.
3) Screening
39g of Potato Dextrose Agar (PDA) culture medium powder and 50-150g of beta-glucan are weighed, distilled water is used for fixing the volume to 1L, the mixture is naturally pH-stabilized, sterilized for 30min at 121 ℃, and poured into a sterile culture dish to be cooled and solidified into a solid culture medium. And (3) taking sterilized culture dishes with the diameter of 90mm, pouring 30ml of culture medium into each culture dish, culturing for 24 hours at 37 ℃, selecting a sterile plate, entering the next working link, and sterilizing and destroying the contaminated plate. The single colony growing rapidly after space mutagenesis is selected and inoculated on a screening plate prepared in advance by a line drawing method.
② culturing: the inoculated plate is put into a mould incubator and cultivated for 5 days at the constant temperature of 30 ℃.
Thirdly, selecting bacterial colonies: picking out single colony with fast growth speed on the plate.
4) Fermentation of
Preparing a potato glucose broth culture medium (PDB culture medium): weighing 35 g of potato dextrose broth culture medium, adding into 1000ml of distilled water, naturally adjusting pH, shaking up, and autoclaving at 121 ℃ for 30 minutes for later use.
Activating the strain on a plate PDA culture medium, culturing at 30 deg.C for 10 days, and selecting about 4mm2The fungus blocks with the sizes are inoculated into 250mL PDB culture medium and cultured for 10d at 30 ℃ at 180r/min by shaking.
5) Measurement of
Mycelium biomass and extracellular crude polysaccharide content determination: culturing according to the above test method for several days, collecting fermentation liquid, centrifuging at 8000r/min for 20min, collecting precipitate to obtain mycelium, washing with distilled water for 3 times, freeze drying, and weighing to obtain mycelium biomass. The units are calculated in g/L.
Centrifuging the fermentation liquor, taking supernatant, measuring the volume of the supernatant, slowly adding 4 times of volume of absolute ethyl alcohol, uniformly stirring, standing at 4 ℃ for precipitating with ethanol for 12h, centrifuging at 8000r/min for 20min, collecting precipitate, washing the precipitate with ethanol of the same concentration for 2 times, adding distilled water to the precipitate for fully dissolving, heating to volatilize the ethanol, and freeze-drying to obtain the amount of extracellular crude polysaccharide. The results of the measurement of crude extracellular polysaccharides of the mutant strain and the starting strain are shown in Table 2.
And (3) determining the content of the exopolysaccharide: and (3) carrying out alcohol precipitation on the fermentation liquor, taking a certain amount of supernatant, and measuring extracellular polysaccharide by adopting a phenol-sulfuric acid method. The units are calculated in g/L. The measurement results of exopolysaccharides of the mutant strain and the original strain are shown in Table 2.
6) Results
TABLE 2 influence of spatial mutagenesis on the growth and exopolysaccharide content of the mycelia of a strain of Ganoderma lucidum
The test results show that the quantity of mycelium and extracellular polysaccharide produced by the ganoderma lucidum strain is not only related to the strain, but also greatly related to the culture method. Meanwhile, the effect of the mutant strain in producing extracellular crude polysaccharide and extracellular polysaccharide is more obvious than that of the original strain.
Salt resistance test of mutagenized strains
As shown in FIG. 1, colonies of cultured Ganoderma lucidum about 0.2cm by 0.2cm before and after mutagenesis were inoculated on PDA plate medium containing 1% NaCl and cultured at 30 ℃ for 5 days. The mutagenized strain grows more densely than the original strain on a flat plate with 1% salinity, and the hypha content is more abundant.
Genetic Studies of mutagenized strains
To further determine the genetic characteristics of the mutagenized strains, quantitative proteomics analysis was performed on the proteins of the ganoderma lucidum strains before and after the simulated spatial mutagenesis treatment using the isotope labeling relative and absolute quantification (iTRAQ) technique and non-targeted metabonomics analysis was performed using the LC-MS/MS technique.
