CN104845896A - Strain and method for producing Welan gum - Google Patents
Strain and method for producing Welan gum Download PDFInfo
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- CN104845896A CN104845896A CN201410485635.5A CN201410485635A CN104845896A CN 104845896 A CN104845896 A CN 104845896A CN 201410485635 A CN201410485635 A CN 201410485635A CN 104845896 A CN104845896 A CN 104845896A
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- Prior art keywords
- welan gum
- welan
- strain
- fermentative production
- gum
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- 229920002310 Welan gum Polymers 0.000 title claims abstract description 81
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 42
- 238000000855 fermentation Methods 0.000 claims abstract description 16
- 230000004151 fermentation Effects 0.000 claims abstract description 16
- 238000000605 extraction Methods 0.000 claims abstract description 13
- 238000000746 purification Methods 0.000 claims abstract description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000012043 crude product Substances 0.000 claims abstract description 10
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 9
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 9
- 238000001035 drying Methods 0.000 claims abstract description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 6
- 239000000047 product Substances 0.000 claims abstract description 6
- 239000012535 impurity Substances 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 17
- 230000001580 bacterial effect Effects 0.000 claims description 16
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 15
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 12
- 238000012262 fermentative production Methods 0.000 claims description 12
- 239000001301 oxygen Substances 0.000 claims description 12
- 229910052760 oxygen Inorganic materials 0.000 claims description 12
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 claims description 9
- 230000008569 process Effects 0.000 claims description 9
- 239000002243 precursor Substances 0.000 claims description 8
- 239000002904 solvent Substances 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 7
- 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 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 238000002474 experimental method Methods 0.000 claims description 6
- 235000012054 meals Nutrition 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- -1 urase Proteins 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 230000033228 biological regulation Effects 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 5
- 235000015097 nutrients Nutrition 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 4
- 230000001476 alcoholic effect Effects 0.000 claims description 4
- 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 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 239000003002 pH adjusting agent Substances 0.000 claims description 4
- 235000019319 peptone Nutrition 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 238000011084 recovery Methods 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 3
- 108010082340 Arginine deiminase Proteins 0.000 claims description 3
- 108010010803 Gelatin Proteins 0.000 claims description 3
- 241000726221 Gemma Species 0.000 claims description 3
- 108010048581 Lysine decarboxylase Proteins 0.000 claims description 3
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 3
- 102000052812 Ornithine decarboxylases Human genes 0.000 claims description 3
- 108700005126 Ornithine decarboxylases Proteins 0.000 claims description 3
- 241001052560 Thallis Species 0.000 claims description 3
- 240000008042 Zea mays Species 0.000 claims description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 3
- WQZGKKKJIJFFOK-PQMKYFCFSA-N alpha-D-mannose Chemical compound OC[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-PQMKYFCFSA-N 0.000 claims description 3
- 108010005774 beta-Galactosidase Proteins 0.000 claims description 3
- 235000005822 corn Nutrition 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 3
- 235000019800 disodium phosphate Nutrition 0.000 claims description 3
- 239000008273 gelatin Substances 0.000 claims description 3
- 229920000159 gelatin Polymers 0.000 claims description 3
- 235000019322 gelatine Nutrition 0.000 claims description 3
- 235000011852 gelatine desserts Nutrition 0.000 claims description 3
- 230000007062 hydrolysis Effects 0.000 claims description 3
- 238000006460 hydrolysis reaction Methods 0.000 claims description 3
- 150000002475 indoles Chemical class 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 229940045641 monobasic sodium phosphate Drugs 0.000 claims description 3
- 235000006408 oxalic acid Nutrition 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000002203 pretreatment Methods 0.000 claims description 3
- 230000035484 reaction time Effects 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 2
- 235000003276 Apios tuberosa Nutrition 0.000 claims description 2
- 235000010777 Arachis hypogaea Nutrition 0.000 claims description 2
- 235000010744 Arachis villosulicarpa Nutrition 0.000 claims description 2
- 241000255789 Bombyx mori Species 0.000 claims description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 2
- 229920001353 Dextrin Polymers 0.000 claims description 2
