CN106367372A - Enterobacter aerogenes clx-74 bacterial strain of strains produced diosgenin and application thereof - Google Patents
Enterobacter aerogenes clx-74 bacterial strain of strains produced diosgenin and application thereof Download PDFInfo
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- CN106367372A CN106367372A CN201610826958.5A CN201610826958A CN106367372A CN 106367372 A CN106367372 A CN 106367372A CN 201610826958 A CN201610826958 A CN 201610826958A CN 106367372 A CN106367372 A CN 106367372A
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- 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
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- 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
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Abstract
The invention provides enterobacter aerogenes clx-74 bacterial strain of strains produced diosgenin, and belongs to enterobacter aerogenes. The bacterial strain is preserved in China Center for Type Culture Collection; the address is Wuhan University, Wuhan City, China; the postcode is 430072; the preservation date is September 7, 2016; the preservation number is CCTCC NO: M2016465; the 16S rDNA sequence of the bacterial strain is as shown in SEQ ID NO.1. The enterobacter aerogenes clx-74 bacterial strain can be used for fermentation preparation of diosgenin. A culture medium formula adopted for microbial fermentation is as follows: 12 g/L saccharose, 15 g/L bean pulp, and 15 g/L MgC12, the optimum cultural condition is as follows: the pH value is 6.0, the culture temperature is 34 DEG C, and the shaking table revolution is 150 r/min.
Description
Technical field
The present invention relates to a kind of clostridium perfringen bacterial strain, this bacterial strain can produce diosgenin, belong to microbial technology field.
Background technology
The research of endophyte of plant is gradually taken seriously at present, and the various endophytes of different plants are found in succession.Interior life
The multiformity of strain class also gives the multiformity of its metabolite, and this also imply that excavates biological, change using microbial metabolism
The new resources such as, medicine have huge potentiality.Substantial amounts of research shows, endophyte of plant can produce identical with host plant or
Similar active substance and precursor, this discovery has pushed directly on the wide of endophyte of plant especially medicinal plants endophyte research
General expansion.With the progressively research to medicinal plants endophyte for the people, using the interior life being capable of the metabolism corresponding active component of generation
Bacterium, to produce new, efficient, inexpensive active medicine, solves the present situation of rare Chinese medicinal plant resource scarcity, will become by
Carry out the emphasis of medicinal plants endophyte research.
Although endophyte of plant especially medicinal plants endophyte was increasingly paid close attention to by numerous scholars in the last few years,
It is the endophyte research still only having carried out hundreds of kind of plant at present, and the product medicinal ingredient endophyte with regard to rare Rhizoma Paridis
Research even more substantially only existing in domestic.Filter out from Rhizoma Paridis plant can metabolism produce Rhizoma Paridis saponin, dioscin and
The research of the endophyte of sapogenin provides a new approach for solving Rhizoma Paridis medicinal ingredient present situation in short supply, and at present one
A little scholars have filtered out the endophyte that some produce saponin or its analog, but bacterial strain quantity and species are all few, continue to filter out
Some new bacterial strains producing saponin or sapogenin, have to the research carrying out the approach that fermentable produces saponin or sapogenin in a deep going way
Significant.
Old little wait quietly from magnificent Rhizoma Paridis tuber separate obtain 107 plants of endogenetic bacterias, wherein have 6 plants can produce dioscin
Or its analog, it is belonging respectively to Derxia, bacillus, Planococcus, Enterobacter.Zhang Xiaojie etc. is from Hua Chong
Separate in building and obtain 16 plants of endophytes, produce Rhizoma Paridis saponin through the bacterial strain energy metabolism that screening finds numbering snus-1 or it is similar
Thing, and it is accredited as pseudomonas thomasii.The interior life that the separation from magnificent Rhizoma Paridis such as Ren Zhi obtains 2 plants of energy metabolisms generation dioscins is thin
Bacterium rz03 and rz07.Wei is superfine to filter out 4 plants of endogenetic fungus being capable of metabolism generation diosgenin from magnificent rhizoma paris rhizome
(wd6h, wd7h, xiazx and a221h), is belonging respectively to beancurd sheet spore mycete, Fusarium equiseti, point armful Fusarium spp., Elaphomycetaceae
Monascuses.
