CN104762344A - Method for continuously preparing fructo-oligosaccharide by using immobilized enzyme microreactor - Google Patents
Method for continuously preparing fructo-oligosaccharide by using immobilized enzyme microreactor Download PDFInfo
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- CN104762344A CN104762344A CN201510195846.XA CN201510195846A CN104762344A CN 104762344 A CN104762344 A CN 104762344A CN 201510195846 A CN201510195846 A CN 201510195846A CN 104762344 A CN104762344 A CN 104762344A
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- inulin
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- 238000000034 method Methods 0.000 title claims abstract description 32
- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 17
- 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 7
- 229940107187 fructooligosaccharide Drugs 0.000 title abstract description 7
- 238000006243 chemical reaction Methods 0.000 claims abstract description 43
- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(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(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 claims abstract description 29
- 229920001202 Inulin Polymers 0.000 claims abstract description 28
- 229940029339 inulin Drugs 0.000 claims abstract description 28
- 239000007788 liquid Substances 0.000 claims abstract description 18
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- 108090000790 Enzymes Proteins 0.000 claims description 29
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- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 24
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- 229910010413 TiO 2 Inorganic materials 0.000 claims description 10
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
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- 238000006047 enzymatic hydrolysis reaction Methods 0.000 abstract 1
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- 229930006000 Sucrose Natural products 0.000 description 7
- 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 description 7
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- VAWYEUIPHLMNNF-OESPXIITSA-N 1-kestose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 VAWYEUIPHLMNNF-OESPXIITSA-N 0.000 description 1
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- ODEHMIGXGLNAKK-OESPXIITSA-N 6-kestotriose Chemical group 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 1
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- 241000186660 Lactobacillus Species 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
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- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention discloses a method for continuously preparing fructo-oligosaccharide by using an immobilized enzyme microreactor. The method comprises the steps of fixing endo-inulinase in the microreactor, injecting a substrate, namely an inulin aqueous solution into the microreactor, wherein the flow rate of the substrate is 0.1-1mL/min, and the reaction temperature is 55-65 DEG C, the retention time of circulation and continuous flow of reaction feed liquid in the microreactor is 2-6 hours, and degrading the inulin solution to the fructo-oligosaccharide. The method has the advantages that the microreactor is used for enzymatic hydrolysis of inulin to generate the fructo-oligosaccharide, the reaction efficiency is greatly improved, and the energy consumption is reduced; meanwhile, the content of nystose in the product can be remarkably increased by controlling operating conditions, and the effects of the fructo-oligosaccharide on prevention and treatment of cardiovascular diseases are improved. A micro-reaction chip of the microreactor can be replaced, the microreactor is simple in structure, as long as the number of the microreactor is increased, the production capacity can be expanded in an equal proportion manner, and therefore, the method has good industrialization prospect.
Description
Technical field
The present invention relates to biocatalysis field, be specifically related to a kind of method utilizing microreactor enzymolysis inulin continuous production oligofructose.
Background technology
Oligofructose (Fructo oligosaccharide, FOS) be a kind of natural active matter, oligofructose is also known as fructooligosaccharide, be be combined by 2 ~ 9 fructosyls the sugarcane fruit polysaccharide mixture generated by the fructosyl of β (2-1) glycosidic link in sucrose, sugariness is 0.3-0.6 times of sucrose.It maintains the pure sweet taste of sucrose, salubriouser than sucrose again; Be have regulating intestinal canal flora, propagation bifidus bacillus, promotes the absorption of calcium, adjusting blood lipid, immunomodulatory, the novel sweetener of the nourishing functions such as anti-dental caries.Oligofructose is described as the additive of new generation of most potentiality after the microbiotic epoch---growth-promoting material; Be called as plasmosin (PPE) in France, apply in the numerous food such as milk-product, lactobacillus drink, solid beverage, candy, biscuit, bread, jelly, cold drink.