- 239000004375 Dextrin Substances 0.000 claims description 2
- 235000019733 Fish meal Nutrition 0.000 claims description 2
- 229930091371 Fructose Natural products 0.000 claims description 2
- 239000005715 Fructose Substances 0.000 claims description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 2
- 244000068988 Glycine max Species 0.000 claims description 2
- 235000010469 Glycine max Nutrition 0.000 claims description 2
- 229930195725 Mannitol Natural products 0.000 claims description 2
- 229910002651 NO3 Inorganic materials 0.000 claims description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims description 2
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 2
- 229920002472 Starch Polymers 0.000 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims description 2
- 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 description 2
- 229920002494 Zein Polymers 0.000 claims description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 2
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 2
- 239000004202 carbamide Substances 0.000 claims description 2
- 235000013877 carbamide Nutrition 0.000 claims description 2
- 235000012343 cottonseed oil Nutrition 0.000 claims description 2
- 235000019425 dextrin Nutrition 0.000 claims description 2
- 239000004467 fishmeal Substances 0.000 claims description 2
- 235000011187 glycerol Nutrition 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 239000000594 mannitol Substances 0.000 claims description 2
- 235000010355 mannitol Nutrition 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 235000013379 molasses Nutrition 0.000 claims description 2
- 229910017604 nitric acid Inorganic materials 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 210000001082 somatic cell Anatomy 0.000 claims description 2
- 238000005507 spraying Methods 0.000 claims description 2
- 235000019698 starch Nutrition 0.000 claims description 2
- 239000008107 starch Substances 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- 238000001291 vacuum drying Methods 0.000 claims description 2
- 239000005019 zein Substances 0.000 claims description 2
- 229940093612 zein Drugs 0.000 claims description 2
- 244000105624 Arachis hypogaea Species 0.000 claims 1
- 238000001556 precipitation Methods 0.000 abstract description 2
- 238000009630 liquid culture Methods 0.000 abstract 2
- 241000051314 Sphingomonas sp. WG Species 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 abstract 1
- 238000004321 preservation Methods 0.000 abstract 1
- 108090000623 proteins and genes Proteins 0.000 description 11
- 239000002609 medium Substances 0.000 description 10
- 108020004465 16S ribosomal RNA Proteins 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000007400 DNA extraction Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000008719 thickening Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 244000133018 Panax trifolius Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- AEMOLEFTQBMNLQ-WAXACMCWSA-N alpha-D-glucuronic acid Chemical compound O[C@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-WAXACMCWSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- MGIYRDNGCNKGJU-UHFFFAOYSA-N isothiazolinone Chemical compound O=C1C=CSN1 MGIYRDNGCNKGJU-UHFFFAOYSA-N 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
Classifications
-
- 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
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- 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
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Materials Engineering (AREA)
- Polymers & Plastics (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a Welan gum producing marine strain, which belongs to Sphingomonas sp.WG, and has a preservation number of CCTCC No: M2013161. According to the present invention, the strain can be cultured in a liquid culture medium so as to obtain the Welan gum, and the production efficiency is high; the requirements of the strain of the present invention on the carbon source and the nitrogen source are low; the fermentation process comprises: (1) seed liquid culture and (2) fermentation production of Welan gum; the extraction and purification steps comprise: (1) fermentation broth pretreatment, (2) crude product precipitation, (3) impurity removing, and (4) drying so as to obtain the Welan gum product; with application of the marine strain to produce the Welan gum in the fermentation manner, the yield can be up to 33 g/L, and the extraction and purification yield can be up to 95%; and with the strain and the method of the present invention, the low-cost, high-efficiency and high-quality Welan gum production can be achieved.
Description
Technical field
The present invention relates to one plant height produce Welan gum marine bacteria strain Sphingol single-cell (
sphingomonassp. WG) and utilize this bacterial strain to carry out the processing method of the fermentation of Welan gum high yield and extraction purification, belong to industrial microbial technology field.
Background technology
Welan gum (Welan gum) is a kind of mixed polysaccharide by Microbe synthesis, is a kind of high molecular polymer, and its skeleton structure is made up of the unit of D-Glucose, D-glucuronic acid, D-Glucose and L-rhamnosyl.Side chain is made up of the L-seminose of strand or the L-rhamnosyl of strand.