Rhizoma Paridis endophyte just starts to be paid close attention to by people in recent years, on the whole, with regard to Rhizoma Paridis endophyte especially
The research being capable of the metabolism corresponding steroidal saponin composition of generation is still relatively fewer, and is largely focused on magnificent Rhizoma Paridis aspect, and
The research of Rhizoma Paridis endophyte is also only limited to endogenetic fungus, rarely has the report of the Rhizoma Paridis endogenetic bacteria with regard to producing saponin constituent.
Content of the invention
The present invention solves the problems, such as in background technology, there is provided a kind of clostridium perfringen clx-74 of product diosgenin
Bacterial strain, this bacterial strain can produce diosgenin.
The present inventor screens one plant of novel strain, and this bacterial strain is named as clx-74, belongs to a kind of clostridium perfringen
(enterobacter aerogenes), this bacterial strain is preserved in China typical culture collection center, address: China, and Wuhan is military
Chinese university；Postcode 430072, preservation date is September in 2016 7, and deposit number is: cctcc no:m2016465；This bacterial strain
16s rdna sequence as shown in seq id no.1.
Above-mentioned clostridium perfringen clx-74 bacterial strain can be used in fermentation and prepares diosgenin.Fermentable is adopted
Culture medium prescription is: sucrose 12g/l, bean cake 15g/l, mgcl215g/l, optimal culture conditions are: ph value 6.0, cultivation temperature
34 DEG C, shaking speed 150r/min.
Compared with prior art, the invention has the advantages that clostridium perfringen clx-74 bacterial strain provided by the present invention
Metabolism can produce diosgenin.The bacterial strain that the present invention provides can carry out large scale fermentation culture in a short time, and ferments
Cost is relatively low, and is not limited by conditions such as time domain, seasons, naturally provides therefore, it is possible to solve Rhizoma Paridis by the approach of fermentable
The increasingly deficient present situation in source is it is ensured that the long-term sustainable of Rhizoma Paridis medicine resource utilizes.After additionally providing optimization in the present invention simultaneously
Condition of microbe fermentation and culture medium formula, the yield of diosgenin is higher.
Brief description
Fig. 1 is clostridium perfringen clx-74 bacterial strain first time hplc detection figure；
Fig. 2 is second hplc detection figure of clostridium perfringen clx-74 bacterial strain；
Fig. 3 is the LC-MS chromatograph detection figure of clostridium perfringen clx-74 bacterial strain.
Specific embodiment
With reference to specific embodiment, detailed specific description is done to the present invention, but protection scope of the present invention not office
It is limited to following examples.
The separation of bacterial strain, culture
In the present embodiment, the fresh seeds buying are carried out surface sterilization 20min with 75% ethanol, then with aseptic
Water cleans 3-5 time, puts in the standby mortar of prior sterilizing, plus 5ml sterilized water grinds to form white suspension.Take 300 μ l suspended
Liquid, in each pda culture medium flat plate, is smoothened with aseptic painting rod, with sealed membrane sealing after air-drying, in 37 DEG C of constant incubators
Middle culture 5～9d, observes in time, and in the 3rd, 5,7,9d Taking Pictures recording respectively, if funguses cover with flat board, no longer carry out separating,
And sterilization treatment.Carry out purification repeatedly with the antibacterial growing on toothpick picking flat board to lb solid medium flat board, until
To single bacterium colony.
Tuber is cleaned simultaneously, remove the surface hard thing part of necrosis, then crosscutting one-tenth 1cm2Thin slice fritter, first use
75% alcohol-pickled 3～5min, then with sterile water wash 2～3 times, wash away the ethanol of remnants, then soak about 5 with sterilized water
Minute, blot surface moisture with the filter paper of sterilizing after taking-up.Fritter after processing is labelled on pda solid plate, each flat board
On put 5, sealed membrane sealing 37 DEG C of culture 5-9d in constant incubator, daily observe, record, the antibacterial growing is carried out
Purification repeatedly.
