CN101368195A - Preparation method for high purity fructo-oligosaccharide - Google Patents
Preparation method for high purity fructo-oligosaccharide Download PDFInfo
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- CN101368195A CN101368195A CNA2008100303329A CN200810030332A CN101368195A CN 101368195 A CN101368195 A CN 101368195A CN A2008100303329 A CNA2008100303329 A CN A2008100303329A CN 200810030332 A CN200810030332 A CN 200810030332A CN 101368195 A CN101368195 A CN 101368195A
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- fructo oligosaccharides
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- 238000002360 preparation method Methods 0.000 title claims abstract description 48
- FTSSQIKWUOOEGC-RULYVFMPSA-N fructooligosaccharide Chemical compound OC[C@H]1O[C@@](CO)(OC[C@@]2(OC[C@@]3(OC[C@@]4(OC[C@@]5(OC[C@@]6(OC[C@@]7(OC[C@@]8(OC[C@@]9(OC[C@@]%10(OC[C@@]%11(O[C@H]%12O[C@H](CO)[C@@H](O)[C@H](O)[C@H]%12O)O[C@H](CO)[C@@H](O)[C@@H]%11O)O[C@H](CO)[C@@H](O)[C@@H]%10O)O[C@H](CO)[C@@H](O)[C@@H]9O)O[C@H](CO)[C@@H](O)[C@@H]8O)O[C@H](CO)[C@@H](O)[C@@H]7O)O[C@H](CO)[C@@H](O)[C@@H]6O)O[C@H](CO)[C@@H](O)[C@@H]5O)O[C@H](CO)[C@@H](O)[C@@H]4O)O[C@H](CO)[C@@H](O)[C@@H]3O)O[C@H](CO)[C@@H](O)[C@@H]2O)[C@@H](O)[C@@H]1O FTSSQIKWUOOEGC-RULYVFMPSA-N 0.000 title abstract description 8
- 229940107187 fructooligosaccharide Drugs 0.000 title abstract description 8
- 229940088598 enzyme Drugs 0.000 claims abstract description 87
- 102000004190 Enzymes Human genes 0.000 claims abstract description 84
- 108090000790 Enzymes Proteins 0.000 claims abstract description 84
- 238000000034 method Methods 0.000 claims abstract description 57
- 108010015776 Glucose oxidase Proteins 0.000 claims abstract description 43
- 239000004366 Glucose oxidase Substances 0.000 claims abstract description 43
- 229940116332 glucose oxidase Drugs 0.000 claims abstract description 43
- 235000019420 glucose oxidase Nutrition 0.000 claims abstract description 43
- 108010042889 Inulosucrase Proteins 0.000 claims abstract description 38
- 238000004519 manufacturing process Methods 0.000 claims abstract description 34
- 229930006000 Sucrose Natural products 0.000 claims abstract description 31
- 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 31
- 108010093096 Immobilized Enzymes Proteins 0.000 claims abstract description 11
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical class OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 53
- 229920001661 Chitosan Polymers 0.000 claims description 32
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical class OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 29
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical group O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 27
- 239000005720 sucrose Substances 0.000 claims description 27
- 230000000694 effects Effects 0.000 claims description 24
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- 238000006243 chemical reaction Methods 0.000 claims description 22
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- 239000004005 microsphere Substances 0.000 claims description 20
- 238000005406 washing Methods 0.000 claims description 19
- 235000010443 alginic acid Nutrition 0.000 claims description 18
- 229920000615 alginic acid Polymers 0.000 claims description 18
- 238000003756 stirring Methods 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 239000000843 powder Substances 0.000 claims description 15
- 239000000047 product Substances 0.000 claims description 15
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 claims description 13
- 229940072056 alginate Drugs 0.000 claims description 13
- 239000003795 chemical substances by application Substances 0.000 claims description 13
- 238000002156 mixing Methods 0.000 claims description 10
- 230000008569 process Effects 0.000 claims description 10
- 238000000926 separation method Methods 0.000 claims description 9
- 238000005342 ion exchange Methods 0.000 claims description 8
- 229910052751 metal Inorganic materials 0.000 claims description 8
- 239000002184 metal Substances 0.000 claims description 8
- 150000004032 porphyrins Chemical class 0.000 claims description 8
- 241000228245 Aspergillus niger Species 0.000 claims description 7
- 241001507822 Aspergillus terricola Species 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 7
- 229920005989 resin Polymers 0.000 claims description 7
- 239000011347 resin Substances 0.000 claims description 7
- 238000005507 spraying Methods 0.000 claims description 7
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 6
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 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
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 6
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 6
- 229910052791 calcium Inorganic materials 0.000 claims description 6
- 239000011575 calcium Substances 0.000 claims description 6
- 238000012412 chemical coupling Methods 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 239000000661 sodium alginate Substances 0.000 claims description 6
- 235000010413 sodium alginate Nutrition 0.000 claims description 6
- 229940005550 sodium alginate Drugs 0.000 claims description 6
- 230000005587 bubbling Effects 0.000 claims description 5
- 239000012153 distilled water Substances 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 238000011084 recovery Methods 0.000 claims description 5
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 claims description 4
- 238000010521 absorption reaction Methods 0.000 claims description 4
- 239000011324 bead Substances 0.000 claims description 4
- 239000000648 calcium alginate Substances 0.000 claims description 4
- 235000010410 calcium alginate Nutrition 0.000 claims description 4
- 229960002681 calcium alginate Drugs 0.000 claims description 4
- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 claims description 4
- 230000008878 coupling Effects 0.000 claims description 4
- 238000010168 coupling process Methods 0.000 claims description 4
- 238000005859 coupling reaction Methods 0.000 claims description 4
- 210000000582 semen Anatomy 0.000 claims description 4
- 240000006439 Aspergillus oryzae Species 0.000 claims description 3
- 235000002247 Aspergillus oryzae Nutrition 0.000 claims description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 3
- 239000008187 granular material Substances 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 230000002572 peristaltic effect Effects 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- 229930091371 Fructose Natural products 0.000 claims description 2
- 239000005715 Fructose Substances 0.000 claims description 2
- 238000010923 batch production Methods 0.000 claims description 2
- 238000004132 cross linking Methods 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 239000011543 agarose gel Substances 0.000 claims 2
- 238000012544 monitoring process Methods 0.000 claims 2
- 239000012295 chemical reaction liquid Substances 0.000 claims 1
- 238000011010 flushing procedure Methods 0.000 claims 1
- 235000012204 lemonade/lime carbonate Nutrition 0.000 claims 1
- 229920000642 polymer Polymers 0.000 claims 1
- 229960004793 sucrose Drugs 0.000 abstract description 26
