CN107686822A - A kind of method for producing low pigment xanthans of fermenting - Google Patents
A kind of method for producing low pigment xanthans of fermenting Download PDFInfo
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- CN107686822A CN107686822A CN201710916896.1A CN201710916896A CN107686822A CN 107686822 A CN107686822 A CN 107686822A CN 201710916896 A CN201710916896 A CN 201710916896A CN 107686822 A CN107686822 A CN 107686822A
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- xanthomonas campestris
- xanthans
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- fermentation
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- 229920001285 xanthan gum Polymers 0.000 title claims abstract description 42
- 239000000049 pigment Substances 0.000 title claims abstract description 16
- 238000004519 manufacturing process Methods 0.000 title claims description 10
- 241000589636 Xanthomonas campestris Species 0.000 claims abstract description 37
- 210000004027 cell Anatomy 0.000 claims abstract description 30
- 238000000855 fermentation Methods 0.000 claims abstract description 28
- 230000004151 fermentation Effects 0.000 claims abstract description 28
- 238000000034 method Methods 0.000 claims abstract description 20
- 230000002950 deficient Effects 0.000 claims abstract description 18
- 210000002421 cell wall Anatomy 0.000 claims abstract description 17
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 13
- 229930006000 Sucrose Natural products 0.000 claims abstract description 10
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 10
- 239000005720 sucrose Substances 0.000 claims abstract description 10
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229910000388 diammonium phosphate Inorganic materials 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims abstract description 7
- 230000002906 microbiologic effect Effects 0.000 claims abstract description 4
- 230000001580 bacterial effect Effects 0.000 claims description 23
- 239000002609 medium Substances 0.000 claims description 19
- 238000011534 incubation Methods 0.000 claims description 15
- 239000007787 solid Substances 0.000 claims description 15
- 229920001817 Agar Polymers 0.000 claims description 12
- 239000008272 agar Substances 0.000 claims description 12
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 7
- 239000001963 growth medium Substances 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 6
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 238000011081 inoculation Methods 0.000 claims description 6
- 210000000170 cell membrane Anatomy 0.000 claims description 5
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 4
- 239000001110 calcium chloride Substances 0.000 claims description 4
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 4
- 238000012549 training Methods 0.000 claims description 4
- 108010046845 tryptones Proteins 0.000 claims description 4
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 3
- 229910052564 epsomite Inorganic materials 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 claims description 2
- 238000004043 dyeing Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000002504 physiological saline solution Substances 0.000 claims 1
- 239000000725 suspension Substances 0.000 claims 1
- 239000012530 fluid Substances 0.000 abstract description 10
- 238000006243 chemical reaction Methods 0.000 abstract description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 5
- 229910052799 carbon Inorganic materials 0.000 abstract description 5
- 239000002994 raw material Substances 0.000 abstract description 3
- 238000005259 measurement Methods 0.000 abstract description 2
- 235000019838 diammonium phosphate Nutrition 0.000 abstract 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 21
- 239000000047 product Substances 0.000 description 13
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 238000001556 precipitation Methods 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 150000004676 glycans Chemical class 0.000 description 6
- 229920001282 polysaccharide Polymers 0.000 description 6
- 239000005017 polysaccharide Substances 0.000 description 6
- 238000002329 infrared spectrum Methods 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 239000000230 xanthan gum Substances 0.000 description 5
- 229940082509 xanthan gum Drugs 0.000 description 5
- 235000010493 xanthan gum Nutrition 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 239000004094 surface-active agent Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- GJCOSYZMQJWQCA-UHFFFAOYSA-N 9H-xanthene Chemical compound C1=CC=C2CC3=CC=CC=C3OC2=C1 GJCOSYZMQJWQCA-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 229920002558 Curdlan Polymers 0.000 description 3