The method of suitability for industrialized production oligofructose has two kinds.One is that sucrose is prepared through fructose-transferring enzyme catalysis, and product oligomeric fructose is CFn type; Two is utilize inulinase directionally hydrolyzing synanthrin to prepare, and product oligomeric fructose is the mixed type of CFn and Fn.Prepare in oligofructose at fructose-transferring enzyme catalysing sucrose, byproduct of reaction glucose is the inhibitor of enzyme, low conversion rate, and containing a large amount of dextrose plus saccharose in product, oligofructose only accounts for less than 55 ~ 60%, and complex process cost is high.It is a step list enzyme reaction that endo-inulinase directionally hydrolyzing inulin prepares oligofructose, and technique is simple, and in product, oligofructose content is up to more than 80%.Therefore, be that material, enzyme method one one-step hydrolysis synanthrin wherein produces oligofructose with inulin, have the advantages that technique is simple, transformation efficiency is high, by product is few.
There are some researches show, GF3 has very strong anti-oxidizing activities, to the prevention and therapy successful of cardiovascular disorder; Related article in 2014 at International Journal of Food Science and Technology 2014,49,1500 – 1505 deliver, and title is " In vitro anti-hydroxyl radical activity of thefructooligosaccharides 1-kestose and nystose using spectroscopic and computationalapproaches ".
Endo-inulinase, is mainly derived from feverfew tissue, and the former Substratspezifitaet is comparatively strong, only for inulin.Endo-inulinase is a kind of hydrolysis of inulin enzyme, and its action characteristic is: cut off glycosidic link at random from synanthrin intramolecule, and its product is GF
nand F
ndeng oligofructose, n=2 ~ 9.
Microreactor is a kind of microchannel formula reactor be based upon on continuous flow basis, the passage of its process fluid in micron level, in order to alternative traditional reactor.Because it has larger specific surface area, mass-and heat-transfer efficiency is high, and residence time destribution is narrow, and reaction conditions can accurately control, and security is high, is easy to amplify, and can the advantage such as continuous seepage receive much concern in recent years.Type conventional in microreactor is micro passage reaction, manufactures by PDMS, PMMA, glass round tube etc.And the size and dimension of passage can be simulated by CFD.
Immobilized enzyme has many good qualities, and especially stability and reusability are widely applied in a lot of field.The process for fixation such as glutaraldehyde are lived to enzyme sometimes has serious loss, such as blocking activities center, induced conformational change etc.Along with deepening continuously of immobilization technology, fixing carrier is also weeded out the old and bring forth the new thereupon, no longer being confined to traditional inorganic materials or polysaccharide material is carrier, and the new type functional material being tending towards selecting to improve immobilized enzyme performance is carrier, such as mesoporous material, nano material, magnetic microsphere etc.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of method utilizing immobilized enzyme micro-reactor continuous production oligofructose, low to solve the synthesis of oligonucleotides fructose response intensity existed in prior art, can not the problem such as continuous prodution, realize the production model controlled in order of oligofructose simultaneously.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of method utilizing immobilized enzyme micro-reactor continuous production oligofructose, endo-inulinase is fixed in microreactor, the substrate inulin aqueous solution is injected in microreactor, substrate flow velocity is 0.1-1mL/min, temperature of reaction is 55-65 DEG C, circulate in the microreactor retention time of continuous flow of reaction feed liquid is 2 ~ 6h, makes inulin solution degradation be oligofructose.
Wherein, the described substrate inulin aqueous solution, solution ph is 3.5-5.5 (preferred pH4.6), and the concentration of solute inulin is 5-30wt%.
Wherein, described microreactor, be made up of cover plate and Sptting plate, containing disconnectable micro-channel chip in the groove at Sptting plate center, micro-channel chip closely wriggles and is carved with passage, the upper surface of passage opens wide, the lower surface of passage and side-closed, passage overall length is 50-200mm, and the tangent plane of passage is class ladder structure wide at the top and narrow at the bottom, and the trapezoidal long limit of class is 0.5-1.5mm, the trapezoidal minor face of class is 0.1 ~ 0.4mm, the trapezoidal height of class is 0.3-1.0mm, and trapezoidal four angles of class are according to corners process, and corners diameter is 0.1mm; Be provided with retaining clip between the cover plate of described reactor and Sptting plate to leak to prevent solution side, wherein, the material of cover plate is glass or stainless steel, and Sptting plate material is stainless steel.
