CN107034164A - It is a kind of to move Sphingomonas and its application less - Google Patents
It is a kind of to move Sphingomonas and its application less Download PDFInfo
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- CN107034164A CN107034164A CN201710379552.1A CN201710379552A CN107034164A CN 107034164 A CN107034164 A CN 107034164A CN 201710379552 A CN201710379552 A CN 201710379552A CN 107034164 A CN107034164 A CN 107034164A
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- exocellular polysaccharide
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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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
- 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
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Abstract
The present invention separates and screened the bacterial strain for obtaining being capable of high-yield extracellular polysaccharide from Huanglong's tangerine orange blade of Nanping City in Fujian province Shunchang County, the bacterial strain moves the method that Sphingomonas FJAT 10272 prepares exocellular polysaccharide using this less to move Sphingomonas FJAT 10272 (Sphingomonas paucimobilis) less, and disclosing.The advantage of the invention is that：A kind of new Sphingomonas FJAT 10272 (Sphingomonas paucimobilis) dynamic less is provided, and the yield for preparing exocellular polysaccharide using the bacterial strain is higher, so as to add the source that exocellular polysaccharide produces bacterial strain；And it is simple, easily operated using the present invention process that dynamic Sphingomonas FJAT 10272 (Sphingomonas paucimobilis) prepares exocellular polysaccharide less.
Description
【Technical field】
The present invention relates to a kind of microorganism and its application, and in particular to a kind of to move Sphingomonas less and its answer
With.
【Background technology】
Polysaccharide is using monose as basic composition unit, by the polymer of certain way repeated arrangement.For food plus
The polysaccharide of work is commonly called as colloid, i.e. exocellular polysaccharide.Exocellular polysaccharide can derive from animal, plant, microorganism, seaweeds exocellular polysaccharide
Mainly include agar and carragheen and alginic acid and its derivative.Plant exocellular polysaccharide includes Arabic gum, cellulose gum
And its derivative, konjaku flour etc..Microbial exopolysaccharides mainly include xanthans, gellan gum, carragheen etc., with adherence,
The features such as stability, emulsibility and gelation, suffer from being widely applied in terms of food, field of medicaments, it is especially near
Nian Lai, with the introducing of Protocols in Molecular Biology, polysaccharide is in terms of anticoagulation, anti-aging, anti-cancer and cancer-preventing, disease immune
Function is also gradually revealed.By its purposes, exocellular polysaccharide can be classified as thickener and gelling agent again, can be used as food additive
Plus agent, coagulating agent, antistaling agent etc..Reported can the microorganism Pseudomonas of extracellular polysaccharide have Sphingomonas
(Sphingomonas), Agrobacterium (Agrobacterium), xanthomonas (Xanthomonas) etc..
【The content of the invention】
The technical problems to be solved by the invention are that offer is a kind of and move Sphingomonas less, and the bacterial strain is few dynamic
Sphingomonas FJAT-10272 (Sphingomonas paucimobilis), was preserved on 01 14th, 2016
China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Institute of Microorganism, Academia Sinica, deposit number be CGMCC NO.11997, and disclose prepared using the bacterial strain it is extracellular many
Sugar.
The present invention is to solve above-mentioned technical problem by the following technical programs：
The applicant separates from Huanglong's tangerine orange blade of Nanping City in Fujian province Shunchang County and screens that obtain being capable of high yield born of the same parents
The bacterial strain of exo polysaccharides, by carrying out physio-biochemical characteristics measure and aliphatic acid Sherlock-MIDI network analyses to the bacterial strain, most
It is identified as moving a bacterial strain of Sphingol single-cell eventually less.