- 239000001879 Curdlan Substances 0.000 description 3
- 102000016943 Muramidase Human genes 0.000 description 3
- 108010014251 Muramidase Proteins 0.000 description 3
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 241000589634 Xanthomonas Species 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 229940078035 curdlan Drugs 0.000 description 3
- 235000019316 curdlan Nutrition 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 239000004325 lysozyme Substances 0.000 description 3
- 229960000274 lysozyme Drugs 0.000 description 3
- 235000010335 lysozyme Nutrition 0.000 description 3
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 241000067595 Cissus campestris Species 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- HYBBIBNJHNGZAN-UHFFFAOYSA-N furfural Chemical compound O=CC1=CC=CO1 HYBBIBNJHNGZAN-UHFFFAOYSA-N 0.000 description 2
- 239000013028 medium composition Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 210000001938 protoplast Anatomy 0.000 description 2
- 235000005040 sarson Nutrition 0.000 description 2
- 244000128879 sarson Species 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 229920001187 thermosetting polymer Polymers 0.000 description 2
- WTLKTXIHIHFSGU-UHFFFAOYSA-N 2-nitrosoguanidine Chemical compound NC(N)=NN=O WTLKTXIHIHFSGU-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- JBQLQIMCKFDOHK-UHFFFAOYSA-N Stephanol Natural products CC(O)C1(O)CCC2(O)C3(O)CC=C4CC(O)CCC4(C)C3C(O)C(O)C12C JBQLQIMCKFDOHK-UHFFFAOYSA-N 0.000 description 1
- 241000219094 Vitaceae Species 0.000 description 1
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 description 1
- 238000004026 adhesive bonding Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000012267 brine Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical class CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 235000021021 grapes Nutrition 0.000 description 1
- 239000009153 huxin Substances 0.000 description 1
- -1 initially PH 6.5~7.5 Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 229940066779 peptones Drugs 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000004260 plant-type cell wall biogenesis Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- C—CHEMISTRY; METALLURGY
- 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
- C12P19/06—Xanthan, i.e. Xanthomonas-type heteropolysaccharides
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention discloses a kind of method that low pigment xanthans is produced using the cell wall defective type cell fermentation of xanthomonas campestris (Xanthomonas campestris) 1.1781, belongs to technical field of bioengineering.The present invention is starting strain using China General Microbiological collection xanthomonas campestris (Xanthomonas campestris) 1.1781, cell wall defective type mutant strain is obtained by continuous passage mode, sucrose is used as carbon source, add the raw materials such as dusty yeast, diammonium hydrogen phosphate, in the case where aerobic condition and pH maintain 5.5 7.5 environment, fermented and cultured obtains product xanthans in 80~100 hours.Liquid fermentation and culture yield can reach 32g/L zymotic fluids, and raw material Sucrose conversion can be more than 90%.OD after being diluted to cell wall defective type cell fermentation liquid560Value measures, and is as a result 0.051, and uses original strain zymotic fluid OD560Measurement result is 0.296, and the result shows that pigment content significantly declines.
Description
Technical field
Lacked the invention discloses one kind using xanthomonas campestris (Xanthomonas campestris) 1.1781 cell membranes
Damage type cell fermentation produces the method for low pigment xanthans and discloses the method for obtaining the strain cell wall wall deficient cell.
Fermentation produces low pigment xanthan gluing method more particularly to the culture medium prescription and fermentation condition to the strain fermentation culture.
Background technology
Xanthans (Xanthan gum) is to utilize carbohydrate through xanthomonas campestris or Xanthomonas camp stris P V. campestris
A kind of acid exocellular polysaccharide obtained for main fermenting raw materials.It is a kind of high molecular polymer, mainly sweet by D-Glucose, D-
Dew sugar and D-Glucose furfural are with 2:2:1 ratio composition, simultaneously containing 2% -6% pyruvic acid and 4.5% acetic acid.
Xanthans has unique rheological behavior because of its unique molecular structure, good water solubility, and acidproof
Alkali, high temperature resistant, because the various good characteristics of xanthans make it more in food, papermaking, beverage, chemical industry, cosmetics, oil exploitation etc.