Wherein, described microreactor is in advance through following process: spread upon inside cover plate by polydimethylsiloxane colloidal sol, then with sieve plate by SH-TiO
2powder is evenly distributed on polydimethylsiloxane colloidal sol, puts into 60-80 DEG C of baking oven and toasts 100-200min, colloidal sol is solidified.
Wherein, described SH-TiO
2equal proportion zooms in or out and prepares as follows: by 1g TiO
2be scattered in the toluene of 30-50ml, add 5 ~ 10ml 3-mercaptopropyl trimethoxysilane, at 110 DEG C of oil bath backflow 6-10h, by sample suction filtration, toluene wash; Sample will be leached and be placed in Soxhlet extractor, with ethanol extracting 12-24h under reflux temperature, to remove unreacted coupling agent; Take out sample after suction filtration and can obtain SH-TiO after dry 24-48h at 60 DEG C
2.Preferably, by 1g TiO
2be scattered in the toluene of 50ml, add 5ml 3-mercaptopropyl trimethoxysilane, at 110 DEG C of oil bath backflow 8h, by sample suction filtration, toluene wash; Sample will be leached and be placed in Soxhlet extractor, with ethanol extracting 24h under reflux temperature, to remove unreacted coupling agent; Take out sample after suction filtration and can obtain SH-TiO after dry 24h at 60 DEG C
2.
Wherein, described hole diameter of sieve (perforated) plate 800 order.
Wherein, the method be fixed on by endo-inulinase in microreactor is as follows: endo-inulinase liquid is injected disconnectable micro-channel chip (3), and flow rate control is at 1-100 μ L/min, and retention time is 1-5 hour, with the enzyme liquid that ultrapure water is unnecessary, realize enzyme immobilizatio.
Wherein, described endo-inulinase, 200-300U/mg more alive than enzyme, the supported quantity of microreactor is 50-200mg.Enzyme analytical procedure alive is as follows: l mL enzyme liquid adds the inulin liquid of 4mL3% (with 0.lmol/L, pH4.5 acetate buffer solution is prepared), react 30min at 50 DEG C, boiling water bath boils 5min, after get 0.5ml reaction solution, add 1.5mL water and 1.5mL3,5 one edlefsen's reagents, boiling water bath heating 5min, cooling is immediately settled to 25ml, measure absorbancy under 540nm, contrast fructose typical curve calculates the reducing sugar amount that reaction generates; Under the same conditions, the enzyme amount transformed needed for generation 1 μm of ol reducing sugar with per minute is an enzyme activity unit.
Wherein, in microreactor, the enzyme of endo-inulinase is lived as 4.2KU, supported quantity is 20mg, the substrate inulin aqueous solution is injected in microreactor, substrate flow velocity is 0.1-1mL/min, temperature of reaction is 55-65 DEG C, substrate pH is at 3.5-5.5, circulate in the microreactor retention time of continuous flow of reaction feed liquid is 2h, adjustment reaction flow velocity is at 0.1mL/min again, adjustment temperature of reaction is 65 DEG C, adjustment substrate pH is 3.5, circulate in the microreactor retention time of continuous flow of reaction feed liquid is 4h, in the oligofructose prepared, the concentration of GF3 is at more than 60wt%.
Beneficial effect: Selection utilization microreactor enzymolysis inulin of the present invention generates oligofructose, and reaction efficiency improves greatly, power consumption reduces; Meanwhile, controlling by becoming operational condition, the content of GF3 in product can be significantly improved, the effect of strengthening oligofructose in prevention and therapy cardiovascular disorder.Device of the present invention can change micro-anti-chip, and structure is simple, and only needs simply to be amplified by number to get final product expanding production capacity of equal proportion, has good industrial prospect.
Accompanying drawing explanation
Fig. 1 is the structural representation of microreactor of the present invention.
Fig. 2 replaceable reaction chip enlarged view.
Fig. 3 is that Fig. 2 draws a circle place's passage tangent plane structural representation.