- 102000016938 Catalase Human genes 0.000 abstract description 8
- 108010053835 Catalase Proteins 0.000 abstract description 8
- 102000003992 Peroxidases Human genes 0.000 abstract 3
- 229910052739 hydrogen Inorganic materials 0.000 abstract 3
- 239000001257 hydrogen Substances 0.000 abstract 3
- 125000004435 hydrogen atom Chemical class [H]* 0.000 abstract 3
- 230000003278 mimic effect Effects 0.000 abstract 3
- 108040007629 peroxidase activity proteins Proteins 0.000 abstract 3
- 150000001875 compounds Chemical class 0.000 abstract 1
- 238000010924 continuous production Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 29
- 238000004128 high performance liquid chromatography Methods 0.000 description 10
- 238000011218 seed culture Methods 0.000 description 9
- 238000009736 wetting Methods 0.000 description 9
- 239000012531 culture fluid Substances 0.000 description 8
- 239000008367 deionised water Substances 0.000 description 7
- 229910021641 deionized water Inorganic materials 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000002386 leaching Methods 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 238000001728 nano-filtration Methods 0.000 description 6
- 229920001542 oligosaccharide Polymers 0.000 description 6
- 150000002482 oligosaccharides Chemical class 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 238000004448 titration Methods 0.000 description 6
- ODEHMIGXGLNAKK-OESPXIITSA-N 6-kestotriose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@@H]1[C@@H](O)[C@H](O)[C@](CO)(O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)O1 ODEHMIGXGLNAKK-OESPXIITSA-N 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 5
- 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 description 5
- 241000219095 Vitis Species 0.000 description 5
- 235000009754 Vitis X bourquina Nutrition 0.000 description 5
- 235000012333 Vitis X labruscana Nutrition 0.000 description 5
- 235000014787 Vitis vinifera Nutrition 0.000 description 5
- 239000008272 agar Substances 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- -1 ferrous porphyrin Chemical class 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 238000000465 moulding Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- 241000125121 Aspergillus carbonarius Species 0.000 description 3
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 3
- RGHNJXZEOKUKBD-SQOUGZDYSA-N Gluconic acid Natural products OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000008121 dextrose Substances 0.000 description 3
- 235000013681 dietary sucrose Nutrition 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
- 239000000174 gluconic acid Substances 0.000 description 3
- 235000012208 gluconic acid Nutrition 0.000 description 3
- 125000003051 glycosyloxy group Chemical group 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000009288 screen filtration Methods 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 101710128063 Carbohydrate oxidase Proteins 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 235000009973 maize Nutrition 0.000 description 2
- 239000003094 microcapsule Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000003825 pressing Methods 0.000 description 2
- 230000003252 repetitive effect Effects 0.000 description 2
- 238000005201 scrubbing Methods 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 150000003538 tetroses Chemical class 0.000 description 2
- 150000004043 trisaccharides Chemical class 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 238000000207 volumetry Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- GDIUQZNEUVFHHD-UHFFFAOYSA-N [Fe+3].N1C(C=C2N=C(C=C3NC(=C4)C=C3)C=C2)=CC=C1C=C1C=CC4=N1 Chemical group [Fe+3].N1C(C=C2N=C(C=C3NC(=C4)C=C3)C=C2)=CC=C1C=C1C=CC4=N1 GDIUQZNEUVFHHD-UHFFFAOYSA-N 0.000 description 1
- JQRLYSGCPHSLJI-UHFFFAOYSA-N [Fe].N1C(C=C2N=C(C=C3NC(=C4)C=C3)C=C2)=CC=C1C=C1C=CC4=N1 Chemical group [Fe].N1C(C=C2N=C(C=C3NC(=C4)C=C3)C=C2)=CC=C1C=C1C=CC4=N1 JQRLYSGCPHSLJI-UHFFFAOYSA-N 0.000 description 1
- LPQOADBMXVRBNX-UHFFFAOYSA-N ac1ldcw0 Chemical compound Cl.C1CN(C)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN3CCSC1=C32 LPQOADBMXVRBNX-UHFFFAOYSA-N 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 229940045110 chitosan Drugs 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 235000019621 digestibility Nutrition 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- XITQUSLLOSKDTB-UHFFFAOYSA-N nitrofen Chemical compound C1=CC([N+](=O)[O-])=CC=C1OC1=CC=C(Cl)C=C1Cl XITQUSLLOSKDTB-UHFFFAOYSA-N 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
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- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
Classifications
-
- 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
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/06—Enzymes or microbial cells immobilised on or in an organic carrier attached to the carrier via a bridging agent
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a preparation method of high purified fructo-oligosaccharide, in particular to a method for preparing the high purified fructo-oligosaccharide by using immobilized enzyme. The preparation method of the invention prepares immobilized fructosyltransferase, immobilized glucose oxidase and immobilized mimic hydrogen peroxidase; then prepared enzymes are used to prepare the high purified fructo-oligosaccharide through an interrupted or continuous production method. In the preparation method, cheap metalporphyrin compounds are used as the mimic hydrogen peroxidase to replace expansive catalase; the fructosyltransferase, the glucose oxidase and the mimic hydrogen peroxidase are all immobilized and all can be recycled and reused; the stability and the operating factor of the enzymes are improved; the production cost for preparing the high purified fructo-oligosaccharide is greatly reduced. The preparation method can use one step method to directly produce the high purified fructo-oligosaccharide from cane sugar.
Description
Technical field
The present invention relates to a kind of production method of high-purity fructo oligosaccharides, relate in particular to a kind of method of utilizing immobilized enzyme to produce high-purity fructo oligosaccharides.
Background technology
Oligofructose (Fructooligosaccharide, FOS), claiming fructooligosaccharide or Nutriflora P again, is the mixture that is combined kestose, GF3 and the GF4 etc. that generate by 1～3 fructosyl by β (2-1) glycosidic link and the fructosyl in the sucrose.Wherein kestose, GF3 and GF4 are non-digestibility sugar, have bifidus bacillus propagation function, are the effective constituent of functional oligose.Up to now, the oligofructose product on the market is divided into two kinds: a kind of is that the oligofructose of oligosaccharide contg about 50% is (hereinafter referred to as FOS
50), wherein oligose (trisaccharide+tetrose+pentasaccharides) content is about 50%, and all the other are dextrose plus saccharose, and diabetics and obese person can not accept; Another kind is a high-purity fructo oligosaccharides, and promptly oligosaccharide contg reaches 75% or higher oligofructose, is comparatively ideal oligofructose product.