Welan gum has good thermostability, suspension, emulsifying property and thickening property, can be used as all respects that thickening material, suspension agent, emulsifying agent, stablizer, lubricant, membrane-forming agent and tackiness agent are applied to industrial or agricultural, be particularly with a wide range of applications in the industry such as food, concrete, oil and ink.
At present, Welan gum mainly passes through Production by Microorganism Fermentation, kelco company of the U.S. has now become the maximum Welan gum production and supply business in the whole world, the domestic research to Welan gum is still in the starting stage, Nanjing University of Technology, University Of Nanchang, Southern Yangtze University and Hebei Xin He Chemical Co., Ltd. are engaged in the research of this respect, but produce bacterial strain and zymotechnique different, have no other relevant reports in addition.
Summary of the invention
One object of the present invention is to provide a kind of Sphingol single-cell, and it can synthesize Welan gum in the liquid nutrient medium of low cost.
Another object of the present invention is the fermentation manufacturing technique providing a kind of Welan gum.
Last object of the present invention is the extraction and purification process providing a kind of Welan gum.
A kind of bacterial strain producing Welan gum of the present invention, is characterized in that described bacterial strain is Sphingol single-cell WG(
sphingomonassp. WG), be preserved in China typical culture collection center (Wuhan University) on April 26th, 2013, preserving number is: CCTCC No:M2013161; Sphingol single-cell WG(provided by the invention
sphingomonassp
.wG) obtain by being separated in Jiaozhou Bay of Qingdao ooze, its single bacterium colony is in yellow, circular, center projections, and thickness is opaque, neat in edge, smooth surface; Sphingol single-cell WG(provided by the invention
sphingomonassp
.wG) strain morphology feature: Gram-negative, thalline is rod-short, amphitrichous, without gemma; Sphingol single-cell WG(provided by the invention
sphingomonassp.WG) physiological and biochemical property: growth temperature is 20 ~ 40 DEG C, growth pH is 4 ~ 9, NaCl tolerance is 3%; Oxydase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urase, beta-galactosidase enzymes and citric acid utilize experiment to be the positive, and VP, indoles produce, gelatin hydrolysis is tested, H
2s produces experiment for negative.
Sphingol single-cell WG(provided by the invention
sphingomonassp.WG) 16S rRNA gene sequence characteristic: Sphingol single-cell WG is inoculated in LB substratum, 30 DEG C of 180 rpm cultivates 16 ~ 18 h, bacterial genomes DNA extraction kit (Tian Gen biochemical technology company limited) is utilized to extract its genomic dna, adopt upstream primer 5 '-AGAGTTTGATCMTGGCTCAG-3 ' and downstream primer 5 '-TACGGYTACCTTGTTACGACTT-3 ' to carry out pcr amplification, obtain its 16S rRNA gene fragment.PCR condition is as follows: 94 DEG C of 5 min; 94 DEG C of 45 s, 56 DEG C of 45 s, 72 DEG C of 90 s, 30 circulations; 72 DEG C of 10 min.After the 16S rRNA gene fragment purifying obtained by pcr amplification, being connected to pMD18-T carrier with connecting test kit soon, connecting product conversion
e. colidH5 α, at the dull and stereotyped enterprising row filter of LB containing 100 μ g/mL penbritins, obtains positive colony through PCR qualification, mono-clonal is sent to Shanghai Sheng Gong biotechnology company limited, utilize pMD18-T carrier universal sequencing primer thing to check order.Sequencing result shows, this bacterial strain 16S rRNA gene order length is 1434 bp.
A fermentation manufacturing technique for Welan gum, comprises the steps:
(1) actication of culture: by Sphingol single-cell in YPD solid medium in 20 ~ 40 DEG C of constant temperature culture 2 ~ 6 days; (2) seed liquor preparation: the single colony inoculation activated in step (1) is cultivated in the YPD liquid nutrient medium of fresh sterile, adopt afterwards and amplify the mode spread cultivation step by step and carry out, culture temperature is 20 ~ 40 DEG C, and incubation time is 10 ~ 26 hours; (3) fermentative production Welan gum: the seed liquor obtained spreading cultivation step by step in step (2) is inoculated in fermention medium with the inoculative proportion of 1% ~ 15%, by realizing the fermentative production of Welan gum to the regulation and control of fermenting process, the Welan gum of high yield can be obtained, reach as high as 33 g/L.