Embodiment
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, the content described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
Embodiment 1:
As shown in Figure 1, microreactor of the present invention, be made up of cover plate 1 and Sptting plate 2, containing disconnectable micro-channel chip 3 in the groove at Sptting plate center, as shown in Figure 2 micro-channel chip 3 closely wriggles and be carved with passage, the upper surface of passage opens wide, the lower surface of passage and side-closed, passage overall length is 50-200mm, and the tangent plane of passage is class ladder structure (Fig. 3) wide at the top and narrow at the bottom, and the trapezoidal long limit of class is 0.5-1.5mm, minor face is 0.1 ~ 0.4mm, height is 0.3-1.0mm, and trapezoidal four angles of class are according to corners process, and corners diameter is 0.1mm; Be provided with retaining clip between the cover plate 1 of described reactor and Sptting plate 2 to leak to prevent solution side, wherein, the material of cover plate is glass or stainless steel, and Sptting plate material is stainless steel.
Embodiment 2:
By 1g TiO
2be scattered in the toluene of 50ml, add 5ml 3-mercaptopropyl trimethoxysilane, at 110 DEG C of oil bath backflow 8h.By sample suction filtration, toluene wash for several times; Sample will be leached and be placed in Soxhlet extractor, with ethanol extracting under reflux temperature, 24h, to remove unreacted coupling agent; Take out sample after suction filtration and can obtain SH-TiO after dry 24h at 60 DEG C
2.
Polydimethylsiloxane colloidal sol is spread upon inside cover plate, then uses aperture 800 object sieve plate by SH-TiO
2powder is evenly distributed on polydimethylsiloxane colloidal sol, puts into 60-80 DEG C of baking oven and toasts 100-200min, colloidal sol is solidified.
Endo-inulinase liquid is injected disconnectable micro-channel chip 3, and flow rate control is at 1-100 μ L/min, and retention time is 1-5 hour, with the enzyme liquid that ultrapure water is unnecessary, realizes enzyme immobilizatio.Described endo-inulinase, 200-300U/mg more alive than enzyme, the supported quantity of microreactor is 50-200mg.
Adopt SH-TiO
2be fixed on the inwall of micro passage reaction, the reaction times of micro-anti-middle endo-inulinase is increased greatly, this immobilized enzyme micro-reactor can use more than 10 times continuously, greatly strengthen stability and the work-ing life of enzyme, thus reduces the consumption of enzyme largely.
Embodiment 3:
Apparatus structure such as embodiment 1,5ml inulin content is the aqueous solution of 5wt%, and sulfuric acid adjusts pH to 4.6, and this reaction system is pumped in microreactor by syringe pump continuous circulation with the flow velocity of 100 μ L/min.In microreactor, runner is class ladder structure wide at the top and narrow at the bottom, long limit is 0.5mm, minor face is 0.1mm, height 0.5mm, passage length 50mm, the enzyme of endo-inulinase is lived as 4.2KU, and supported quantity is 20mg, control temperature of reaction 55 DEG C, circulate the in the reactor retention time of continuous flow of reaction feed liquid is 4h.The yield preparing oligofructose with HPLC detection enzymatic degradation is 99.5%.
Embodiment 4:
Apparatus structure such as embodiment 1,50ml inulin content is the aqueous solution of 10wt%, and sulfuric acid adjusts pH to 4.6, and this reaction system is pumped in microreactor by syringe pump continuous circulation with the flow velocity of 500 μ L/min.In microreactor, runner is class ladder structure wide at the top and narrow at the bottom, and long limit is 1mm, and minor face is 0.22mm, height 0.3mm, passage length 100mm, and the enzyme of endo-inulinase is lived as 24.5KU, and supported quantity is 120mg.Control temperature of reaction 60 DEG C, circulate the in the reactor retention time of continuous flow of reaction feed liquid is 2h.The yield preparing oligofructose with HPLC detection enzymatic degradation is 98.3%.
Embodiment 5:
Apparatus structure such as embodiment 1,100ml inulin content is the aqueous solution of 20wt%, and sulfuric acid adjusts pH to 4.6, and this reaction system is pumped in microreactor by syringe pump continuous circulation with the flow velocity of 1mL/min.In microreactor, runner is class ladder structure wide at the top and narrow at the bottom, and long limit is 1.5mm, and minor face is 0.4mm, height 0.8mm, passage length 150mm, and the enzyme of endo-inulinase is lived as 52.3KU, and supported quantity is 250mg.Control temperature of reaction 60 DEG C, circulate the in the reactor retention time of continuous flow of reaction feed liquid is 6h.The yield preparing oligofructose with HPLC detection enzymatic degradation is 98.7%.
Embodiment 6:
Apparatus structure such as embodiment 1,100ml inulin content is the aqueous solution of 30wt%, and sulfuric acid adjusts pH to 4.6, and this reaction system is pumped in microreactor by syringe pump continuous circulation with the flow velocity of 1mL/min.In microreactor, runner is class ladder structure wide at the top and narrow at the bottom, and long limit is 1.5mm, and minor face is 0.4mm, height 1.0mm, passage length 200mm, and the enzyme of endo-inulinase is lived as 66.4KU, and supported quantity is 330mg.Control temperature of reaction 65 DEG C, circulate the in the reactor retention time of continuous flow of reaction feed liquid is 6h.The yield preparing oligofructose with HPLC detection enzymatic degradation is 95.6%.
Embodiment 7:
(apparatus structure is as embodiment 1 to utilize immobilization endo-inulinase microreactor of the present invention, in microreactor, runner is class ladder structure wide at the top and narrow at the bottom, long limit is 0.5mm, minor face 0.1mm, height 0.5mm, passage length 50mm), adopt alternating temperature, become the coordinated regulation of pH and unsteady flow speed, greatly can improve the content of GF3 in product, realize the controllable polymerization degree preparation of oligofructose.Be the solution of 5wt% by 5ml inulin content, sulfuric acid adjusts pH to 4.6, control temperature of reaction 60 DEG C, this reaction system is pumped into immobilization endo-inulinase microreactor with the flow velocity of 100 μ L/min by syringe pump continuous circulation, and (enzyme of endo-inulinase is lived as 4.2KU, supported quantity is 20mg), after running 2h, the transformation efficiency of inulin to oligofructose reaches more than 80%; Afterwards according to the orthogonal experiment condition of table 1, after continuing to run 4h in microreactor, the content of GF3 in product is analyzed, the results are shown in Table 1.
The orthogonal experiment L of GF3 concentration relationship in each operational condition of table 1 and product
9(3
3) result
By method of the present invention, realize the controlled degradation of inulin, reach on 99.5% basis at transformation efficiency, greatly improve the concentration of GF3 in oligofructose.As shown in Table 1, in the preparation process of oligofructose, each factor primary and secondary order affecting GF3 concentration is followed successively by flow velocity (factor C) > temperature (factor B) > pH (factor A).By the Collaborative Control of these three factors, the concentration of GF3 in oligofructose can be brought up to more than 60%.
According to the operational condition of the preferred embodiment A1B3C1 of orthogonal experiment, the transformation efficiency of inulin is greater than 99.5%, and wherein the content of GF3 reaches 62%.
Embodiment 8:
Apparatus structure is as embodiment 1, and in microreactor, runner is class ladder structure wide at the top and narrow at the bottom, and long limit is 0.5mm, minor face 0.1mm, height 0.5mm, passage length 50mm, and the enzyme of endo-inulinase is lived as 4.2KU, and supported quantity is 20mg.5ml inulin content is the solution of 5wt%, and sulfuric acid adjusts pH to 4.6, and this reaction system to be pumped in microreactor by syringe pump continuous circulation with the flow velocity of 100 μ L/min reacts 2h, controls temperature of reaction 60 DEG C; Regulate reaction system pH to 3.5 subsequently, enter in microreactor to react 4h with the flow pump of 100 μ L/min, control temperature of reaction 65 DEG C.The yield preparing oligofructose with HPLC detection enzymatic degradation is 99.5%, and main ingredient is kestose, GF3 and a small amount of GF4, and wherein GF3 content accounts for 62% of oligofructose total amount.
