CN110511876A - A kind of korean epimedium herb endophyte, cultural method and its metabolite - Google Patents
A kind of korean epimedium herb endophyte, cultural method and its metabolite Download PDFInfo
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Abstract
The invention proposes isolated strains Ek-Endogenous-S10, culture medium, cultural method, separation method and its secondary metabolites of a kind of korean epimedium herb endophyte, a kind of isolated strains Ek-Endogenous-S10 of korean epimedium herb endophyte, the isolated strains are being biologically pure.The isolated bacterial strain of the method for the present invention can produce and chemical component identical in host cell, and have many advantages, such as not by time season limit quickly, orientation, it is easily separated, the structure of the fungi is relatively easy, Metabolite is simple, stablizes, orientation, compared with traditional Chinese medicine, the extraction separative efficiency gesture of chemical component is also greatly improved.
Description
Technical field
The present invention relates to microorganisms technical fields, and in particular to a kind of korean epimedium herb endophyte, cultural method and its generation
Thank to product.
Background technique
In the prevalence of endophyte in medicinal plant body, the growth of medicinal plant, genuineness, yield and quality, effect at
Point, resistance etc. all generate important influence；On the other hand, it is living also to generate some physiology for medicinal plant endophyte itself
Property substance, new drug development, medicinal plant cultivation production, bacterial manure exploitation, biocontrol microorganisms research and development, soil ecology reparation, industrialization enzyme
Production application etc. all shows huge potentiality to be exploited.
Korean epimedium herb (EpimediumkoreanumNakai) is Berberidaceae Epimedium herbaceos perennial, is me
The distinctive natural resources of Chinese medicinal materials in the Northeast, state has warming and invigorating kidney Yang, strengthening the bones and muscles, wind-damp dispelling and other effects.Artificial cultivation is difficult, dosage has
The trend that increasing nothing subtracts makes korean epimedium herb, and wild resource is reduced year by year.Therefore, expand korean epimedium herb introducing and planting and
It is urgently to be resolved to find its substitute.
The acquisition of the existing flavones ingredient coarse extraction from plant mostly, later by enzyme decompose, conversion, separation etc. into
The purification of one step, not only growth cycle is long during plant culture, and artificial field management etc. is at high cost, the separation in later period conversion etc. at
This also should not be underestimated.Simultaneously as Chinese medicine composition complexity, either traditional water extraction, alcohol extracting method or modern film
Separation, new material extractive technique are no more than 50% to effective component extraction efficiency in Chinese medicine, and probably 30% or so, remaining is
With discarded dregs of a decoction processing, not only waste of resource waste, which is dealt with improperly, also be can cause environmental pollution.The structure of fungi is relatively easy,
Metabolite is simple, stablizes, orientation, and compared with traditional Chinese medicine, the extraction separative efficiency of chemical component will certainly be mentioned greatly
It is high.On the other hand, medicinal plant needs longer growth cycle mostly, and genuineness is more demanding to environment, growth conditions etc.,
Fungi is quite different, it is only necessary to enough illumination, carbon source, nitrogen source can benign growths, do not influenced by season time, target product
Output capacity multiplication.
Chinese patent CN10471130A and CN106995829A disclose the method for generating barren wort total chromocor, mainly
Total extract is extracted from medicinal material, purer flavone aglycone is obtained by the method for enzymatic isolation method, conversion, but such method generates Huang
Ketones component is by time season limit and is difficult to separate.
Summary of the invention
In order to solve the above technical problems, the present invention provides a kind of korean epimedium herb endophyte, cultural method and its generation
Thank to product, it is intended that providing a kind of korean epimedium herb endophyte, the structure of the fungi is relatively easy, Metabolite is simple,
Stablize, orientation, compared with traditional Chinese medicine, the extraction separative efficiency gesture of chemical component is also greatly improved.
The present invention provides a kind of isolated strains Ek-Endogenous-S10 of korean epimedium herb endophyte, in 2019
July 1 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms collection, deposit number are as follows: CGMCC
NO.18131。
As further improvement of the invention, the isolated strains are being biologically pure.
The present invention further protects the culture medium of korean epimedium herb endophyte Ek-Endogenous-S10 a kind of, the training
Feeding base is divided into fluid nutrient medium and solid medium, the Liquid Culture based formulas: peptone 10.0g, yeast extract 5.0g,
NaCl 10.0g, distilled water 1000mL, 7.0,121 DEG C of high pressure steam sterilization 20min of pH value, the solid medium is in institute
Addition adds agar 20.0g/L on the basis of stating Liquid Culture based formulas, which is characterized in that is additionally added carbon source and nitrogen source.
