CN104726371B - A kind of Xanthomonas axonopodis bacterial strain and application - Google Patents

A kind of Xanthomonas axonopodis bacterial strain and application Download PDF

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CN104726371B
CN104726371B CN201510101981.3A CN201510101981A CN104726371B CN 104726371 B CN104726371 B CN 104726371B CN 201510101981 A CN201510101981 A CN 201510101981A CN 104726371 B CN104726371 B CN 104726371B
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bacterial strain
xanthomonas axonopodis
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fjat
polysaccharide colloid
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CN104726371A (en
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朱育菁
郑梅霞
刘波
黄素芳
潘志针
陈峥
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Crop Research Institute Of Fujian Academy Of Agricultural Sciences Fujian Provincial Germplasm Resources Center
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Institute of Agricultural Biological Resources of Fujian Academy of Agricultural Sciences
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/64—Xanthomonas
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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 bacterial strain that the present invention separates from the shaddock blade of the yellow twig erythrocyte growth model of Shunchang, Fujian Province and screens to obtain by producing polysaccharide colloid, the bacterial strain is Xanthomonas axonopodis bacterial strain FJAT 10151 (Xanthomonas axonopodis), and prepares a kind of polysaccharide colloid of good performance using Xanthomonas axonopodis bacterial strain FJAT 10151；The polysaccharide colloid hardness is small, elasticity, adherence it is good, adhesivity and chewiness are good, additionally with preparation process it is simple the characteristics of.

Description

A kind of Xanthomonas axonopodis bacterial strain and application
【Technical field】
The present invention relates to microorganism and its application, more particularly to a kind of Xanthomonas axonopodis bacterial strain and its preparation of application it is more Carbohydrate gum matter.
【Background technology】
Polysaccharide is the polymer that is formed by certain way repeated arrangement using monose as basic composition unit.For food plus The polysaccharide of work is commonly called as colloid, i.e. polysaccharide colloid.Polysaccharide colloid can derive from animal, plant, microorganism, seaweeds polysaccharide colloid Mainly include agar and carragheen and alginic acid and its derivative.Plant polysaccharide colloid includes Arabic gum, cellulose gum And its derivative, konjaku flour etc..Microbial polysaccharide colloid mainly includes xanthans, gellan gum, carragheen etc., have adherence, The features such as stability, emulsibility and gelation, suffer from being widely applied in food, field of medicaments etc., it is especially near Nian Lai, with the introducing of Protocols in Molecular Biology, polysaccharide is in anticoagulation, anti-aging, anti-cancer and cancer-preventing, disease immune etc. Function is also gradually revealed.By its purposes, polysaccharide colloid can be classified as thickener and gelling agent again, can be used as food additive Add agent, coagulating agent, antistaling agent etc..The microorganism Pseudomonas for producing polysaccharide colloid reported has Sphingomonas (Sphingomonas), Agrobacterium (Agrobacterium), xanthomonas (Xanthomonas) etc., but have no carpet Straw colour monad is as the application for preparing polysaccharide colloid.
【The content of the invention】
The technical problems to be solved by the invention are to provide a kind of Xanthomonas axonopodis bacterial strain FJAT-10151 (Xanthomonas axonopodis), Xanthomonas axonopodis bacterial strain FJAT-10151 (Xanthomonas Axonopodis China Committee for Culture Collection of Microorganisms's common micro-organisms center) was preserved on 01 04th, 2015, Address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and deposit number is CGMCC No.10271, and uses the carpet straw colour Aeromonas strain FJAT-10151 prepares that a kind of hardness is small and elastic and the preferable polysaccharide colloid of adherence.
The present invention is that solve above-mentioned technical problem by the following technical programs：
This laboratory separates from the shaddock blade of the yellow twig erythrocyte growth model of Shunchang, Fujian Province and sieved by producing polysaccharide colloid Select obtained bacterial strain, and the bacterial strain its physicochemical property etc. is identified, be finally identified as Xanthomonas axonopodis Pseudomonas One bacterial strain.
Further, prepare that a kind of hardness is small and elastic and the preferable polysaccharide colloid of adherence using the bacterial strain, Its preparation method comprises the following steps that：
(1) activation of the Xanthomonas axonopodis bacterial strain FJAT-10151 described in claim 1：With oese by right It is required that the Xanthomonas axonopodis bacterial strain FJAT-10151 described in 1 is lined 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 for the Xanthomonas axonopodis bacterial strain FJAT-10151 that step (1) is obtained is connect Kind is placed in constant temperature oscillation shaking table in seed culture medium and cultivates 16h, 30 DEG C of temperature, rotating speed 170rpm/min；
(3) bacterial strain fermentation liquor is prepared：The seed liquor that step (2) is obtained is transferred to sterilization by 2% inoculum concentration Cultivated in fermentation medium, 30 DEG C, rotating speed 190rpm/min of temperature, culture 72h obtains bacterial strain fermentation liquor；
(4) preparation of polysaccharide colloid：Bacterial strain fermentation liquor obtained by step (3) is centrifuged, collects supernatant, is used Ice ethanol settles polysaccharide colloid, and the volume ratio of ice ethanol and supernatant is 3：1；Stood overnight after at 4 DEG C, then carry out Centrifuge, the precipitation 3 times of centrifugation gained is washed using absolute ethyl alcohol, nitrogen drying, finally crushes and produces the polysaccharide colloid 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；The component of the seed culture medium is：Sucrose 20gL-1, peptone 5g·L-1, yeast extract 5gL-1、pH 7.0；Fermentation medium：Glucose 30gL-1, peptone 3gL-1, yeast extract 2g L-1、KH2PO41g·L-1、MgSO40.1g·L-1, micro- salting liquid 1mL/L, pH 7.0；Wherein, micro- salting liquid Component be：MnCl2·4H2O 1.8g·L-1, FeSO4·7H2O 2.4g·L-1, H3BO30.283g·L-1, CuCl20.0027g·L-1, CoCl2·6H2O 0.0074g·L-1。
The beneficial effects of the present invention are：
A kind of Xanthomonas axonopodis bacterial strain FJAT-10151 is provided, and uses the Xanthomonas axonopodis bacterial strain FJAT-10151 prepares polysaccharide colloid；The polysaccharide colloid hardness is small, and elasticity, adherence are good, and adhesivity and chewiness are good, additionally With preparation process it is simple the characteristics of.
【Brief description of the drawings】
The invention will be further described in conjunction with the embodiments with reference to the accompanying drawings.
Fig. 1 is the bacterial strain FJAT-10151 of 16S rDNA sequence constructs phylogenetic tree.
【Embodiment】
A kind of Xanthomonas axonopodis bacterial strain FJAT-10151 (Xanthomonas axonopodis) of the present invention is from good fortune The bacterial strain for being separated on the shaddock blade of the yellow twig erythrocyte growth model of Jian Sheng Shunchang Counties and screening to obtain by producing polysaccharide colloid, and pass through this Bacterial strain obtains the polysaccharide colloid that a kind of hardness is small, colloidality is good.
1st, bacterial strain FJAT-10151 separation：
(1) shaddock blade (the shaddock blade is in the north bottom of whole red heart grapefruit) 5g of yellow twig erythrocyte growth model is weighed, Shaddock blade is rinsed well with running water first, blots the water of shaddock blade surface, after soaking 30s in 75% alcohol, then Take out with 10% sodium hypochlorite immersion 5 minutes, then with rinsed with sterile water shaddock blade 3 times；
(2) in order to examine the effect that shaddock blade surface sterilizes, the shaddock treated through step (1) is clipped with sterilized tweezers Blade simultaneously makes its surface contact 2min with NA flat boards, and NA flat boards then are placed on into 30 DEG C of culture 2d, if finding do not have bacterium on flat board Drop out existing, it was demonstrated that shaddock blade surface sterilizing is thorough, can be used in follow-up operation；
(3) take step (2) to be sterilized thoroughly shaddock blade after examining, in shaddock blade is shredded to mortar under sterile working and add Enter 45mL sterilized waters, be fully ground homogenate and be made 10-1Sample stoste, stand 30min；It is dilute that sample stoste is subjected to gradient successively Release, be 10 from dilution factor-2、10-3；Take 200 μ L dilutions to be added separately on NA flat boards, and be applied to flat board drying, repeat 3 It is secondary；NA flat boards after coating are placed in 28 DEG C of incubator and cultivate 24h, it is observation colonial morphology, color, rim condition, transparent The features such as degree, surface dry and wet state；The representative single bacterium colony in each position of picking, and count, purify, -80 DEG C of 20% glycerine Preserve, so as to isolated bacterial strain, describe for convenience, be bacterial strain FJAT-10151 by separating obtained Strain Designation.
2. bacterial strain FJAT-10151 identification:
Morphological Identification：
Physicochemical property to bacterial strain FJAT-10151 etc. identifies, obtain bacterial strain FJAT-10151 Main Morphology and Physiology and biochemistry is as follows：Bacterium colony is smaller, circular, faint yellow, surface is smooth and moistening, micro- grand, neat in edge, it is opaque, have and move Shifting property, Gram-negative, aesculin, ONPG, semi-solid agar, glucose are semi-solid, Gluconic Acid Ammonium salt and ornithine decarboxylase are The positive, MR-VP, malonate, urease, mannitol, sorbierite, dulcitol, lactose, xylose, salicin, rhamnose, cotton seed Sugar, xylose gelatin, Xi Mengshi citrates, lysine decarboxylase and tryptophan meat soup are feminine gender.
Molecular biology identification：
DNA extractions are carried out to bacterial strain FJAT-10151 using phenol chloroform method；To extract obtained bacterial strain DNA as mould Plate, with bacterial universal primers (such as SEQ ID NO：1st, shown in 2：- the GAGTTTGATCCTGGCTCAG-3 ' of forward primer 5 ', reversely - ACGGCTACCTTGTTA the CGACTT-3 ' of primer 5 ') it is that primer pair enters performing PCR amplification：
Pcr amplification reaction system (25 μ L systems)：1 μ L templates, 0.5 μ L 0.01molL-1dNTP、2.5μL 10× Buffer, primer each 1 μ L and 0.3 μ L (5U μ L-1) Taq enzyme；
Pcr amplification reaction program：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 45s, 72 DEG C of extension 1min 30s, 35 circulations；Last 72 DEG C of extensions 10min.
The sequencing and analysis of PCR primer：Pcr amplification product is directly delivered to the progress of Shanghai Bo Shang Bioisystech Co., Ltd It is sequenced (sequence measured is as shown in SEQ ID NO.3), its nucleotides sequence shows 1412bp.By the sequence measured through NCBI ratios Right, phylogenetic tree is shown in Fig. 1.By Fig. 1 it will be evident that bacterial strain FJAT-10151 and Xanthomonas axonopodis category (Xanthomonas axonopod) is closest on evolution position.
Pass through above-mentioned identification, it may be determined that bacterial strain FJAT-10151 belongs to Xanthomonas axonopodis category (Xanthomonas axonopod)。
3. the specific preparation process of polysaccharide colloid includes following operation：
(1) Xanthomonas axonopodis bacterial strain FJAT-10151 activation：With oese by Xanthomonas axonopodis bacterial strain FJAT-10151 is lined on NA flat boards, and 48h is cultivated in constant incubator, and cultivation temperature is 30 DEG C；
(2) seed liquor is prepared：The single bacterium colony for the Xanthomonas axonopodis bacterial strain FJAT-10151 that step (1) is obtained is connect Kind is placed in constant temperature oscillation shaking table in seed culture medium and cultivates 16h, 30 DEG C of temperature, rotating speed 170rpm/min；
(3) bacterial strain fermentation liquor is prepared：The seed liquor that step (2) is obtained is transferred to sterilization by 2% inoculum concentration Cultivated in fermentation medium, 30 DEG C, rotating speed 190rpm/min of temperature, culture 72h obtains bacterial strain fermentation liquor；
(4) preparation of polysaccharide colloid：Bacterial strain fermentation liquor obtained by step (3) is centrifuged, collects supernatant, is used Ice ethanol settles polysaccharide colloid, and the volume ratio of ice ethanol and supernatant is 3：1；Stood overnight after at 4 DEG C, then carry out Centrifuge, the precipitation 3 times of centrifugation gained is washed using absolute ethyl alcohol, nitrogen drying, finally crushes and produces the polysaccharide colloid Product.
Wherein, the component of NA flat boards is：Beef extract 3gL-1, peptone 5gL-1, glucose 10gL-1, yeast extract 1g·L-1, agar powder 17gL-1、pH 7.2；The component of seed culture medium is：Sucrose 20gL-1, peptone 5gL-1, yeast Cream 5gL-1、pH 7.0；Fermentation medium：Glucose 30gL-1, peptone 3gL-1, yeast extract 2gL-1、 KH2PO41g·L-1、MgSO40.1g·L-1, micro- salting liquid 1mL/L (i.e. contain the micro- of 1mL in every liter of fermentation medium Secondary element salting liquid), pH 7.0；Wherein, the component of micro- salting liquid is：MnCl2·4H2O 1.8g·L-1, FeSO4· 7H2O 2.4g·L-1, H3BO30.283g·L-1, CuCl20.0027g·L-1, CoCl2·6H2O 0.0074g·L-1。
4. polysaccharide colloid specificity analysis is tested：
Test method：
Survey the viscosity of polysaccharide colloid of the present invention under the conditions of 1% concentration, 60r/min.The mechanical property of polysaccharide colloid uses matter The most frequently used test pattern Texture profile analysis (TPA) in the test of structure instrument, the texture parameter of correlation can be obtained Have：Hardness, coherency, elasticity, recovery, adhesiveness, chewiness and fragility etc.；And using commercially available gellan gum and xanthans as pair According to.
Sample preparation：Polysaccharide colloid of the present invention, gellan gum, xanthan gum powder it will dissolve in right amount at room temperature, stirring is equal After even, packing in a mold, is immediately placed in cooling gel in brine ice, is used to carry out TPA surveys after 24h is placed in 4 DEG C of refrigerators Examination.
And the test parameter of TPA tests is：Precompressed speed is 1.0mm/s, and it is 1.0mm/s to push speed, replys speed and is 1.0mm/s, depression distance 4mm, the residence time between two second compressions is 5s, trigger force 5g.Every group of test is done 5 times and put down Row measure, experimental result are averaged.
Result of the test：
The polysaccharide colloid that bacterial strain FJAT-10151 of the present invention is produced is soluble in water, jelly, thick；Polysaccharide colloid, knot The viscosity of cold glue and xanthans is respectively 408mpa/s, 266mpa/s and 436mpa/s, i.e. polysaccharide colloid viscosity and xanthans Quite, it is 1.5 times of gellan gum, shows that the colloidality of the polysaccharide colloid is good；And compression-the stretching for testing to obtain according to TPA is bent The texture parameter that line obtains correlation is as shown in table 1.
The viscosity of the polysaccharide of table 1 and related texture parameter
Tackness reflection is due to that sticking together for test sample acts on consumed work(；The broken colloidal state of adhesivity reflection is to can Required energy during chewing state；Chewiness is reflection from chewable state to the energy that can be swallowed required for state；By table 1 Understand that the present invention prepares the polysaccharide colloid of gained compared with commercially available xanthans, gellan gum：Hardness is small, elasticity and adherence compared with It is good, and adhesivity and chewiness are best.
To sum up, the invention provides a kind of Xanthomonas axonopodis bacterial strain FJAT-10151, and the carpet straw colour is used Aeromonas strain FJAT-10151 prepares polysaccharide colloid；The polysaccharide colloid hardness is small, and elasticity, adherence are good, adhesivity and chewing Property is good, additionally with preparation process it is simple the characteristics of.

Claims (3)

  1. A kind of 1. Xanthomonas axonopodis bacterial strain, it is characterised in that：The bacterial strain is Xanthomonas axonopodis bacterial strain FJAT- 10151(Xanthomonas axonopodis), Chinese microorganism strain preservation management committee was preserved on 01 04th, 2015 Member's meeting common micro-organisms center, deposit number is CGMCC No.10271.
  2. A kind of 2. polysaccharide colloid, it is characterised in that：Its preparation method concretely comprises the following steps：
    (1) activation of the Xanthomonas axonopodis bacterial strain FJAT-10151 described in claim 1：With oese by claim Xanthomonas axonopodis bacterial strain FJAT-10151 described in 1 is lined on NA flat boards, and 48h is cultivated in constant incubator, And cultivation temperature is 30 DEG C；
    (2) seed liquor is prepared：The single bacterium colony for the Xanthomonas axonopodis bacterial strain FJAT-10151 that step (1) is obtained is inoculated in In seed culture medium, and it is placed in constant temperature oscillation shaking table and cultivates 16h, 30 DEG C of temperature, rotating speed 170rpm/min；
    (3) bacterial strain fermentation liquor is prepared：The seed liquor that step (2) is obtained is transferred to the fermentation of sterilization by 2% inoculum concentration Cultivated in culture medium, 30 DEG C, rotating speed 190rpm/min of temperature, culture 72h obtains bacterial strain fermentation liquor；
    (4) preparation of polysaccharide colloid：Bacterial strain fermentation liquor obtained by step (3) is centrifuged, supernatant is collected, with ice second Alcohol settles polysaccharide gum matter, and the volume ratio of ice ethanol and supernatant is 3：1；Stood overnight after at 4 DEG C, then centrifuged Separation, the precipitation 3 times of centrifugation gained is washed using absolute ethyl alcohol, nitrogen drying, finally crushes the production for producing the polysaccharide colloid Thing.
  3. A kind of 3. polysaccharide colloid according to claim 2, it is characterised in that：The component of the NA flat boards is：Beef extract 3g·L-1, peptone 5gL-1, glucose 10gL-1, yeast extract 1gL-1, agar powder 17gL-1、pH 7.2；The kind The component of sub- culture medium is：Sucrose 20gL-1, peptone 5gL-1, yeast extract 5gL-1、pH 7.0；Fermentation medium：Portugal Grape sugar 30gL-1, peptone 3gL-1, yeast extract 2gL-1、KH2PO41g·L-1、MgSO40.1g·L-1, trace element salt Solution 1mL/L, pH 7.0；
    Wherein, the component of micro- salting liquid is：MnCl2·4H2O 1.8g·L-1, FeSO4·7H2O 2.4g·L-1, H3BO30.283g·L-1, CuCl20.0027g·L-1, CoCl2·6H2O 0.0074g·L-1。
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