The method of suitability for industrialized production high-purity fructo oligosaccharides adopts two-step approach both at home and abroad so far always, promptly obtains FOS in microorganism (enzyme) method
50On the basis, adopt chromatographic separation method or nanofiltration membrane separation method that dextrose plus saccharose is separated again, obtain the high-purity fructo oligosaccharides product.Wherein external how the separation with dextrane gel SephadexG-10 resin, its separating effect is fine, but this resin price very expensive (about 1200 dollars/kilogram) makes equipment investment cost too high, and enterprise is difficult to realize.It is that the membrane module of 300Da carries out nanofiltration separation that the enterprise of domestic production high-purity fructo oligosaccharides adopts molecular weight cut-off mostly, because the membrane module of 300Da is except filtering dextrose plus saccharose, also to also filter the kestose of nearly half quantity, consequently the rate of recovery of oligofructose and product yield are all very low, approximately need to consume 3～4 tons of FOS
50Could obtain the high-purity fructo oligosaccharides product (FOS of 1 ton of oligosaccharide contg about 95%
95), because of FOS
50Market price be 8000 yuan per ton, the light raw materials cost is just up to 2.4～30,000 yuan/ton, it is long to add operation hours, energy consumption is too high, makes production cost high.
Chinese invention patent ZL 00130200.0 employing molecular weight cut-off is that the membrane module of 200Da carries out nanofiltration separation, though can avoid the loss of kestose, but this membrane module can only filter monose, most of sucrose is trapped, therefore need to increase an enzyme reaction and transform sucrose, and need to increase a desalination, decoloration process simultaneously, and carry out the nanofiltration second time then, could obtain FOS
95Product, its technology are too complicated, and the cost that is equivalent to twice production low-purity oligofructose product adds the cost of twice nanofiltration, its total cost and 300Da membrane module nano filtering process height about the same.
Except chromatography and nanofiltration separation method, also there were double-enzyme method or mixed enzyme method to produce the exploratory study report of high-purity fructo oligosaccharides both at home and abroad.Yun J.W equals report in 1993,1994, and the two enzymes that add glucose oxidase with fructosyl transferase are to obtain the FOS product more than 90.05%; But these tests are owing to need to use catalase supporting, and catalatic cost is too high, can not reclaim again, do not have industrial applications to be worth.Domestic also someone attempted double-enzyme method; The Jiang Bo of Southern Yangtze University etc. were reported in and adopt aspergillus niger list enzyme process in 1997 is on the basis of 56.64%FOS with sucrose inversion, second step added the catalase synergy with glucose oxidase, with conversion of glucose is gluconic acid, remove gluconic acid with ion exchange method then, can obtain 87% FOS.The weakness of this double-enzyme method is that suitability for industrialized production is difficult to realize, reason is not recycling of glucose oxidase, catalatic expensive.According to the quotation in 2008 of Sigma company, article No. is that the catalase price of C3515 is 785.07 yuan/10mg.
In view of the above-mentioned defective of prior art, existing necessity provides a kind of method that can be significantly reduced to this suitability for industrialized production high-purity fructo oligosaccharides.
Summary of the invention
The invention provides a kind of method that can be significantly reduced to this suitability for industrialized production high-purity fructo oligosaccharides.The present invention utilizes reusable immobilized enzyme production of high purity in next life oligofructose.
The preparation method of high-purity fructo oligosaccharides of the present invention comprises following two steps:
(1) preparation immobilized fructosyltransferase, immobilized glucose oxidase and immobilization hydrogen peroxide analogue enztme;
(2) utilize the enzyme of step (1) preparation to produce high-purity fructo oligosaccharides with the production method of intermittent type or continous way.
Wherein, the preparation of immobilized fructosyltransferase:
Selection has the strain excellent that can secrete fructosyl transferase, cultivates in suitable medium, and a large amount of mycelium that cultivation is obtained carry out broken wall then, with centrifuging enzyme are separated, and with immobilized reagent enzyme are fixed up.
Recited above have the strain excellent that can secrete fructosyl transferase and experimentize and screen through the inventor, following several requirement that reaches is arranged, and they are: aspergillus oryzae (Aspergillus oryzae), aspergillus niger (Aspergillus niger) and aspergillus terricola (Aspergillus terricola).
Suitable medium recited above comprises following several material: 1-10% sucrose, 1-5% bean cake powder, 0.1-1% Semen Maydis powder, its cultural method is at 25-35 ℃, and ventilating ratio 1:0.1-1:1 is under the stirring velocity 100-300rpm condition, through 15-45 hour, obtain a large amount of mycelium.
The mycelium that obtains above must carry out broken wall earlier, collect the intracellular enzyme of these somatic cells, just obtain fructosyl transferase preferably, described thalline broken wall can obtain the mixed solution of fructosyl transferase with known method such as machinery broken wall law, freeze-thaw method, ultrasonic disruption method, bacteriolyze enzyme process etc., will contain the solution separating of enzyme again with centrifuging.
Recited above with the method for reagent with enzyme immobilization, adopt chemical coupling method, immobilized reagent comprises carrier and linking agent, and the carrier that is used for fixing enzyme is a macroporous resin, and used linking agent is a glutaraldehyde.Utilize linked reaction can make between the fructosyl transferase that is adsorbed on the carrier and connect, reach immobilized purpose with chemical bond.Used glutaraldehyde concentration is crossed the low or too high immobilized effect that all influences at 0.1-0.9%.
In the immobilization process, the proportional range of enzyme and carrier is that every gram carrier needs enzyme 15-95u, when the consumption of enzyme＜15u/g carrier, the enzyme of immobilized enzyme is lived too low, enzymatic action is difficult to bring into play, but lives when enzyme〉during the 95u/g carrier, the immobilization efficiency of enzyme is too low, part is the resolvase state, just is lost after the use once.