The composition of the fermention medium described in step (3), in percent weight in volume, carbon source 1 ~ 15%, nitrogenous source 0.2 ~ 5.0%, magnesium sulfate 0.02 ~ 0.2%, dipotassium hydrogen phosphate 0.02 ~ 0.4%, Sodium phosphate dibasic 0.3 ~ 1.0%, SODIUM PHOSPHATE, MONOBASIC 0.1 ~ 0.5%, pH value is 4.5 ~ 8.5.
Described carbon source is one or more among glucose, sucrose, maltose, fructose, molasses, glycerine, starch and dextrin.
Described nitrogenous source is one or more among peptone, yeast extract, corn steep liquor, soybean cake powder, cottonseed meal, Zein powder, groundnut meal, biological nitrogen, fish meal, dried silkworm chrysalis meal, urea, ammonium sulfate and nitrate.
The regulation and control of the fermenting process described in step (3) are divided into three phases: the first stage: 0 ~ 24 hour, and this stage is the thalli growth stage, progressively improve the dissolved oxygen level in fermentor tank, control more than 20%; Subordinate phase: 24 ~ 48 hours, this stage is that precursor adds the stage, and in control fermentor tank, dissolved oxygen level is more than 30%, and adds precursor substance; Phase III, 48 ~ 90 hours, this stage was feed phase, and in control fermentor tank, dissolved oxygen level is more than 30%, and added the production that a certain proportion of Carbon and nitrogen sources maintains Welan gum.
Described precursor substance is one or more in seminose, rhamnosyl, N.F,USP MANNITOL and acetate.
An extraction and purification process for Welan gum, comprises the steps:
(1) fermentation liquor pretreatment: adopt degerming method to destroy or to remove somatic cells, and suppress Welan gum to be degraded; (2) crude product is separated out: the acid alcoholic solvent after being regulated with pH adjusting agent by fermented liquid good for pre-treatment in step (1) mixes, volume ratio is 1:1 ~ 1:5, mixed solution final pH is 2 ~ 7, reaction times is greater than 10 minutes, Welan gum is separated out gradually, adopts the methods such as centrifugal, filtration to obtain Welan gum crude product; (3) removal of impurity: the Welan gum crude product cleaning solvent in step (2) is carried out washing impurity-removing, obtains Welan gum work in-process; (4) dry: adopt drying means to dehydrate the Welan gum work in-process obtained in step (3), obtain Welan gum finished product, total recovery reaches as high as 95%.
Degerming method described in step (1) comprise heating, add sterilant, centrifugal and filter in one or more.
Alcoholic solvent described in step (2) is one or more in methyl alcohol, ethanol and Virahol.
PH adjusting agent described in step (2) is one or more in hydrochloric acid, sulfuric acid, nitric acid, acetic acid, phosphoric acid, oxalic acid, citric acid, sodium hydroxide, potassium hydroxide and ammoniacal liquor.
Cleaning solvent described in step (3) is one or more in water, methyl alcohol, ethanol, Virahol and acetone.
Drying means described in step (4) comprises one or more in vacuum-drying, spraying dry and lyophilize.
Advantage of the present invention:
1. Sphingol single-cell provided by the invention is separated and obtains from Jiaozhou Bay of Qingdao ooze, has good tolerance to hypersaline environment, can utilize the Carbon and nitrogen sources fermentative production Welan gum of multiple cheapness.
2. adopt effective fermentation control means, Welan gum fermenting process is divided into three stage controls, for the synthesis of thalli growth and Welan gum provides suitable dissolved oxygen, carbon source, nitrogenous source and precursor substance, final fermentation yield is up to 33 g/L, exceed the output of about 20 g/L of other bibliographical informations, achieve the High-efficient Production of Welan gum.
3. have employed fermentation liquor pretreatment, crude product precipitation, removal of impurities and dry 4 step process extraction purification Welan gum from fermented liquid, initially add sterilant at whole leaching process, effectively suppress Welan gum to be degraded further, total recovery can reach 95%, technique is simple, is applicable to suitability for industrialized production.
Accompanying drawing explanation
Single colonial morphology of bacterial strain WG when Fig. 1 is 30 DEG C of cultivation 24 h on LB substratum.
Fig. 2 is Welan gum fermenting process curve.