As further improvement of the invention, the solid medium could alternatively be PDA culture medium, buckwheat, sorghum training
Base is supported, carbon source and nitrogen source are additionally incorporated.
As further improvement of the invention, the carbon source is alpha-lactose, and the nitrogen source is glycine.
The present invention further protects the separation method of korean epimedium herb endophyte Ek-Endogenous-S10 a kind of, including
Following steps: taking clean korean epimedium herb blade, handles 4-5min with liquor natrii hypochloritis after being cleaned with tap water；Sterile water
Residual sterile water is sucked with aseptic filter paper after rinsing 4 times；Rhizome 4-5min is handled with ethanol solution again；Then rinsed with sterile water is used
4 times and suck residual sterile water；With sterile razor blade will treated that be cut into side length be about 1-2mm, it is planted on culture medium；It will training
It supports and is based on 23-25 DEG C constant temperature incubation 3-5 days, after growing bacterium colony, the fresh culture is forwarded to oese picking thallus
It is purified on base, until 3 bacterium colonies after purification are purebred；It is spare that purebred bacterium is transferred to preservation on slant medium.
As further improvement of the invention, liquor natrii hypochloritis's concentration is 5% (v/v), and the ethanol solution is dense
Degree is 75% (v/v).
The present invention further protects the cultural method of korean epimedium herb endophyte Ek-Endogenous-S10 a kind of, including
Following steps: the strain for hiding of going bail for is crossed on a small amount of strain to fresh culture of picking and is activated 3-5 days in 23-25 DEG C of constant temperature,
The single colonie of activation is forwarded in the culture medium of Fresh, is cultivated under proper culture conditions.
As further improvement of the invention, the suitable condition of culture is that temperature is 25 DEG C, light application time 12h, pH
Value is 6.
The present invention further protects the secondary metabolite of korean epimedium herb endophyte Ek-Endogenous-S10 a kind of,
Main icariin, Epimedin A, Epimedin B and epimedin C including being generated in host cell.
The invention has the following beneficial effects: the isolated bacterial strain of the method for the present invention can produce it is identical with host cell
Chemical component, and have many advantages, such as not by time season limit quickly, orientation, it is easily separated, the structure of the fungi is relatively easy, generation
Simple ingredient, stabilization, orientation are thanked, compared with traditional Chinese medicine, the extraction separative efficiency gesture of chemical component is also greatly improved.It should
Technology can be the exploitation of medicinal fungi, and it is also endophyte and its host cell effective component that the protection of medicinal plant, which provides thinking,
The correlation of synthesis provides scientific basic.
Detailed description of the invention
Fig. 1 is korean epimedium herb endophyte Ek-Endogenous-S10 mode of appearance figure；
Fig. 2 is the aspect graph of korean epimedium herb endophyte Ek-Endogenous-S10 bacterial strain under different condition；
Fig. 3 is the influence diagram that carbon source grows korean epimedium herb endophyte Ek-Endogenous-S10；
Fig. 4 is the influence diagram that nitrogen source grows korean epimedium herb endophyte Ek-Endogenous-S10；
Fig. 5 is the influence diagram that pH grows korean epimedium herb endophyte Ek-Endogenous-S10；
Fig. 6 is the influence diagram that illumination grows korean epimedium herb endophyte Ek-Endogenous-S10；
Fig. 7 is the influence diagram that temperature grows korean epimedium herb endophyte Ek-Endogenous-S10
Fig. 8 is HPLC standard items figure；
Fig. 9 is the HPLC figure of korean epimedium herb endophyte Ek-Endogenous-S10 secondary metabolite.
Specific embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, the embodiment described is the embodiment of part of representative of the invention, rather than whole embodiments, this field are general
Other all embodiments obtained belong to protection of the invention to logical technical staff without making creative work
Range.
The separation method of 1 korean epimedium herb endophyte of embodiment and identification
Clean korean epimedium herb blade is taken, handles 4- with 5% (v/v) liquor natrii hypochloritis after being cleaned with tap water
5min；Residual sterile water is sucked with aseptic filter paper after rinsed with sterile water 4 times；Rhizome 4- is handled with 75% (v/v) ethanol solution again
5min；Then with rinsed with sterile water 4 times and residual sterile water is sucked.With sterile razor blade will treated that be cut into side length be about 1-
2mm is planted in PDA culture medium；Culture is based on 23-25 DEG C constant temperature incubation 3-5 days, after growing bacterium colony, is chosen with oese
It takes thallus to be forwarded on fresh PDA culture medium to be purified, until 3 bacterium colonies after purification are purebred；Purebred bacterium is transferred to
It is saved backup on PDA slant medium.