Claims (9)
1. one kind utilizes the method for immobilized enzyme micro-reactor continuous production oligofructose, it is characterized in that, endo-inulinase is fixed in microreactor, the substrate inulin aqueous solution is injected in microreactor, substrate flow velocity is 0.1-1mL/min, temperature of reaction is 55-65 DEG C, and circulate in the microreactor retention time of continuous flow of reaction feed liquid is 2 ~ 6h, makes inulin solution degradation be oligofructose.
2. the method utilizing immobilized enzyme micro-reactor continuous production oligofructose according to claim 1, is characterized in that, the described substrate inulin aqueous solution, and solution ph is 3.5-5.5, and the concentration of solute inulin is 5-30wt%.
3. the method utilizing immobilized enzyme micro-reactor continuous production oligofructose according to claim 1, it is characterized in that, described microreactor, be made up of cover plate (1) and Sptting plate (2), containing disconnectable micro-channel chip (3) in the groove at Sptting plate center, upper closely the wriggling of micro-channel chip (3) is carved with passage, the upper surface of passage opens wide, the lower surface of passage and side-closed, passage overall length is 50-200mm, the tangent plane of passage is class ladder structure wide at the top and narrow at the bottom, the trapezoidal long limit of class is 0.5-1.5mm, the trapezoidal minor face of class is 0.1 ~ 0.4mm, the trapezoidal height of class is 0.3-1.0mm, be provided with retaining clip between the cover plate (1) of described reactor and Sptting plate (2) to leak to prevent solution side, wherein, the material of cover plate is glass or stainless steel, and Sptting plate material is stainless steel.
4. the method utilizing immobilized enzyme micro-reactor continuous production oligofructose according to claim 3, is characterized in that, described microreactor is in advance through following process: spread upon inside cover plate by polydimethylsiloxane colloidal sol, then with sieve plate by SH-TiO
2powder is evenly distributed on polydimethylsiloxane colloidal sol, puts into 60-80 DEG C of baking oven and toasts 100-200min, colloidal sol is solidified.
5. the method utilizing immobilized enzyme micro-reactor continuous production oligofructose according to claim 4, is characterized in that, described SH-TiO
2equal proportion zooms in or out and prepares as follows: by 1g TiO
2be scattered in the toluene of 30-50ml, add 5 ~ 10ml 3-mercaptopropyl trimethoxysilane, at 110 DEG C of oil bath backflow 6-10h, by sample suction filtration, toluene wash; Sample will be leached and be placed in Soxhlet extractor, with ethanol extracting 12-24h under reflux temperature, to remove unreacted coupling agent; Take out sample after suction filtration and can obtain SH-TiO after dry 24-48h at 60 DEG C
2.
6. the method utilizing immobilized enzyme micro-reactor continuous production oligofructose according to claim 4, is characterized in that, described hole diameter of sieve (perforated) plate 800 order.
7. the method utilizing immobilized enzyme micro-reactor continuous production oligofructose according to claim 1 or 4, it is characterized in that, the method be fixed on by endo-inulinase in microreactor is as follows: endo-inulinase liquid is injected disconnectable micro-channel chip (3), flow rate control is at 1-100 μ L/min, retention time is 1-5 hour, with the enzyme liquid that ultrapure water is unnecessary, realize enzyme immobilizatio.
8. the method utilizing immobilized enzyme micro-reactor continuous production oligofructose according to claim 1 or 4, is characterized in that, described endo-inulinase, and 200-300U/mg more alive than enzyme, the supported quantity of microreactor is 50-200mg.
9. the method utilizing immobilized enzyme micro-reactor continuous production oligofructose according to claim 1 or 4, it is characterized in that, in microreactor, the enzyme of endo-inulinase is lived as 4.2KU, supported quantity is 20mg, the substrate inulin aqueous solution is injected in microreactor, substrate flow velocity is 0.1-1mL/min, temperature of reaction is 55-65 DEG C, substrate pH is at 3.5-5.5, circulate in the microreactor retention time of continuous flow of reaction feed liquid is 2h, adjustment reaction flow velocity is at 0.1mL/min again, adjustment temperature of reaction is 65 DEG C, adjustment substrate pH is 3.5, circulate in the microreactor retention time of continuous flow of reaction feed liquid is 4h, in the oligofructose prepared, the concentration of GF3 is at more than 60wt%.
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