Immobilized condition is: pH4-8, temperature 0-10 ℃, stirring velocity 15-60rpm.The immobilization operation: fructosyl transferase is after separating, (HPLC) measures its enzyme activity with high performance liquid chromatography, add carrier in certain enzyme/carrier ratio, behind mixing and absorption 5～24h, add certain density glutaraldehyde solution, under these conditions, crosslinking reaction 10-24h, thorough washing, centrifuge dripping can being fixed fructosyl transferases, in 0-10 ℃ of preservation.
In additional embodiments of the present invention, the preparation method of immobilized fructosyltransferase can repeat no more according to disclosed method preparation among the ZL01128345.9 herein.
The preparation of immobilized glucose oxidase among the present invention:
The glucose oxidase that will dissociate is crosslinked with chemical coupling method and carrier, and used carrier is macroporous resin, sepharose, allyl group dextrane gel or chitosan gel rubber etc., and used linking agent is a glutaraldehyde.
Immobilized condition is: the proportioning of enzyme and carrier is that every gram carrier needs enzyme 100～300U, at the final concentration of pH5.0～7.0,10～50 ℃ of temperature, stirring velocity 10～100r/min, glutaraldehyde is to react 3～10h under 0.05～0.3% the condition.
Used free glucose oxidase can be a commercial enzyme, also can use Aspergillus niger strain (Aspergillus carbonarius OLG 2) from row filter and mutagenesis, after obtaining thalline through seed culture and fermentation culture, the glucose oxidase that extracts through broken wall, centrifugation again.It is (in g/L) that the fermention medium of this bacterial strain is formed: sucrose 30～50, agar 10～20, NaNO
3, MgSO
47H
2O, KCI, FeSO
44H
2O and K
2HPO
4Each 0.01～3.0, regulating pH is 5.5～7.5.
The preparation of immobilized glucose oxidase also can adopt the interface of chitosan and alginates to solidify-crosslinked coupling immobilization method.The sodium alginate soln that will contain whipping agent earlier splashes into calcium chloride solution, forms a kind of calcium alginate gel beads with a lot of micropores; The porous calcium alginate gel beads forms porous microsphere with chitosan reaction again.Owing on the chitosan molecule chain a large amount of primary aminos is arranged, and a large amount of carboxyls is arranged on the molecular chain of alginates, so chitosan and sodium alginate can form polyelectrolyte film by the attraction of positive and negative electric charge, its ball degree of Zhi Bei microcapsule and smooth finish are fine like this, and have a lot of micropores, its specific surface is bigger, is well suited for the fixation support as glucose oxidase, has so at first solved fixation support and has selected and the moulding problem.Secondly, because the surface of each micropore is one deck chitosan gel rubber in this porous microsphere, utilize the bi-functional cross-linking agent glutaraldehyde that has two aldehyde radicals to react by Schiff with chitosan and glucose oxidase and enzyme is fixed on each micropore of microcapsule again.Enzyme is very firm with combining of carrier like this, is difficult for running off, and only is distributed in the surface of micropore in the porous microsphere, and the resistance to mass transfer of space barrier and enzymatic reaction is all very little, has improved the activity of immobilized enzyme.
Immobilized condition is:
1.0～2.0% (massfraction) sodium alginate soln that will contain 0.01～0.20% NaHCO3 (massfraction) adds at peristaltic pump depresses the CaCl that splashes into 1.0～2.0% (massfractions) by φ 1～2mm syringe needle
2Solution, room temperature is placed 2～4h, obtains the alginate calcium porous microsphere of white.Chitosan solution with 5% acetum preparation 0.1～0.5% (massfraction), press 2 parts of solution: 1 part of microballoon (mass ratio) drops into the alginate calcium porous microsphere, in 30～40 ℃, the constant temperature shaking table of 200rpm, shake 20～40min, with the CaCl of 0.5～1.0% (massfraction)
2Solution washing obtains alginate calcium-chitosan porous microsphere.Get alginate calcium-chitosan porous microsphere 100g, add 5L 5% glutaraldehyde solution, shake 4～6h in 25～35 ℃, the constant temperature shaking table of 200rpm, the distilled water repetitive scrubbing is to remove remaining glutaraldehyde.Need glucose oxidase 200～500U by every gram alginate calcium-chitosan porous microsphere carrier, in 25～35 ℃, the constant temperature shaking table of 200rpm, shake 2～4h, put into refrigerator in 4 ℃ of following standing over night, centrifugal, precipitation, wash repeatedly with distilled water, filter and promptly get immobilized glucose oxidase.
The preparation of immobilization hydrogen peroxide analogue enztme among the present invention:
Metal porphyrins protohemine or oxyphorase is crosslinked with chemical coupling method and carrier, and used carrier is sepharose, allyl group dextrane gel or chitosan gel rubber etc.; Used linking agent is a glutaraldehyde.Immobilized condition is: pH8.0～12.0,10～50 ℃ of temperature, stirring velocity 10～100r/min, the mass ratio of metal porphyrins and carrier are that every gram carrier needs metal porphyrins 0.1～0.3 gram, the final concentration of linking agent glutaraldehyde is 0.05～0.3%, reaction times 3～10h.Reaction washes with water, and measures its catalase activity after finishing.
Catalatic active centre is iron (III) porphyrin.As the catalase analogue enztme, its advantage is with the natural extract protohemine (ferrous porphyrin) with iron porphyrin ring or oxyphorase: 1, with the catalase structural similitude, the performance of analogue enztme is good; 2, belong to natural product, safety non-toxic is particularly suitable for the use of foodstuffs industry; 3, material source is in animal blood, aboundresources, and extraction process is easy, and price is not high.
Production method with intermittent type or continous way is produced high-purity fructo oligosaccharides, and the method that wherein step batch process is produced high-purity fructo oligosaccharides is:
In retort, the sucrose solution that adds 20～55% (mass percents), the enzyme amount that adds 1000～4000U immobilized fructosyltransferase, 1000～4000U immobilized glucose oxidase and 1000～4000U immobilization hydrogen peroxide analogue enztme by every Kg sucrose (doing) simultaneously, with the flow velocity bubbling air of 1.0～3.0L/min, use CaCO
3The pH value of powder control reaction solution, it is maintained between 4.0～7.0, temperature is to react 10～50h under the condition of 50～200r/min 10～50 ℃ and stirring velocity, with HPLC detect oligofructose content up to standard after, use the screen filtration reaction solution, Separation and Recovery immobilized enzyme (immobilized fructosyltransferase, immobilized glucose oxidase and immobilization hydrogen peroxide analogue enztme), and make the usefulness of the production of next batch raw material behind the water thorough washing.Filtrate becomes the high-purity fructo oligosaccharides product through smart filter, ion-exchange and spraying drying.