(1) Proteomics research
As shown in fig. 2, a total of 14893 peptides and 3783 proteins were identified in proteomics analysis, and 982 differential proteins were identified, 490 proteins were up-regulated and 492 were down-regulated. There were 268 cytoplasmic proteins, 224 mitochondrial proteins, 195 extracellular proteins, 174 nuclear proteins, 54 plasma membranes, 34 cytoplasmic and nuclear proteins, 26 cytoskeletons, 5 cytoplasmic and mitochondrial proteins, and 2 peroxisome bodies.
(2) Metabonomic research
As shown in table 3, metabolomics analysis results showed significant upregulation of inosine, indole-3-acetic acid, biotin, which was significantly associated with growth, whereas jasmonic acid was downregulated, and ribitol, which was associated with the pentose phosphate pathway, was upregulated. Results of proteomics and metabonomics explain the reason why mutagenic strains grow faster and crude extracellular polysaccharide content is increased.
TABLE 3
The foregoing description is only exemplary of the preferred embodiments of the application and is provided for the purpose of illustrating the general principles of the technology and the like. Meanwhile, the scope of the invention according to the present application is not limited to the technical solutions in which the above-described technical features are combined in a specific manner, and also covers other technical solutions in which the above-described technical features or their equivalent are combined arbitrarily without departing from the inventive concept described above. For example, the above features may be replaced with (but not limited to) features having similar functions disclosed in the present application.
Claims (4)
1. A ganoderma lucidum mutant strain is characterized in that: ganoderma lucidum Ganoderma lucidum SBL-LZ-KJYB-5 with preservation number of CGMCC NO. 23055.
2. The mutant strain of ganoderma lucidum as claimed in claim 1, wherein: the application of the mutant strain is that the ganoderma lucidum mutant strain is fermented to produce extracellular crude polysaccharide and extracellular polysaccharide.
3. The mutant strain of ganoderma lucidum as claimed in claim 2, wherein: the content of extracellular crude polysaccharide of the ganoderma lucidum mutant strain is 3.06g/L-3.56g/L after the liquid fermentation of a potato glucose broth culture medium of a conventional ganoderma lucidum fermentation culture medium for 10 days, which is improved by 41.01-52.79 percent compared with the original strain.
4. The mutant strain of ganoderma lucidum as claimed in claim 2, wherein: the ganoderma lucidum mutant strain is subjected to liquid fermentation for 10 days in a potato glucose broth culture medium of a conventional ganoderma lucidum fermentation culture medium, the content of extracellular polysaccharide reaches 16.77g/L-20.77g/L, and is increased by 39.52% -59.52% compared with the original strain.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111093569.3A CN113717865B (en) | 2021-09-17 | 2021-09-17 | Ganoderma lucidum mutant strain and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111093569.3A CN113717865B (en) | 2021-09-17 | 2021-09-17 | Ganoderma lucidum mutant strain and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113717865A true CN113717865A (en) | 2021-11-30 |
| CN113717865B CN113717865B (en) | 2023-06-09 |
Family
ID=78684216
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111093569.3A Active CN113717865B (en) | 2021-09-17 | 2021-09-17 | Ganoderma lucidum mutant strain and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113717865B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102465160A (en) * | 2010-11-04 | 2012-05-23 | 王刚强 | Method for breeding high-yield extracellular polysaccharide bacterial strain by ganoderma lucidum protoplast mutagenesis |
| CN103548556A (en) * | 2013-06-04 | 2014-02-05 | 神舟太空产品高科技成果推广中心集团有限公司 | Efficient space-mutagenesis ganoderma lucidum, application thereof and preparation method of capsule preparation of efficient space mutagenesis ganoderma lucidum |
| CN105219657A (en) * | 2015-10-22 | 2016-01-06 | 江苏安惠生物科技有限公司 | Rainbow conk liquid fermenting high polysaccharide bacterial strain and selection thereof |
| CN109943488A (en) * | 2019-02-19 | 2019-06-28 | 中国科学院合肥物质科学研究院 | A kind of ganoderma lucidum polysaccharide superior strain RWHBW-1 and its application |