The antibacterial picking that purification on flat board is completed to the 1.5ml ep pipe equipped with 600 μ l lb fluid mediums, sealing
Film seals, and then in 37 DEG C of constant-temperature tables after 190r/min shaking table culture 1d, adds isopyknic 50% glycerol, after mixing
Preserve in -80 DEG C of ultra cold storage freezers, each bacterial strain at least preserves 3 parts.
The strain of preservation is taken 8 μ l to be inoculated in 600 μ l lb fluid mediums, lives in 37 DEG C of constant-temperature table 190r/min
Change culture, fermented in the lb fluid medium to triangular flask for the strain taking 8 μ l activation after 24h, liquid amount 60ml/
100ml, 37 DEG C, 190r/min shaking table culture 7d.After above fermentation culture, collect fermentation liquid.
The screening of bacterial strain
Diosgenin in each bacterial strain fermentation liquor sample is carried out detect for the first time, by its spectrogram, data and mark product data
Compare, you can substantially determine in sample whether contain corresponding diosgenin, further according to the standard of corresponding diosgenin
Curve, you can calculate the corresponding Determination of Diosgenin in respective sample.For the interference avoiding other materials may bring, changing
After becoming chromatograph testing conditions, second confirmatory qualitative detection, bag are carried out to the positive strain fermentation liquid of first time detection gained
Include the detection of mark product and the detection of sample.
After above detection, screening obtains the Rhizoma Paridis endophyte of one plant of product diosgenin, is named as clx-74 bacterium
Strain.In the fermentation liquid of this bacterial strain, the first time hplc detection of diosgenin is as shown in figure 1, the Rhizoma Paridis saponin vi of clx-74 bacterial strain
Average product be: 0.22mg/l.In the fermentation liquid of this bacterial strain, second hplc detection of diosgenin is as shown in Figure 2.
It is found that corresponding ion stream, as shown in figure 3, further determining that in the LC-MS detection collection of illustrative plates of the fermentation liquid of clx-74 bacterial strain
Clx-74 bacterial strain metabolism can produce diosgenin.
Molecular Identification is carried out to this bacterial strain
The clx-74 strain deposited of going bail for is inoculated in the 10ml culture bottle equipped with 5ml lb fluid medium, 37 DEG C, 190r/
Min shaking table culture 3d.
The extraction of endogenetic bacteria dna and electrophoresis detection
The primer sequence of 16srdna:
Forward primer: 16f (5 '-agagtttgatcatggctcag-3 ')
Downstream primer: 16r (5 '-tacggttaccttgttacgactt-3 ')
Reclaimed using the purification that sigma na1020pcr product QIAquick Gel Extraction Kit carries out 16srdna pcr product.
16srdna fragment and the connection of cloning vehicle
By reclaim 16s rdna fragment be connected with pmd 18-t vector carrier, after flicking mixing, 16 DEG C connection 6h or
4 DEG C of connection 12h.
Connection product transformed competence colibacillus cell
The clone's detection of bacterium solution pcr
Bacterium solution pcr is carried out using universal primer, determines purpose fragment whether successful conversion, screening positive clone.
Primer sequence: m13r:caggaaacagctatgacc
M13f:tgtaaaacgacggccagt
Using conventional amplification reaction system and amplification program, wherein in reaction system, replace former 3 μ l templates with 2 μ l bacterium solution.
Take 10 μ l amplified productions to enter row agarose gel electrophoresis detection, subpackage, conservation are carried out to the corresponding positive colony obtaining.