Individual field is utilized extensively.
Xanthans producing strains are screened the 1950s by Jeanes et al. and obtained, the beginning of the sixties, by the U.S.
C.P.Kelco company trades.1969, United States food and drag administration (FDA) approval xanthans can be used as food to add
Agent；Nineteen eighty-three, the World Food Programme and health organization are ratified it and used in world wide.Xanthans is in global total output within 2010
Reach 110,000 tons, and Chinese total output reaches 7.4 ten thousand tons, xanthans has become the microorganism that output is maximum and purposes is most wide
Polysaccharide.
Xanthan gum fermentation bacterial strain breeding research background：Sutherland IW report that screening Cell wall synthesis defect bacterial strain can
Xanthans superior strain can be obtained.The reports such as Bai Xianfang are using microwave screening non-pigment xanthans producing strains.Wang Guilan etc. is reported
Xanthan gum fermentation output increased can be promoted by adding surfactant in the fermentation medium.Traditional theory thinks, surfactant
The synthesis of cell membrane fat acid can be suppressed, so as to change the constituent of cell membrane and its permeability, tunning is more held
Easily infiltrate, and then improve fermentation production rate.The reports such as Diao Huxin obtain production xanthans Strain Protoplast by lysozyme, should
Protoplast is cultivated in sucrose is the hypertonic medium of substrate, can be synthesized and be secreted xanthans.
This item patent is introduced a kind of by continuous training method acquisition cell wall defective type xanthomonas campestris mutant strain
Method, this method without using surfactant, also be not used lysozyme.
The source of continuous passage training strategy scheme is carried out to bacterial strain：Inventor's thermosetting property since 2014 is more
Sugar research, Tokuya Harada are taught in an entitled " the story ofResearch into Curdlan and the
Mentioned in Bacteria Produing it " paper, what fermentation production can arrive right glue original strain 10C3 most primiparity is a kind of water
Soluble polysaccharide, after the strain Secondary Culture of 2 years, bacterial strain is undergone mutation, and tunning becomes a kind of water-insoluble polysaccharide,
That is curdlan.
Attempted during xanthan gum fermentation research is carried out to bacterial strain sarson Huang unit cell this research department
(Xanthomonas campestris) 1.1781 carries out continuous passage culture, and whether exploration is possible to ferment thermosetting property xanthan
Glue；We fail to find matching somebody with somebody for strain passage culture during Japanese Tokuya Harada professors team research curdlan
Side；We select be by preliminary medium optimization and can output xanthans culture medium prescription, during the research
It was found that by continuous culture in 5-6 months, there is defect in the cell membrane of the bacterium, is embodied in after Gram's staining carefully
Born of the same parents' form is become round by shaft-like.Low pigment xanthans can be obtained using the cell defect type strain fermentation and substrate conversion efficiency can
Up to more than 90%.
The content of the invention
One object of the present invention, it is a kind of that xanthomonas campestris (Xanthomonas is prepared by way of continuous passage
Campestris) the method for 1.1781 cell wall defective type bacterial strains.It is characteristic of the invention that obtained by way of continuous passage
The cell wall defective type bacterial strain of xanthomonas campestris (Xanthomonascampestris) 1.1781, the bacterial strain contaminate through gram
Cell is circular rather than shaft-like (going out bacterium germination gram-Negative bacillus) under simple microscope after color.Lured different from conventional ultra-violet
Change, nitrosoguanidine mutagenesis, also without the material such as addition surfactant, EDTA, SDS or lysozyme, the present invention is obtains cell
Wall deficient xanthomonas campestris provides a kind of new method.This method comprises the following steps：
1st step, inclined-plane culture：By purchased from the xanthomonas campestris of China General Microbiological preservation administrative center
(Xanthomonas campestris) 1.1781 bacterial strain streak inoculation is cultivated in slant medium.In slant medium
Include following component：20g/L glucose, 5g/L dusty yeasts, 5g/L tryptones, 20g/L agar, pH 6.5~7.5；Culture temperature
28~32 DEG C of degree, incubation time 50~80 hours.