Embodiment
embodiment 1sphingol single-cell provided by the invention
sphingomonasthe physiology and morphology biochemical identification of sp.WG bacterial strain
Sphingol single-cell WG(provided by the invention
sphingomonassp.WG) obtaining by being separated in Jiaozhou Bay of Qingdao ooze, being coated with flat board through continuous several times dilution and obtaining pure growth.By gramstaining, morphologic observation, rich inspection gram negative bacterium identification systems, its strain morphology and biochemical reactions are identified.
Sphingol single-cell WG(
sphingomonassp.WG) on LB flat board, 30 DEG C of cultivation 24 h can grow single bacterium colony, and its single bacterium colony is in yellow, circular, center projections, and thickness is opaque, neat in edge, smooth surface (see accompanying drawing 1); This bacterial strain Gram-negative, thalline is rod-short, amphitrichous, without gemma; Its growth temperature is 20 ~ 40 DEG C, and growth pH is 4 ~ 9, NaCl tolerance is 3%; Oxydase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urase, beta-galactosidase enzymes and citric acid utilize experiment to be the positive, and VP, indoles produce, gelatin hydrolysis is tested, H
2s produces experiment for negative.
embodiment 2sphingol single-cell WG(provided by the invention
sphingomonasthe clone of 16S rRNA gene sp.WG) and sequencing
Sphingol single-cell WG(provided by the invention
sphingomonassp.WG) 16S rRNA gene sequence characteristic: by WG inoculation in LB substratum, 30 DEG C of 180 rpm cultivates 16 ~ 18 h, bacterial genomes DNA extraction kit (Tian Gen biochemical technology company limited) is utilized to extract its genomic dna, adopt upstream primer 5 '-AGAGTTTGATCMTGGCTCAG-3 ' and downstream primer 5 '-TACGGYTACCTTGTTACGACTT-3 ' to carry out pcr amplification, obtain its 16S rRNA gene fragment.PCR condition is as follows: 94 DEG C of 5 min; 94 DEG C of 50 s, 56 DEG C of 45 s, 72 DEG C of 90 s, 30 circulations; 72 DEG C of 10 min.After the 16S rRNA gene fragment purifying obtained by pcr amplification, being connected to pMD18-T carrier with connecting test kit soon, connecting product conversion
e. colidH5 α, at the dull and stereotyped enterprising row filter of LB containing 100 μ g/mL penbritins, obtains positive colony through PCR qualification, mono-clonal is sent to Shanghai Sheng Gong biotechnology company limited, utilize pMD18-T carrier universal sequencing primer thing to check order.Sequencing result shows, this bacterial strain 16S rRNA gene order length is 1434 bp.
embodiment 3the preparation of seed liquor
Solid medium: yeast extract paste 10 g/L, peptone 20 g/L, glucose 20 g/L, agar 20 g/L, pH7.5.
Liquid nutrient medium: yeast extract paste 10 g/L, peptone 20 g/L, glucose 20 g/L, pH7.5.
The bacterial classification of Sphingol single-cell WG is lined 30 DEG C of constant temperature culture at YPD solid medium within 4 days, activate, yellow color colonies to be grown, at 2 ~ 4 DEG C, short term storage is stand-by.Be equipped with in 250 ml triangular flasks of YPD liquid nutrient medium by good for activation single bacterium colony access, triangular flask liquid amount is 50 ml, 30 DEG C, shaking speed be 230 rpm under constant temperature culture 18 h, obtain primary seed solution.Primary seed solution is inoculated in the 500 ml triangular flasks containing 100 ml liquid YPD medium with 1.5% inoculum size, 30 DEG C, shaking speed be 230 rpm under constant temperature culture 15 h, obtain secondary seed solution.
embodiment 4fermentative production Welan gum in fermentor tank (20 L)
Liquid fermentation medium: maltose 5%, yeast extract paste 0.4%, corn steep liquor 0.2%, biological nitrogen 1.5%, magnesium sulfate 0.8%, dipotassium hydrogen phosphate 0.2%, Sodium phosphate dibasic 0.75%, SODIUM PHOSPHATE, MONOBASIC 0.25%.121 DEG C of real tank sterilizings 20 minutes.