Further, exocellular polysaccharide is prepared using the bacterial strain, its preparation method is concretely comprised the following steps：
(1) activation for moving Sphingomonas FJAT-10272 less described in claim 1：It will be weighed with oese
Profit requires that the Sphingomonas FJAT-10272 that moves less described in 1 is lined on NA flat boards, and in constant incubator
48h is cultivated, and cultivation temperature is 30 DEG C；
(2) seed liquor is prepared：The single bacterium colony for moving Sphingomonas FJAT-10272 less that step (1) is obtained
It is inoculated in seed culture medium, and is placed in constant temperature oscillation shaking table and cultivates 16h, 30 DEG C of temperature, rotating speed 170rpmmin-1；
(3) zymotic fluid is prepared：The seed liquor that step (2) is obtained is transferred to by 2% inoculum concentration by percent by volume
Fermented and cultured 72h in the fermentation medium of sterilization, 30 DEG C of temperature, rotating speed 190rpmmin-1, produce zymotic fluid；
(4) extraction of exocellular polysaccharide：(at room temperature, rotating speed is centrifuged in zymotic fluid obtained by step (3)
6000rpm·min-1Centrifuge 10min), supernatant is collected, ice ethanol, and the body of ice ethanol and supernatant are added into supernatant
Product is than being 3：1, after standing overnight sedimentation exocellular polysaccharide at 4 DEG C；Then (room temperature is centrifuged in the solution after standing
Under, rotating speed 6000rpmmin-1Centrifuge 10min), collect sediment；Using absolute ethyl alcohol washing precipitate 3 times, then nitrogen
Drying, finally crushes and produces the exocellular polysaccharide product.
Further, the component of the NA flat boards is：Beef extract 3gL-1, peptone 5gL-1, glucose 10gL-1、
Yeast extract 1gL-1, agar powder 17gL-1、pH 7.2。
Further, the component of the seed culture medium is：Sucrose 20gL-1, peptone 5gL-1, yeast extract 5gL-1、pH 7.0。
Further, the component of the fermentation medium is：Glucose 30gL-1, peptone 3gL-1, yeast extract
2g·L-1、KH2PO4 1g·L-1、MgSO4 0.1g·L-1, micro- salting liquid 1mLL-1、pH 7.0；
Wherein, the component of micro- salting liquid is：MnCl2·4H2O 1.8g·L-1、FeSO4·7H2O 2.4g·L-1、
H3BO3 0.283g·L-1、CuCl2 0.0027g·L-1、CoCl2·6H2O 0.0074g·L-1。
The beneficial effects of the present invention are：
A kind of new Sphingomonas FJAT-10272 (Sphingomonas dynamic less are provided
Paucimobilis Sphingomonas FJAT-10272), and using this is moved less and prepares exocellular polysaccharide, and prepares born of the same parents
The yield of exo polysaccharides is higher, i.e., this move less Sphingomonas FJAT-10272 can high-yield extracellular polysaccharide, so as to increase
Exocellular polysaccharide produces the source of bacterial strain；And Sphingomonas FJAT-10272 is moved using the present invention less
The process that (Sphingomonas paucimobilis) prepares exocellular polysaccharide is simple, easily operated.
【Brief description of the drawings】
The utility model will be further described in conjunction with the embodiments with reference to the accompanying drawings.
Fig. 1 is the fatty acid profile of bacterial strain FJAT-10272 in the present invention.
【Embodiment】
A kind of Sphingomonas that moves less of the present invention is less dynamic Sphingomonas FJAT-10272
(Sphingomonas paucimobilis) is separated and screened from Huanglong's tangerine orange blade of Nanping City in Fujian province Shunchang County
To being capable of the bacterial strain of high-yield extracellular polysaccharide, and disclose moved less using this Sphingomonas FJAT-10272 prepare it is extracellular
The method of polysaccharide.
In order to clearly illustrate the present invention, the applicant gives following several specific embodiments.
The separation of embodiment 1, bacterial strain FJAT-10272：
(1) the yellow twig tangerine orange blade 5g of Nanping City in Fujian province Shunchang County is weighed respectively, and blade is rinsed with running water
Totally, the water of blade surface is blotted, 30s is soaked in 75% alcohol afterwards, then 5min is soaked with 10% sodium hypochlorite, then use
Rinsed with sterile water blade 3 times；
(2) in order to examine the effect of surface sterilizing, the blade after being handled through step (1) is clipped with sterilized tweezers, and
Blade surface is contacted 2min with NA flat boards, then NA flat boards are placed at 30 DEG C and cultivate 2d, if finding there is no bacterium on NA flat boards
Drop out existing, it was demonstrated that blade surface sterilizing is thorough, can be used as subsequent operation, otherwise giving up to use.