2nd step, continuous passage culture：Take in the step of a ring the 1st inclined-plane seed to be seeded to HF-1 solid mediums to be cultivated；HF-1
Following component is included in solid medium：30g/L glucose, 1-2g/L dusty yeasts, 1-2g/L (NH4)2HPO4, 0.5-1g/L paddy
Propylhomoserin, 1-2g/L CaCO3, 0.5g/L MgSO4·7H2O, 20g/L agar, pH 6.5~7.5,28~32 DEG C of cultivation temperature, training
Support 2~4 days time, continuous passage incubation time about 5-6 months.
Another object of the present invention, there is provided utilize xanthomonas campestris (Xanthomonas campestris)
1.1781 cell wall defective type bacterial strains carry out the method that fermented and cultured produces low pigment xanthans.Xanthans is through sarson Huang unit cell
A kind of exocellular polysaccharide caused by bacterium or Xanthomonas camp stris P V. campestris fermentation, and it is currently one kind that production scale is maximum in the world
Polysaccharide.Uranidin can be produced during due to producing xanthans in fermentation, and then increases extraction cost.Therefore, fermentation production is found
The method of low pigment xanthans has very important significance.On the other hand, substrate conversion efficiency is improved, similarly helps to reduce life
Produce cost, this patent report is a kind of conversion ratio can be reached more than 90% method, through analysis, conversion ratio can reach 90%
The reason for be improve individual cells production xanthans ability.This method comprises the following steps：
1st step, inclined-plane culture：Bacterial strain streak inoculation is cultivated in slant medium, slant medium composition：20g/
L sucrose, 5g/L dusty yeasts, 5g/L tryptones, 20g/L agar, pH 6.5~7.5；28~32 DEG C of cultivation temperature, incubation time
40~70 hours, the culture medium contained abundant carbon source and nitrogen source, is advantageous to thalli growth.
2nd step, fermented and cultured：Ring species access fermentation medium HF-2 is taken to be cultivated from inclined-plane, the culture medium includes：
35g/L sucrose, 0.5-1g/L dusty yeasts, 0.5-1g/L (NH4)2HPO4, 0.5-1g/L Pidolidones, 1g/L CaCl2, initially
PH 6.5~7.5, liquid amount 30ml are per 250ml triangular flasks, 28~32 DEG C, rotating speed 180-200rpm/min of cultivation temperature, culture
Hour time 80-100；
3rd step, extraction product：20ml zymotic fluids are taken, the dilution of 40ml pure water is added and mixes, centrifuged under the conditions of 9000rpm
20min, collect supernatant.3 times of ethanol of volume 95% are added in supernatant and separate out xanthans, product is collected, is dried under the conditions of 60 DEG C
To constant weight.
4th step, dry cell weight measure：20ml zymotic fluids are taken, the dilution of 40ml pure water is added and mixes, centrifuged under the conditions of 9000rpm
20min, collect precipitation.After 2 times of mL normal saline washings are added in precipitation, 20min is centrifuged under the conditions of 9000rpm, is collected thin
Born of the same parents are precipitated, and constant weight is dried under the conditions of 60 DEG C.
Brief description of the drawings
Fig. 1 is tunning infrared spectrum.
Fig. 2 is photo under cell transmission electron microscope, and cell week is with substantial amounts of tunning.Cell under transmission electron microscope
Form is rod-short, different with rounded form under Gram's staining ordinary electronic microscope.Reason is Gram's staining
There is the step of Ethanol Treatment cell in step, and Ethanol Treatment cell is not used in transmission electron microscope cell processing procedure.