The method that the pillar reaction method of continous way (being single stage method) is produced high-purity fructo oligosaccharides is:
Immobilized fructosyltransferase, immobilized glucose oxidase and immobilization hydrogen peroxide analogue enztme are pressed enzyme lives than 1.0～4.0:1.0～3.0:1.0～4.0 mixing; again with the calcium carbonate granule mixing of 2～8% (mass percents); pack in the column reactor; with the sucrose solution of 10～60% (mass percents) in pH4.0～7.0; under 10～50 ℃ of the temperature repeatedly by this post; detect oligofructose content after-filtration up to standard with HPLC, become the high-purity fructo oligosaccharides product through ion-exchange and spraying drying.
Compare with the preparation method of existing high-purity fructo oligosaccharides, fructosyl transferase of the present invention, glucose oxidase and hydrogen peroxide analogue enztme all pass through immobilization and handle, and have improved the stability of enzyme, and can reuse, and have improved the utilising efficiency of enzyme.In addition, replace expensive catalase with cheap metal porphyrins as the hydrogen peroxide analogue enztme, it is catalatic 1/10th that its cost has only approximately, can reduce the production cost of preparation high-purity fructo oligosaccharides so significantly.Moreover, with the method for preparing high-purity oligomer fructose of external existing two-step approach relatively, preparation method of the present invention can be with single stage method from the highly purified oligofructose of sucrose direct production.Brief, the technology maturation, workable of technical process of the present invention, and production cost reduces greatly, is suitable for suitability for industrialized production.
Illustrate: the alleged sugared content of (1) the present invention is mass percent, and oligosaccharide contg refers to the summation of trisaccharide+tetrose+pentasaccharides, and the content of each component sugars is calculated and got by the normalization method of high performance liquid chromatography (HPLC).
(2) alleged " the low-purity oligofructose " of the present invention is meant oligosaccharide contg about 50%, and alleged " high-purity fructo oligosaccharides " is meant that oligosaccharide contg (is containing) more than 75%.
(3) the enzymic activity definition difference that the present invention contacts three kinds of enzymes is as follows:
Fructosyl transferase (fructosytransferase, abbreviation FTS, enzyme classification EG2.41.9): measuring method is by the appendix A of national light industry standard QB 2583-2003, enzyme activity definition is under the condition according to the best enzyme reaction of enzyme supplier sign, to be an enzyme activity unit (U) with sucrose inversion for the oligofructose per minute produces the required enzyme amount of 1 μ mol kestose.
Glucose oxidase (glucose oxidase, abbreviation GOD, enzyme classification EC1.13.4): the enzyme activity determination method (sees that Wu shows chapter work " industrial production technology of enzyme " by volumetry, Jilin science tech publishing house, 1988 49～53 pages), enzyme activity definition be under the condition of 35 ℃ temperature of reaction and defined per minute to produce the required enzyme amount of 1 μ mol gluconic acid be an enzyme activity unit (U).
The hydrogen peroxide analogue enztme: the enzyme activity determination method (is seen B. Shi Teer Mach work by volumetry, Qian Jiayuan translates " measuring method of enzyme ", China Light Industry Press, first version in 1992), enzyme activity definition be under the condition of 35 ℃ temperature of reaction and defined per minute to decompose the required enzyme amount of 1 μ mol hydrogen peroxide be an enzyme activity unit (U).
Introduce the preparation method of high-purity fructo oligosaccharides of the present invention in detail below in conjunction with embodiment.Following embodiment only is used for explaining the present invention, and should not limit protection scope of the present invention with this, and the equivalent variations of doing according to essence of the present invention still belongs to protection scope of the present invention.
Embodiment
Embodiment one
(1) preparation of immobilized fructosyltransferase:
Choose the access of aspergillus terricola (Aspergillus terricola) bacterial classification and contain in 200 liters of fermentor tanks of 5% sucrose, 3% bean cake powder, 0.3% maize powder medium,, get seed culture fluid in 33 ℃ of fermentations 24 hours.In 5000 liters of fermentor tanks, add culture medium solution, wherein contain sucrose 5%, bean cake powder 3%, Semen Maydis powder 0.3%.In 121 ℃ of sterilization 40min, add 150 liters of seed culture fluids, under 33 ℃, the condition of 220rpm, cultivate 24h, obtain containing mycelial fermented liquid, centrifugal through whizzer, the separated and collected mycelium, washing, broken wall, centrifuging and taking enzyme liquid after HPLC measures enzyme activity, add the ratio of 1g carrier in every 40u enzyme liquid, in 2000 liters of retort, add enzyme liquid and macroporous resin, stir, be cooled to 8 ℃, absorption 12h.Add glutaraldehyde, making its ultimate density is 0.2%, reacts 16 hours down at 8 ℃, crosses the leaching solids, thorough washing, and centrifuge dripping moisture promptly gets the immobilized fructosyltransferase preparation.
(2) preparation of immobilized glucose oxidase:
Mutagenesis aspergillus niger (Aspergillus carbonarius OLG 2) bacterial classification inserts and contains 2～3% sucrose, 1～2% agar, 1～2% yeast extract and NaNO
3, MgSO
47H
2O, KCl, FeSO
44H
2O and K
2HPO
4In 100 liters of seed fermentation jars of each 0.001～0.3% substratum, regulating pH is 5.5～7.0, in 33 ℃ of cultivation 16h, gets seed culture fluid.In 3000 liters of fermentor tanks, add culture medium solution, wherein contain 3～5% sucrose, 1～2% agar, NaNO
3, MgSO
47H
2O, KCl, FeSO
44H
2O and K
2HPO
4Each is 0.001～0.3% years old, in 120 ℃ of sterilization 30min, add 80 liters of seed culture fluids, at 33 ℃, cultivate 30h under the condition of 200r/min, must contain mycelial fermented liquid, mycelium is collected in centrifugation, washing, broken wall, centrifuging and taking enzyme liquid, ultrafiltration and concentration obtains the oxidasic crude enzyme liquid of glucose, with its enzyme activity of titration measuring.