The Sphingol single-cell secondary seed solution obtained in embodiment 3 be inoculated in the 20 L fermentor tanks containing 15 L liquid fermentation mediums with 10% inoculum size, starting condition: pH is 7.5, and rotating speed is 300 rpm, and air flow is 7.2 L/min, and temperature is 30 DEG C.
Welan gum fermentation is carried out to the regulation and control of three phases.First stage: 0 ~ 24 hour, progressively improved the dissolved oxygen level in fermentor tank by raising rotating speed, the mode such as tank pressure, interpolation oxygen carrier that increases, controlling dissolved oxygen is all the time greater than 20%; Subordinate phase: 24 ~ 48 hours, in control fermentor tank, dissolved oxygen level is more than 30%, and adds precursor substance seminose and rhamnosyl, and the two ratio is 1:1, and final additional amount is 1.2 g/L; Phase III, 48 ~ 90 hours, to control in fermentor tank dissolved oxygen level more than 30%, and add glucose and yeast extract paste to maintain the production of Welan gum, the two ratio is 10:1, and final additional amount is respectively 40 g/L and 4 g/L.Welan gum output reaches 33.3 g/L, and fermentating liquid volume reaches 17 L, and ultimate production is 566 g.Fermenting process curve is shown in accompanying drawing 2.
embodiment 5the extraction purification of Welan gum
Its final concentration is made to be 0.2 g/L by adding isothiazolinone in the Welan gum fermented liquid obtained in embodiment 4, to kill Sphingol single-cell in fermented liquid, and destroy the key of protein, thus the activity of multiple enzyme in T suppression cell liquid, ensure that Welan gum is not degraded.
Mixed by the acidic ethanol that fermented liquid good for pre-treatment and oxalic acid regulate, volume ratio is 1:1, and mixed solution final pH is 5.2, and in 20 minutes reaction times, Welan gum is separated out gradually, adopts the method for Plate Filtration to obtain Welan gum crude product.
Welan gum crude product is carried out washing 3 times with acetone and pure water successively, Welan gum: acetone is 1:1(mass volume ratio), Welan gum: water is 1:2(mass volume ratio), to remove unnecessary foreign protein, cell debris and salt, adopt the method for Plate Filtration to obtain Welan gum work in-process, acetone is recycled.
Welan gum work in-process are carried out drying as in low temperature continuous vacuum dryer, and drying temperature is 80 DEG C, is greater than 1 hour time of drying, and to water content lower than 10%, through pulverizing final acquisition Welan gum finished product after taking-up, total recovery is 95.4%.
Claims (15)
1. produce a Sphingol single-cell WG bacterial strain for Welan gum, be preserved in China typical culture collection center, preserving number is CCTCC NO:M2013161.
2. the bacterial strain of production Welan gum according to claim 1, is characterized in that:
Sphingol single-cell WG(provided by the invention
sphingomonassp.WG) obtain by being separated in Jiaozhou Bay of Qingdao ooze, its single bacterium colony is in yellow, circular, center projections, and thickness is opaque, neat in edge, smooth surface;
Sphingol single-cell WG(provided by the invention
sphingomonassp. WG) strain morphology feature: Gram-negative, thalline is rod-short, amphitrichous, without gemma;
Sphingol single-cell WG(provided by the invention
sphingomonassp.WG) physiological and biochemical property: growth temperature is 20 ~ 40 DEG C, growth pH is 4 ~ 9, NaCl tolerance is 3%; Oxydase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urase, beta-galactosidase enzymes and citric acid utilize experiment to be the positive, and VP, indoles produce, gelatin hydrolysis is tested, H
2s produces experiment for negative.
3. a method for fermentative production Welan gum, the actication of culture that it is characterized in that the method comprises the steps: (1): by bacterial strain according to claim 1 in YPD solid medium in 20 ~ 40 DEG C of constant temperature culture 2 ~ 6 days; (2) seed liquor preparation: the single colony inoculation activated in step 1) is cultivated in the YPD liquid nutrient medium of fresh sterile, adopt afterwards and amplify the mode spread cultivation step by step and carry out, culture temperature is 20 ~ 40 DEG C, and incubation time is 10 ~ 26 hours; (3) fermentative production Welan gum: by step 2) in spread cultivation step by step the seed liquor that obtains with 1% ~ 15% inoculative proportion be inoculated in fermention medium, by realizing the fermentative production of Welan gum to the regulation and control of fermenting process, the Welan gum of high yield can be obtained.