The Liquid Culture based formulas: peptone 10.0g, yeast extract 5.0g, NaCl 10.0g, alpha-lactose 10g are sweet
Propylhomoserin 5g, 1000mL, pH7.0,121 DEG C of high pressure steam sterilization 20min of distilled water；
Solid medium is to add agar 20.0g/L in above-mentioned formula.
The colonial morphology villiform, yellow-white, spore is high-visible, is in yellowish-brown.Bacterium colony size expands to entire solid
Culture medium is in concentric wheel stripe shape.Through extracting genome DNA, the amplification of 16SrDNA specific primer PCR, amplified production purifying, DNA
Sequencing, sequence alignment.The bacterial strain is accredited as Ilyonectria, is Ilyonectria cyclaminicola.
Table 1
Footnote: 1. depositary institution's titles: China Committee for Culture Collection of Microorganisms's common micro-organisms collection, ground
Location: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation time: on June 19th, 2019.
The inoculation method of 2 korean epimedium herb endophyte of embodiment
The slant strains for hiding of going bail for are crossed on picking a small amount of strain to fresh PDA culture medium and are activated in 23-25 DEG C of constant temperature
3-5 days.The single colonie of activation is forwarded in improvement PDA (nitrogen source, carbon source etc. is added) culture medium of Fresh.It is raw on basis
On long culture medium, by single-factor variable method, the lifes of the conditions to bacterial strain such as temperature, illumination, carbon source, nitrogen source, pH value are investigated respectively
The influence of long situation.As a result referring to attached drawing 3-7.
The result shows that in terms of carbon source bacterial strain to alpha-lactose using best, best, most thermophilic is utilized to glycine in terms of nitrogen source
When degree is 25 DEG C, most suitable illumination is the 12h illumination of 12h dark, optimum pH 6.
The quality of 3 secondary metabolite of embodiment is studied
Chromatographic condition: chromatographic columnODS-3 (4.6mm × 250mm, 5 μm)；Mobile phase water (A)-acetonitrile (B)；
Gradient elution journey 0-37min, 24%B；37-47,24%-38%B；47-60min, 38%-24%B；Detection wavelength 360nm；Column
30 DEG C of temperature；10 μ L of sample volume.
The preparation of reference substance solution: it is appropriate that precision weighs icariin, Epimedin A, Epimedin B, epimedin C reference substance,
Methanol is added 0.105mgmL-1 containing icariin, Epimedin A 0.106mgmL-1 to be respectively prepared, towards leaves of pulse plants B0.101mgmL-
1, the mixed solution of epimedin C 0.108mgmL-1 to get.
The preparation of test solution: taking the bacterial strain powder about 0.2g, accurately weighed, sets in stuffed conical flask, and precision is added
Diluted Alcohol 20mL, ultrasonic (250W, 30KHz) handle 1h, then weighed weight, the weight of less loss are supplied with Diluted Alcohol, is shaken up, and filter
Cross, take subsequent filtrate to get.
Measurement: precision draws mixed reference substance solution and 10 μ L of test solution injects high performance liquid chromatograph.
The results are shown in attached figure 8-9.Fig. 8 is HPLC standard items figure；Wherein 1.-be 4. followed successively by Epimedin A, Epimedin B, fixed towards the leaves of pulse plants
C and icariin；Fig. 9 is the HPLC figure of korean epimedium herb endophyte Ek-Endogenous-S10 secondary metabolite, according to figure
9, determine 1.-be 4. followed successively by Epimedin A, Epimedin B, epimedin C and icariin.The result shows that bacterium generation is thin with host
The similar ingredient of cell phase, predominantly flavones ingredient.
It is compared, is shown in the bacterium secondary metabolite containing big by the method and known standard items of efficient liquid phase
Amount known flavonoid glycoside ingredient and some other unknown flavones ingredients, be flavones ingredient mass production without by from
Offer is extracted in plant may.That is protection wild resource saves the costs such as time, material again.
Compared with prior art, the isolated bacterial strain of the method for the present invention can produce it is identical with host cell chemistry at
Point, and have many advantages, such as not by time season limit quickly, orient, it is easily separated, the structure of the fungi is relatively easy, Metabolite
Simply, stablize, orient, compared with traditional Chinese medicine, the extraction separative efficiency gesture of chemical component is also greatly improved.The technology can
Protection for the exploitation of medicinal fungi, medicinal plant provides thinking, also synthesizes for endophyte with its host cell effective component
Correlation provides scientific basic.
Those skilled in the art is not under conditions of departing from the spirit and scope of the present invention that claims determine, also
Various modifications can be carried out to the above content.Therefore the scope of the present invention is not limited in above explanation, but by
The range of claims determines.