In 100 liters of retort, under 30～40 ℃, the ratio in every gram carrier (wetting) adding 100～300U enzyme liquid adds the chitosan gel rubber (wetting) of 10Kg and the enzyme liquid of 100～3,000,000 U, add glutaraldehyde behind the stirring and evenly mixing, making its final concentration is 0.05～0.2%, and at pH 5.0～7.0,30～40 ℃ are reacted 4～6h down, cross the leaching solids, use the deionized water thorough washing, centrifuge dripping, moulding promptly get immobilization grape glycosyloxy zymin.
(3) preparation of immobilization hydrogen peroxide analogue enztme
In 100 liters of retort, add 0.1mol/L sodium hydroxide solution 50L, 30～40 ℃ add ferrous porphyrin 2.5Kg down and are stirred to dissolving fully.Press ferrous porphyrin: the mass ratio of chitosan gel rubber=1.0～3.0:10.0, the chitosan gel rubber (wetting) that adds 10～25Kg, add glutaraldehyde behind the stirring and evenly mixing, making its final concentration is 0.05～0.2%%, and at pH 9.0～12.0,30～40 ℃ are reacted 4～8h down, cross the leaching solids, use the deionized water thorough washing, centrifuge dripping, moulding promptly get immobilization hydrogen peroxide analogue enztme.With its enzyme activity of titration measuring.
(4) immobilized enzyme method is produced high-purity fructo oligosaccharides
At 2.2M
3Retort in, add 50～55% (mass percent) sucrose solution 2000Kg, add immobilized fructosyltransferase preparation 125～3,800,000 U, immobilization grape glycosyloxy zymin 100～3,000,000 U and immobilization hydrogen peroxide analogue enztme 150～4,000,000 U, with the flow velocity bubbling air of 1.0～3.0L/min, use CaCO
3The pH value of powder control reaction solution, it is maintained between 5.0～7.0, be to react 12～30h under the condition of 50～200r/min 30～45 ℃ and stirring velocity, with HPLC detect oligofructose content up to standard after, use the screen filtration reaction solution, Separation and Recovery immobilized fructosyltransferase, immobilized glucose oxidase and immobilization hydrogen peroxide analogue enztme, and make the usefulness of the production of next batch raw material behind the water thorough washing.Filtrate becomes the high-purity fructo oligosaccharides product through smart filter, ion-exchange and spraying drying.
Embodiment two
(1) preparation of immobilized fructosyltransferase
Choose the access of aspergillus terricola (Aspergillus terricola) bacterial classification and contain in 200 liters of fermentor tanks of 5% sucrose, 3% bean cake powder, 0.3% maize powder medium,, get seed culture fluid in 33 ℃ of fermentations 24 hours.In 5000 liters of fermentor tanks, add culture medium solution, wherein contain sucrose 5%, bean cake powder 3%, Semen Maydis powder 0.3%.In 121 ℃ of sterilization 40min, add 150 liters of seed culture fluids, under 33 ℃, the condition of 220rpm, cultivate 24h, obtain containing mycelial fermented liquid, centrifugal through whizzer, the separated and collected mycelium, washing, broken wall, centrifuging and taking enzyme liquid after HPLC measures enzyme activity, adds the ratio of 1g carrier in every 40u enzyme liquid, in 2000 liters of retort, add enzyme liquid and macroporous resin, stir, be cooled to 8 ℃, absorption 12h.Add glutaraldehyde, making its ultimate density is 0.2%, reacts 16 hours down at 8 ℃, crosses the leaching solids, thorough washing, and centrifuge dripping moisture promptly gets the immobilized fructosyltransferase preparation.
(2) preparation of immobilized glucose oxidase:
Mutagenesis aspergillus niger (Aspergillus carbonarius OLG2) bacterial classification inserts and contains 2～3% sucrose, 1～2% agar, 1～2% yeast extract and NaNO
3, MgSO
47H
2O, KCl, FeSO
44H
2O and K
2HPO
4In 100 liters of seed fermentation jars of each 0.001～0.3% substratum, regulating pH is 5.5～7.0, in 33 ℃ of cultivation 16h, gets seed culture fluid.In 3000 liters of fermentor tanks, add culture medium solution, wherein contain 3～5% sucrose, 1～2% agar, NaNO
3, MgSO
47H
2O, KCl, FeSO
44H
2O and K
2HPO
4Each is 0.001～0.3% years old, in 120 ℃ of sterilization 30min, add 80 liters of seed culture fluids, at 33 ℃, cultivate 30h under the condition of 200r/min, must contain mycelial fermented liquid, mycelium is collected in centrifugation, washing, broken wall, centrifuging and taking enzyme liquid, ultrafiltration and concentration obtains the crude enzyme liquid of grape carbohydrate oxidase, with its enzyme activity of titration measuring.
To contain 0.01～0.20% NaHCO
31.0～2.0% (massfraction) sodium alginate soln of (massfraction) adds at peristaltic pump and to depress the CaCl that splashes into 1.0～2.0% (massfractions) by φ 1～2mm syringe needle
2Solution, room temperature is placed 2～4h, obtains the alginate calcium porous microsphere of white.Chitosan solution with 5% acetum preparation 0.1～0.5% (massfraction), press 2 parts of solution: 1 part of microballoon (mass ratio) drops into the alginate calcium porous microsphere, in 30～40 ℃, the constant temperature shaking table of 200rpm, shake 20～40min, with the CaCl of 0.5～1.0% (massfraction)
2Solution washing obtains alginate calcium-chitosan porous microsphere.Get this microballoon 100g, add the 5L5% glutaraldehyde solution, shake 4～6h in 25～35 ℃, the constant temperature shaking table of 200rpm, the distilled water repetitive scrubbing is to remove remaining glutaraldehyde.
In 100 liters of retort, under 30～40 ℃, the ratio that adds 200～500U enzyme liquid in every gram porous microsphere carrier (wetting), add the porous microsphere carrier (wetting) of 10Kg and the enzyme liquid of 200～5,000,000 U, in 25～35 ℃, the constant temperature shaking table of 200rpm, shake 2～4h, put into refrigerator-freezer in 4 ℃ of following standing over night, centrifugal, precipitate with deionized water is washed repeatedly, and centrifuge dripping promptly gets the immobilized glucose oxidase preparation.
(3) preparation of immobilization hydrogen peroxide analogue enztme
In 100 liters of retort, add deionized water 50L, 30～40 ℃ add oxyphorase 3Kg down and are stirred to dissolving fully.Press oxyphorase: the mass ratio of chitosan gel rubber=2.0～3.0:10.0, the chitosan gel rubber (wetting) that adds 10～15Kg, add glutaraldehyde behind the stirring and evenly mixing, making its final concentration is 0.05～0.2%%, ℃ following reaction 4～8h in pH5.0～7.0,30～40, cross the leaching solids, use the deionized water thorough washing, centrifuge dripping, moulding promptly get immobilization hydrogen peroxide analogue enztme.With its enzyme activity of titration measuring.