Embodiment
It is that the continuous passage culture of xanthomonas campestris (Xanthomonas campestris) 1.1781 obtains carefully below
The method of cell wall deficient cell and the embodiment that low pigment xanthans research is produced using the cell fermentation.But the present invention is not
It is limited to listed several examples.
The strain passage culture of embodiment 1
By purchased from the xanthomonas campestris of China General Microbiological preservation administrative center (Xanthomonas campestris)
1.1781 bacterial strain streak inoculations are cultivated in slant medium.Following component is included in slant medium：20g/L grapes
Sugar, 5g/L dusty yeasts, 5g/L tryptones, 20g/L agar, pH 6.5~7.5,28~32 DEG C of cultivation temperature, incubation time 50
~80 hours.
Continuous passage culture：Take a ring inclined-plane seed to be seeded to HF-1 solid mediums to be cultivated；In HF-1 solid mediums
Include following component：30g/L glucose, 1-2g/L dusty yeasts, 1-2g/L (NH4)2HPO4, 0.5-1g/L glutamic acid, 1-2g/L
CaCO3, 0.5g/L MgSO4·7H2O, 20g/L agar, pH 6.5~7.5,28~32 DEG C of cultivation temperature, incubation time 2~4
My god, continuous passage incubation time 5-6 months, it is thin to obtain xanthomonas campestris (Xanthomonas campestris) 1.1781
Cell wall deficient bacterial strain.
Experimental result：
A ring bacterial strain on HF-1 flat boards is taken, is mixed after adding sterile saline and it carries out Gram's staining, it is common aobvious
Micro- Microscopic observation cellular morphology.Form of the bacterial strain under simple microscope is circular rather than shaft-like, and a diameter of 0.8 ---
2.0um, cellular colours are red, are gram-Negative bacillus.Outside has a light blue circle, for xanthans dyeing knot
Fruit；
The cell wall defective type cell of the xanthomonas campestris of embodiment 2 (Xanthomonas campestris) 1.1781 in
HF-2 solid medium cultures
The cell wall defective type cell of a ring xanthomonas campestris (Xanthomonas campestris) 1.1781 is taken in HF-2
Solid medium culture, HF-2 culture mediums are obtained after being optimized on HF-1 medium bases, compared with HF-1 culture mediums,
Carbon source has changed sucrose into from glucose, and HF-2 culture mediums have higher carbon-nitrogen ratio.HF-2 solid culture based components：35g/L
Sucrose, 0.5-1g/L dusty yeasts, 0.5-1g/L (NH4)2HPO4, 0.5-1g/L Pidolidones, 1g/L CaCl2, 20g/L agar,
PH 6.5~7.5, cultivation temperature are 28~32 DEG C, and incubation time 4 days, agar surface forms product.
Experimental result
The cell wall defective type cell of xanthomonas campestris (Xanthomonas campestris) 1.1781 is trained in HF-2 solids
Colonial morphology after being cultivated 4 days in base is supported, bacterium colony is rounded, color is sticky for white, surface；
Embodiment 3HF-2 solid-substrate fermentations yield determines
Product after HF-2 solid culture flat boards culture 4 days is taken, medication spoon is collected tunning and thalline in culture dish and (is careful not to
Scratch agar), with distillation water washing culture dish surface residual xanthans product and thalline and it is collected into beaker.In collected production
After adding distilled water diluting (2-4 times of volume) in thing, centrifuged 20 minutes under the conditions of 9000rpm.Thalline is precipitated as, supernatant is hair
Ferment product.After cell precipitation brine, centrifuged 20 minutes under the conditions of 9000rpm, collect cell precipitation again, 60 DEG C
Under the conditions of be dried to constant weight；Remove and 2 times of ethanol of volume 95% are added in thalline tunning, product forms precipitation and provoked with glass rod
After collection, washed with 1 times of ethanol of volume 95%, constant weight is dried under the conditions of 60 DEG C.