Claims (10)
1. a kind of isolated strains Ek-Endogenous-S10 of korean epimedium herb endophyte is preserved on July 1st, 2019
China Committee for Culture Collection of Microorganisms's common micro-organisms collection, deposit number are as follows: CGMCC NO.18131.
2. a kind of isolated strains Ek-Endogenous-S10 of korean epimedium herb endophyte according to claim 1, feature
It is, the isolated strains are being biologically pure.
3. a kind of culture medium of korean epimedium herb endophyte Ek-Endogenous-S10, the culture medium are divided into fluid nutrient medium
And solid medium, the Liquid Culture based formulas: peptone 10.0g, yeast extract 5.0g, NaCl 10.0g, distilled water
1000mL, 7.0,121 DEG C of high pressure steam sterilization 20min of pH value, the solid medium is in the Liquid Culture based formulas
On the basis of addition plus agar 20.0g/L, which is characterized in that be additionally added carbon source and nitrogen source.
4. a kind of culture medium of korean epimedium herb endophyte Ek-Endogenous-S10, feature exist according to claim 3
In the solid medium could alternatively be PDA culture medium, buckwheat, sorghum culture medium, additionally incorporate carbon source and nitrogen source.
5. special according to a kind of culture medium of korean epimedium herb endophyte Ek-Endogenous-S10 of claim 3 or 4
Sign is that the carbon source is alpha-lactose, and the nitrogen source is glycine.
6. a kind of separation method of korean epimedium herb endophyte Ek-Endogenous-S10, which is characterized in that including following step
It is rapid: to take clean korean epimedium herb blade, handle 4-5min with liquor natrii hypochloritis after being cleaned with tap water；Rinsed with sterile water 4
Residual sterile water is sucked with aseptic filter paper after secondary；Rhizome 4-5min is handled with ethanol solution again；Then rinsed with sterile water is used 4 times simultaneously
Suck residual sterile water；With sterile razor blade will treated that be cut into side length be about 1-2mm, it is planted on culture medium；By culture medium
In 23-25 DEG C constant temperature incubation 3-5 days, after growing bacterium colony, be forwarded to oese picking thallus on the fresh culture medium
It is purified, until 3 bacterium colonies after purification are purebred；It is spare that purebred bacterium is transferred to preservation on slant medium.
7. separation method according to claim 6, which is characterized in that liquor natrii hypochloritis's concentration is 5% (v/v), institute
Stating ethanol solution concentration is 75% (v/v).
8. a kind of cultural method of korean epimedium herb endophyte Ek-Endogenous-S10, which is characterized in that including following step
Rapid: the strain for hiding of going bail for is crossed on a small amount of strain to fresh culture of picking and is activated 3-5 days in 23-25 DEG C of constant temperature, will activate
Single colonie be forwarded in the culture medium of Fresh, cultivate under proper culture conditions.
9. a kind of cultural method of korean epimedium herb endophyte Ek-Endogenous-S10 according to claim 8, feature
It is, the suitable condition of culture is that temperature is 25 DEG C, light application time 12h, pH value 6.
10. a kind of secondary metabolite of korean epimedium herb endophyte Ek-Endogenous-S10, which is characterized in that main packet
Include the icariin generated in host cell, Epimedin A, Epimedin B and epimedin C.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114836366A (en) * | 2022-04-28 | 2022-08-02 | 云南龙藏生物科技有限公司 | Suspension culture method for callus of epimedium dauricum |
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| CN106047715A (en) * | 2016-05-13 | 2016-10-26 | 贵州大学 | Nothapodytes pittosporoides endophyte trichoderma sp. strain and method thereof for extracting camptothecin |
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| CN114836366A (en) * | 2022-04-28 | 2022-08-02 | 云南龙藏生物科技有限公司 | Suspension culture method for callus of epimedium dauricum |
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| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Xiao Jinglei Inventor after: Li Ruotong Inventor after: Jiang Dacheng Inventor after: Du Xinglin Inventor after: Wang Yingzhe Inventor after: Li Jing Inventor after: Liu Yucui Inventor after: Qin Jianchun Inventor after: Yan Li Inventor after: Guo Junjie Inventor after: Nian Minghui Inventor after: Luan Hanyu Inventor before: Xiao Jinglei Inventor before: Jiang Dacheng Inventor before: Du Xinglin Inventor before: Li Jing Inventor before: Liu Yucui Inventor before: Qin Jianchun Inventor before: Yan Li Inventor before: Guo Junjie Inventor before: Nian Minghui Inventor before: Luan Hanyu Inventor before: Li Ruotong |