(4) immobilized enzyme method is produced high-purity fructo oligosaccharides
At 3.5M
3Retort in, add 50～55% (mass percent) sucrose solution 3000Kg, add immobilized fructosyltransferase preparation 200～5,500,000 U, immobilization grape glycosyloxy zymin 150～4,500,000 U and immobilization hydrogen peroxide analogue enztme 220～5,600,000 U, with the flow velocity bubbling air of 1.0～2.0L/min, use CaCO
3The pH value of powder control reaction solution maintains between 4.5～7.0, be to react 12～28h under the condition of 100～200r/min 30～45 ℃ and stirring velocity, with HPLC detect oligofructose content up to standard after, use the screen filtration reaction solution, Separation and Recovery immobilized enzyme-immobilized fructosyltransferase, immobilized glucose oxidase and immobilization hydrogen peroxide analogue enztme, and make the usefulness of the production of next batch raw material after washing with water.Filtrate becomes the high-purity fructo oligosaccharides product through smart filter, ion-exchange and spraying drying.
Embodiment three
(1) preparation of immobilized fructosyltransferase, embodiment one operation of pressing ZL 01128345.9.
(2) preparation of immobilized glucose oxidase:
Fixation support is pressed the preparation of embodiment two methods, get the crude enzyme liquid of commercially available commercial grape carbohydrate oxidase, behind the titration measuring enzyme activity, in 100 liters of retort, under 30～40 ℃, the ratio that adds 200,000～500,000 U enzyme liquid in every Kg porous microsphere carrier (wetting), add the porous microsphere carrier (wetting) of 20Kg and the enzyme liquid of 400,000～1,000,000 U, in 25～35 ℃, the constant temperature shaking table of 200rpm, shake 2～4h, put into refrigerator-freezer in 4 ℃ of following standing over night, centrifugal, precipitation is washed repeatedly with deionized water, centrifuge dripping promptly gets the immobilized glucose oxidase preparation.
(3) preparation of immobilization hydrogen peroxide analogue enztme
In 200 liters of retort, add 0.1mol/L sodium hydroxide solution 100L, 30～40 ℃ add ferrous porphyrin 5Kg down and are stirred to dissolving fully.Press ferrous porphyrin: the mass ratio of chitosan gel rubber=1.0～3.0:10.0, the chitosan gel rubber (wetting) that adds 20～50Kg, add glutaraldehyde behind the stirring and evenly mixing, making its final concentration is 0.05～0.2%%, and at pH 9.0～12.0,30～40 ℃ are reacted 4～8h down, cross the leaching solids, use the deionized water thorough washing, centrifuge dripping, moulding promptly get immobilization hydrogen peroxide analogue enztme.With its enzyme activity of titration measuring.
(4) immobilized enzyme method is produced high-purity fructo oligosaccharides
With immobilized fructosyltransferase; the enzyme of immobilized glucose oxidase and immobilization hydrogen peroxide analogue enztme is lived than pressing: mix 1.2～3.8:1.0～3.0:1.5～4.0; again with the calcium carbonate granule mixing of 2～8% (mass percents); pack in the column reactor; with the sucrose solution of 10～50% (mass percents) in pH4.8～7.0; under 30～50 ℃ of the temperature repeatedly by this post; with HPLC detect oligofructose content up to standard after; the filtered and recycled immobilized fructosyltransferase; immobilized glucose oxidase and immobilization hydrogen peroxide analogue enztme, filtrate becomes the high-purity fructo oligosaccharides product through ion-exchange and spraying drying.
Claims (20)
1. the production method of a high-purity fructo oligosaccharides, it may further comprise the steps:
(1) preparation immobilized fructosyltransferase, immobilized glucose oxidase and immobilization hydrogen peroxide analogue enztme;
(2) utilize the enzyme of step (1) preparation to produce high-purity fructo oligosaccharides with the production method of intermittent type or continous way.
2. the production method of high-purity fructo oligosaccharides as claimed in claim 1, it is characterized in that, the described method for preparing immobilized fructosyltransferase is: select to have the strain excellent that can secrete fructosyl transferase, in suitable medium and culture condition, cultivate, the mycelium that cultivation is obtained carries out broken wall then, with centrifuging enzyme is separated, and with immobilized reagent enzyme is fixed up, wherein, described strain excellent is aspergillus oryzae, aspergillus niger or aspergillus terricola.
3. the production method of high-purity fructo oligosaccharides as claimed in claim 2, it is characterized in that, described suitable medium comprises following several material 1～10% (massfraction) sucrose, 1～5% (massfraction) bean cake powder,～1% 0.1 (massfraction) Semen Maydis powder, described culture condition comprises 25～35 ℃ of culture temperature, and ventilating ratio is 1:0.1～1:1, stirring velocity 100～300rpm.
4. the production method of high-purity fructo oligosaccharides as claimed in claim 2, it is characterized in that, describedly enzyme is fixed up with immobilized reagent, used process for fixation is a chemical coupling method, described immobilized reagent comprises carrier and linking agent, wherein said carrier is a macroporous resin, and described linking agent is a glutaraldehyde.
5. the production method of high-purity fructo oligosaccharides as claimed in claim 4, it is characterized in that, the proportional range of enzyme and carrier is the enzyme of every gram carrier correspondence 15～95u in the immobilization process, fixing condition is pH4～8,0～10 ℃ of temperature, stirring velocity 15～60rpm, wherein immobilization comprises following operation steps: fructosyl transferase is measured its enzyme activity after separating, add carrier in above-mentioned enzyme/carrier ratio, behind mixing and absorption 5～24h, add the glutaraldehyde solution of 0.1～0.9% concentration, under these conditions, crosslinking reaction 10～24h, washing, centrifuge dripping can being fixed fructosyl transferase, in 0～10 ℃ of preservation.
6. the production method of high-purity fructo oligosaccharides as claimed in claim 1, it is characterized in that, immobilized glucose oxidase is that the free glucose oxidase is crosslinked with chemical coupling method and carrier, and used carrier is natural or the synthetic superpolymer, and used linking agent is a glutaraldehyde.