(3) sterilized thoroughly blade after step of learning from else's experience (2) inspection, and under sterile working, blade is shredded to mortar simultaneously
45mL sterilized waters are added, homogenate is fully ground and is made 10-1Sample stoste, stands 30min；Sample stoste is subjected to ladder successively afterwards
Degree dilution, is 10 from dilution factor-2, 10-3Dilution；
(4) take the dilution of 200 μ L steps (3) selection and be added separately on NA flat boards, be applied to flat board drying, experiment weight
It is multiple 3 times；NA flat boards after coating are placed in the bacterium grown after 28 DEG C of constant incubator culture, culture 24h on observation NA flat boards
The form fallen, picking surface compact, color are golden yellow single bacterium colony,；And the single bacterium colony of institute's picking is inoculated in seed liquor training
Support in base, cultivated at 30 DEG C and seed liquor is obtained after 16h；Gained seed liquor is accessed in fermentation medium and carried out in fermented and cultured, hair
Ferment culture 72h, 30 DEG C of temperature；(at room temperature, rotating speed 6000rpmmin is centrifuged in fermentation gained zymotic fluid-1Centrifugation
10min), supernatant is collected, ice ethanol is added into supernatant, and the volume ratio of ice ethanol and supernatant is 3：1, after 4
Sedimentation exocellular polysaccharide is stood overnight at DEG C；Then (at room temperature, rotating speed 6000rpm is centrifuged in the solution after standing
min-1Centrifuge 10min), collect sediment；Using absolute ethyl alcohol washing precipitate 3 times, then nitrogen is dried up, and is finally crushed and is produced
Exocellular polysaccharide product, claims dry weight；Compare the exocellular polysaccharide product yield of different strains, filter out exocellular polysaccharide product yield highest
Bacterial strain, and by the Strain Designation be bacterial strain FJAT-10272.The identification of embodiment 2, bacterial strain FJAT-10272
(1) physio-biochemical characteristics are identified：
The measure of the bacterial strain FJAT-10272 progress physio-biochemical characteristics obtained will be screened, obtain the Main Morphology of the bacterial strain
Learn feature as follows：Bacterium colony is smaller, circular, yellow, smooth surface and moistening, micro- grand, neat in edge, it is opaque, with migration.
(2) Sherlock-MIDI system identifications：The microorganism automatic identifying system produced using MIDI companies of the U.S.
(Sherlock Microbial Identification System) is analyzed bacterial strain FJAT-10272 aliphatic acid, point
Analysis result shows that the bacterial strain FJAT-10272 aliphatic acid and Sherlock-MIDI systems Plays move Sphingol single-cell less
The similarity factor of the aliphatic acid of bacterial strain is up to 0.609；Bacterial strain FJAT-10272 aliphatic acid figure is as shown in Figure 1.
The qualification result of 2 kinds of methods of summary, then judge bacterial strain FJAT-10272 to move Sphingol single-cell less
(Sphingomonas paucimobilis)。
Embodiment 3, using bacterial strain FJAT-10272 prepare exocellular polysaccharide
Bacterial strain FJAT-10272 activation：With oese Sphingol single-cell bacterium will be moved less described in claim 1
Strain FJAT-10272 is lined on NA flat boards, and cultivates in constant incubator 48h, and cultivation temperature is 30 DEG C；
Prepare seed liquor：Bacterial strain FJAT-10272 is activated to the Sphingomonas FJAT-10272 dynamic less obtained
Single bacterium colony be inoculated in seed culture medium, and be placed in constant temperature oscillation shaking table and cultivate 16h, 30 DEG C of temperature, rotating speed
170rpm·min-1, obtain seed liquor；
Prepare zymotic fluid：The seed liquor obtained is transferred to sterilization by 2% inoculum concentration by percent by volume
Fermented and cultured 72h in fermentation medium, 30 DEG C of temperature, rotating speed 190rpmmin-1, produce zymotic fluid；
The extraction of exocellular polysaccharide：(at room temperature, rotating speed 6000rpmmin is centrifuged in the zymotic fluid of gained-1From
Heart 10min), supernatant is collected, ice ethanol is added into supernatant, and the volume ratio of ice ethanol and supernatant is 3：1, after
Sedimentation exocellular polysaccharide is stood overnight at 4 DEG C；Then (at room temperature, rotating speed 6000rpm is centrifuged in the solution after standing
min-1Centrifuge 10min), collect sediment；Using absolute ethyl alcohol washing precipitate 3 times, then nitrogen is dried up, and is finally crushed and is produced
The exocellular polysaccharide product.
Embodiment 4, exocellular polysaccharide produced by the present invention are determined
The content of neutral polysaccharide in exocellular polysaccharide product made from embodiment 3 is determined using phend-sulphuric acid：Example
Exocellular polysaccharide product accurately configures 2gL made from 3-1Exocellular polysaccharide solution, measure the exocellular polysaccharide that 0.4mL prepared molten
Liquid, and 1mL is complemented to using ultra-pure water, it is subsequently added into the phenol solutions of 0.5mL 6% and the 3mL concentrated sulfuric acids, boiling water after vibration is mixed
20min is bathed, room temperature is subsequently cooled to, absorbance is determined most at 490nm wavelength, using glucose as control, the result of measure
Such as table 1 below.
The content of neutral sugar in the exocellular polysaccharide product of table 1
As shown in Table 1, the content of neutral polysaccharide is up to 0.5817g/L in exocellular polysaccharide product produced by the present invention, therefore,
Indicating being capable of high-yield extracellular polysaccharide using bacterial strain FJAT-10272 of the present invention.
It should be noted that in the present invention, the component of NA flat boards is：Beef extract 3gL-1, peptone 5gL-1, Portugal
Grape sugar 10gL-1, yeast extract 1gL-1, agar powder 17gL-1、pH 7.2；The component of seed culture medium is：Sucrose 20gL-1, peptone 5gL-1, yeast extract 5gL-1、pH 7.0；The component of fermentation medium is：Glucose 30gL-1, peptone
3g·L-1, yeast extract 2gL-1、KH2PO41g·L-1、MgSO4 0.1g·L-1, micro- salting liquid 1mLL-1、pH
7.0；The component of micro- salting liquid is in fermentation medium：MnCl2·4H2O 1.8g·L-1、FeSO4·7H2O 2.4g·
L-1、H3BO30.283g·L-1、CuCl2 0.0027g·L-1、CoCl2·6H2O 0.0074g·L-1.And without specified otherwise
In the case of, the percentage in the present invention is mass percent.
To sum up, the present invention provides a kind of new Sphingomonas FJAT-10272 (Sphingomonas dynamic less
Paucimobilis Sphingomonas FJAT-10272), and using this is moved less and prepares exocellular polysaccharide, and prepares born of the same parents
The yield of exo polysaccharides is higher, i.e., this move less Sphingomonas FJAT-10272 can high-yield extracellular polysaccharide, so as to increase
Exocellular polysaccharide produces the source of bacterial strain；And Sphingomonas FJAT-10272 is moved using the present invention less
The process that (Sphingomonas paucimobilis) prepares exocellular polysaccharide is simple, easily operated.
Claims (5)
1. a kind of move Sphingomonas less, it is characterised in that：The bacterial strain is dynamic Sphingomonas FJAT- less
10272 (Sphingomonas paucimobilis), Chinese microorganism strain preservation management was preserved on 01 14th, 2016
Committee's common micro-organisms center, deposit number is CGMCC NO.11997.
2. a kind of preparation method of exocellular polysaccharide, it is characterised in that：The preparation method specifically includes following steps：
(1) activation for moving Sphingomonas FJAT-10272 less described in claim 1：Will by right with oese
Ask the Sphingomonas FJAT-10272 that moves less described in 1 to line on NA flat boards, and cultivated in constant incubator
48h, and cultivation temperature is 30 DEG C；
(2) seed liquor is prepared：The single bacterium colony inoculation for moving Sphingomonas FJAT-10272 less that step (1) is obtained
In seed culture medium, and it is placed in constant temperature oscillation shaking table and cultivates 16h, 30 DEG C of temperature, rotating speed 170rpmmin-1；
(3) zymotic fluid is prepared：The seed liquor that step (2) is obtained is transferred to by 2% inoculum concentration by percent by volume and sterilized
Fermented and cultured 72h in the fermentation medium of sterilizing, 30 DEG C of temperature, rotating speed 190rpmmin-1, produce zymotic fluid；
(4) extraction of exocellular polysaccharide：Zymotic fluid obtained by step (3) is centrifuged, supernatant is collected, into supernatant
Ice ethanol is added, and the volume ratio of ice ethanol and supernatant is 3：1, after standing overnight sedimentation exocellular polysaccharide at 4 DEG C；Then
Solution after standing is centrifuged, sediment is collected；Using absolute ethyl alcohol washing precipitate 3 times, then nitrogen is dried up,
Finally crush and produce the exocellular polysaccharide product.
3. a kind of preparation method of exocellular polysaccharide according to claim 2, it is characterised in that：The component of the NA flat boards
For：Beef extract 3gL-1, peptone 5gL-1, glucose 10gL-1, yeast extract 1gL-1, agar powder 17gL-1、pH
7.2。
4. a kind of preparation method of exocellular polysaccharide according to claim 2, it is characterised in that：The group of the seed culture medium
It is divided into：Sucrose 20gL-1, peptone 5gL-1, yeast extract 5gL-1、pH 7.0。
5. a kind of preparation method of exocellular polysaccharide according to claim 2, it is characterised in that：The group of the fermentation medium
It is divided into：Glucose 30gL-1, peptone 3gL-1, yeast extract 2gL-1、KH2PO4 1g·L-1、MgSO4 0.1g·L-1, it is micro-
Secondary element salting liquid 1mLL-1、pH 7.0；
Wherein, the component of micro- salting liquid is：MnCl2·4H2O 1.8g·L-1、FeSO4·7H2O 2.4g·L-1、H3BO3
0.283g·L-1、CuCl2 0.0027g·L-1、CoCl2·6H2O 0.0074g·L-1。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113142547A (en) * | 2021-04-20 | 2021-07-23 | 广东药科大学 | Gellan gum/curdlan compound microbial food gum and preparation method thereof |
| CN113957030A (en) * | 2021-11-05 | 2022-01-21 | 南开大学 | Sphingomonas strain with characteristic of efficiently synthesizing Wzy type extracellular polysaccharide and construction method and application thereof |
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| CN1970738A (en) * | 2006-09-12 | 2007-05-30 | 山东大学 | Sphingomonaspaucimobilis of high-yield gellan gum and use therefor |
| CN102747016A (en) * | 2012-06-28 | 2012-10-24 | 福建省农业科学院农业生物资源研究所 | Gellan gum efficient production strain and its application |
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| CN1970738A (en) * | 2006-09-12 | 2007-05-30 | 山东大学 | Sphingomonaspaucimobilis of high-yield gellan gum and use therefor |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113142547A (en) * | 2021-04-20 | 2021-07-23 | 广东药科大学 | Gellan gum/curdlan compound microbial food gum and preparation method thereof |
| CN113957030A (en) * | 2021-11-05 | 2022-01-21 | 南开大学 | Sphingomonas strain with characteristic of efficiently synthesizing Wzy type extracellular polysaccharide and construction method and application thereof |
| CN113957030B (en) * | 2021-11-05 | 2024-01-05 | 南开大学 | Sphingomonas strain with characteristic of synthesizing Wzy type extracellular polysaccharide, construction method and application thereof |
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