Experimental result
Thalline is collected from 1 piece of solid culture ware, it is 6.8mg that dry cell weight is measured after drying；Tunning is collected, adds 2 times
It is tunning a large amount of gelatinous precipitates occur after the ethanol of volume 95%, in beaker, and measuring yield of xanthan gum after washing and drying is
229mg.I.e. in flat board incubation, 1mg somatic cells, which can ferment, produces 33.7mg xanthans.
The infrared spectrum analysis tunning structure of embodiment 4
Take to collect in example 3 and obtain tunning, using decay total reflection (ATR) method measure product infrared spectrum.By the xanthan
Glue sample is close on atr crystal, the scanning acquisition product infrared spectrogram (Fig. 1) in 400-4000cm-1 sections.Using U.S.
Thermo companies of state, Nexus type infrared chromatographs.
As shown in figure 1, the product infrared spectrum feature is consistent with xanthans infrared spectrum.889cm-1 shows the sugar by β glycosidic bonds
Connection, 3415.7cm-1 O-H, 2917.8cm-1 C-H.
Embodiment 5HF-2 liquid shake-flask fermentations produce low pigment xanthans
Fermentation strain is the cell wall defective type bacterial strain of xanthomonas campestris (Xanthomonas campestris) 1.1781, is entered
The method that row fermented and cultured produces low pigment xanthans, comprises the following steps：
1st step, inclined-plane culture：Bacterial strain streak inoculation is cultivated in slant medium, slant medium composition：20g/L
Sucrose, 5g/L dusty yeasts, 5g/L peptones, 20g/L agar, pH 6.5~7.5；28~32 DEG C of cultivation temperature, incubation time 50
~70 hours, the culture medium contained abundant carbon source and nitrogen source, is advantageous to thalli growth.
2nd step, fermented and cultured：Ring species access fermentation medium HF-2 is taken to be cultivated from inclined-plane, liquid amount 30ml is every
250ml triangular flasks, 30 DEG C of cultivation temperature, rotating speed 180rpm/min, aerobic incubation time 80-100 hours；
3rd step, extraction product：20ml zymotic fluids are taken, the dilution of 40ml pure water is added and mixes, centrifuged under the conditions of 9000rpm
20min, collect supernatant.3 times of ethanol of volume 95% are added in supernatant and separate out xanthans, product is collected, is dried under the conditions of 60 DEG C
To constant weight.
4th step, dry cell weight measure：20ml zymotic fluids are taken, the dilution of 40ml pure water is added and mixes, centrifuged under the conditions of 9000rpm
20min, collect precipitation.After 2 times of mL normal saline washings are added in precipitation, 20min is centrifuged under the conditions of 9000rpm, is collected thin
Born of the same parents are precipitated, and constant weight is dried under the conditions of 60 DEG C.
The sterilising temp of culture medium is 121 DEG C, 20min.Carbon source dispenses after individually sterilizing.
Experimental result
Tunning 645mg is extracted from 20ml zymotic fluids, it is 7.4mg to collect and obtain dry cell weight.20ml zymotic fluids contain bottom
Thing 700mg, i.e. substrate conversion efficiency are 92.1%；Per mg thalline can product 87.2mg, i.e. individual cells gum yield exceedes cell
80 times of weight.Pigment detection：Zymotic fluid is diluted 50 times, and to OD560Value measures, and is as a result 0.051, and uses former
Beginning bacterial strain fermentation liquor OD560Measurement result is 0.296, and zymotic fluid pigment content greatly declines.
Claims (2)
1. a kind of prepare xanthomonas campestris (Xanthomonas campestris) 1.1781 by way of continuous passage
The method of cell wall defective type mutant strain, it is characterised in that comprise the following steps：
1st step, inclined-plane culture：By purchased from the xanthomonas campestris of China General Microbiological preservation administrative center
(Xanthomonas campestris) 1.1781 bacterial strain streak inoculation is cultivated in slant medium.In slant medium
Include following component：20g/L glucose, 5g/L dusty yeasts, 5g/L tryptones, 20g/L agar, pH 6.5~7.5；Culture temperature
28~32 DEG C of degree, incubation time 50~80 hours.