7. the production method of high-purity fructo oligosaccharides as claimed in claim 6 is characterized in that, described carrier is cross-linked agarose gel, crosslinked allyl group dextrane gel or cross-linked chitosan gel.
8. as the production method of claim 6 or 7 described high-purity fructo oligosaccharides, it is characterized in that, the proportioning of enzyme and carrier is the glucose oxidase of every gram carrier correspondence 100～300U in the immobilization process, and immobilization is that the final concentration in pH 5.0～7.0,10～50 ℃ of temperature, stirring velocity 10～100r/min, glutaraldehyde is 0.05～0.3% and reacts under the condition of 3～10h and carry out.
9. the production method of high-purity fructo oligosaccharides as claimed in claim 1 is characterized in that, the preparation method of immobilized glucose oxidase is: solidify-crosslinked coupling immobilization method at the interface of chitosan and alginates.
10. the production method of high-purity fructo oligosaccharides as claimed in claim 9, it is characterized in that, the interface of described chitosan and alginates solidifies-and crosslinked coupling immobilization method is: and the sodium alginate soln that will contain whipping agent earlier splashes into calcium chloride solution, forms a kind of calcium alginate gel beads with a lot of micropores; The porous calcium alginate gel beads forms the microballoon with a lot of micropores with chitosan reaction again; Again with linking agent glutaraldehyde and chitosan and glucose oxidase by the Schiff reaction and enzyme is fixed on the micropore of microballoon.
11. the production method of high-purity fructo oligosaccharides as claimed in claim 10 is characterized in that, and the interface of described chitosan and alginates solidifies-and crosslinked coupling immobilization method detailed process is: will contain 0.01～0.20%NaHCO
31.0～2.0% (massfraction) sodium alginate soln of (massfraction) adds at peristaltic pump depresses the CaCl that splashes into 1.0～2.0% (massfractions)
2Solution, room temperature is placed 2～4h, obtains the alginate calcium porous microsphere of white; Chitosan solution with 5% acetum preparation 0.1～0.5% (massfraction), press 2 parts of solution: 1 part of microballoon (mass ratio) drops into the alginate calcium porous microsphere, in 30～40 ℃, the constant temperature shaking table of 200rpm, shake 20～40min, with the CaCl of 0.5～1.0% (massfraction)
2Solution washing obtains alginate calcium-chitosan porous microsphere; Get alginate calcium-chitosan porous microsphere 100g, add 5L 5% (massfraction) glutaraldehyde solution, shake 4～6h in 25～35 ℃, the constant temperature shaking table of 200rpm, distilled water wash is removed remaining glutaraldehyde; Need the amount of glucose oxidase 200～500U to add glucose oxidase by every gram alginate calcium-chitosan porous microsphere carrier, in 25～35 ℃, the constant temperature shaking table of 200rpm, shake 2～4h, put into refrigerator in 4 ℃ of following standing over night, centrifugal, the precipitation distilled water flushing filters and promptly gets immobilized glucose oxidase.
12. the production method of high-purity fructo oligosaccharides as claimed in claim 1, it is characterized in that, the preparation method of immobilization hydrogen peroxide analogue enztme is: metal porphyrins is crosslinked with chemical coupling method and carrier, wherein used carrier is natural or synthetic high polymer, and used linking agent is a glutaraldehyde.
13. the production method of high-purity fructo oligosaccharides as claimed in claim 12, it is characterized in that, described carrier is cross-linked agarose gel, crosslinked allyl group dextrane gel or cross-linked chitosan gel, and described metal porphyrins is protohemine or oxyphorase.
14. the production method of high-purity fructo oligosaccharides as claimed in claim 12, it is characterized in that the preparation of immobilization hydrogen peroxide analogue enztme is at pH 8.0～12.0,10～50 ℃ of temperature, carry out under stirring velocity 10～100r/min condition, wherein, the mass ratio of metal porphyrins and carrier is that every gram carrier needs metal porphyrins 0.1～0.3 gram, and the final concentration of linking agent glutaraldehyde is 0.05～0.3%, reaction times 3～10h, after reaction finished, enzymic activity was surveyed in washing.
15. the preparation method of high-purity fructo oligosaccharides as claimed in claim 1, it is characterized in that, described batch production method is: in retort, add sucrose solution, add immobilized fructosyltransferase simultaneously, immobilized glucose oxidase and immobilization hydrogen peroxide analogue enztme, bubbling air, the pH value of control reaction solution, under certain temperature and stirring velocity, react 10～50h, the content of oligofructose in the monitoring reaction liquid, after fructose content is up to standard, filtering reacting liquid, the Separation and Recovery immobilized enzyme, the further smart filter of filtrate then, ion-exchange and spraying drying become the high-purity fructo oligosaccharides product.
16. the preparation method of high-purity fructo oligosaccharides as claimed in claim 15, it is characterized in that the sucrose that adds in the retort and the content of immobilized enzyme are: every Kg sucrose adds 1000～4000U immobilized fructosyltransferase, 1000～4000U immobilized glucose oxidase and 1000～4000U immobilization hydrogen peroxide analogue enztme.
17. the preparation method of high-purity fructo oligosaccharides as claimed in claim 16 is characterized in that, the amount of described bubbling air is 1.0～3.0L/min.
18. the preparation method of high-purity fructo oligosaccharides as claimed in claim 15 is characterized in that, is to use CaCO
3The pH value of controlling reaction solution is between 4.0～7.0.
19. the preparation method of high-purity fructo oligosaccharides as claimed in claim 1; it is characterized in that; described continuous reaction method is: with immobilized fructosyltransferase; immobilized glucose oxidase and immobilization hydrogen peroxide analogue enztme are mixed in proportion; again with the calcium carbonate granule mixing; pack in the column reactor; with the sucrose solution of 10～30% (mass percents) in pH4.0～7.0; under 10～40 ℃ of the temperature repeatedly by this post; the content of monitoring oligofructose; after oligofructose content is up to standard; filtering reacting liquid becomes the high-purity fructo oligosaccharides product through ion-exchange and spraying drying.
20. the preparation method of high-purity fructo oligosaccharides as claimed in claim 19, it is characterized in that, in the column reactor, the ratio of immobilized fructosyltransferase, immobilized glucose oxidase and immobilization hydrogen peroxide analogue enztme is: 1.0～4.0:1.0～3.0:1.0～4.0; The mass percent of described lime carbonate is 2～8%.
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