Sequencing and sequence alignment, analysis
Positive colony gives certain Bioisystech Co., Ltd to be sequenced, the 16s rdna sequence such as seq id no.1 institute of this bacterial strain
Show.Sequencing result carries out blast similarity analysis in genbank nucleic acid database, divides through blast sequence alignment and cladogram
Analysis, the homology of clx-74 and enterobacter aerogenes strain lrc134 is 99%, in conjunction with phylogenetic analysis,
Determine that it is clostridium perfringen (enterobacter aerogenes).
The preservation of bacterial strain:
This bacterial strain is preserved in China typical culture collection center, address: China, Wuhan, Wuhan University；Postcode
430072, preservation date is September in 2016 7, and deposit number is: cctcc no:m2016465.
The optimization of fermentation condition
The fermentation medium component of microorganism allows for meeting the needs of bacterial strain bacterial strain fast-growth breeding, simultaneously suitable
Fermentation culture conditions ensure that a large amount of accumulation of its secondary metabolites again.The present invention passes through single factor test and orthogonal test experiment
The fermentation medium components of clx-74 bacterial strain are optimized, the optimum medium formula after optimization is: sucrose 12g/l, bean
Dregs of rice 15g/l, mgcl2 15g/l.After determining nutrient media components and corresponding concentration, further potato is produced to this bacterial strain metabolism
The most suitable starting ph of Chinese yam sapogenin, cultivation temperature and shaking speed are optimized, and finally determine the most suitable of clx-74 bacterial strain
Condition of culture is: starting ph 6.0,34 DEG C of cultivation temperature, shaking speed 150r/min.
Claims (3)
1. clostridium perfringen (enterobacter aerogenes) the clx-74 bacterial strain of one plant of product diosgenin, this bacterial strain is protected
It is hidden in China typical culture collection center, address: China, Wuhan, Wuhan University；Postcode 430072, preservation date is 2016
On September 7, deposit number is: cctcc no:m2016465, and the 16s rdna sequence of this bacterial strain is as shown in seq id no.1.
2. clostridium perfringen clx-74 bacterial strain described in claim 1 purposes it is characterised in that: prepare dioscin for fermentation
Unit.
3. purposes according to claim 2 it is characterised in that: the culture medium prescription that fermentable is adopted is: sucrose
12g/l, bean cake 15g/l, mgcl215g/l, condition of culture is: ph value 6.0,34 DEG C of cultivation temperature, shaking speed 150r/min.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610826958.5A CN106367372B (en) | 2016-09-18 | 2016-09-18 | The clostridium perfringen clx-74 bacterial strain and application thereof of one plant of production diosgenin |
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| CN201610826958.5A CN106367372B (en) | 2016-09-18 | 2016-09-18 | The clostridium perfringen clx-74 bacterial strain and application thereof of one plant of production diosgenin |
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| CN106367372B CN106367372B (en) | 2019-03-12 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106399170A (en) * | 2016-09-18 | 2017-02-15 | 中南民族大学 | Bacillus amyloliquefaciens clx-60 bacterial strain producing polyphyllin VI and application thereof |
| CN107629980A (en) * | 2017-09-30 | 2018-01-26 | 中南民族大学 | The Ludwig enterobacteria SXZ N5 bacterial strains and purposes of one plant of production huperzine and Huperzine B |
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| CN102475710A (en) * | 2010-11-24 | 2012-05-30 | 东北师范大学 | Application of diosgenin and derivative thereof for preparing tumor chemotherapy sensitization medicines |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399170A (en) * | 2016-09-18 | 2017-02-15 | 中南民族大学 | Bacillus amyloliquefaciens clx-60 bacterial strain producing polyphyllin VI and application thereof |
| CN106399170B (en) * | 2016-09-18 | 2019-06-07 | 中南民族大学 | The bacillus amyloliquefaciens clx-60 bacterial strain and application thereof of one plant of production polyphyllin Ⅵ |
| CN107629980A (en) * | 2017-09-30 | 2018-01-26 | 中南民族大学 | The Ludwig enterobacteria SXZ N5 bacterial strains and purposes of one plant of production huperzine and Huperzine B |
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