2nd step, continuous passage culture：Take in the step of a ring the 1st inclined-plane seed to be seeded to HF-1 solid mediums to be cultivated；HF-1
Following component is included in solid medium：30g/L glucose, 1-2g/L dusty yeasts, 1-2g/L (NH4)2HPO4, 0.5-1g/L paddy
Propylhomoserin, 1-2g/L CaCO3, 0.5g/L MgSO4·7H2O, 20g/L agar, pH 6.5~7.5,28~32 DEG C of cultivation temperature, training
Support 2~4 days time, continuous passage incubation time about 5-6 months.
The discovery of 3rd step, cell wall defective type bacterial strain：After continuous passage incubation time about 5-6 months, the bacterial strain is in HF-1 solids
After being cultivated 2~4 days in culture medium, take a ring thalline to be placed in 1ml physiological saline, after mixing, take a drop bacteria suspension to carry out gram
Dyeing.Its form under simple microscope is circular (starting strain is that Gram-negative is shaft-like), red, and diameter is about
0.8 --- 2.0um, cell week are with light blue circle.
2. using bacterial strain xanthomonas campestris (Xanthomonas campestris) 1.1781 cell membranes described in claim 1
The method that deficient cell fermentation prepares low pigment xanthans.
It is characterized in that：
(1) flat board culture production xanthans：After the bacterial strain is cultivated into 2~4 in HF-2 solid mediums, circular colonies, face are produced
Color is transparent, surface is sticky.Following component is included in HF-2 solid mediums：35g/L sucrose, 0.5-1g/L dusty yeasts, 0.5-1g/
L(NH4)2HPO4, 0.5-1g/L Pidolidones, 1g/L CaCl2, 20g/L agar, initial pH 6.5~7.5, cultivation temperature is
28~32 DEG C, incubation time 2~4 days.
(2) liquid fermentation and culture：A ring inclined-plane inoculation is taken in HF-2 liquid fermentation mediums.Fermentation medium includes such as
Lower composition：35g/L sucrose, 0.5-1g/L dusty yeasts, 0.5-1g/L (NH4)2HPO4, 0.5-1g/L Pidolidones, 1g/L
CaCl2, initial pH 6.5~7.5,28~32 DEG C of cultivation temperature, incubation time 80~100 hours.
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Cited By (4)
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| CN110777105A (en) * | 2019-11-20 | 2020-02-11 | 上海交通大学 | A high-yield engineering strain of white gum and its construction and application |
| CN114015612A (en) * | 2021-11-30 | 2022-02-08 | 山东省食品发酵工业研究设计院 | Xanthomonas citrii and application thereof in fermentation production of xanthane gum |
| CN117286082A (en) * | 2023-11-24 | 2023-12-26 | 内蒙古工业大学 | Xanthomonas campestris and method for producing low-viscosity xanthan gum by fermentation |
| CN117535202A (en) * | 2023-12-21 | 2024-02-09 | 内蒙古工业大学 | Xanthomonas campestris for producing high-transparency xanthan gum by fermentation and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110777105A (en) * | 2019-11-20 | 2020-02-11 | 上海交通大学 | A high-yield engineering strain of white gum and its construction and application |
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| CN117286082B (en) * | 2023-11-24 | 2024-01-30 | 内蒙古工业大学 | Xanthomonas campestris and method for producing low-viscosity xanthan gum through fermentation |
| CN117535202A (en) * | 2023-12-21 | 2024-02-09 | 内蒙古工业大学 | Xanthomonas campestris for producing high-transparency xanthan gum by fermentation and application thereof |
| CN117535202B (en) * | 2023-12-21 | 2024-04-02 | 内蒙古工业大学 | Xanthomonas campestris for producing transparent xanthan gum by fermentation and application thereof |
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