| US20190352345A1 (en) * | 2017-04-08 | 2019-11-21 | Xitian Zhang | Mutants of recombinant immunoregulatory protein of Ganoderma lucidum and applications thereof |
-
2021
- 2021-09-17 CN CN202111093569.3A patent/CN113717865B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102465160A (en) * | 2010-11-04 | 2012-05-23 | 王刚强 | Method for breeding high-yield extracellular polysaccharide bacterial strain by ganoderma lucidum protoplast mutagenesis |
| CN103548556A (en) * | 2013-06-04 | 2014-02-05 | 神舟太空产品高科技成果推广中心集团有限公司 | Efficient space-mutagenesis ganoderma lucidum, application thereof and preparation method of capsule preparation of efficient space mutagenesis ganoderma lucidum |
| CN105219657A (en) * | 2015-10-22 | 2016-01-06 | 江苏安惠生物科技有限公司 | Rainbow conk liquid fermenting high polysaccharide bacterial strain and selection thereof |
| US20190352345A1 (en) * | 2017-04-08 | 2019-11-21 | Xitian Zhang | Mutants of recombinant immunoregulatory protein of Ganoderma lucidum and applications thereof |
| CN109943488A (en) * | 2019-02-19 | 2019-06-28 | 中国科学院合肥物质科学研究院 | A kind of ganoderma lucidum polysaccharide superior strain RWHBW-1 and its application |
Non-Patent Citations (1)
| Title |
|---|
| 刘广建 等: "诱变筛选云芝高产多糖菌株及其液体培养条件优化", 《食用菌》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113717865B (en) | 2023-06-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107629973B (en) | Pichia kudriavzevii strain for producing β -phenethyl alcohol and application thereof | |
| CN102220270B (en) | Screening method for producing chondroitin sulfate bacterial strain and application of bacterial strain fermentation method in production of chondroitin sulfate | |
| CN108220169B (en) | Separation screening and identification method of strain for degrading polystyrene | |
| CN109294933B (en) | Saccharomyces cerevisiae and method for improving quality of traditional fermented food by co-culturing same with ester-producing yeast | |
| CN111484954B (en) | Pseudomonas nigricans for producing alginate lyase | |
| CN108753625B (en) | Polysaccharide-producing space Hericium coralloides ST21-3 and application thereof in improving biological immune activity | |
| CN109294954B (en) | Amylose streptomycete and application thereof | |
| CN117887591B (en) | Boletus edulis growth promoting strain CxBeF11 and application | |
| CN107937297A (en) | Mortifier stress tolerance saccharomyces cerevisiae more than one plant and preparation method, application | |
| CN117025491B (en) | Larens estuary pseudomonas with salt tolerance and growth promoting functions and application thereof | |
| CN113502241B (en) | Rose fungus and application thereof | |
| CN105755091B (en) | A method of it co-cultures using general bacterium raw in rice and chlorella and improves chlorella lipid-producing | |
| CN107513504B (en) | Saccharomyces cerevisiae mutant strain and mutagenesis and screening method thereof | |
| CN113717865B (en) | Ganoderma lucidum mutant strain and application thereof | |
| CN110835619A (en) | Acetobacter pasteurianus mutant strain and mutagenesis and screening method thereof | |
| CN107299062B (en) | Fusarium rubrum (Fusarium incarnatum) A17 for producing unsaturated fatty acid | |
| CN107557311B (en) | Lactobacillus acidophilus and application thereof in fermentation production of antibacterial polypeptide | |
| CN114045247B (en) | Bacillus seawater and application thereof in salinized farmland production | |
| CN112725208B (en) | Tibetan-derived yeast strain capable of producing oil at low temperature and application thereof | |
| CN112143656B (en) | Preparation method of yellow tumor spore powder | |
| CN114058522A (en) | Saccharomyces cerevisiae for high yield of squalene, application and breeding method thereof | |
| CN113881581A (en) | High-ribonucleic acid saccharomyces cerevisiae strain and application thereof | |
| CN108676728B (en) | Polysaccharide-producing space coralloid hericium ST21-2 and application thereof in improving biological oxidation resistance | |
| CN105296379A (en) | Pseudonocardia sp. and application thereof | |
| CN110643552A (en) | Bacterial strain for preparing seaweed syrup by using soluble starch and application thereof |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |