CN106399170B - The bacillus amyloliquefaciens clx-60 bacterial strain and application thereof of one plant of production polyphyllin Ⅵ - Google Patents

The bacillus amyloliquefaciens clx-60 bacterial strain and application thereof of one plant of production polyphyllin Ⅵ Download PDF

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CN106399170B
CN106399170B CN201610826957.0A CN201610826957A CN106399170B CN 106399170 B CN106399170 B CN 106399170B CN 201610826957 A CN201610826957 A CN 201610826957A CN 106399170 B CN106399170 B CN 106399170B
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bacterial strain
clx
bacillus amyloliquefaciens
polyphyllin
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CN106399170A (en
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张鹏
宋发军
于鹏飞
周兰
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South Central University for Nationalities
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RINDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00Microorganisms ; Processes using microorganisms
    • C12R2001/01Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07Bacillus
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, 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
    • C12N1/20Bacteria; Culture media therefor
    • C12N1/205Bacterial isolates
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00Preparation of steroids
    • C12P33/20Preparation of steroids containing heterocyclic rings

Abstract

The present invention provides a kind of bacillus amyloliquefaciens CLX-60 bacterial strains for producing polyphyllin Ⅵ, belong to a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens), which is preserved in China typical culture collection center, address: China, Wuhan, Wuhan University;Postcode 430072, the deposit date is on September 7th, 2016, deposit number are as follows: CCTCC NO:M2016463;The 16S rDNA sequence of the bacterial strain is as shown in SEQ ID NO.1.Above-mentioned bacillus amyloliquefaciens clx-60 bacterial strain can be used in fermentation and prepare polyphyllin Ⅵ.Culture medium prescription used by microbial fermentation are as follows: sucrose 12g/L, dregs of beans 15g/L, MgCl215g/L, optimal culture conditions are as follows: pH value 6.0,34 DEG C of cultivation temperature, shaking speed 150r/min.

Description

The bacillus amyloliquefaciens clx-60 bacterial strain and application thereof of one plant of production polyphyllin Ⅵ
Technical field
The present invention relates to a kind of Bacillus amyloliquefaciens strain, which can produce polyphyllin Ⅵ, belong to microbial technique Field.
Background technique
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 diversity of strain class also gives the diversity of its metabolite, this, which is also implied, excavates biology using microbial metabolism, changes The new resources such as, medicine have huge potentiality.It is a large amount of studies have shown that endophyte of plant can produce it is identical as host plant or Similar active material and precursor, this discovery have pushed directly on the wide of endophyte of plant especially medicinal plant endophyte research General expansion.Gradually research with people to medicinal plant endophyte, utilization can be metabolized the interior life for generating corresponding active constituent Bacterium produces novel, efficient, inexpensive active medicine, solves the status of rare Chinese medicinal plant resource scarcity, and will become will Carry out the emphasis of medicinal plant endophyte research.
Although the concern increasingly by numerous scholars in recent years of endophyte of plant especially medicinal plant endophyte, It is the current endophyte research for still only having carried out several hundred kinds of plants, and the production medicinal ingredient endophyte about rare Rhizoma Paridis Research be even more substantially only existing in the country.Filtered out from Paris polyphylla plant metabolizable generation chonglou saponin, Dioscin and The research of the endophyte of sapogenin provides a new approach to solve Paris polyphylla medicinal ingredient status in short supply, has one at present A little scholars have filtered out some production saponin(es or the endophyte of its analog, but bacterial strain quantity and type are all few, continue to filter out Some new bacterial strains for producing saponin(e or sapogenin, have the research for carrying out the approach that microbial fermentation produces saponin(e or sapogenin in a deep going way It is significant.
It is old it is small wait isolated 107 plants of endogenetic bacterias from magnificent Paris polyphylla stem tuber quietly, wherein there is 6 plants can generate Dioscin Or its analog, it is belonging respectively to Derxia, bacillus, Planococcus, Enterobacter.Zhang Xiaojie etc. is from Hua Chong Isolated 16 plants of endophytes in building, the bacterial strain energy metabolism through screening discovery number SNUS-1 generates chonglou saponin or its is similar Object, and it is accredited as pseudomonas thomasii.The interior life for appointing isolated 2 plants of energy metabolisms from magnificent Paris polyphylla such as intelligence to generate Dioscin is thin Bacterium RZ03 and RZ07.Wei is superfine to filter out 4 plants of endogenetic fungus that can be metabolized generation diosgenin from magnificent rhizoma paris rhizome (WD6H, WD7H, XIAZX and A221H) is belonging respectively to Beancurd sheet spore mould, Fusarium equiseti, point and embraces sickle-like bacteria, Elaphomycetaceae Monascus.
Paris polyphylla endophyte just starts to be concerned by people in recent years, on the whole, especially about Paris polyphylla endophyte Can be metabolized generate corresponding steroid saponin ingredient research it is still relatively fewer, and in terms of being largely focused on magnificent Paris polyphylla, and The research of paris polyphylla endophyte is also only limited to endogenetic fungus, rarely has the report about the paris polyphylla endogenetic bacteria for producing saponin constituent.
Summary of the invention
The present invention solves the problems in background technique, provides a kind of bacillus amyloliquefaciens for producing polyphyllin Ⅵ CLX-60 bacterial strain, the bacterial strain can produce polyphyllin Ⅵ.
The present inventor screens one plant of novel strain, which is named as clx-60, belongs to a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens), which is preserved in China typical culture collection center, address: China, military The Chinese, Wuhan University;Postcode 430072, the deposit date is on September 7th, 2016, deposit number are as follows: CCTCC NO:M2016463;It should The 16S rDNA sequence of bacterial strain is as shown in SEQ ID NO.1.
Above-mentioned bacillus amyloliquefaciens clx-60 bacterial strain can be used in fermentation and prepare polyphyllin Ⅵ.Microbial fermentation is adopted Culture medium prescription are as follows: sucrose 12g/L, dregs of beans 15g/L, MgCl215g/L, optimal culture conditions are as follows: pH value 6.0, culture 34 DEG C of temperature, shaking speed 150r/min.
Compared with prior art, the invention has the following advantages that bacillus amyloliquefaciens CLX-60 provided by the present invention Bacterial strain can be metabolized generation polyphyllin Ⅵ.Bacterial strain provided by the invention can carry out large scale fermentation culture in a short time, and Fermentation costs are lower, and are not limited by conditions such as time domain, seasons, therefore Paris polyphylla can be solved by the approach of microbial fermentation certainly The increasingly deficient status of right resource guarantees that the long-term sustainable of Paris polyphylla medicine resource utilizes.It is additionally provided in the present invention simultaneously excellent The formula of condition of microbe fermentation and culture medium after change, the yield of polyphyllin Ⅵ are higher.
Detailed description of the invention
Fig. 1 is bacillus amyloliquefaciens clx-60 bacterial strain first time HPLC detection figure;
Fig. 2 is second of HPLC detection figure of bacillus amyloliquefaciens clx-60 bacterial strain;
Fig. 3 is that the LC-MS chromatography of bacillus amyloliquefaciens clx-60 bacterial strain detects figure.
Specific embodiment
Detailed specific description done to the present invention combined with specific embodiments below, but protection scope of the present invention not office It is limited to following embodiment.
Separation, the culture of bacterial strain
The fresh seeds bought are subjected to surface sterilization 20min with 75% ethyl alcohol in the present embodiment, then with sterile Water cleans 3-5 times, is put into the spare mortar that sterilizes in advance, 5mL sterile water is added to be ground into white suspension.Take 300 μ L suspended Liquid is smoothened with sterile painting stick, is sealed after air drying with sealed membrane, in 37 DEG C of constant incubators in each PDA culture medium plate 5~9d of middle culture is observed in time, and is photographed to record respectively in the 3rd, 5,7,9d, is no longer separated if fungi covers with plate, And sterilization treatment.It is purified repeatedly on the bacterium to LB solid medium tablets grown on toothpick picking plate, until To single bacterium colony.
Stem tuber is cleaned simultaneously, removes the hard object part in surface of necrosis, it is then crosscutting at 1cm2Thin slice fritter, first use 75% alcohol impregnates 3~5min, then with sterile water wash 2~3 times, washes away remaining alcohol, then impregnate about 5 with sterile water Minute, surface moisture is blotted with the filter paper of sterilizing after taking-up.By treated, fritter is labelled on PDA solid plate, each plate On put 5, in 37 DEG C of culture 5-9d in constant incubator, observation daily, record, the bacterium progress to growing after sealed membrane sealing It purifies repeatedly.
By the bacterium picking for purifying completion on plate to being equipped in the 1.5mL EP pipe of 600 μ L LB liquid mediums, seal Film sealing, then in 37 DEG C of constant-temperature tables after 190r/min shaking table culture 1d, is added 50% isometric glycerol, after mixing It is saved in -80 DEG C of ultra low temperature freezers, each bacterial strain at least saves 3 parts.
8 μ L are taken to be inoculated into 600 μ L LB liquid mediums the strain of preservation, it is living in 37 DEG C of constant-temperature table 190r/min Change culture, the strain for taking 8 μ L to activate afterwards for 24 hours ferments in the LB liquid medium into triangular flask, liquid amount 60ml/ 100ml, 37 DEG C, 190r/min shaking table culture 7d.After the above fermented and cultured, fermentation liquid is collected.
The screening of bacterial strain
To in each bacterial strain fermentation liquor sample chonglou saponin I, II, VI, VII carry out first time detection, by its spectrogram, data with Whether mark product data are compared, can substantially determine containing corresponding chonglou saponin in sample, further according to the standard of corresponding saponin(e Curve can calculate the corresponding saponin content in respective sample.To avoid the possible interference of other materials, we are changing After chromatographic test strip part, second of confirmatory qualitative detection is carried out to resulting positive strain fermentation liquid is detected for the first time, including The detection of mark product and the detection of sample.
Screening obtains the Paris polyphylla endophyte of one plant of production polyphyllin Ⅵ after detecting above, is named as CLX-60 bacterium Strain.First time HPLC of polyphyllin Ⅵ detects the chonglou saponin VI as shown in Figure 1, clx-60 bacterial strain in the fermentation liquid of the bacterial strain Average product are as follows: 0.238mg/L.Second of HPLC detection of polyphyllin Ⅵ is as shown in Figure 2 in the fermentation liquid of the bacterial strain. Corresponding ion stream is had found in the LC-MS test map of the fermentation liquid of clx-60 bacterial strain, as shown in figure 3, further determining that Clx-60 bacterial strain, which can be metabolized, generates chonglou saponin VI.
Molecular Identification is carried out to the bacterial strain
The CLX-60 strain deposited of going bail for is inoculated into the 10mL culture bottle equipped with 5mL LB liquid medium, 37 DEG C, 190r/ Min shaking table culture 3d.
The extraction and electrophoresis detection of endogenetic bacteria DNA
The primer sequence of 16SrDNA:
Upstream primer: 16F (5 '-AGAGTTTGATCATGGCTCAG-3 ')
Downstream primer: 16R (5 '-TACGGTTACCTTGTTACGACTT-3 ')
The purification and recovery of 16SrDNAPCR product is carried out using Sigma NA1020PCR product QIAquick Gel Extraction Kit.
The connection of 16SrDNA segment and cloning vector
The 16S rDNA segment of recycling is connect with pMD 18-T Vector carrier, after flicking mixing, 16 DEG C of connection 6h or 4 DEG C of connection 12h.
Connection product transformed competence colibacillus cell
Bacterium solution PCR-clone's detection
Using universal primer carry out bacterium solution PCR, determine target fragment whether successful conversion, screening positive clone.
Primer sequence: M13R:CAGGAAACAGCTATGACC
M13F:TGTAAAACGACGGCCAGT
Using conventional amplification reaction system and amplification program, former 3 μ L templates wherein are replaced with 2 μ L bacterium solutions in reaction system. It takes 10 μ L amplified productions to carry out agarose gel electrophoresis detection, the corresponding positive colony of acquisition is dispensed, conservation.
Sequencing and sequence alignment, analysis
Positive colony gives certain Bioisystech Co., Ltd to be sequenced, the 16S rDNA sequence of the bacterial strain such as SEQ ID NO.1 institute Show.Sequencing result carries out Blast similarity analysis in GenBank nucleic acid database, through Blast sequence alignment and chadogram point The homology of analysis, clx-60 and Bacillus amyloliquefaciens strain SWM1 are 99%, with Bacillus The homology of amyloliquefaciens strain W16 is 99%, in conjunction with chadogram, determines that it is bacillus amyloliquefaciens (Bacillus amyloliquefaciens strain)。
The preservation of bacterial strain:
The bacterial strain is preserved in China typical culture collection center, address: China, Wuhan, Wuhan University;Postcode 430072, the deposit date is on September 7th, 2016, deposit number are as follows: CCTCC NO:M2016463.
The optimization of fermentation condition
The fermentation medium component of microorganism allows for the needs for meeting the breeding of bacterial strain bacterial strain fast-growth, while most suitable Fermentation culture conditions can guarantee a large amount of accumulation of its secondary metabolites again.The present invention is tested by single factor test and orthogonal test The fermentation medium components of clx-60 bacterial strain are optimized, the optimum medium formula after optimization are as follows: sucrose 12g/L, beans The dregs of rice 15g/L, MgCl2 15g/L.Nutrient media components and corresponding concentration has been determined and then further the bacterial strain has been metabolized and has generated weight Most suitable initial pH value, cultivation temperature and the shaking speed of building saponin(e VI are optimized, final to determine the most suitable of clx-60 bacterial strain Condition of culture are as follows: initial pH value 6.0,34 DEG C of cultivation temperature, shaking speed 150r/min.

Claims (3)

1. bacillus amyloliquefaciens (Bacillus amyloliquefaciens) clx-60 bacterial strain of one plant of production polyphyllin Ⅵ, The bacterial strain is preserved in China typical culture collection center, address: China, Wuhan, Wuhan University;Postcode 430072, preservation day Phase is on September 7th, 2016, deposit number are as follows: CCTCC NO:M2016463, the 16S rDNA sequence such as SEQ ID of the bacterial strain Shown in NO.1.
2. the purposes of bacillus amyloliquefaciens clx-60 bacterial strain described in claim 1, it is characterised in that: prepare Paris polyphylla for fermentation Saponin(e VI.
3. purposes according to claim 2, it is characterised in that: used by bacillus amyloliquefaciens clx-60 strain fermentation Culture medium prescription are as follows: sucrose 12g/L, dregs of beans 15g/L, MgCl215g/L, condition of culture are as follows: pH value 6.0,34 DEG C of cultivation temperature, Shaking speed 150r/min.
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