4. the method for fermentative production Welan gum according to claim 4, it is characterized in that the composition of the fermention medium described in step 3), in percent weight in volume, carbon source 1 ~ 15%, nitrogenous source 0.2 ~ 5.0%, magnesium sulfate 0.02 ~ 0.2%, dipotassium hydrogen phosphate 0.02 ~ 0.4%, Sodium phosphate dibasic 0.3 ~ 1.0%, SODIUM PHOSPHATE, MONOBASIC 0.1 ~ 0.5%, pH value is 4.5 ~ 8.5.
5. the method for fermentative production Welan gum according to claim 4, is characterized in that described carbon source is one or more among glucose, sucrose, maltose, fructose, molasses, glycerine, starch and dextrin.
6. the method for fermentative production Welan gum according to claim 4, is characterized in that described nitrogenous source is one or more among peptone, yeast extract, corn steep liquor, soybean cake powder, cottonseed meal, Zein powder, groundnut meal, fish meal, dried silkworm chrysalis meal, urea, ammonium sulfate and nitrate.
7. the method for fermentative production Welan gum according to claim 4, it is characterized in that the regulation and control of described fermenting process are divided into three phases: the first stage: 0 ~ 24 hour, this stage is the thalli growth stage, progressively improves the dissolved oxygen level in fermentor tank, controls more than 20%; Subordinate phase: 24 ~ 48 hours, this stage is that precursor adds the stage, and in control fermentor tank, dissolved oxygen level is more than 30%, and adds precursor substance; Phase III, 48 ~ 90 hours, this stage was feed phase, and in control fermentor tank, dissolved oxygen level is more than 30%, and added the production that a certain proportion of Carbon and nitrogen sources maintains Welan gum.
8. method according to claim 8, is characterized in that described precursor substance is one or more in seminose, rhamnosyl, N.F,USP MANNITOL and acetate.
9. the method for fermentative production Welan gum according to claim 4, is characterized in that the output of described Welan gum is up to 33 g/L.
10. a method for extraction purification Welan gum, is characterized in that the method comprises the steps:
(1) fermentation liquor pretreatment: adopt degerming method to destroy or to remove somatic cells, and suppress Welan gum to be degraded; (2) crude product is separated out: mixed with alcoholic solvent by fermented liquid good for pre-treatment in step (1), volume ratio is 1:1 ~ 1:5, pH of mixed is regulated with pH adjusting agent, its final pH is made to be 2 ~ 7, reaction times is greater than 10 minutes, Welan gum is separated out gradually, adopts the methods such as centrifugal, filtration to obtain Welan gum crude product; (3) removal of impurity: the Welan gum crude product cleaning solvent in step (2) is carried out washing impurity-removing, obtains Welan gum work in-process; (4) dry: adopt drying means to dehydrate the Welan gum work in-process obtained in step (3), obtain Welan gum finished product, total recovery reaches as high as 95%.
The method of 11. extraction purification Welan gums according to claim 11, it is characterized in that the degerming method described in step 1) comprise heating, add sterilant, centrifugal and filter in one or more.
The method of 12. extraction purification Welan gums according to claim 11, is characterized in that step 2) described in alcoholic solvent be one or more in methyl alcohol, ethanol and Virahol.
The method of 13. extraction purification Welan gums according to claim 11, is characterized in that step 2) described in pH adjusting agent be one or more in hydrochloric acid, sulfuric acid, nitric acid, acetic acid, phosphoric acid, oxalic acid, citric acid, sodium hydroxide, potassium hydroxide and ammoniacal liquor.
The method of 14. extraction purification Welan gums according to claim 11, is characterized in that the cleaning solvent described in step 3) is one or more in water, methyl alcohol, ethanol, Virahol and acetone.
The method of 15. extraction purification Welan gums according to claim 11, is characterized in that one or more that the drying means described in step 4) comprises in vacuum-drying, spraying dry and lyophilize.
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| CN112553106A (en) * | 2020-12-09 | 2021-03-26 | 鄂尔多斯市中轩生化股份有限公司 | Sphingomonas and process for producing high-quality welan gum by using same |
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